Activity Report 2007/08 - Heinrich-Pette

Transcription

Tätigkeitsbericht
Tätigkeitsbericht 200 7/ 2008
2007/2008
Stiftung bürgerlichen Rechts
Martinistraße 52 · 20251 Hamburg
Tel.: +49 (0) 40 480 51-0 · Fax: +49 (0) 40 480 51-103
hpi@hpi.uni-hamburg.de · www.hpi-hamburg.de
Impressum
Verantwortlich
für den Inhalt
Prof. Dr. Thomas Dobner
Dr. Heinrich Hohenberg
Redaktion
Dr. Angela Homfeld
Dr. Nicole Nolting
Grafik & Layout
AlsterWerk MedienService GmbH
Hamburg
Druck
Hartung Druck + Medien GmbH
Hamburg
Titelbild
Neu gestaltete Fassade
des Seuchenlaborgebäudes
Tätigkeitsbericht
2007/2008
Heinrich-Pette-Institut
für Experimentelle Virologie und Immunologie
an der Universität Hamburg
Postfach 201652 · 20206 Hamburg
Telefon: + 49 - 40 /4 80 51- 0
Telefax: + 49 - 40 /4 80 51-103
E-Mail: hpi@hpi.uni-hamburg.de
Internet:www.hpi-hamburg.de
Das Heinrich-Pette-Institut ist Mitglied
der Leibniz-Gemeinschaft (WGL)
Internet:www.wgl.de
Inhaltsverzeichnis
Allgemeiner Überblick
Vorwort ....................................................................................................
Die Struktur des Heinrich-Pette-Instituts . .............................................
Modernisierung des HPI erfolgreich abgeschlossen .............................
60 Jahre HPI...............................................................................................
Offen für den Dialog ...............................................................................
Preisverleihungen und Ehrungen ...........................................................
Personelle Veränderungen in den Jahren 2007 und 2008 . ..................
Wissenschaftliche Highlights ..................................................................
Hohes Drittmittelaufkommen am HPI ...................................................
HPI fördert wissenschaftlichen Nachwuchs . .........................................
Gleichstellungsinitiative am HPI . ...........................................................
Das „Leibniz Center for Infection“ (LCI) . ...............................................
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Programmbereich „Virus-Wirts-Wechselwirkungen“
Program Area “Virus-Host Interaction”*
Introduction ............................................................................................. 27
Research Projects...................................................................................... 29
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elper function of wild-type p53 in Simian virus 40 induced
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cellular transformation ............................................................................
Role of c-Met in SV40-induced mammary tumorigenesis ........................
Mutant p53 gain of function in mammary carcinogenesis . .....................
A preclinical model for monitoring treatment and prevention
of metastasis in mammary carcinoma .....................................................
Induction of a cellular immune response against SV40
induced mammary carcinoma in transgenic mice ....................................
Steroid dependent control of HIV replication . .........................................
Control of viral mRNA export by adenovirus regulatory proteins .............
Identification of a new substrate of the adenovirus/E1B-55K/E4orf6
ubiquitin ligase complex .........................................................................
Human adenovirus E4 region orf3/4 protein is required
for efficient virus replication . ..................................................................
Localization and importance of the adenovirus E4orf4 protein
during lytic infection ...............................................................................
Intrinsic disorder in the amino terminus of human adenovirus
type 5 E1B-55K and its related proteins ..................................................
A novel 49K isoform of the adenovirus type 5 early region
1B 55K protein is sufficient to support viral replication ...........................
Daxx-mediated negative regulation of adenovirus
replication is counteracted by the adenovirus E1B-55K protein ...............
Posttranscriptional processing of HIV-1 mRNA . .......................................
*Titel im Inhaltsverzeichnis sind z. T. gekürzt aufgelistet.
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ombating drug resistance in HIV-1 infection .........................................
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Eradication of HIV-1 proviral DNA from patient cells ...............................
Rapid response kinetics to HLA-B57/58 Gag-restricted CTL epitopes
is associated with slow disease progression in chronic HIV-1 infection . ...
Determinants of successful antiviral treatment of concomitant
acute HCV/HIV coinfection . ....................................................................
Crossing the Species Barrier: Role of Env-Receptor Interactions
in Gammaretrovirus Infections ................................................................
Requirement of Activation for Hepatitis B Virus Infection ........................
Enzymatic treatment of duck hepatitis B virus: topology of the
surface proteins for virions and noninfectious subviral particles . .............
Entry of Duck Hepatitis B Virus into Primary Duck Liver
and Kidney Cells .....................................................................................
Elucidation of the SV40-induced DNA damage signaling cascades
and their components utilized by SV40 ...................................................
Role of the host’s p53 status in SV40 life cycle . ......................................
Elucidation of the molecular mechanisms that underlie
SV40-induced polyploidization ................................................................
Inhibition of duck hepatitis B virus infection of liver cells
by combined treatment with viral e antigen and carbohydrates ..............
Solution structure of stem-loop alpha of the hepatitis B virus
post-transcriptional regulatory element ...................................................
Analysis of Duck Hepatitis B Virus core protein phosphorylation
and dephosphorylation............................................................................
Transactivation activity of the X and PreS protein of duck hepatitis B
virus are dispensible for DHBV infection in vitro and in vivo ....................
Polyomavirus-encoded miRNAs ...............................................................
A Global Analysis of Evolutionary Conservation Among Known
and Predicted Gammaherpesvirus microRNAs .........................................
Monitoring the Epigenetic Fate of Gammaherpesvirus Genomes . ...........
A requirement for Drosha in the Maintenance of Kaposi's
sarcoma-associated Herpesvirus Latency . ................................................
A FACS-based FRET assay to analyze interactions between viral
and cellular proteins in living cells ...........................................................
Inhibition of Transferrin-Receptor Uptake by SIV and HIV Nef-alleles .......
Inefficient Nef-Mediated Downmodulation of CD3 and MHC-I
Correlates with Loss of CD4+ T Cells in Natural SIV Infection . ................
Single Nef Proteins from HIV-1 Subtypes C and F Fail
to Up-Regulate Invariant Chain Cell Surface Expression . .........................
Human Immunodeficiency Virus Type 1 Nef Expression
Prevents AP-2-Mediated Internalization of the Major
Histocompatibility Complex Class II-Associated Invariant Chain ...............
Siglec expression on chimpanzee T-cells might contribute to
the asymptomatic course of lentiviral infection in human primates .........
HIV-1 budding and persistence in macrophages ......................................
A serine phosphorylation site in HIV-1 Vpu is required
for efficient counteraction of tetherin and replication in macrophages . ..
Viral determinants for AIDS-progression in pediatric HIV-1
subtype B and C infections ...................................................................
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eplication of ICP0-Null Mutant Herpes Simplex Virus Type 1
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Is Restricted by both PML and Sp100 ......................................................
Duck Hepatitis B Virus Requires Cholesterol
for Endosomal Escape during Virus Entry ................................................
Strong and selective inhibitors of hepatitis B virus replication among
novel N4-hydroxy- and 5-methyl-beta-L-deoxycytidine analogues . ..........
Assembly and budding of a hepatitis B virus is mediated by
a novel type of intracellular vesicles ........................................................
Avian Hepatitis B Viruses: Molecular and Cellular Biology,
Phylogenesis and Host Tropism ...............................................................
Efficiency of DHBV infection is restricted by an abortive entry pathway . .
Secretion of duck hepatitis B virus occurs via exocytosis ..........................
Naturally occurring adefovir resistance mutation rtI233V
selected under adefovir therapy ..............................................................
Cellular Determinants of HBV Replication ...............................................
High resolution electron microscopical (EM)-analysis of the distribution
of TNF receptor 1 . ..................................................................................
Cryotomography Ultrastructural analysis of cytomegalovirus
tegument protein interactions . ...............................................................
Dynamic processes in organic and biological samples
investigated with pump-probe experiments ............................................
Size and Surface Effects on the MRI Relaxivity
of Manganese Ferrite Nanoparticle Contrast Agents ...............................
Real-time magnetic resonance imaging and quantification
of lipoprotein metabolism in vivo using nanocrystals ...............................
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Programmbereich „Zelluläre Dysregulation“
Research Program “Cellular Dysregulation“
Introduction ............................................................................................. 91
Research Projects .....................................................................................
A. Cellular defense mechanisms
A1. Cellular target genes of viral transformation
■ Cell transformation by human adenoviruses . ..........................................
■ RNA interference as a tool to study adenovirus
E1A/ E1B-mediated cell transformation ...................................................
■ Merkel cell polyomavirus (MCPyV) in Merkel cell carcinoma cell lines ......
A2. Apoptosis, Senescence, and Autophagy
p53-Rb2/p130 crosstalk in cellular senescence ......................................
■ Dissecting the mechanism of apoptosis-resistance
in hematopoietic malignancies expressing wild-type p53 ......................
■ FLASH meets nuclear bodies: CD95 receptor signals
via a nuclear pathway............................................................................
■ Functional p53 is required for effective telomerase inhibition
in BCR-ABL-positive CML cells in vitro ..................................................
■ Invasin- and β1-Integrin-dependent uptake of bacteria
is coupled to the activation of autophagy .............................................
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A3. Mechanisms DNA repair
■ Regulation and function of the stress-induced polymerase
α-primase-wtp53 complex ....................................................................
■ Repair fidelity of UV-induced lesions depends on ATR and
the p53 status ......................................................................................
■ In UV-damaged S-phase cells, delayed origin loading of
Pol α depends on the ATR-∆p53-p21-cyclin A-Cdk2 pathway ...............
■ Dissection of the role of SPOC1 in DNA repair ......................................
■ A role for E1B-AP5 in ATR signalling pathways during
adenovirus infection . ............................................................................
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B. Cellular controls of self-renewal, proliferation, and differentiation
B1. Gene transcription and chromatin modulation
■ Modulation of gene expression in U251 glioblastoma cells by
binding of mutant p53 R273H to intronic and intergenic sequences ..... 112
■ Mutant and wild-type p53 as modulator
of global chromatinorganization ........................................................... 113
■ P53 as a potential epigenetic modulator of gene expression ................. 113
■ Oncogenicity and expression profiling of SPOC1 in normal
and tumor tissues ................................................................................. 114
■ SPOC1: A novel PHD containing protein modulating
chromatin structure and mitotic chromosome condensation ................. 115
■ Enhanced SPOC1 expression induces chromatin instability .................... 116
■ SPOC1: A novel chromatin remodeling protein? ................................... 117
■ The RUNX1 transcription factor: A gatekeeper in acute leukaemia . ...... 119
B2. Regulators of stem cells, proliferation, and differentiation
The MADS transcription factor MEF2C is an important regulator
of monopoiesis .....................................................................................
■ Aldehyde dehydrogenase 1 (ALDH1) as a functional marker
of cancer stem cells in a mammary carcinoma mouse model ................
■ Hierarchy and p53-dependent differentiation of embryonal
stem/progenitor cells in mouse F9 teratocarcinoma cell line ..................
■ Spontaneous F9 cell differentiation in a capillary-based 3D cell culture ....
■ SPOC1 is essential for spermatogonial stem cells ..................................
■ Activated receptor tyrosine kinases and their downstream
pathways in acute myelogenous leukemia ............................................
■ Identification of genetic mutations that cooperate with constitutively
active FLT3 in the induction of acute lymphoblastic leukemia (ALL) .......
■ Differential regulation of human TERT gene expression and telomerase
activity during liver regeneration and liver cell differentiation ................
■ Regulation of human and mouse TERT promoter activity during
progression of mammary carcinogenesis ...............................................
■ Physiological role of cellular prion protein in insulin producing β-cells ...
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C. Molecular mechanisms for “fine tuning” normal cell processes
C1. Post-transcriptional and translational control mechanisms
■ Dissecting nucleocytoplasmic transport of CD83 mRNA ........................ 134
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ualitative and quantitative image analysis of ultra structural
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changes in sarcomere assembly of cMyBP-C knock-in mice . ................. 135
Translational Elongation of Cyclin D1 requires an RNA Chaperone ........ 136
The fusion protein OTT-MAL induces R-loop formation
and genomic instability ......................................................................... 137
C2. Functional regulation by protein modifications
Adenovirus type 5 early region 1 B 55-kDa oncoprotein is modified
by different isoforms of the small ubiquitin-related modifier (SUMO) . .....
■ The adenovirus E1B-55K oncoprotein induces SUMO modification of p53 ...
■ Characterization of posttranslational modifications of SPOC1 ...............
■ The ubiquitin-specific protease USP7 is a target
of the adenovirus E1B-55K oncoprotein . ..............................................
■ The MDM2 ubiquitination signal in the DNA-binding domain of p53
forms a docking site for calcium calmodulin kinase superfamily members . .
■ Homeodomain-interacting protein kinase 2 is the ionizing
radiation-activated p53 serine 46 kinase and is regulated by ATM ........
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Leibniz Center for Infection (LCI)
Ultrastructural investigation of the in vitro life cycle of Schistosoma
mansoni, analysed by cryo-techniques and TEM-tomography . .............. 143
■ Electron microscopical dissection of the exoerythrocytic
forms of Plasmodium berghei in situ ..................................................... 144
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Personelle Zusammensetzung der Wissenschaftlichen
Abteilungen, Forschungs- und Nachwuchsgruppen . .........................
Veröffentlichungen . ..............................................................................
Habilitationen, Dissertationen, Diplomarbeiten,
Masterarbeiten, Bachelorarbeiten .......................................................
Lehrtätigkeit ..........................................................................................
Gutachtertätigkeit .................................................................................
Organisation von Meetings und Chairman-Tätigkeiten . ...................
Mitherausgabe wissenschaftlicher Zeitschriften ................................
Drittmittelprojekte ................................................................................
Erfindungsmeldungen und Patente .....................................................
Finanzielle Förderung . ..........................................................................
Seminare . ...............................................................................................
Mitglieder des Wissenschaftlichen Beirats ..........................................
Mitglieder des Kuratoriums . ................................................................
Kaufmännische Abteilung ....................................................................
Personal der Kaufmännischen Abteilung und Allgemeine Dienste ....
Beauftragte ............................................................................................
Organigramm . .......................................................................................
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Gemälde von Heinrich Pette. Es schmückt nun das Foyer des HPI im
neuen Ersatz- und Erweiterungsbau II.
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Vorwort
Dieser Tätigkeitsbericht des Heinrich-Pette-Instituts (HPI) stellt die aktuellen
Entwicklungen der wissenschaftlichen Programme und der Aktivitäten der Mit
arbeiter in den Jahren 2007 und 2008 detailliert dar. Zugleich informiert das HPI
mit diesem Bericht über seine wichtigsten Forschungsergebnisse, seine zentra
len Ziele und die zukünftigen Pläne im Rahmen seiner Forschungsprogramme.
Ergänzt wird dies durch Erläuterungen zur Struktur, zu Aufgaben und Visionen
des Instituts. Ferner soll der Bericht dazu beitragen, die Ergebnisse unserer Arbeit,
die uns bewegenden dringlichen Fragestellungen und auch die Faszination virologischer Grundlagenforschung der Öffentlichkeit zu vermitteln.
Wir sind zuversichtlich, dass die in diesem Bericht dokumentierte Leistungsbilanz
der vergangenen zwei Jahre unsere Zuwendungsgeber, die Mitglieder des Kura
toriums und des Wissenschaftlichen Beirats sowie die Freunde und Förderer
des HPI von der wissenschaftlichen und gesellschaftlichen Bedeutung unseres
Instituts und unserer Forschungsarbeiten überzeugen wird.
Hamburg, im April 2009
Vorsitzender des Kollegiums
Status- und Funktionsbezeichnungen, die in diesem Dokument in der männlichen oder weiblichen Sprachform verwendet
werden, schließen die jeweils andere Sprachform ein.
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Die Struktur des Heinrich-Pette-Instituts
Das Institut ist als Stiftung bürgerlichen Rechts eine gemeinnützige und selbstständige Forschungseinrichtung und seit 1995 selbstständiges Mitglied der
Leibniz-Gemeinschaft (WGL), einem Verbund von derzeit 86 außeruniversitären Forschungsinstituten, die sich durch ihre überregionale und gesellschafts
relevante Bedeutung in Deutschland auszeichnen.
Das Institut wird anteilig finanziert durch das Bundesministerium für Gesundheit
(BMG) und die gemeinsame Forschungsförderung der Länder, vertreten durch
die Behörde für Wissenschaft und Forschung (BWF) der Freien und Hansestadt
Hamburg. Ein nicht unerheblicher Anteil des Forschungsetats wird zudem im
wettbewerblichen Verfahren eingeworben. Die Gelder stammen aus staatlichen
Forschungsprogrammen, privaten Stiftungen und der Industrie.
Das HPI hat vier permanente wissenschaftliche Abteilungen (Molekulare Viro
logie, Allgemeine Virologie, Zellbiologie und Virologie, Tumorvirologie), zwei
Forschungsgruppen (Elektronenmikroskopie und Mikrotechnologie, Molekulare Pathologie) sowie derzeit zwei unabhängige Nachwuchsgruppen (Zelluläre
Virusabwehr, Virus-Pathogenese). Eine dritte Nachwuchsgruppe (InfluenzaPathogenese) wurde Anfang 2009 besetzt und wird im Mai 2009 ihre Arbeit
aufnehmen. Daneben gibt es innerhalb der Abteilungen eine Vielzahl von
Arbeitsgruppen, die sich durch Drittmittel weitgehend selbst finanzieren. Die
Forschungsabteilungen stellen die strukturell-thematischen Basiseinheiten dar,
die in Bezug auf bestimmte humanpathogene Viren und ein spezifisches
Methodenspektrum jeweils unterschiedliche Schwerpunkte setzen und damit
eine sowohl thematische als auch technologische Kernfunktion innehaben.
Die Mischung permanenter und zeitlich begrenzter Organisationseinheiten
gewährleistet eine breite Themen- und Technologiebasis als Kernstruktur des
Instituts, die mit Forschungstätigkeiten zu aktuellen Fragen ergänzt wird.
Hierdurch erreicht das HPI ein hohes Maß an Flexibilität, ohne die Kontinuität
seines Forschungsprofils zu vernachlässigen. Diese Flexibilität begünstigt das
rasche Aufgreifen neuer Entwicklungen, was gerade im Bereich der virusasso
ziierten Infektionskrankheiten wegen des häufigen Auftretens neuer oder ver
änderter Erreger von Bedeutung ist. Die Einteilung in wissenschaftliche
Abteilungen und Gruppen wird durch zwei abteilungsübergreifende Pro
grammbereiche („Virus-Wirts-Wechselwirkungen“ und „Zelluläre Dysregu
lation“) zu einer flexiblen Matrix zusammengefasst, die den interdisziplinären
Austausch und die Kooperationen innerhalb des Instituts maßgeblich prägt.
Prof. Joachim Hauber, Abteilung „Zellbiologie und Virologie“, ist für den Pro
grammbereich „Virus-Wirts-Wechselwirkung“ verantwortlich und Dr. Carol
Stocking, Forschungsgruppe „Molekulare Pathologie“, für den Programmbereich
„Zelluläre Dysregulation“.
Gemäß seiner Satzung wird das HPI durch einen Vorstand geleitet, der zum einen
aus einem Abteilungs- oder Forschungsgruppenleiter besteht, welcher als Wissen
schaftlicher Direktor alle fünf Jahre neu gewählt wird, und dem zum anderen der
Leiter der kaufmännischen Abteilung als ein permanentes Mitglied angehört. Die
jeweiligen Vertreter komplettieren den Vorstand. Im Berichtszeitraum ist Herr
Dr. Heinrich Hohenberg wissenschaftlicher Direktor, sein Stellvertreter ist Herr
Prof. Thomas Dobner. Von der kaufmännischen Seite gehört dem Vorstand als
Leiter der Abteilung Herr Dr. Volker Uhl an, stellvertreten durch Herrn Jörg
Schinkel.
Der Vorstand legt gegenüber dem Kuratorium Rechenschaft ab, der Wissen
schaftliche Beirat sorgt für die externe Beratung, das Kollegium für die interne
wissenschaftliche Koordination. Das Kuratorium führt unter dem Vorsitz des
Kurators die Aufsicht über das Direktorium und überwacht die Geschäfte der
Stiftung im Hinblick auf Rechtmäßigkeit, Zweckmäßigkeit und Wirtschaftlichkeit.
Es berät das Direktorium in Fragen der Forschungsziele und Struktur
angelegenheiten sowie mittelfristig in der Finanz- und Wirtschaftsplanung und
der Bestellung der Mitglieder des Wissenschaftlichen Beirats. Ein externer
Wissenschaftlicher Beirat evaluiert regelmäßig die Leistungen des HPI und berät
in Fragen der wissenschaftlichen Ausrichtung bzw. der Programmbudgets. Als
internes Beratungsgremium fungiert das Kollegium, welches in einem Turnus
von etwa zwei Monaten tagt. Es ist verantwortlich für die wissenschaftliche
Entwicklung des Instituts, unterstützt bei der Erstellung der Forschungsberichte
und wirkt bei der Berufung von Abteilungs-, Forschungs- und Nachwuchs
gruppenleitern mit. Derzeitiger Leiter des Kollegiums ist Prof. Thomas Dobner,
sein Stellvertreter ist Herr Dr. Michael Schindler.
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Modernisierung des HPI erfolgreich
abgeschlossen
Im vorliegenden Berichtszeitraum konnte das Heinrich-Pette-Institut eine lange
Phase reger Bautätigkeiten und Modernisierungen erfolgreich abschließen. Zu
seinem 60-jährigen Jubiläum im Jahr 2008 präsentierte sich das Institut mit
modernen, gut ausgestatteten und lichtdurchfluteten Labor- und Büroflächen.
Auch das äußere Erscheinungsbild entspricht jetzt modernen ästhetischen und
architektonischen Standards. Der neue Ersatz- und Erweiterungsbau war bereits
im September 2006 eingeweiht worden. Während der knapp zweijährigen Bauphase konnten auf Grund der günstigen Baukonjunktur deutliche Einsparungen
erzielt werden, so dass der für den Neubau vorgesehene Investitionsrahmen von
16,15 Mio. Euro nicht ausgeschöpft werden musste. Daher bewilligten die Zuwendungsgeber, das Bundesministerium für Gesundheit und der Senat der Freien
und Hansestadt Hamburg, abschließend nochmals 1,8 Mio. Euro für die Sanierung
des alten Seuchenlaborgebäudes des Heinrich-Pette-Instituts. So konnte der dringend notwendige Modernisierungsprozess vollendet werden.
Im Inneren des alten Seuchenlaborgebäudes entstanden auf 460 qm modernste
Laborflächen, die neuesten L2-Sicherheitsstandards entsprechen. Außerdem wurden die Dachfläche und die Fassade des Gebäudes aus den 60er Jahren saniert
und dem Erscheinungsbild der neuen Institutsteile angepasst. Die Wärmedämmung der Außenflächen zur effizienteren Energieeinsparung war dem
Institut ein besonderes Anliegen. Der damalige Wissenschaftssenator Jörg
Dräger bestätigte im Jahr 2007: „Mit der Sanierung des alten Gebäudes wird der
Erneuerungsprozess der Gebäude des Heinrich-Pette-Instituts abgeschlossen.
Damit verfügt diese exzellente Forschungseinrichtung dann über eine moderne,
erstklassige Forschungsinfrastruktur.“
Das renovierte Seuchenlaborgebäude mit neu gestalteter Fassade
Unser herzlicher Dank gilt unseren Zuwendungsgebern, die die Modernisierung
des Heinrich-Pette-Instituts auch in wirtschaftlich schwierigen Zeiten unterstützt
und begleitet haben. Diese vertrauensvolle Unterstützung bestärkt uns darin,
auch in Zukunft das hohe wissenschaftliche Ansehen des Heinrich-Pette-Instituts
weiter auszubauen und die Infektionsforschung in der Metropolregion Hamburg
entscheidend mitzuprägen.
Wir danken auch der Fördergemeinschaft Kinderkrebs-Zentrum Hamburg e.V.,
die sich im Rahmen einer Public-Privat-Partnership an den Kosten des Neubaus
beteiligt hatte, und dort seit 2006 in 540 qm Laborfläche eingezogen ist. Seit zwei
Jahren hat sich mit dem Forschungsinstitut Kinderkrebs-Zentrum eine äußerst
erfolgreiche Partnerschaft im wissenschaftlichen, organisatorischen und administrativen Bereich entwickelt: zwei Forschungskooperationen entstanden, Schlüsseltechnologien werden gemeinsam genutzt und öffentlichkeitswirksame Veranstaltungen, wie die Hamburger „Nacht des Wissens“, gemeinsam durchgeführt.
60 Jahre HPI
Prof. Rudolf Jaenisch hält
die Pette-Lecture 2008
Im Jahr 2008 konnte das Heinrich-Pette-Insitut
auf sechzig erfolgreiche Jahre seiner Institutsgeschichte zurückblicken und feierte dieses Jubiläum im Rahmen einer exzellenten Pette-Lecture
mit Herrn Prof. Rudolf Jaenisch und einem
großen Sommerfest für alle Mitarbeiterinnen
und Mitarbeiter, Freunde des Instituts und deren
Familien. Das HPI ehrte Prof. Rudolf Jaenisch,
Gründungsmitglied des „Whitehead“ Instituts in
Cambridge, USA, für seine herausragenden
Arbeiten im Bereich des therapeutischen Klonens, transgener Mausmodelle und der Stammzellforschung mit der Vergabe der HeinrichPette-Lecture 2008.
Prof. Rudolf Jaenisch (1975 bis 1985 auch Abteilungsleiter am HPI) war nach
Prof. Peter K. Vogt (2006) und Prof. Thomas Shenk (2007) der dritte Ehrengast
der seit 2006 jährlich stattfindenden Heinrich-Pette-Lecture.
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Im Jubiläumssommer 2008 besuchte
auch die Hamburger Wissenschaftssenatorin Frau Dr. Herlind Gundelach das Heinrich-Pette-Institut,
um sich vor Ort umfassend über
die Forschungsschwerpunkte, die
Labore, Technologien, Strukturen
und Visionen des Traditionsinstituts
zu informieren. Begleitet wurde
sie von Staatsrat Bernd Reinert
und Mitarbeitern der Behörde für
Wissenschaft und Forschung. Die
Besuchergruppe zeigte sich beeindruckt von der Neustrukturierung
des Instituts mit hochmodernen
Technologie- und Methodenplatt- Besuch der Wissenschaftssenatorin Dr. Herlind Gundelach am
(von li nach re Dr. H. Hohenberg, Senatorin Dr. H. Gundelach,
formen und neuen attraktiven HPI
Staatsrat B. Reinert)
Arbeitsbedingungen für Nachwuchsforscher. Ein wichtiger Aspekt war die intensivierte Vernetzung wissenschaftlicher Arbeiten nicht nur innerhalb des HPI, sondern vor allem mit regionalen, nationalen und internationalen Partnern. Die Senatorin: „Forschung
funktioniert heute nur im Verbund, und ich sehe, dass Sie hier am HPI exzellente
Netzwerke knüpfen und national und international ein starker Partner in innovativen Forschungsverbünden sind.“
Offen für den Dialog
Das Heinrich-Pette-Institut stellt seine Forschung im Dialog mit der Öffentlichkeit
transparent dar und hat seine Presse- und Öffentlichkeitsarbeit seit 2007 weiter
ausgebaut. Insgesamt berichteten nationale und internationale Printmedien
im Berichtszeitraum 214mal über Forschungsergebnisse am HPI, Online-Medien
735mal und 22mal wurden wissenschaftliche Highlights des HPI in Rundfunk und
Fernsehn porträtiert. Insbesondere
die Pressemeldung zur „Molekularen
Schere gegen HIV-1“ fand weltweit
große Beachtung.
Darüber hinaus öffnet das HPI seine
Türen für interessierte Mitbürger. So
besuchten bei der 2. Hamburger
„Nacht des Wissens“ im Juni 2007
wieder mehr als 900 interessierte
Gäste, Familien, Schülerinnen und
Schüler das Institut und waren
Bericht zum 60-jährigen
Jubiläum des HPI im
NDR-Fernsehn
begeistert bei vielen Mitmachangeboten und Präsentationen unter dem Titel
„Faszination Virusforschung“ dabei. Als einen der Gäste konnte das HPI gemeinsam mit dem Forschungsinstitut Kinderkrebs-Zentrum auch den damaligen
Wissenschaftssenator Jörg Dräger begrüßen.
Bereits zweimal war der Hamburger Zweig der international tätigen Initiative
„Common Purpose“ bei Prof. Joachim Hauber am HPI zu Gast. Führungskräfte
Hamburger Firmen sowie aus Behörden und gemeinnützigen Organisationen
kamen im Rahmen eines mehrmonatigen Matrix-Programmes, um sich über
aktuelle Forschungsprojekte im Bereich HIV/AIDS und deren gesellschaftliche
Bedeutung zu informieren.
Gäste bei der „Nacht des Wissens“ am Elektronenmikroskop
Auch naturwissenschaftlich interessierte junge Talente konnten bei zwei weiteren
Veranstaltungen gezielt angesprochen werden: Das HPI beteiligte sich im
September 2007 an einem von 1100 Schülern besuchten Schülerkongress zu den
Themen Molekularbiologie, Biomedizin und Biotechnologie in Hamburg. Im
Sommer 2008 konnten dann Schülerinnen, Schüler und Studenten auf Einladung
des Heinrich-Pette-Instituts an einem Symposium zur Stammzellforschung teilnehmen. Sie lernten aktuelle Forschungsarbeiten dreier internationaler Wissenschaftler zur Leukämieforschung, Immuntherapie gegen Krebs und zu modernen
Bildgebenden Verfahren in der Stammzellforschung kennen. Der Schüler- und
Studententag fand im Rahmen des internationalen Wilsede-Meetings „Modern
Trends in Human Leukemia“ statt (mitorganisiert von Dr. Carol Stocking, HPI)
und wurde mit einem persönlichen Gespräch der jungen Menschen mit Prof. Sir
Timothy Hunt, Nobelpreisträger der Medizin 2001, abgeschlossen.
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8
Preisverleihungen und Ehrungen
Das HPI-Forschungsprojekt „Eradikation proviraler HIV-1 DNA aus Patientenzellen“ war beim GO-Bio Wettbewerb 2007 des Bundesministeriums für Bildung
und Forschung (BMBF) erfolgreich. Das Projekt unter der Leitung von Prof.
Joachim Hauber gehört zu den sieben Siegern, die im Juli 2007 bei der zweiten
Förderrunde des Wettbewerbs
aus 85 Mitbewerbern nominiert
wurden. Für die nächsten drei
Jahre stehen den Forscherteams
insgesamt 20 Millionen Euro zur
Verfügung, um eine wissenschaftliche Idee zu einem marktfähigen Produkt entwickeln zu
können; davon gehen 2,2 Mio.
Euro an das Projekt von Prof.
Hauber und seine Kooperationspartner. Die Antragstellung des
HPI profitierte insbesondere von
einer guten und engen Zusammenarbeit mit der PatentManagement-Agentur Ascenion.
Ascenion hat sich klar auf das
Gebiet der „Life-Sciences“ fokussiert und betreut das HPI in allen
Aspekten der Patentierung und
Preisverleihung zum GoBio Wettbewerb 2007 durch Ministerialrat
Kommerzialisierung ihrer For- E. Warmuth (BMBF, im Bild li) an Prof. J. Hauber (im Bild re) bei der
Biotechnica 2007
schungsergebnisse.
Prof. Joachim Hauber erhielt 2008 die Ehre, in den wissenschaftlichen Beirat der
Robert-Koch-Stiftung berufen zu werden, der u. a. unter Schirmherrschaft des
Bundespräsidenten jährlich den renommierten Robert-Koch-Preis verleiht.
Außerdem wurde er für die nächsten fünf Jahre in den Universitätsrat der
„Veterinärmedizinischen Universität Wien“ berufen.
Prof. Wolfgang Deppert wurde damit ausgezeichnet, die „Centers of Excellence
Lecture“ 2007 an der Keio University Tokio mit dem Titel „Mutant p53 Gain of
Function: in vivo Phenotype and in vitro Molecular Analyses“ halten zu können.
Er erhielt diese Ehrung für seine grundlegenden Studien zur Funktion des
Tumorsuppressors P53 und dessen mutierte Varianten bei der Kontrolle des
Zellzyklus und der Tumorgenese sowie für die Entwicklung eines transgenen
Mausmodells zur Mammakarzinogenese.
Dr. Heike Helmbold wurde im Juli 2007 mit dem Promotions-Preis des Freundesund Fördervereins Chemie der Universität Hamburg ausgezeichnet. Sie erhielt den
Preis für ihre Studien zur Dysregulation der G1-Phase, die sie im Rahmen ihrer
Promotionsarbeit in der Abteilung „Tumorvirologie“ erfolgreich abschließen konnte. Die Dysregulation der G1-Phase ist häufig der Einstieg in die Tumorentstehung.
Fast immer sind dabei
der p16INK4A-pRb und/
oder der ARF-p53 Signalweg gestört, meistens
durch Mutation oder
Deletion der Tumorsuppressorgene p16INK4A, ARF und p53. Die
Reaktivierung der Signalwege durch Rekonstitution der inaktivierten
Proteine könnte eine
Mög lichkeit sein, die
Kontrolle über die entgleiste Proliferation wieVerleihung des Promotions-Preises durch Prof. Heck (UKE) an Dr. Heike Helmbold
der herzustellen.
Im Jahr 2007 wurde erstmals der neu ausgelobte Nachwuchspreis des HeinrichPette-Instituts verliehen. Der Preis soll zukünftig aktuelle hochrangige Publikationen von Diplom- bzw. Promotionsarbeiten des Instituts fördern. Ausgezeichnet
wurden für die besten Publikationen im Jahr 2006 Barbara Fries für ihre Arbeit
„Analysis of Nucleocytoplasmic Trafficking of the HuR Ligand APRIL and its
Influence on CD83 Expression“ im Journal of Biological Chemistry und Dr. Heike
Helmbold für ihre Publikation „Regulation of cellular senescence by Rb2/p130“ in
Oncogene. Wir beglückwünschen beide für ihre hervorragenden Publikationen und
je 500 Euro Preisgeld. Das Auswahlverfahren für 2008 ist derzeit noch nicht abgeschlossen.
Dr. Gabriela B. Iwanski gewann den Jürgen Lüthje-Promotionspreis 2007. Die
Doktorandin von Dr. Carol Stocking und Prof. Klaus Harbers hatte über „Untersuchungen zur Rolle des Transkriptionsfaktors C/EBPalpha bei der normalen und
aberranten Hämatopoese mit Hilfe der RNA-Interferenz Methode“ gearbeitet und
diese Arbeiten im März 2007 mit der Promotion erfolgreich abgeschlossen. Der
vom Freundes- und Förderkreis des Universitätsklinikums verliehene und mit
1250 Euro dotierte Preis wurde am 14. Dezember 2007 überreicht.
Dr. Afra Engelmann (Forschungsgruppe „Molekulare Pathologie“) wurde 2007 mit
dem Mildred-Scheel-Stipendium der Mildred-Scheel-Stiftung für Krebsforschung
ausgezeichnet.
Prof. Vladimir Prassolow, Gastwissenschaftler der Forschungsgruppe „Molekulare
Pathologie“, gewann 2007 den EMBO Journal-Cover Contest für das beste Titelfoto
des EMBO-Journals.
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10
Personelle Veränderungen in den Jahren
2007 und 2008
Nach einer Phase begrenzter und beengter Büro- und Laborflächen in den Vorjahren, bedingt durch umfassende Baumaßnahmen, konnten die Abteilungen
und Forschungs- sowie Nachwuchsgruppen am Heinrich-Pette-Institut ab 2007
wieder vollständig besetzt werden. Insgesamt stehen dem HPI nun 30 Prozent
mehr hochmoderne Laborflächen zur Verfügung.
Adenovirusforschung am HPI etabliert
Mit Herrn Prof. Thomas Dobner gelang es dem HPI 2006 einen international
anerkannten Experten für Adenoviren zu berufen. So konnte Prof. Dobner die
Ende 2006 neu eingerichtete Abteilung „Molekulare Virologie“ mit dem Forschungsschwerpunkt humaner Adenoviren im Berichtzeitraum erfolgreich etablieren. Humane Adenoviren sind für meist harmlos verlaufende Infektionen der
oberen Atemwege und der Bindehaut des Auges verantwortlich. In Nagern können Adenoviren jedoch Tumore erzeugen. Seit Jahrzehnten dienen Adenoviren
insbesondere als Modell für grundlegende Fragestellungen zur Funktion viraler
Kontrollproteine in der Steuerung des produktiven Replikationszyklus und in der
Virus-vermittelten Zelltransformation. In ihrer Veröffentlichung im Fachjournal
Journal of Virology beschrieb die Abteilung erstmals eine Beteiligung des adenoviralen E1B-156R Proteins bei der Entartung von Nagetierzellen (J. Virol. 2007,
81: 95-105).
Ein weiterer Schwerpunkt der Abteilung liegt auf Untersuchungen der virusregulierten Vorgänge des Kerntransports und Proteinabbaus sowie auf Analysen zur
Rolle der PML-NB Zellkerndomänen (PML-NBs) in der viralen und zellulären Proliferationskontrolle. Prof. Thomas Dobner
und Dr. Barbara Härtl zeigten
erstmals, dass ein Proteinkomplex, der normalerweise Brüche
im Erbgut repariert und wichtige
Mechanismen in Zellen steuert,
während der Tumorbildung durch
Adenoviren manipuliert wird Verteilung von Mre11 (grün) und E1B-55K (rot); Wildtyp (links) und
(Oncogene, 2008, 27: 3673-3684). Mutante (rechts)
Mre11, ein Teil dieses Reparaturkomplexes, ist zentrales Ziel der Adenoviren.
Prof. Dobner ist als Mitglied der Fakultät für Mathematik, Informatik und Naturwissenschaften (MIN) seit 2007 ebenfalls im neuen Bachelor-/Masterstudiengang
der Universität Hamburg eingebunden. Die ersten Bachelorstudenten konnten am
HPI im Rahmen des Vertiefungsmoduls „Methoden der molekularen Virologie und
Zellbiologie“ bereits ihre Fallstudien und Abschlussarbeiten fertigstellen.
Nachwuchsgruppe „Virus-Pathogenese“
Ende 2007 konnte eine zweite Nachwuchsgruppe mit dem Arbeitsschwerpunkt
„Virus-Pathogenese“ am Heinrich-Pette-Institut starten. Das HPI freut sich
hierfür Dr. Michael Schindler, einen mehrfach ausgezeichneten Jungforscher aus
Ulm, an das Institut geholt zu haben. Ermöglicht wurde dies durch Fördergelder
aus dem Wettbewerbsverfahren „Pakt für Forschung und Innovation“.
Dr. Michael Schindler beschäftigt sich seit mehreren Jahren mit der Frage,
warum HI-Viren beim Menschen AIDS hervorrufen, während Infektionen mit nahe verwandten
SI-Viren bei Affen meist harmlos verlaufen. Bereits
2007 erhielt er den renommierten Paul-Ehrlich- und
Ludwig-Darmstaedter Nachwuchspreis, gefolgt vom
Deutschen AIDS-Preis für seine Erkenntnis, dass
das virale Nef-Protein hierfür verantwortlich sein
könnte. Neben der Rolle des Virusproteins Nef untersucht die Nachwuchsgruppe auch die Funktion der
akzessorischen viralen Proteine Vpu und Vpr. Da
das pathogene Potenzial eines Virus nicht nur von
viralen, sondern auch von zellulären Faktoren abDr. Michael Schindler
hängt, und die Entstehung von AIDS eine Summe
diverser Wechselwirkungen ist, versucht die Forschungsgruppe zudem grundlegende Unterschiede im Immunsystem infizierter Wirte zu identifizieren, die
den Krankheitsverlauf beeinflussen.
Wissenschaftliche Koordination und Öffentlichkeitsarbeit
Im Jahr 2007 konnte das HPI eine Stelle für einen Wissenschaftlichen Koordinator/
Vorstandsreferenten schaffen. Sie wurde im April 2007 mit Dr. Jörg MaxtonKüchenmeister besetzt, der zum Januar 2008 als Generalsekretär an die Hamburger Akademie der Wissenschaften wechselte. Im Hinblick auf zunehmende
Aufgaben im Bereich der Öffentlichkeitsarbeit und wissenschaftskommunikativen Aktivitäten hat das HPI die Vorstandsreferentenstelle im Jahr 2008 halbiert
und so zusätzlich eine halbe Stelle für Presse- und Öffentlichkeitsarbeit eingerichtet. Die verbliebene halbe Vorstandsreferentenstelle und eine halbe LCIKoordinatorenstelle, deren Finanzierung die Stadt Hamburg zunächst für zwei
Jahre übernahm, hat seit 2008 Frau Dr. Nicole Nolting in Personalunion inne.
Frau Dr. Nolting war vorher als wissenschaftliche Mitarbeiterin an den Universitäten Bochum und Göttingen beschäftigt und hat dort u. a. an der Entwicklung der Graduate School-Konzepte mitgewirkt und die Bewerbung innerhalb
der Exzellenzinitiativen unterstützt. Als Referentin für Presse- und Öffentlichkeitsarbeit konnte das HPI Frau Dr. Angela Homfeld gewinnen, die diesen Bereich
am Institut bereits seit 2004 als freie wissenschaftliche Mitarbeiterin betreut hatte.
Das Institut strebt eine Wiederaufstockung der Vorstandsreferentenstelle auf eine
volle Stelle an, ohne die Stellen für Öffentlichkeitsarbeit/wissenschaftskommunikative Aktivitäten und für die LCI-Koordination zu beschneiden.
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12
Wissenschaftliche Highlights
Grundstein für neuartige HIV-Therapie
Das Highlight des Jahres 2007 war zweifellos die hervorragende Arbeit von Dr.
Ilona Hauber und Prof. Joachim Hauber sowie ihren Kooperationspartnern zur
„Eradikation proviraler HIV-1 DNA aus Patientenzellen“ mittels einer maßgeschneiderten Rekombinase. Die in Science (Sarkar et al., 2007, 316: 1912-1915)
veröffentlichte Studie ermöglicht erstmals die quantitative Entfernung chromosomal-integrierter Proviren aus HIV-infizierten Zellen und bietet somit ein neuartiges biotechnologisches Verfahren in der HIV-Therapie. Besondere Beachtung
fand die Publikation auch in Form eines von Alan Engelmann geschriebenen
zweiseitigen Kommentars in Science.
Das Ausgangsmaterial für die neue Methode war eine natürlich vorkommende
Cre-Rekombinase des Bakteriophagen P1. Sie katalysiert spezifische symmetrische Sequenzen von 34 bp Länge, die dem HIV-Erbgut nur entfernt ähnlich
sind. Der Kooperationspartner am Max-Planck-Institut für Molekulare Zellbiologie und Genetik in Dresden,
Dr. Frank Buchholz, entwickelte
daraus mit Hilfe der molekularen Evolution in 126 Zyklen
eine neue hochspezifische TreRekombinase, die eine natürlich
vorkommende asymmetrische
HIV-1 LTR-Sequenz erkennt und
rekombiniert. Die Gruppe am
HPI zeigte, dass die gentherapeutisch eingeschleuste TreRekombinase in infizierten Zellkulturen etwa zehn Wochen
benötigt, um alle Proviren aus Dr. Frank Buchholz, Dr. Ilona Hauber und Prof. Joachim
(von li nach re)
der Kultur zu entfernen.
Hauber
1
HIV Lebenszyklus
2
HIV Entfernung
Infektion
Tre Rekombinase
3
Integration
Therapierte Wirtszelle
Virusvermehrung
Eradikation proviraler HIV-1 DNA aus infizierten Zellen (Grafik: Dr. F. Buchholz, MPI Dresden)
Blockade bei HIV-Infektionen entschlüsselt
Wissenschaftler des Heinrich-Pette-Instituts entdeckten eine Blockade in ruhenden Blutzellen, die eine Vermehrung des AIDS-Erregers HIV-1 unterbindet. Der
zentrale Schalter dieser Blockade ist ein zelluläres Protein, der Glucocorticoidrezeptor (GR). Dr. Klaus Wiegers, Dr. Wolfgang Bohn und Kollegen des HPI
veröffentlichten ihre Ergebnisse in der online-Ausgabe des Fachjournals Virology
(Wiegers et al., 2008, 375: 73-84). Während das HI-Provirus in aktivierten
Blutzellen in den Zellkern transportiert und im Erbgut eingebaut wird, bleiben
die Proviren in ruhenden Blutzellen im Zytoplasma und werden nach wenigen
Stunden abgebaut. Die HPI-Forscher überwanden diese frühe Blockade der HIVInfektion in ruhenden, nicht ausgereiften Blutzellen, indem sie den Glucocorticoid-Rezeptor aktivierten. Sie zeigten erstmals, dass Steroide die Blockade in
diesen Zellen aufheben können, und vermuten, dass sich bei Stress und Steroidtherapien die Anzahl der Blutzellen erhöht, die für eine produktive HIV-Infektion
empfänglich sind.
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14
Nef schützt Affen vor T-Zell-Verlust
Während HIV-1 bei Menschen die Immunschwäche AIDS hervorruft, verlaufen
Infektionen mit dem nahe verwandten SI-Virus in Mangabenaffen meist harmlos. In seinen früheren Studien zeigte Dr. Michael Schindler (Nachwuchsgruppe
„Virus-Pathogenese“), dass hierfür das virale Nef-Protein mitverantwortlich ist.
Beide Virusarten besitzen dessen Erbinformation, Nef-Allele aus HIV und SIV
kodieren aber offensichtlich für unterschiedliche Funktionen. Im Juli 2008 beschrieben Dr. Michael Schindler und Prof. Frank Kirchhoff (Universität Ulm) im
renommierten online-Magazin PLoS Pathogens (2008;doi:10.1371/ppat.1000107),
wie Nef vor einer tödlichen Immunschwäche schützt. Außerdem zeigten sie erstmals, dass dieser Schutz tatsächlich eine Rolle spielt, wenn Mangabenaffen in
freier Wildbahn infiziert werden. Nur Nef aus SIV-infizierten Tieren mit einem
stabilen Immunsystem kann vor einem Verlust von T-Zellen schützen.
MEF2C moduliert Blutzellen
Veränderungen in Genen für Transkriptionsfaktoren sind maßgeblich an der Entstehung von akuten Leukämien beteiligt. Diese Transkriptionsfaktoren werden
u.a. durch Zytokine gesteuert und regulieren im Knochenmark die Entstehung
von reifen Blutzellen aus hämatopoetischen Vorläufer- oder Stammzellen. Dr.
Carol Stocking und Mitarbeiter ihrer Forschungsgruppe „Molekulare Pathologie“
entdeckten, dass die Regulation des Gens für den MEF2C-Transkriptionsfaktor in
akut myeloischen Leukämien des Kindesalters verändert ist. MEF2C reguliert
normalerweise die Entwicklung muskulärer und kardiovaskulärer Gewebe. In
Blood (2008, 111: 4532-4541) zeigte die Forschungsgruppe, dass das MEF2C-Gen
auch in hämatopoetischen Stammzellen, bei der Differenzierung reifer myeloischer und lymphoider Blutzellen sowie in bestimmten AML-Zellen aktiviert
ist. In diesen AML-Zellen steht der Transkriptionsfaktor unter der Kontrolle
des leukämischen Fusionsproteins MLL-ENL.
Hepatitis B Viren und intrazelluläre Vesikel
Dr. Mouna Mhamdi, Dr. Hüseyin Sirma und Kollegen des HPI konnten massive
Umstrukturierungen nachweisen, die im Verlauf einer hepadnaviralen Infektion in
Hepatozyten entstehen. Dies äußert sich in der Bildung zahlreicher
intrazellulärer Vesikel (VCV) unterschiedlicher Größe. Diese intrazellulären Vesikel sind dynamische Strukturen und unterliegen sowohl
homo- als auch heterotypischer Fusion. Ein Teil der VCV leitet sich
wahrscheinlich von der äußeren Kernmembran und vom rauhen
endoplasmatischen Retikulum (rER) ab. Die HPI-Forscher zeigten, dass
ein Teil der VCV sowohl Virionen als auch subvirale Partikel (SVPs)
enthalten. Erstmals konnte die lang diskutierte Frage beantwortet werden, ob die für Hepatitis B Viren typischen SVPs über denselben
Morphogeneseweg formiert und warum sie im Überschuss produziert Intrazelluläre Vesikel
(Bild: Dr. H. Hohenberg)
werden. Die Daten wurden in Hepatology (2007, 46: 95-106) publiziert.
Transgenes Mausmodell zu Mammakarzinomen
Wissenschaftler der Abteilung „Tumorvirologie“ entwickelten ein transgenes
Mausmodell, mit dem die Rolle von Punktmutationen im p53-Gen während einzelner Stadien der Mammakarzinogenese und der Entstehung von Metastasen
untersucht werden kann. Dieses Modell, in dem in erwachsenen Mäusen durch
Anschalten der Tumor-auslösenden SV40 Proteine Brusttumore induziert werden
können, ermöglicht erstmals quantitative Aussagen zur Funktion von p53-Mutanten in einer definierten isogenen Kohorte. In ca. 30% aller Brusttumore bei
Menschen finden Kliniker veränderte dysfunktionelle P53-Proteine, denen definierte Punktmutationen im p53-Gen zugrunde liegen. Mit Hilfe ihres neuen
Mausmodells können Prof. Wolfgang Deppert und seine Kollegen untersuchen, wie
sich analoge Punktmutationen in Brustdrüsen der Maus auf den Krankheitsverlauf
und die Bildung von Metastasen auswirken. Die Forscher stellten hierfür in transgenen Mäusen p53-Mutanten unter Kontrolle eines Brustdrüsen-spezifischen
Promotors und kreuzten diese Mäuse in die oben beschriebenen SV40-Mäuse ein.
Erste funktionelle und histopathologische Befunde zeigten, dass p53-Mutanten bei
der Progression der Mammakarzinome eine wichtige Rolle spielen und mit einem
erhöhten metastatischen Potenzial der Tumorstammzellen korrelieren. Die
Wissenschaftler konnten ihre Studien im International Journal of Cancer veröffentlichen (Heinlein et al., 2008, 122: 1701-1709).
Darstellung funktionalisierter Nanosomen
Dr. Heinrich Hohenberg und Dr. Rudolf Reimer ist erstmals die Darstellung so
genannter funktionalisierter aggregierter Nanopartikel im Transmissions-Elektronenmikroskop gelungen. In einem Gemeinschaftsprojekt unter Leitung von Prof.
Horst Weller (Universität Hamburg) wurden spezifizierte superparamagnetische
MnFe2O4-Nanopartikel für die Magnetresonanz-Tomograpie entwickelt. Die HPIForscher konnten diese mit Liposomen und Micellen aggregierten Nanosomen in
ultradünnen Carbo-Cell-Mikrokapillaren elektronenmikroskopisch in ihrer Dynamik darstellen. Die Ergebnisse wurden im Juli 2007 in Nanoletters publiziert
(Tromsdorf et al., 2007, 7: 2422-2427).
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Hohes Drittmittelaufkommen am HPI
Das hohe Drittmittelaufkommen des HPI bewegt sich in den letzten Jahren durchschnittlich auf einem Niveau von 22% bezogen auf die Grundfinanzierung. Nach
Eliminierung der Bauinvestitionen erreicht das Institut sogar einen Drittmittelanteil
von fast 30%, so dass es damit einen Spitzenplatz innerhalb der Leibniz-Gemeinschaft erreicht. Drittmittelprojekte, die in den Jahren 2007 und 2008 besondere
Beachtung gefunden haben, sind im Folgenden aufgelistet.
HIV-Forschung
Prof. Joachim Hauber gewann 2007 den vom BMBF geförderten GO-Bio-Wettbewerb und konnte für die nächsten drei Jahre 2,2 Mio. Euro für das Kooperationsprojekt „Eradikation proviraler HIV-1 DNA aus Patientenzellen“ einwerben. Mit Hilfe der Fördergelder soll die neu entwickelte „Molekulare Schere“, die
Tre-Rekombinase, optimiert und von der Grundlagenforschung in ein anwendbares Produkt weiter entwickelt werden. Zunächst wird die Herstellung der
Tre-Rekombinase vereinfacht und optimiert. Da die Rekombinase mittels somatischer Gentherapie in Zellen eingebracht wird, müssen hierfür außerdem geeignete Vektoren gefunden und verbessert werden. Anschließend wird das Enzym im
Tiermodell auf seine Verträglichkeit und Wirksamkeit hin untersucht. Ziel ist es,
eine Methode zu entwickeln, durch die hämatopoetische Stammzellen aus AIDSPatienten isoliert, mit dem Rekombinasegen-Vektor behandelt und anschließend
wieder in den Blutkreislauf der Patienten zurückgeführt werden können.
Prof. Joachim Hauber ist neben
Prof. Tim Brümmendorf Koordinator des Verbundvorhabens
„Combating Drug Resistance
in Chronic Myeloid Leukemia
and HIV-1 Infection“, für das im
BMBF-Förderverfahren „Innovative Therapieverfahren auf
molekularer und zellulärer Basis“
für die nächsten drei Jahre ebenfalls 2,1 Mio. Euro bewilligt
wurden. Teilnehmer an diesem Projektgruppe „Combating Drug Resistance“
Forschungsverbund sind neben
dem HPI Arbeitsgruppen des Universitätsklinikums Hamburg-Eppendorf und
der Universitäten Hamburg und Lübeck.
Ein kürzlich bewilligter DFG-Forschungsantrag der Nachwuchsgruppe „VirusPathogenese“ unter Leitung von Dr. Michael Schindler mit einem Umfang
von etwa 300T Euro soll klären, wie HIV-1 im Gegensatz zu nahe verwandten
SI-Viren und HIV-2 die Eisenaufnahme infizierter Zellen verstärkt.
Mechanismen der Onkogenese
Dr. Carol Stocking, Leiterin der Forschungsgruppe „Molekulare Pathologie“ am
HPI, erhält in einer neuen Förderrunde 0,3 Mio. Euro von der José Carreras
Stiftung. Seit der ersten Förderrunde im Jahr 2002 würdigt und unterstützt die
Stiftung ihre Forschungsarbeit mit annähernd 1 Mio. Euro. Inhalt des aktuellen
Projekts ist die Bedeutung der MEF2C und MEF2D Transkriptionsfaktoren bei
der Entstehung der akuten Leukämie.
Eine der häufigsten molekularen Veränderungen in der Akuten Myeloischen
Leukämie (AML) ist die FLT3-Mutation. Frau Dr. Stocking und ihre Kollegen
der Forschungsgruppe „Molekulare Pathologie“ sind Teil eines nun verlängerten
Verbundprojektes, in dem Onkogen-Netzwerke und FLT3-abhängige Signalwege
in der AML entschlüsselt werden sollen. Das Verbundprojekt wird bis 2011 mit
insgesamt 2,76 Mio. Euro gefördert.
Die Erich und Gertrud Roggenbuck-Stiftung zur Förderung der Krebsforschung
und die Wilhelm-Sander-Stiftung unterstützen die Abteilung „Molekulare Virologie“ des HPI mit jeweils einer Promotionsstelle zuzüglich Sachmitteln zum
Studium molekularer Mechanismen der viralen Onkogenese.
Die „German Israeli Foundation“
fördert ein Projekt der Abteilung
„Tumorvirologie“ unter Leitung von
Prof. Wolfgang Deppert zu weiteren
Studien von mutantem p53; der Forschungsverbund der Krebshilfe fördert Arbeiten zur „Beteiligung von
Tumorstamm-/Progenitorzellen am
Aufbau ihrer zellulären Umgebung
und der metastatischen Nische“.
TIME (Disseminated Tumor Cells
as Targets for Inhibiting Metastasis
of Epithelial Tumors) heißt ein neues
vom Verein zur Förderung der KrebsTIME-Projektgruppe
forschung e.V. gefördertes Verbundprojekt unter Leitung des Universitätsklinikums Hamburg-Eppendorf (UKE).
Prof. Wolfgang Deppert erhält als einer der Partner über drei Jahre eine zusätzliche Wissenschaftlerstelle sowie Sachmittel.
Nanotechnologie in der Tumordiagnostik
„TOMCAT“ ist ein interdisziplinäres Konsortium aus sechs Hamburger und
Schleswig-Holsteiner Forschungsinstituten und einem Industriepartner (Philips
Forschungslaboratorien, Hamburg), an dem die Forschungsgruppe „Elektronenmikroskopie und Mikrotechnologie“ unter Leitung von Dr. Heinrich Hohenberg
beteiligt ist. Es hat sich zum Ziel gesetzt, unter Einsatz neuester nanotechnologischer Verfahren in der Magnetresonanztomographie, hochspezifische, zielge-
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richtete Kontrastmittel zu entwickeln, die auf molekularer Ebene im Millimetermaßstab kleinste Gewebeveränderungen oder Metastasen erkennen können. Die
Wissenschaftler verwenden hierfür so genannte SPIOs. Das sind superparamagnetische Eisenoxid-Partikel, die nur wenige Nanometer groß sind und an spezifische Tumormarker gekoppelt werden können. Im Rahmen des Projektes untersucht die HPI-Forschungsgruppe, wie diese Nanopartikel an Zellen und Gewebe
binden, wie sie abgebaut oder eingelagert werden. So soll neben der makroskopischen Sichtbarmachung der Tumoren mittels MRT auch die Wirkungsweise der
Kontrastmittel im Mikro- und Nanokosmos auf zellulärer und molekularer
Ebene analysiert werden. Das Bundesministerium für Bildung und Forschung
(BMBF) fördert „TOMCAT“ mit 2,8 Mio. Euro, von denen allein 400T Euro ans
HPI zur Forschungsgruppe von Herrn Dr. Hohenberg fließen.
Antikörper-gekoppelte SPIOs für die Krebsdiagnostik
(Grafik: Roche, modifiziert durch R. Ittrich, UKE)
Bildgebende Verfahren am HPI
Im Rahmen des Ausbaus der strukturanalytischen Forschung im Bereich Infektionskrankheiten am Standort Hamburg erhält das HPI ein tomographisches
Cryo-Elektronenmikroskop (CTEM), mit dem Krankheitserreger in intakten
Zellen und Geweben lebensnah und räumlich dargestellt werden können. Das
rund 800T Euro teure Großgerät soll 2009 in Betrieb gehen. Dr. Heinrich Hohenberg konnte das Gerät im Rahmen des „Paktes für Forschung und Innovation“
erfolgreich einwerben.
HPI fördert wissenschaftlichen Nachwuchs
Die Ausbildung wissenschaftlichen Nachwuchses nimmt am HPI traditionsgemäß einen wichtigen Platz ein. Der Schwerpunkt liegt dabei bei der Qualifizierung
Postgraduierter. Seit 2007 hat sich Prof. Thomas Dobner darüber hinaus erfolgreich am Bachelor-/Masterprogramm der MIN-Fakultät der Universität Hamburg
eingebracht und bietet innerhalb des Lehrplans Module zur Molekularen Virologie an. Mit einer deutlich gestiegenen Betreuung von Praktikanten (39 in den
Jahren 2007 und 2008) kommt das Institut dem gestiegenen Interesse junger
Menschen entgegen, die vor ihrer Entscheidung für eine naturwissenschaftliche
Ausbildung Einblick in den Laboralltag nehmen möchten.
Neue Aus- und Weiterbildungskonzepte werden im Folgenden erläutert:
Mobilitätsprogramm
Im Jahr 2007 hat der Vorstand des HPI ein Mobilitätsprogramm für Doktoranden initiiert. Im Rahmen dieses Programms gewährt das HPI Stipendien von
5000 Euro, um Doktoranden einen Forschungsaufenthalt in einem ausländischen
Labor zu ermöglichen. Ein weiteres Ziel des Mobilitätsprogramms ist es, jungen
Forschern aus dem Ausland die Möglichkeit zu geben, für begrenzte Zeit am
HPI zu arbeiten.
Zwei junge Wissenschaftlerinnen des HPI wurden bereits im Rahmen dieses
Programms gefördert, um in ausländischen Forschungseinrichtungen neueste
Techniken und Methoden zu erlernen: Birte Niebuhr für einen Aufenthalt in Los
Angeles (Arbeiten zur Nutzung der SNP-ChIP-Technologie zur Analyse von Genveränderungen während der Tumorgenese) und Sabrina Schreiner für einen
Aufenthalt in Montreal (genetische und biochemische Untersuchungen eines
adenoviralen Ubiquitinligase-Komplexes). Zusätzlich erhielt Alession Medeyski,
Doktorand an der Universität Neapel, Unterstützung für einen Aufenthalt am
HPI bei Dr. Wolfgang Bohn.
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HPI-Nachwuchsgruppen
Mit der Einrichtung von Nachwuchsgruppen initiierte das HPI ein Programm
zur Förderung von Nachwuchswissenschaftlern, das den Leitern dieser Forschungsgruppen zu einem frühen Zeitpunkt der wissenschaftlichen Karriere die
Möglichkeit bietet, in höchstem Grad selbstständig zu arbeiten. Zudem versetzt
es das Institut zusätzlich in die Lage, flexibel und schnell auf neue strategischwissenschaftliche Anforderungen einzugehen sowie neueste Forschungsmethoden
und Techniken zu etablieren.
Neben Dr. Adam Grundhoff, der die Nachwuchsgruppe „Zelluläre Virusabwehr“
führt, ist es dem Vorstand des HPI 2007 gelungen, Dr. Michael Schindler vom
Institut für Virologie des Universitätsklinikums Ulm als Nachwuchsgruppenleiter
für die neue Arbeitsgruppe „Virus-Pathogenese“ des HPI zu gewinnen. Anfang
des Jahres 2009 konnte auch die dritte Nachwuchsgruppe mit Dr. Gülsah Gabriel,
derzeit an der Universität Marburg (mit Gastaufenthalt an der Universität
Oxford), erfolgreich besetzt werden. Frau Dr. Gabriel wird im Mai 2009 ihre
Arbeit beginnen und trägt dazu bei, das virologische Forschungsfeld des HPI mit
dem Thema Influenzaviren zu erweitern. Ihre wissenschaftlichen Arbeiten belegen ein hohes Maß an wissenschaftlichem Potenzial, und das HPI ist glücklich,
die dritte Nachwuchsgruppe nun unter die Leitung einer Wissenschaftlerin
stellen zu können.
LCI Graduate School
„Modelsystems of Global
Infectious Diseases”
Das HPI ist über seine Beteiligung am Forschungsverbund Leibniz Center for
Infection (LCI) auch in der
LCI Graduate School engagiert und leistet damit einen
wesentlichen Beitrag zu einer
hochqualifizierten Ausbildung
wissenschaftlichen
Nachwuchses. Die interdisziplinäre
Graduiertenausbildung startet
im Mai 2009. Das Programm Die Stipendiaten der Leibniz-Graduate-School mit Dr. Nicole Nolting (li),
vernetzt die führenden Insti- Prof. Dr. B. Fleischer (BNI, 2.v.re.), Dr. Heinrich Hohenberg (re)
tute im Bereich der Infektionsforschung im Raum Hamburg: die drei LeibnizInstitute Bernhard-Nocht-Institut für Tropenmedizin (BNI), Forschungszentrum
Borstel (FZB) und das Heinrich-Pette-Institut (HPI), weltweit anerkannte
Einrichtungen für Grundlagenforschung zu globalen Infektionskrankheiten.
Außerdem beteiligt sich das Universitätsklinikum Hamburg-Eppendorf an der
Graduiertenschule. Die Förderung stammt aus Mitteln des Paktes für Forschung
und Innovation. Die Leibniz-Graduate School bietet den Stipendiaten ein
anspruchsvolles, interdiziplinäres Lehr- und Ausbildungsprogramm, in dem sie
neben ihrem eigenen Forschungsthema intensive Einblicke in Fragestellungen und
Methoden der Parasitologie, Bakteriologie, Virologie und Immunologie erhalten.
Dabei befassen sich die jungen Wissenschaftler mit hochaktuellen und bedeutenden Problemen der Infektionsforschung.
Neben dem engen Mentoring durch zwei Betreuer und neben regelmäßigen
Weiterbildungen in wissenschaftlicher Theorie und Praxis, präsentieren sich die
Stipendiaten bei Fortschrittsberichten und Seminaren in englischer Sprache. Des
Weiteren wird das Programm ergänzt durch Kurse in Projektentwicklung, Drittmitteleinwerbung, Dokumentation, Präsentationstechniken und Wissenschaftskommunikation. Die Graduierten besuchen wissenschaftliche Tagungen und verbringen einen mehrwöchigen Forschungsaufenthalt in einem Labor im Ausland.
Ferdinand-Bergen-Stipendium
Die Ferdinand-Bergen-Stiftung unterstützt seit 2001 die Forschung am HeinrichPette-Institut. Seit 2008 fördert die Ferdinand-Bergen-Stiftung darüber hinaus
besonders begabte und leistungsbereite Doktoranden der Naturwissenschaften
und Medizin im Rahmen eines Ferdinand-Bergen-Stipendiums. Die Stipendiaten
erhalten eine dreijährige finanzielle Grundförderung auf Basis einer halben Stelle
und werden regelmäßige Workshops des HPI sowie des „Leibniz Center for
Infection“ (LCI) besuchen. Nach der Sichtung vieler hervorragender Bewerbungen
wurde das Stipendium an Christine Henning aus der Nachwuchsgruppe „Zelluläre
Virusabwehr“ vergeben.
Das HPI ermuntert Diplomanden und Doktoranden, verstärkt Fachtagungen zu
besuchen, und schafft durch finanzielle Unterstützung einen zusätzlichen Anreiz.
Seit Ende 2008 wird ein Reisekosten-Zuschuss von 250 Euro für die aktive Teilnahme (Poster, Vortrag) an wissenschaftlichen Fachtagungen gewährt, der schon
nach kurzer Zeit zu einer erhöhten Teilnahme von vor allem jungen HPI-Mitarbeitern an internationalen Fachtagungen geführt hat.
HPI-interner Sprachunterricht für deutsche und
ausländische Mitarbeiter
Seit 2007 ermöglicht das HPI seinen Mitarbeitern die Teilnahme an ganzjährigen
Sprachkursen (für Deutsche: Englisch-Sprachkurse, für Ausländer: DeutschSprachkurse), die im Institut abgehalten werden. Der leistungsgestaffelte Sprachunterricht soll die englischsprachige Kommunikation innerhalb des HPI verbessern. Die Deutschkurse helfen interessierten ausländischen Mitarbeitern,
deutsche Spachkenntnisse zu erwerben oder zu erweitern und ihre Kommunikation am Arbeitsplatz zu verbessern.
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Gleichstellungsinitiative am HPI
Das 60-jährige Jubiläum des HPI gab Anlass, die Förderung von Frauen als Mitarbeiterinnen in der Wissenschaft, Verwaltung und Dienstleistung des Instituts
weiter zu stärken. Beim „Frauentag am HPI“ am 29. Mai 2008 wurden Ideen und
Konzepte hierfür entwickelt. Anregung erhielten die Kolleginnen des HPI durch
Sylvie Faverot-Spengler, die Gleichstellungsbeauftragte am DESY, die über ihre
langjährige Erfahrung in der Frauenvertretung und Umsetzung von Fördermaßnahmen am DESY berichtete.
Anlässlich der WGL-Jahrestagung 2008 bekannte sich das Heinrich-Pette-Institut
in Form einer Selbstverpflichtung zu den inhaltlichen Zielen der „forschungsorientierten Gleichstellungsstandards“ der Deutschen Forschungsgemeinschaft.
Der Vorstand des HPI startete in enger Zusammenarbeit mit der neu gewählten
Gleichstellungsbeauftragten, Alicja Iwanski, eine Initiative zur Verbesserung der
Familienförderung und Gleichstellung von Frau und Mann am Arbeitsplatz.
Ein Ziel dieser Aktivitäten war es, sich am 30. Januar 2009 für das „TOTAL
E-QUALITY“ Prädikat zu bewerben. Der Verein „TOTAL E-QUALITY
Deutschland“ zeichnet Unternehmen, Organisationen, Hochschulen und Forschungseinrichtungen, die eine an Chancengleichheit orientierte Personalpolitik verfolgen, mit diesem Prädikat aus.
Das HPI hat hierfür einen Gleichstellungsplan erarbeitet, in dem unter anderem
die Notfallbetreuung für Kinder (0 bis 12 Jahre) gewährleistet wird. Dazu hat das
HPI eine Vereinbarung mit der „Vereinigung Hamburger Kindertagesstätten
GmbH“ abgeschlossen. Seit Juni 2008 werden alle Beschäftigten, die sich in
Elternzeit oder in der Beurlaubung befinden, außerdem auf Wunsch in den
allgemeinen Informationsfluss des HPI eingebunden. Das Informationsangebot
wird aktiv genutzt.
Die HPI-Gleichstellungsbeauftragte Alicja Iwanski (Mitte) mit ihrer Stellvertreterin Ute Neumann (li) und
Leibniz-Präsident Prof. E. Rietschel beim Leibniz-Workshop „Chancengleichheit“
Das „Leibniz Center for Infection“ (LCI)
Das „Leibniz Center for Infection“ ist die erste wissenschaftliche Allianz innerhalb der Leibniz-Gemeinschaft, die Forschungsaktivitäten mehrerer regionaler
Leibniz-Institute unter einem Dach vereint. Das Bernhard-Nocht-Institut, das
Forschungszentrum Borstel sowie das Heinrich-Pette-Institut nehmen durch die
Gründung dieses Forschungsverbundes eine Vorreiterrolle bei der Schaffung
neuer wissenschaftlicher und öffentlichkeitswirksamer Konzepte in der LeibnizGemeinschaft ein. Sie stärken dadurch die Infektionsforschung im norddeutschen Raum, reagieren gemeinsam auf aktuelle infektiologische Probleme und
Fragestellungen und sichern gleichzeitig ihre internationale Spitzenposition auf
diesem Gebiet.
Das LCI koordiniert und unterstützt gemeinsame wissenschaftliche Projekte,
ermöglicht eine gemeinsame Öffentlichkeitsarbeit im Bereich der Infektionsforschung und gewährleistet gleichzeitig die Beibehaltung der individuellen Profile und Forschungsthemen der beteiligten Institute. Innerhalb eines thematisch
zentrierten Leibniz-Zentrums können sich die beteiligten Institute mit mehr
Gewicht und fokussierter in regionalen und deutschlandweiten Verbünden
präsentieren und zudem in neuen Forschungsallianzen wie Exzellenzclustern,
Transregio-Anträgen, SFBs oder zukünftig auch europaweit in Projekten der
EU-Forschungsrahmenprogramme engagieren. Solche Beteiligungen können
durch das LCI zentral, längerfristig und auf infektiologische Themen zugeschnitten organisiert werden und ermöglichen eine institutsübergreifende
strategische Forschungsplanung, mit dem Potenzial, schnell thematisch passende
neue Verbünde zu identifizieren und entsprechend koordiniert aktiv zu werden.
Gemeinsame Projekte des LCI
Seit 2007 finden innerhalb des LCI Workshops statt, die sich mit spezifischen
technologischen Schwerpunkten der Institute beschäftigen und so die Schaffung
institutsübergreifender Technologieplattformen unterstützen. Hierbei geht es um
das Vermitteln und Erlernen von Methoden und Technologien, die für unterschiedliche infektiologische Fragestellungen genutzt werden können. Dadurch wird
nicht nur das wissenschaftstechnologische Arbeits- und Know-how-Spektrum
eines jeden Instituts im Rahmen der Nutzung gemeinsamer Ressourcen erweitert.
Vielmehr tragen institutsübergreifende Technologieplattformen dazu bei, international kompetitive Hochtechnologien und einen entsprechenden Gerätepark
zur Verfügung zu stellen, der z.B. ein weites Spektrum an Imaging- und Screeningverfahren abdeckt.
Aktuell wird das Konzept der gemeinsamen Hochtechnologieplattformen durch
die Installation eines tomographischen Cryo-Elektronenmikroskops (CTEM) im
HPI umgesetzt. Für die Anschaffung dieses Großgerätes wurden, im Rahmen des
„Paktes für Forschung und Innovation“ federführend durch das HPI, in einem
durch gemeinsame Projekte der LCI-Partner unterstützten Forschungsantrag
Mittel in Höhe von 800T Euro eingeworben. Zukünftige Planungen sehen weitere
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Kooperationen im Bereich des High-Throughput-Screening, der konfokalen LaserScanning Mikroskopie und der Zellsorting-Technologien vor. Auch die Bereitstellung und Verwendung unterschiedlichster Modellsysteme und Organismen
(3D-Zell- und Gewebesysteme, Versuchstiermodelle), verbunden mit entsprechender Expertise, sollen zukünftig verstärkt in Angriff genommen werden. Bereits
vorhandene Projekte sind in diesem Tätigkeitsbericht ausgewiesen.
Regelmäßige Treffen der Entscheidungsträger der einzelnen Institute stellen sicher,
dass neben den institutsspezifischen wissenschaftlichen Strategien eine übergreifende LCI-Strategie existiert, die sich vor allem mit den Fragen gemeinsamer
Förderungen und verstärkten Zusammenarbeiten beschäftigt. Die gegenseitige
Beteiligung der Partner an Beiratsbegehungen unterstützt dieses Vorhaben. Aus
dieser Strategie heraus haben sich bereits kürzlich gemeinsame SFB-Anträge
sowie Anträge um Mittel der Exzellenzclusterinitiative der Freien und Hansestadt
Hamburg ergeben. Mittel- und langfristig sollen durch die Abstimmung gemeinsamer LCI-Konzepte einerseits Wettbewerbsvorteile für die einzelnen Institute im
Bereich der Drittmitteleinwerbung entstehen, andererseits sollen die Zuwendungsgeber einen in der Infektionsforschung breit aufgestellten, aber themenfokussierten Verbundpartner in der Infektionsforschung vorfinden. Dieser Verbundpartner
ist mit zukunftsorientierten Konzepten, exzellenten Projekten, modernsten Technologieplattformen und Modellsystemen und auch im internationalen Maßstab
ausgewiesener Exzellenz eine gute Adresse für Fördermaßnahmen im Bereich der
Erforschung weltweit relevanter humanpathogener Infektionskrankheiten.
Zukunftsperspektiven
Projekte, die die Zusammenarbeit von Wissenschaftlern der LCI-Institute in
Zukunft noch verstärken können, sind beispielsweise LCI-Veranstaltungen wie
Retreats, Kooperationsworkshops und gemeinsame thematische Tagungen. Es ist
mit Sicherheit zu erwarten, dass die Leibniz-Graduate School sowie die zunehmende Zusammenarbeit einzelner Forschungsgruppen und beispielsweise ein
institutsübergreifender Publikationspreis für junge Wissenschaftler zu weiteren
gemeinsamen Forschungs- und Planungsinitiativen der beteiligten LCI-Partner
führen werden.
In diesem Sinne ist für die Zukunft vor allem ein gemeinsames öffentliches Auftreten der Leibniz-Partnerinstitute geplant. Zu diesem Zweck soll im Sommer
2009 in einem ersten Schritt eine detaillierte Informationsbroschüre erscheinen,
die nicht nur über das LCI insgesamt, sondern auch über bisherige Zusammenarbeiten und gemeinschaftliche Projekte berichtet. Ergänzt wird die Broschüre
voraussichtlich im Herbst durch einen Internetauftritt des LCI, der vor allem
auch noch einmal die „Graduate School“ darstellt und hier den Studenten als
Informationsplattform für die Ankündigung und Planung von Veranstaltungen
dient. Daneben sollen hier aktuellste wissenschaftliche Höhepunkte der Institute
sowie die Darstellung der gemeinsamen Technologieplattformen und der Kooperationen zu finden sein.
Eine der am weitesten in die Zukunft reichenden LCI-Aktivitäten in der norddeutschen Wissenschaftslandschaft betrifft die Beteiligung des LCI am Zentrum für
strukturelle Systembiologie (Centre for Structural Systems Biology, CSSB), das
ab 2010/2011 auf dem DESY-Campus geplant ist. Ziel dieser Großforschungseinrichtung mit infektionsbiologischem Schwerpunkt ist das Erreichen eines
möglichst umfassenden und detaillierten Verständnisses der diversen molekularen
Wechselwirkungen innerhalb lebender Zellen und Gewebe im Rahmen strukturund systembiologischer Grundlagenforschung. Wichtige Grundlage hierfür ist die
systematische Analyse und Modellierung der molekularen Architektur und Dynamik
biologischer Systeme über sämtliche Auflösungsskalen. Es ist geplant, dass das LCI
sich mit einer gemeinsamen Arbeitsgruppe an dieser interdisziplinären Einrichtung
beteiligt. Daneben werden weitere Forschungsabteilungen von Universitäten und
außeruniversitären Institutionen gemeinsam mit dem DESY dort angesiedelt werden, die komplexe zelluläre Prozesse im Infektionsgeschehen im Rahmen eines
systembiologischen Gesamtkonzepts untersuchen sollen. Auf Grund der immensen
medizinischen Bedeutung der Infektionsforschung ist geplant, frühzeitig Partner
aus der pharmazeutischen Industrie mit einzubinden.
Mit der Errichtung des CSSB als (über)regionalem Forschungscampus wird im
Großraum Hamburg ein weithin sichtbarer „Leuchtturm“ in der biomedizinischen
Forschung mit einem Schwerpunkt in der Infektionsforschung entstehen – eine
optimale Gelegenheit für das LCI, sich nachhaltig zu profilieren und über eine
Zusammenarbeit mit weiteren Infektionsforschern, Systembiologen, Strukturbiologen und Physikern eine internationale Spitzenposition in der Infektionsforschung einzunehmen. Das frühzeitige Engagement des LCI im CSSB wird von
Seiten der Leibniz-Gemeinschaft befürwortet und die zukünftige Gründung einer
gemeinsamen Forschungsabteilung von Seiten des Präsidiums aktiv unterstützt.
Das „Leibniz Center for Infection“, offiziell gegründet im Jahr 2005, durchläuft
eine zunehmend dynamische Entwicklung. Die bisher gemeinsam durchgeführten Projekte und eingeworbenen Mittel zeigen, auf der Basis einer verstärkt
koordinierten Arbeit, bereits Erfolge. Durch die Schaffung einer halben Stelle
für die Betreuung der Interessen der beteiligten Partnerinstitute und die gemeinsame Öffentlichkeitsarbeit stehen seit Mitte 2008 personelle Ressourcen zur
Verfügung, die den gesteigerten Anspruch des LCI an nationaler Sichtbarkeit und
wissenschaftlicher Exzellenz auf der organisatorischen Seite unterstützen können. Trotz der individuellen Forschungsausrichtung jedes Instituts und seines
spezifischen „Institutscharakters“, der sich über viele Jahrzehnte hinweg entwickelt hat, verbindet das BNI, das FZB und das HPI nicht nur die weithin über
die Grenzen Deutschlands hinweg anerkannte wissenschaftliche Leistung zum
Thema „Infektionsforschung“, sondern auch der „Leibniz-Gedanke“, medizinische Grundlagenforschung im Bereich der weltweit wichtigsten und gefährlichsten humanpathogenen Infektionskrankheiten im Hinblick auf gesamtstaatliche Gesundheitsverantwortung zu betreiben. Wir sind zuversichtlich, dass sich
das LCI in Anbetracht dieser Gemeinsamkeiten langfristig zu einer überregional
bekannten Leibniz-Einrichtung und zu einem internationalen Forschungsschwerpunkt für infektionsbiologische Fragen entwickeln wird.
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Programmbereich
„Virus-Wirts-Wechselwirkungen“
Program Area „Virus-Host Interactions“
Head: Prof. Dr. Joachim Hauber
Introduction
According to the World Health Organization (WHO), globally infectious diseases
are by far the leading cause of death. The ongoing HIV/AIDS-pandemic gives an
example for the disastrous socio-economic impact that a single infectious agent
may exert on developing countries, as well as for the financial burden that is
placed on the public health-systems in the industrialized world. The detailed
investigation of the life-cycle of infectious agents is therefore of utmost importance for the successful development of novel and improved anti-microbial
strategies.
Within this research program the life-cycle of various humanpathogenic viruses,
or related animal viruses, are investigated in detail. For example, these include
Hepatitis B Virus (HBV), human and simian immunodeficiency virus (HIV and
SIV), Herpes Simplex-Virus (HSV), Epstein-Barr Virus (EBV), Kaposi-Sarcoma
associated Herpes Virus (KSHV), Simian Virus 40 (SV40), Merkel Cell
Polyomavirus (MCPyV), Adenoviruses and various murine retroviruses.
Viruses are obligatory parasites that can produce progeny only after successful
infection of permissive host cells. Thus, virus replication depends on multiple and
frequently critical interactions involving viral and cellular components. Because
viruses exploit preexisting cellular pathways, they also serve as valuable tools to
understand the molecular basis of fundamental host cell mechanisms such as, for
example, the regulation of immune responses or the induction of cell stress and
carcinogenesis. Therefore, the investigation of different viruses and their interaction with their host cells will not only improve our understanding of the different
strategies that are applied by the respective viruses to secure progeny formation,
but will also explicitly expand our knowledge on cellular metabolism. Finally,
these studies will also reveal novel viral and cellular target structures, that may be
subsequently exploited for the development of advanced antiviral therapies.
As in previous years, the major research activities in 2007-2008 within the program area “Virus-Host Interaction” can be roughly divided into research fields
that focus on early and late aspects of virus infection. Examples for the first
research area are the analysis of the receptor usage of gammaretrovirus envelope
proteins or of the host tropism and entry mechanisms of Hepatitis B Viruses.
Examples for the latter research area are studies of the mechanisms that direct the
nuclear export of adenoviral and lentiviral mRNAs, on Adenovirus- and SV40induced transformation and tumorigenesis, on the activity of Polyomavirus and
Herpes Virus encoded microRNAs, or on the assembly and budding of Hepatitis
Viruses. Furthermore, these projects were complemented by analyses that, for
example, investigated SV40-triggered immune responses or HIV-1-induced
pathogenesis, as well as by the development of novel experimental antiviral
therapies. A detailed description of the individual projects is given further
below.
Within the report period significant progress with respect to improved networking, i.e. by moving away from individual towards inter-institutional research
27
Virus-Host Interaction
28
projects, was made. Particularly the interdisciplinary consortium “Combating
Drug Resistance in Chronic Myeolid Leukemia and HIV-Infection”, which is
funded by the Federal Ministry of Education and Research (BMBF), was initiated
by a project originating in the program area “Virus-Host Interaction”. This consortium represents an outright translational research approach and integrates
laboratories from the University Hospital Hamburg-Eppendorf, the Universities
of Hamburg and Lübeck and the Heinrich-Pette-Institute. By bringing together
preclinical and clinical virus research, biochemistry, structural biology, chemistry
and bioinformatics into a close collaboration, this consortium may also serve as
a model for future proposals within the framework of the upcoming initiative for
excellence in the sciences of the state of Hamburg (Landesexzellenz-Initiative).
In December 2007 the new junior research group “Virus Pathogenesis”, headed by
Dr. Michael Schindler, became operational and the program area “Virus-Host
Interaction” significantly benefitted from this fact. Dr. Schindler’s research primarily focusses on how HIV-1 induces pathogenesis in the infected organism and
how viral factors interfere with host cell components to counteract antiviral host
activities. By recruiting this internationally recognized scientist the HeinrichPette-Institute continued to refocus its direction of research according to its mission statement towards the investigation of the biology of human viruses, the
pathogenesis of virus-induced diseases and antiviral mechanisms of the infected
organism.
While the main load of the straight-forward virus-related projects are clearly
investigated within the program area “Virus-Host Interaction”, it has to be noted
that many projects are closely connected, experimentally as well as intellectually,
with projects of the program area “Cellular Dysregulation”. Finally and as documented in the publication list, an increased research output for the 2007-2008
period within the program area “Virus-Host Interaction” is ultimately reflected in
numerous publications in peer-reviewed internationally recognized scientific
journals, as well as by various patent applications that originated from our investigation of critical virus-host interactions. The latter particularly highlights a
continuous increase in translational research conducted within this program
area, in turn resulting in significantly increased third-party research funds.
Research Projects
Helper function of wild-type p53 in Simian virus 40 induced cellular
transformation
A. Hermannstädter, C. Ziegler, M. Kühl, G.V. Tolstonog, W. Deppert
Heinrich-Pette-Institute, Hamburg, Germany
Abortive infection of matched pairs of mouse fibroblasts differing in p53
gene status (p53 +/+ cells vs. p53 -/- cells) with SV40 revealed a strongly
decreased efficiency for colony formation in soft agar in p53-/- cells compared to p53 +/+ cells. Transformation competence of SV40 in p53 -/- cells
could be fully restored by reconstituting wild-type p53 (wtp53), but not
mutant p53 (mutp53) expression. The helper effect of wtp53 is mediated by
its cooperation with large T antigen (LT), as stable expression of LT in p53+/+
cells resulted in full transformation, while LT expression in p53-/- cells only
enhanced saturation density, but could not promote growth in suspension or
Gene expression analysis in 3T3 LT, 3T3 IG, 10 1 LT and 10 1 IG cells. A: Bar diagram summarizes the total
number of genes up- or down-regulated in 3T3 LT and 10-1 LT cells in comparison to 3T3 IG and 10-1 IG cells,
respectively. Venn diagram illustrates the overlap between the lists of regulated genes. B: Fold change in
Igf1 gene expression relative to non-LT expressing cells. Total RNA isolated from two biological replicates
of 3T3 LT, 3T3 IG, 10 1 LT and 10 1 IG cells was analyzed by Real-time PCR with primers specific for Igf1 and
Gapdh. Each assay was done in triplicate and expression of Igf1 was normalized to Gapdh. Igf1 expression
in LT-expressing cells is reported relative to control cells (3T3 IG, 10 1 IG). Control cells were set at 100% and
calculations revealed following results for Igf1 expression in p53+/+ cells (3T3 LT) 16.1% (+/- 2.3/2.6%) and in
p53-/- cells (10 1 LT) 80.2% (+/- 1.5/1.5%). Calculation of error bars (RQ Min/RQ Max) is based on confidential
level of 95%. C: Table presents the list of ECM (extracellular matrix proteins)-encoding genes down-regulated
in 3T3 LT cells (> 2-fold) comparing to the control 3T3 IG cells. Gene expression values were obtained using
Agilent Whole Mouse Genome Microarray chips.
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30
in soft agar to a significant extent. The p53 helper function resides in the p53
N-terminus, as an N-terminally truncated p53 (ΔNp53) could not rescue the
p53-null phenotype. The p53 N-terminus serves as a scaffold for recruiting
transcriptional regulators like p300/CBP and Mdm2 into the LT-p53 complex.
Consequently, LT affected global gene expression in p53+/+ cells significantly
stronger than in p53-/- cells. In particular, genes related to the expression of a
transformed phenotype, like genes encoding extracellular matrix proteins
were specifically down-regulated in 3T3 LT cells (p53+/+), but not in 10-1 LT
cells (p53-/-). Furthermore, we were able to demonstrate that the Igf1 gene, a
presumed target gene of the LT-p53 complex, is specifically down-regulated
only in 3T3 LT cells, but not in 10-1 LT cells. Our data suggest that recruitment
of transcriptional regulators into the LT-p53 complex serves to modify cellular
gene expression to the needs of cellular transformation.
*Supported by Deutsche Forschungsgemeinschaft (DFG) and EU FP6
Role of c-Met in SV40-induced mammary tumorigenesis
S. Babu (1), C. Heinlein (1), F. Krepulat (1), B. Otto (2), T. Streichert (2),
G.V. Tolstonog (1), W. Deppert (1)
(1) Heinrich-Pette-Institute, Hamburg, Germany
(2) University Medical Center Hamburg-Eppendorf,
Institute of Clinical Chemistry, Hamburg, Germany
We generated and characterized a transgenic mouse model (WAP-T mice) suitable for molecular analysis of initiation and progression of mammary adenocarcinoma. Expression of SV40 early proteins in WAP-T mice is initiated during late
pregnancy, and tumors develop from T-antigen expressing mammary epithelial
progenitor cells that survived involution. Molecular analyses of the tumors by
aCGH and gene expression profiling revealed that the Met gene is amplified and
strongly expressed in nearly all undifferentiated tumors. Therefore, we focused on
the role of c-Met during SV40-induced tumorigenesis.
Double staining for c-Met and SV40 Large T-antigen (LT) in a mammary epithelial
tumor cell line established from a WAP-T tumor showed that c-Met expression
levels were directly proportional to LT expression levels, indicating a role for LT
in regulating Met expression. Ectopic expression of LT in mouse embryo fibroblasts
with wild-type p53 (wtp53) or p53-null background revealed a significant
LT-dependent increase in c-Met expression in cells with wtp53 compared to p53-null
background. The data suggested that c-Met expression is repressed by wtp53, and
that this repression is alleviated by p53 inactivation through complex formation
with LT. To confirm the role of wtp53 in c-Met expression, we investigated c-Met
expression in mouse cG9 cells which express a temperature-sensitive p53 that is
wild-type at 32°C and mutant at 39°C, respectively. We observed increased
expression of c-Met when p53 was in a mutant conformation. The data further
supported the postulated p53-dependent repression of the Met gene and the
action of LT that leads to transcriptional inactivation of p53 by complex formation.
By ChIP assay we detected binding of p53 to the Met promoter, which was abolished
31
Met gene amplification and expression in SV40-induced mouse mammary carcinoma. A: UCSC Genome Browser
view of mouse Met genomic loci on the chromosome 6. B-C: Graphic visualization of gene copy number and
expression data. Cellular RNA and genomic DNA from tumor samples were analyzed using Affymetrix gene
expression microarrays (C) and Agilent whole mouse genome microarrays (B), respectively. A total of 65 samples including SV40-induced mammary carcinomas in mono- (SV40 early region: WAP-T, WAP-NP8) and bi-transgenic mice (SV40/mutp53: WAP-T-H22, WAP-NP8-W10) as well as spontaneous breast tumors were submitted
to the molecular analysis. Expression values were standardized by the z-score method. Array-CGH values are
log2 ratios after segmentation. Most of the poorly differentiated tumors (grades G3 and G4) are characterized
by amplification of Met loci. The amplico boundaries greatly vary between individual tumors. The asymmetrical
distribution of scale of values highlights the strength of gene amplifications. The expression values correlate
with the amplifications.
Virus-Host Interactions
32
when LT was co-expressed. Silencing of c-Met using shRNA in LT transformed
cells abrogated the cells’ ability to form colonies in soft agar, highlighting the role
of c-Met in proliferation and substrate-independent growth. We conclude that
p53 acts as a repressor of the Met gene. Alleviation of this repressor function by
LT is essential for LT-mediated cellular transformation.
Mutant p53 gain of function in mammary carcinogenesis
E. Lenfert (1), C. Heinlein (1), F. Krepulat (1), T. Streichert (2), B. Otto (2),
W. Deppert (1), G.V. Tolstonog (1)
Institute of Clinical Chemistry, Hamburg, Germany
Missense mutations and loss of the wild-type allele in the TP53 gene frequently
accompany tumor formation. The most prevalent missense mutations found in
human breast cancer are at the positions R248 and R273. The p53 mutant allele
encodes a protein (mutp53), which has lost the transcriptional activity towards
pro-apoptotic and growth arrest genes, but has retained the propensity to interact
specifically with nucleic acids and proteins.
To study the influence of mutp53 on the development of SV40-induced mouse
mammary carcinoma we developed bi-transgenic WAP-T (SV40 early region) x
WAP-mutp53 mouse models with equivalent mutations (R245W, R270H).
Mutp53 acts synergistically with the SV40 T/t-antigens, enhances tumor
incidence and aggravates development of undifferentiated carcinomas and
pulmonary metastasis.
To dissect the molecular mechanisms of mutp53 function we performed gene
expression profiling of tumor and normal tissues. By bioinformatic evaluation
of microarray data we identified a small set of genes which expression strongly
correlates with mutp53. We focused on Apobec3 gene and found that the
tumors and involuted glands of WAP-T-mutp53 bi-transgenic animals contained more Apobec3-positive cells than the respective tissues of the WAP-T
mono-transgenic mice. These preliminary results indicate a role of Apobec3 in
tumor progression and aggressiveness. To test the hypothesis that mutp53 may
influence miRNA expression and in this way provide an additional level of
regulation, miRNA expression in tumor samples of 4 mono-transgenic and
4 bi-transgenic mice was quantified on Agilent chips. Analysis of miRNA
expression profiles pointed at functional crosstalk between wild-type and
mutp53, and supported the possibility that mutp53 enhances metastasis via
changes in miRNA expression.
Virus-Host Interactions
A preclinical model for monitoring treatment and prevention of
metastasis in mammary carcinoma
C. Mänz (1), F. Wegwitz (1), A. Diesterbeck (1), A. Preuß (1),
J.S.D. Mieog (2), G.-J. Lieffers (2), W.G.M. Löwik (2), F. Alves (3),
G. Tolstonog (1), W. Deppert (1)
(2) Leiden University, Leiden, The Netherlands
(3) University of Göttingen, Germany
Breast cancer is the most frequently diagnosed type of cancer and the second
leading cause of death among women in the Western world. Once localized breast
tumors spread to other organs (metastasis), breast cancer is often incurable. The
WAP-T and WAP-T x WAP-mutp53 mice developed in our lab adequately model
the slow onset and development of human breast cancer from ductal carcinoma
in situ (DCIS) up to invasive growth with metastasis. With these mice we aim
to mirror the clinically relevant situation of metastatic disease that arises after
excision of the primary tumor.
We have established a cell line from an endogenous, undifferentiated mammary
carcinoma of a bi-transgenic WAP-T-WAP-mutp53 mouse which exhibits characteristics of tumor stem cells (GeTo cells). Orthotopic transplantation into
syngeneic immune-competent mice of < 100 GeTo cells leads to the development of tumors with similar characteristics as primary tumors in WAP-T mice.
Tumors arising from transplanted GeTo cells into WAP-T mice dissemination
into a variety of tissues, blood and bone marrow. Disseminated tumor cells can
be detected with a sensitivity of less than 1 in 105 cells. Some transplanted animals develop visible lung metastases already within the period of tumor growth.
To mimic the situation encountered by human mammary carcinoma patients,
primary tumors arising from transplanted GeTo cells are surgically removed at
an early stage to allow more time for disseminated cells to establish second site
malignancies, which in human patients can take up to ten years. This preclinical
model can be used to test various treatment regimens for the elimination of
disseminated tumor cells and thus for prevention of metastasis in collaboration
with clinical partners. To further refine the analysis, state of the art in vivo
imaging of mice will be performed in collaboration with Prof. W.G.M. Löwik,
Leiden University, Leiden, The Netherlands and Dr. F. Alves, University of
Göttingen, Germany.
*Supported by Deutsche Forschungsgemeinschaft (DFG) and by EU FP6
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Induction of a cellular immune response against SV40 induced
mammary carcinoma in transgenic mice
M. Bruns (1), G. Pilnitz-Stolze (1), A. Diesterbeck (1), A. Düsedau (1),
O. Utermöhlen (2), U. Schumacher (3), W. Deppert (1)
(2) Institute of Immunology, University of Cologne, Germany
(3) Institute of Anatomy II, UKE, Hamburg, Germany
The immune system can also recognize cancer cells as foreign. In order to study
the different facets of immune surveillance during tumor progression, transgenic
BALB/c mice were established containing the whey acidic protein (WAP) promoter upstream of the large tumor antigen (T-Ag) of simian virus 40 (SV40)
(WAP-T lines). Upon induction of the transgene, these mice develop endogenous
mammary carcinomas, which derive from T-Ag expressing mammary epithelial
progenitor cells that survive involution. As BALB/c mice are considered to be “low
responders” towards SV40 T-Ag, a 33bp oligomer of the nucleoprotein (NP) of
lymphocytic choriomeningitis virus (LCMV), encoding the MHC class I H-2d
restricted T-cell epitope, was inserted into the C-terminal coding region of T-Ag
(WAP-T-NP lines). This NP-epitope mediates the strong immune reaction against
LCMV in BALB/c mice. Expression of the hybrid gene thus allows the analysis of
the immune response after LCMV infection of WAP-T-NP mice. While the WAPT-NP lines are tolerant against SV40, they are not tolerant against LCMV, as these
mice mount a strong cellular immune response against the LCMV-NP epitope
upon infection. Likewise, adoptive transfer of immune competent cells from
LCMV-infected BALB/c wild-type mice into WAP-T-NP mice strongly reduced
the number of T-Ag positive cells
not only in lactating mammary
glands but also in cancerous tissues. The data point towards an
immune surveillance, where NP
epitope-specific CD8+ T-cells not
only are capable of virus elimination, but can also efficiently reduce
the number of T-Ag expressing
tumor cells in WAP-T-NP mice.
The data provoked the question,
why this strong immune reaction
is not mounted during endogenous tumor development. As a
possible explanation we analyzed
whether this phenomenon might
relate to the observation that
chronically LCMV-infected mice Comparison of PD-L1-treated (left) and untreated WAP-T-NP8 mice
contain only functional impaired (right). Comparison of WAP-T-NP8 mice treated either with antibodies against PD-L1 (right) or left untreated (left). Formalin-fixed and
virus-specific CD8+ T-cells paraffin embedded sections of mamma carcinomas were stained
(“exhausted” T-cells), which have by antibodies against SV40 T-Ag (above) or PD-L1 (below).
selectively up-regulated the programmed death 1 (PD-1) protein. As demonstrated
for LCMV-carrier mice, in vivo application of antibodies that blocks the interaction of this receptor-molecule on T-cells with its ligand PD-L1 indeed led to an
elimination of T-Ag expressing tumor cells in WAP-T-NP mice. Consequently,
it was found by FACS and immune histological studies that antibody-treatment
of tumor mice with the aim to inhibit the binding of PD-1 to its ligand revealed
a strong reduction of PD-1+ immune cells and at the same time resulted in
a successful elimination of T-Ag and PD-L1 expressing epithelial cells in parallel
(Fig. 1). This finding could provide a powerful new strategy for the elimination
tumor cells via re-vitalization of exhausted immune cells by blocking the
PD-1/PD-L1 interaction.
*Supported by EU FP6
Steroid dependent control of HIV replication
K. Wiegers, D. Schwarck, R. Reimer, W. Bohn
Nuclear transport and integration of the HIV genome require the functional
interaction between cellular and virus associated proteins. This interaction has
significant influence on the efficiency of virus replication. In resting peripheral
mononuclear blood cells (PBMCs) infection with HIV-1 is not productive due to
a block that occurs early after infection prior to provirus formation. We noticed
that in resting PBMCs the status of the glucocorticoid receptor (GR) contributes
to the restriction in virus replication. Treating PBMCs with a GR specific ligand
is sufficient to overcome the block and increases the proportion of integrated
provirus. The ligand mediated effect is only seen when the ligand is given early
after infection. In addition, it requires the presence of the viral protein Vpr. Vpr
is associated with the pre-integration complex and known to bind the nuclear
receptor. The data suggest that the block is based on an inefficient nuclear transport and/or impaired integration of the virus genome. Microscopic data show
that in unstimulated, naïve PBMCs endogenous GR is retained in the cytoplasm,
as is also an ectopic Vpr expressed in these cells. Stimulation with a GR specific
ligand translocates both proteins into the nucleus. Thus, cytoplasmic retention of
the HIV preintegration complex by the non-activated nuclear hormone receptor
seems to be the obstacle for efficient HIV replication in naïve PBMCs.
Glucocorticoid treatment overcomes this restriction by stimulating nuclear
transport of the receptor. In accordance, in a cell line with a constitutive cytoplasmic GR efficient proviral integration requires addition of a GR ligand. The data
suggest that steroids overcome the restriction on HIV provirus formation and
thereby increase the reservoir of virus producing cells.
*Supported in part by Deutsche Forschungsgemeinschaft (DFG)
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Control of viral mRNA export by adenovirus regulatory proteins
O. Koyuncu (1), K. Kindsmüller (1), P. Blanchette (2), P. Groitl (1),
F. Dallaire (2), T. Speiseder (1), P. Branton (2), J. Hauber (1), T. Dobner (1)
(2) McGill University, Department of Biochemistry, Montreal, Canada
Human adenoviruses have small linear double-stranded DNA genomes encoding
only the virus structural polypeptides and a limited number of regulatory proteins. In order to be able to maintain a compact genome size, and hence restricted coding capacity, yet remain an effective pathogen these viruses have evolved
efficient strategies to utilize the host cell machinery for viral gene expression,
DNA replication and formation of progeny virions. Their ability to subvert host
macromolecular synthesis at defined stages of the viral replication cycle is the
result of both the viral program of transcriptional regulation and virus-encoded
functions that control mRNA processing, nucleo-cytoplasmic mRNA transport
and translation. It is the posttranscriptional mechanisms in particular which
serve to facilitate production of large quantities of viral structural polypeptides
and to shut-off host cell protein synthesis at late times of infection. In productively infected cells regulated viral mRNA splicing and viral mRNA export is
controlled by at least three adenovirus proteins E4orf6, E1B-55K and L4-100K.
Ad5 E1B, E4 and L4 proteins are multifunctional regulators of virus replication. Ad E1B, E4 and L4 gene products encompass a diverse collection of functions that modulate viral and cellular growth control at all levels
of the gene expression pathway. In productively infected cells E1B directly or indirectly alters the pattern of
splicing and stability of late viral RNAs. In addition E1B and E4 proteins been implicated in the preferential
nuclear export of viral late RNA transcripts, inhibition of cellular mRNA transport and protein synthesis, while
L4-100K shuts off host cell translation, promotes translation of tripartite leader (TL) containing viral late
mRNAs via ribosome. In addition the E1B/E4orf6 complex enhances the degradation of cellular target proteins
on cytoplasmic and/or nuclear proteasomes.
These directly or indirectly alter the pattern of splicing and stability of late viral
RNAs and have been implicated in the preferential nuclear export of viral late RNA
transcripts, inhibition of cellular mRNA transport and protein synthesis, selective
translation of tripartite leader (TL) containing viral late mRNAs via ribosome
shunting and nuclear localization of Ad5 capsid components, such as hexon. In
addition the E1B-55K/E4orf6 complex promotes the proteasomal degradation of
the tumor suppressor protein p53, DNA Ligase IV and Mre11, a subunit of the
Mre11/Rad50/NBS1 (MRN) DNA double-strand break repair complex. The latter
activity may also involve nuclear depletion and cytoplasmic deposition of p53
and MRN components into aggresomes, which, consistent with their role in the
removal of aggregated proteins from the cytosol, further accelerates proteolytic
degradation of MRN subunits. Collectively available data suggest that most if not
all of the lytic functions ascribed to E1B-55K, E4orf6 and L4-100K during infection
including control of viral mRNA export and translation are achieved through
continuous nucleo-cytoplasmic shuttling and interactions with a variety of host cell
factors mediating nuclear import and export processes.
Efforts to identify cellular targets that link the E1B-55K, E4orf6 and L4-100K to
nuclear export and import pathways showed that each of the viral proteins possesses a single leucine-rich nuclear export signal (NES) of the HIV-1 Rev-type and
can individually shuttle between the nuclear and cytoplasmic compartment via the
export receptor CRM1. In addition, nucleocytoplasmic trafficking of E1B-55K
involves a CRM1-independent nuclear export pathway, which is controlled by
covalent conjugation of the small ubiquitin-related modifier protein 1 (SUMO1).
Thus, SUMO1 conjugation and deconjugation provides a molecular switch that
commits E1B-55K to CRM1-dependent and -independent export pathways. E1B55K, E4orf6 and L4-100K also interact with cellular factors that could bridge the
viral protein complex to other cellular export receptors, such as HuR, pp32, APRIL
and/or TAP/NXF1. Nonetheless, the contribution of these cellular factors to nucleocytoplasmic shuttling, selective viral late RNA export and viral late gene expression
is still unknown.
Nucleo-cytoplasmic shuttling of E1B-55K is regulated by CRM1 and
SUMO1-conjugation. Subcellular distribution of wt (GFP-E1B and
pE1B-55K) and mutant (pE1B-NES) E1B proteins in the absence or
presence of the CRM1 inhibitor LMB. Magnification x 7600.
As part of our studies of E1B-55K/
E4orf6 function during productive infection we found that E4orf6
connects E1B-55K to components
of a cellular E3 ubiquitin ligase,
thereby allowing the proteasomal
degradation of p53, Mre11 and
DNA Ligase IV. E1B-55K associates with this high molecular
weight complex where it is believed
to function primarily to introduce
bound substrates for degradation
via proteasomes. These observations and the finding that nearly
all of E1B-55K present in infected
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CRM1-mediated nuclear export of Ad5 L4-100K is required for efficient virus growth. (A) Top: Multiple amino acid
sequence alignment of L4-100K polypeptides from human Ad serotypes, subgroups A-D. Numbers in brackets
refer to amino acid residues. Residues that are identical in all serotypes are shown in light grey boxes and the
conserved hydrophobic residue is marked with a dark grey box (Φ: hydrophobic amino acid residue, X: any amino
acid residue). Bottom: The amino acid sequence of wt virus H5pg4100 and the NES virus mutant H5pm4165 with
alanine substitutions at positions 383, 386, 390, 392. (B) Virus yield. A549 cells were infected with wt H5pg4100
or H5pm4165 at indicated multiplicities. Viral particles were harvested at indicated time points (h p.i.) and virus
yield was determined by quantitative E2A-72K immunofluorescence staining on K16 cells. The results represent
the averages from three independent experiments. Error bars indicate the standard error of the mean. (C) A549
cells were infected with H5pg4100 (panel a) and H5pm4165 (panel b) viruses at a multiplicity of 10 FFU per cell.
Infected cells were fixed at 36 h post infection (h p.i.) and stained with rat mab 6B10. In all panels nuclei are
indicated by a dotted line. Magnification x 7600.
cells is found associated with E4orf6 raises the possibility that nucleo-cytoplasmic
trafficking and control of mRNA export requires degradation of cellular target proteins by the E1B-55K/E4orf6 E3 ubiquitin ligase activity. We have tested this hypothesis through the generation and phenotypic analysis of a virus mutant in which the
E4orf6 product is unable to form the ligase complex and indeed have found that this
mutant behaves identically to an E4orf6minus virus in production of late viral proteins, growth, and in the export of late viral mRNAs. Thus, preferential nuclear export
of viral late RNA transcripts and simultaneous inhibition of cellular mRNA transport
is intimately linked to the E1B-55K/E4orf6 E3 ubiquitin ligase activity.
A highly interesting result was also obtained when we analyzed the contribution
of CRM1-mediated nuclear export of L4-100K to productive infection. Phenotypic
analyses of a virus mutant (H5pm4165) carrying amino acid exchanges in the
highly conserved NES of L4-100K in different human tumor cell lines revealed
that it is severely defective in late viral gene expression and virus production
compared to the wild-type virus H5pg4100. Localization of the 100K NES mutant
was restricted to the nuclei of the infected cells. A similar result was obtained
when CRM1 was inactivated by leptomycin B (LMB). These data show that
nuclear export of 100K is required for maximal virus growth and suggest that
CRM1 is the major export receptor for L4-100K in virus-infected cells. Remarkably,
these studies also revealed that L4-100K is a substrate of the protein arginine
methylation machinery an emergent post-translational modification system
involved in a growing list of cellular processes, including transcriptional regulation, cell signaling, RNA processing and DNA repair. L4-100K arginine methylation involves protein arginine methyltransferase 1 (PRMT1), which asymmetrically dimethylates arginines embedded in arginine-glycine-glycine (RGG) or
glycine-arginine-rich (GAR) domains. Arginine to glycine exchanges in the RGG
boxes significantly diminish 100K methylation in the course of an infection and
substantially reduced virus growth, demonstrating that L4-100K methylation is
an important host-cell function required for efficient adenovirus replication.
Taken together, results from studies demonstrate for the first time that efficient
late adenoviral gene expression is dependent on PRMT1 and the export receptor
CRM1, the latter being most critical for efficient virus growth in human tumor
cell lines. In fact, these data suggest substantial mechanistic similarities between
adenovirus and complex retroviruses in the control of viral RNA export.
*Supported by Deutsche Forschungsgemeinschaft (DFG)
Identification of a new substrate of the adenovirus/E1B-55K/E4orf6
ubiquitin ligase complex
F. Dallaire (1), P. Blanchette (1), P. Groitl (2), T. Dobner (2), P. Branton (1)
The human adenovirus E4orf6 and E1B-55K proteins promote viral replication
by targeting several cellular proteins for degradation. The E4orf6 product has
been shown by our group and others to form an E3 ubiquitin ligase complex that
contains Elongins B and C and Cullin family member Cul5. In addition to p53,
its first known substrate, the E1B-55K/E4orf6 complex was shown to promote the
degradation of Mre11 and D0NA Ligase IV; however, additional substrates are
believed to exist. This notion is strengthened by the fact that none of these
substrates seem likely to be associated with additional functions shown to be
mediated by the E4orf6-associated E3 ubiquitin ligase complex, including export
of late viral mRNAs and simultaneous inhibition of host cell mRNAs export from
the nucleus. In an attempt to identify new E1B-55K/E4orf6 substrates we undertook a proteomic screen using human p53-null, non-small cell lung carcinoma
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H1299 cells expressing either E4orf6 protein alone or in combination with E1B55K through infection. One cellular protein that appeared to be degraded by
E1B-55K in combination with the E4orf6 protein was a species of molecular mass
∼130kDa that was identified as integrin alpha 3 subunit (Very Late Antigen 3
alpha subunit or VLA-3α). Preliminary analyses suggested that degradation of α3
may play a role in promoting release and spread of progeny virions in the very
late phase of the infectious cycle.
*Supported by Canadian Institutes of Health Research – CIHR
Human adenovirus E4 region orf3/4 protein is required for efficient
virus replication
T. Speiseder (1), P. Groitl (1), T. Dobner (1)
Human adenovirus early region 4 (E4) encodes at least six distinct polypeptides,
defined as E4orf1 to E4orf6/7 according to the order and arrangement of the
corresponding open reading frames (orfs). E4 gene products operate through
a complex network of protein interactions with key viral and cellular regulatory
components involved in transcription (E4orf6/7), protein degradation, nucleo-cytoplasmic transport and DNA repair (E4orf6), as
well as host cell factors that regulate cell signaling (E4orf1), posttranslational
modifications
(E4orf4) and the integrity of PMLcontaining
nuclear
bodies
(E4orf3).
In the context of our studies of E4
function we generated a set of
mutant viruses carrying point
mutations in the E4 genes. The
phenotypic characterization of
these mutants revealed that lesions
affecting individual orfs had no or
only moderate effects on lytic
virus growth. By contrast, Ad
mutants that lack the entire E4
region or fail to express the E4orf6
protein were severely restricted for
growth. Unexpectedly, a virus
mutant unable to express E4orf3
plus E4orf4 was as defective as the
E4-minus mutant viruses. Further,
Ad2/5 E4 transcription unit. Top: ORF maps spanning the entire
E4 region of Ad5. The three reading frames (1, 2 and 3) of the
E4 region sense strand from position 35606 (cap-site) to 32802
(polyA-signal) are shown. Gray shaded regions indicate ORF1 to
ORF7 coding sequences. Vertical lines bisecting each reading-frame
indicate stop codons, whereas small triangles denote methionine
codons. Middle: E4 mRNAs are indicated by lines and arrows;
introns are indicated by spaces and coding regions are indicated
by open rectangles. With the exception of the ORF3/4 mRNA other
mRNAs species which have not been detected [Dix, 1993 #76] are
not included. Late and Early at the right side denote the temporal
class of each individual mRNA species. Bottom: the location of the
ORFs and the splice donor/acceptor combinations relative to their
position in the Ad5 nucleotide sequence. Abbreviations: ITR, inverted terminal repeat; cap, cap-site; poly(A), polyadenylation signal.
more detailed genetic and biochemical analyses demonstrated that the E4orf3/
E4orf4 double mutant exhibits a pleiotropic phenotype and fails to produce the
E4orf3/4 fusion protein, which consists of the N-terminal 33 amino acid residues
from orf3 and the C-terminal 28 amino acid residues from orf4. Consistent with
this a virus mutant lacking E4orf3/4 and E4orf4 replicated to levels nearly equally reduced to the E4-minus mutant. In sum, these results introduce a function for
E4orf3/4 and suggest that E4orf3/4 and E4orf4 provide redundant functions for
efficient viral replication, which is intimately linked to their identical C-terminal
regions.
*Supported by Wilhelm Sander-Stiftung
Localization and importance of the adenovirus E4orf4 protein
during lytic infection
M-J. Miron (1), P. Blanchette (1), P. Groitl (2), F. Dallaire (1), J. G. Teodoro
(1), S. Li (1), T. Dobner (2), P. Branton (1)
The human adenovirus type 5 (Ad5) E4orf4 product has been studied extensively although in most cases as expressed from vectors in the absence of other
viral products. Thus, relatively little is known about its role in the context of an
adenovirus infection. Although considerable earlier work had indicated that the
E4orf4 protein is not essential for replication, a recent study using dl359, an Ad5
mutant believed to produce a nonfunctional E4orf4 protein, suggested that
E4orf4 is essential for virus growth in primary small-airway epithelial cells.
Hence, to examine further the role of E4orf4 during virus infection, we generated
for the first time a set of E4orf4 virus mutants in a common Ad5 genetic background. Such mutant viruses included those that express E4orf4 proteins containing various individual point mutations, those defective entirely in E4orf4
expression, and a mutant expressing wild-type E4orf4 fused to the green fluorescent protein. E4orf4 protein was found to localize primarily in nuclear structures
shown to be viral replication centers, in nucleoli, and in perinuclear bodies.
Importantly, E4orf4 was shown not to be essential for virus growth in either
human tumor or primary cells, at least in tissue culture. Unlike E4orf4-null virus,
mutant dl359 appeared to exhibit a gain-of-function phenotype that impairs
virus growth. The dl359 E4orf4 protein, which contains a large in-frame internal
deletion, clustered in aggregates enriched in Hsp70 and proteasome components.
In addition, the late viral mRNAs produced by dl359 accumulated abnormally in
a nuclear punctate pattern. Altogether, our results indicate that E4orf4 protein is
not essential for virus growth in culture and that expression of the dl359 E4orf4
product interferes with viral replication, presumably through interactions with
structures in the nucleus.
*Supported by Canadian Institutes of Health Research – CIHR
41
42
Intrinsic disorder in the amino terminus of human adenovirus type
5 E1B-55K and its related proteins
T. Sieber (1), R. Scholz (2), M. Spörner (3), H-R. Kalbitzer (3), T. Dobner (1)
(2) Swiss Federal Institute of Technology Zurich, Zurich, Switzerland
(3) Institute for Biophysics and Physical Biochemistry, University of
Regensburg, Germany
Early region 1B (E1B) of human subgroup C adenoviruses generates two major
mRNA species of 22S and 13S with common 5’ and 3’ termini. Both 22S and 13S
mRNAs are derived from a common mRNA precursor by alternative splicing.
Translation of the 22S mRNA from adenovirus type 5 (Ad5) uses discrete, but overlapping reading frames to produce two unrelated polypeptides of 176 amino acid
residues (R) (E1B-19K) and 496R (E1B-55K). The 19K product is also translated
from three alternatively spliced
transcripts of 13S, 14S and 14.5S,
which additionally direct the synthesis of three smaller 55K related
proteins (156R, 94R and 83R)
containing the amino-terminal 79
residues of E1B-55K. E1B-156R
also shares an identical carboxyterminus with 55K, while the 94R
and 83R E1B polypeptides contain
unique carboxy termini. The fact
that four E1B proteins (E1B-55K,
E1B-155R, E1B-94R and E1B-83R) Ad5 E1B region and its gene products. Scale on top of the figure indinucleotide sequence position relative to the left end of the viral
share a common amino terminus cates
genome. The Ad5 E1B mRNAs and protein products predicted from
indicates that this segment plays a DNA sequence and mapping studies have been illustrated. Included
key role in regulating the function are also the relevant nucleotides for translation start and stop sites.
of E1B-55K and its related proteins.
Nevertheless, as yet very little is
known about the function of this
domain and the smaller E1B proteins in infection.
To gain further insight into the role
the amino-terminal 79 amino acids
for E1B function we performed
NMR spectroscopy and other biophysical methods with bacterially
expressed E1B-93R to obtain highresolution structural information
on this protein region in solution.
Our data show that E1B-93R, and,
likely E1B-84R, are mostly unstructured and fulfill the criteria of an
Ad5 E1B-93R is an intrinsically disordered protein. CD-spectrum
of human Ad5 E1B-93R in a range of 195.5 to 260 nm. E1B-93R
is mostly unfolded with a minor content of regular secondary
structure. For comparison the theoretical spectra of pure α-helix,
β-sheet and random coil were adapted to this figure.
intrinsically disordered protein (IDP) with a high degree of dynamics and very little
stable structure. In addition these findings, indicate that E1B-55K and E1B-156R
contain an intrinsically disordered domain (IDD) in their amino-termini. IDPs
comprise a rapidly growing family of proteins which, despite their lack of a well defined
structure, often fulfill essential regulatory functions. The intrinsic lack of structure
confers functional advantages on these proteins, allowing them to adopt multiple
conformations and to bind to different binding partners. Consistent with this we found
that the IDD mediates an interaction between the 55K related proteins and E1B-55K.
*Supported by ReFoMC program, University Regensburg
A novel 49K isoform of the adenovirus type 5 early region 1B 55K
protein is sufficient to support viral replication
S. Schreiner (1), K. Kindsmüller (1), P. Groitl (1), T. Dobner (1)
The adenovirus type 5 (Ad5) E1B-55K protein is a multifunctional regulator of cellcycle independent virus replication that participates in many processes required for
maximal virus production. As part of a study of 55K function, we generated a virus
mutant (H5pm4133) carrying stop codons after the second and seventh codon of the
E1B reading frame, thereby eliminating synthesis of the full-length 55K product.
Unexpectedly, phenotypic studies revealed that H5pm4133 fully exhibits the characteristics of wild-type (wt) Ad5 in all assays tested. Immunoblot analyses demonstrated that H5pm4133, and, significantly, wt Ad5 express low levels of a ~49K
product, lacking the amino-terminal region of E1B-55K. Expression of the 49K isoform was eliminated when the second and third downstream AUG at positions 64
and 75 of the E1B reading frame were converted to valine codons. Moreover, virus
mutants producing the E1B-49K protein only, replicated to levels comparable to wt
Ad5, while mutants lacking both E1B-55K and the 49K isoform were severely defective for virus growth. These data demonstrate that the Ad5 E1B gene produces low
levels of a novel 49K E1B protein species, which is generated by initiation of translation at two downstream, in-frame AUGs of the E1B mRNA, and is sufficient to
proceed through the productive infection in human tumor cell lines.
Daxx-mediated negative regulation of adenovirus replication is
counteracted by the adenovirus E1B-55K protein
S. Schreiner (1), P. Wimmer (1), H. Sirma (1), R. Everett (2),
P. Blanchette (3), T. Dobner (1)
(2) MRC Virology Unit, Glasgow, Scotland, UK
The death-associated protein Daxx found in PML nuclear bodies (PML-NBs), is
involved in transcriptional regulation and cellular intrinsic immune defense
43
44
against incoming viruses. We found that knock-down of Daxx using siRNA techniques in a non-transformed human hepatocyte cell line results in significantly
increased in adenoviral (Ad) replication, including enhanced viral mRNA synthesis and viral protein expression. This Daxx restriction imposed upon adenovirus
growth is apparently counteracted by early protein E1B-55K, a multifunctional
regulator of cell-cylce independent Ad replication. Here, we show that the Ad
protein binds to Daxx and likely induces its degradation through a proteasomedependent pathway. Remarkably, this process is independent of Ad E4orf6,
known to promote the proteasomal degradation of cellular p53, Mre11 and DNA
Ligase IV in combination with E1B-55K. These results illustrate the importance
of PML-NBs and associated factors in virus growth restriction and suggest that
two Ad early proteins (E1B-55K and E4orf3) synergistically antagonize the innate
antiviral activities of PML-NBs to stimulate the viral replication program.
*Supported by Erich und Gertrud Roggenbuck-Stiftung
Posttranscriptional processing of HIV-1 mRNA
D. Hoffmann (1), M. Krepstakies (1), I. Hauber (1), B. Abel (1),
H. Schaal (2), R.H. Kehlenbach (3), J. Hauber (1)
(2) University of Düsseldorf – Institute of Virology, Düsseldorf, Germany
(3) University of Göttingen – Centre of Biochemistry and Molecular Cell
Biology, Göttingen, Germany
During replication of the human immunodeficiency virus type 1 (HIV-1) the
viral (+)RNA genome is reverse transcribed and integrated into the host cell
genome. Transcription of this provirus by the cellular RNA polymerase II generates a polycistronic pre-mRNA that contains at least four 5’ splice sites and eight
3’ splice sites that enable alternative splicing of more than 40 different mRNAs.
These transcripts can be grouped into the three classes of RNA: The 2 kb-class is
multiply-spliced and encodes the regulatory proteins Tat, Nef or Rev. The 4 kbclass is characterized by a single splicing event and encodes Env or the accessory
proteins Vif, Vpr or Vpu. Finally, the 9-kb class consists of unspliced primary
transcripts that either are used for Gag and Pol expression or act as genomes in
the formation of progeny viruses.
The transport of the 4 kb class and 9 kb class of these RNAs is promoted by the
retroviral Rev regulatory protein while, in contrast, nuclear export of the 2 kb
class occurs independent of Rev. Rev is a nucleocytoplasmic shuttle protein that
binds directly to the viral Rev response element (RRE) RNA sequence, which is
present on all unspliced and singly-spliced HIV-1 transcripts (4 kb and 9 kb
class). Subsequently, Rev interacts with multiple cellular factors that mediate the
translocation of the Rev-bound viral RNA across the nuclear envelope.
The nucleocytoplasmic translocation of Rev-containing ribonucleoprotein (RNP)
complexes is mediated by the general export receptor CRM1, which interacts in
presence of RanGTP with a specific domain in Rev that has been termed the
nuclear export signal (NES). However, efficient Rev-mediated nuclear RNA
export also requires factors in addition to CRM1 and Ran. These apparently
include, for example, eukaryotic initiation factor 5A (eIF-5A), the Src-associated
protein in mitosis Sam68 and DEAD Box RNA Helicase DDX3.
In collaboration with Prof. Heiner Schaal (University of Düsseldorf) we investigated
the translation efficiency of Env-encoding messages as well as the activation of
alternatively used HIV-1 splice acceptors.
Translation of HIV-1 mRNAs generally follows the scanning mechanism. However,
by using subgenomic env expression vectors, we found translation of glycoprotein
from polycistronic mRNAs were inconsistent with leaky scanning. Close inspection
of the leader sequences suggested expression of Env was modulated by an upstream
open reading frame (uORF) and a minimal uORF consisting only of a start and
stop codon that overlaps with the vpu start site. By altering the strength of upstream
AUGs, the length of their coding region and the intercistronic distance, we
demonstrate that each of these parameters influenced downstream translation. The
minimal uORF is conserved throughout almost all HIV-1 subtypes, despite residing
in a non-coding region of the genomic RNA. Mutating the translational start and
stop codons of this uORF resulted in up to five fold reduction in Env expression.
Removing the vpu uORF and increasing the strength of the authentic vpu initiation
sequence abolished Env expression
from subgenomic constructs and
replication of HIV-1, whereas an
identical increase in the strength of
the minimal uORF initiation site
did not alter env-expression.
Distribution of splicing regulatory elements in the HIV-1 pre-mRNA.
(A) Schematic depiction of the HIV-1 genome. (B) Distribution of 5’
splice sites (D1-D5) and 3’ splice sites (A1-A7) within the genomic
HIV-1 RNA. The position of exon 5 is indicated (dark grey box). (C)
Enlargement of the 3’ half of the pre-mRNA depicts the location of
the known enhancer (green) and silencer elements (red) regulating
viral pre-mRNA splicing. Exon 5 is flanked by 5’ splice site D4 and
3’ splice site A5 and contains the GAR ESE in the 5’ part (Cartoon
by courtesy of H. Schaal).
Furthermore, the functional
characterization of the mode of
action of the guanosine-adenosinerich exonic splicing enhancer
(GAR ESE) in exon 5 of the HIV-1
mRNA revealed, that this ESE
fulfils a dual splicing regulatory
function. By synergistically mediating exon recognition through its
individual SR protein-binding
sites and by conferring 3’ splice site
selectivity within the splice site
cluster preceding exon 5. As the
GAR ESE-mediated activation of
the upstream 3’ splice site cluster
also is essential for the processing of
intron-containing vpu/env-mRNAs,
the GAR enhancer substantially
contributes to the regulation of viral
replication.
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46
Another aspect of this project
focussed on the nuclear uptake of
the HIV-1 Rev RNA transport factor. In particular we investigated in
collaboration with PD Dr. Ralph
Kehlenbach
(University
of
Göttingen) the requirements for
soluble and stationary components
of the nucleocytoplasmic transport
machinery for nuclear import of
Rev in living cells. The respective
data suggested that transportin is
the major import receptor for HIV-1
Rev in vivo. Furthermore, nuclear
import of Rev strongly depended
on Nup358, a component of the
cytoplasmic filaments of the nuclear
pore complex. Moreover, Nup358
appears to act as a docking station
for transportin-containing import
complexes on their way into the
nucleus, since nuclear import of
other transportin-dependent substrates (e.g. hnRNP M) was also
impaired in Nup358-depleted cells.
Transcription of the integrated proviral DNA (Provirus) produces
full-length unspliced ~ 9 kb RNA, which is used for biosynthesis of
Gag and Pol. In addition, this RNA species serves also as genome in
the formation of progeny viruses. Splicing of the ~ 9 kb transcript
results in RNAs of the single-spliced ~ 4 kb class, encoding Env, Vif,
Vpu and Vpr, and of the multiple-spliced ~ 2 kb class, encoding
Tat, Rev and Nef. Nuclear export and thereby translation of the
~ 9 kb and ~ 4 kb class of RNAs depends on the retroviral Rev
trans-activator, which directly interacts with the Rev Response
Element (not shown) present in these RNA species.
In addition to mediating nuclear
export of HIV-1 mRNA, also Rev
activities on the splicing, half-life
and translation of these transcripts
has been reported in the literature. In that respect an important and often overlooked
feature of Rev is its ability to form homo-multimers. Various studies have demonstrated that Rev binds as a monomer to its high-affinity RRE binding site and subsequently multimerizes on its RNA target. Although it has been unambiguously established that multimer formation is indeed required for Rev function, it is to date
unclear which Rev activity depends on Rev multimer-formation. To address this
question we also generated biologically inactive multimerization-deficient but shuttling-competent Rev mutant proteins. By fusing these mutants to heterologous oligomerization structures we were able to demonstrate that formation of a Rev homodimer is sufficient to rescue the biological activity of these otherwise inactive Rev
proteins. Furthermore, we were able to show that the cytoplamsic accumulation of
RRE-containing retroviral transcripts depends on Rev multimer-formation. These
findings support the notion that cellular cofactors in addition to CRM1 are required
for Rev-regulated nucleocytoplasmic transport of HIV-1 RRE-containing mRNAs.
*Supported in part by Deutsche Forschungsgemeinschaft (DFG) and in part by
Deutsche Krebshilfe
Combating drug resistance in HIV-1 infection
P. Priyadarshini (1), P. Hartjen (2), I. Hauber (1), S. Balabanov (3),
T. Brümmendorf (3), R. Hilgenfeld (4), C. Meier (5), M. Rarey (6),
U. Holzgrabe (7), J. van Lunzen (1,2), J. Hauber (1)
(2) University Medical Center Hamburg-Eppendorf –
Infectious Diseases Unit, Hamburg, Germany
Department of Oncology and Hematology, Hamburg, Germany
(4) University of Lübeck – Institute for Biochemistry, Lübeck, Germany
(5) University of Hamburg – Institute of Organic Chemistry,
Hamburg, Germany
(6) University of Hamburg – Center for Bioinformatics, Hamburg, Germany
(7) University of Würzburg – Institute of Pharmacy and Food Chemistry,
Würzburg, Germany
The introduction of highly active
antiretroviral therapy (HAART)
into clinical practice has profoundly improved the morbidity
and mortality among HIV-1infected patients. However, in a
growing number of infected subjects long-term HAART is accompanied by the occurrence of multidrug-resistant (HAART-resistant)
virus. Moreover, the transmission
of multidrug-resistant HIV strains
is becoming a growing problem
among newly infected persons. In
fact, recent studies have shown
that 10-12% of viruses harbour
mutations conferring resistance to
one or more drugs or drug classes
which are currently used in clinical practice to treat HIV-1 infection. Thus, novel and advanced
strategies have to be developed,
particularly to combat the so far
Pathway of eIF-5A biosynthesis. The unusual amino acid hypusine largely unmet medical problem of
is a postranslational modification of eIF-5A and necessary for
its activity. Hypusine is formed in two steps under catalysis of drug resistance in HIV-patients.
deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase
(DOHH). DHS transfers an aminobutyl moiety from spermidine
to a specific lysine residue in the eIF-5A precursor. The resulting
intermediate is then hydroxylated by DOHH, generating active
eIF-5A. CNI-1493 and JK8-2 are experimental small-molecular
weight inhibitors of DHS and DOHH, respectively (Cartoon by
courtesy of U. Holzgrabe).
Previous studies revealed that
eukaryotic initiation factor 5A
(eIF-5A) is a critical cellular cofactor of the HIV-1 Rev regulatory
protein, that primarily mediates
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the translocation of incompletely-spliced and unspliced retroviral mRNAs across
the nuclear envelope. This Rev cofactor is the only known cellular protein that
contains the amino acid hypusine, a modification that is required for eIF-5A
activity. The hypusine formation is a highly specific, spermidine-dependent posttranslational reaction that is catalyzed by two enzymes. In the first step, the aminobutyl moiety of spermidine is transferred to the ε-NH2 group of a single lysine
residue at position 50 within the human 154-amino-acid eIF-5A protein. This
reaction is catalyzed by deoxyhypusine synthase (DHS). The resulting eIF-5A
intermediate is then further modified by the metalloenzyme deoxyhypusine
hydroxylase (DOHH), generating the active form of eIF-5A. Therefore, inhibition
of the essential Rev cofactor eIF-5A by interference with hypusine-formation
should provide a novel strategy to block the HIV-1 life cycle.
We previously identified the multivalent guanylhydrazone CNI1493 by high-throughput screening assay to be a novel smallmolecular weight inhibitor of the
eIF-5A-modifying enzyme DHS.
We demonstrated that this compound indeed efficiently inhibits
Rev activity and thereby replication of HIV-1, including the replication of viruses that are other- Inhibition of HIV-1 NL4/3 replication by the experimental DOHH
JK8-2. HIV-1 NL4/3-infected PM1 cells were cultured in
wise fully resistant to classical inhibitor
the presence of the indicated concentrations of JK8-2, or DMSO
HAART. Furthermore, in collabo- as solvent control. Gag p24 antigen levels and cell viabilities
ration with Prof. Ulrike Holzgrabe (alamarBlue assays) were determined at day 6 and 9 post-infection.
The percentage of inhibition of virus replication in the drug-treated
(University of Würzburg) we cell culture, compared to the untreated controls, is shown. The
characterized the antiretroviral analysis of the respective cell viabilities (not shown) revealed that
antiviral effect was not caused by deleterious effects of JK8-2
activity of a novel DOHH inhibi- the
on the host cell.
tors (e.g. JK8-2). In sum, these
data provided the scientific rational (i.e. proof-of-principle) to establish an interinstitutional research network, including scientists from the Heinrich-PetteInstitute, the University Medical Center Eppendorf and the Universities of
Hamburg and Lübeck, to investigate cellular eIF-5A function and to develop
novel DHS/DOHH inhibitors. Since the hypusine-containing protein eIF-5A has
also been identified to be a potential target for therapy of chronic myeloid leukemia (CML), various aspects of this interdisciplinary project also directly contribute to the program area “Cellular Dysregulation” (for details see below). Thus,
novel small molecular-weight DHS/DOHH inhibitors may allow to overcome
drug-resistance in patients suffering from CML or HIV-1 infection.
During the initial project period the existing inhibitor lead-compounds were
further characterized in vitro and in appropriate disease models. In particular, the
antiretroviral activities of these compounds were investigated in various HIV-1
models. Furthermore, studies to analyze the potential influence of these compounds on the functional properties of T cells (i.e. HIV-specific CD4 and CD8 T
cell immune reponses) were initiated. Other aspects of this interdisciplinary
project included the structural analysis of the hypusine-modifying enzymes, the
computational molecular design of inhibitor analogues, the de novo inhibitor
synthesis, and the detailled analysis of the cellular function of the hypusinecontaining protein eIF-5A.
*Supported by Bundesministerium für Bildung und Forschung (BMBF) and in
part by Deutsche Forschungsgemeinschaft (DFG)
Eradication of HIV-1 proviral DNA from patient cells
I. Hauber (1), I. Sarkar (2), J. Chemnitz (1), H. Hofmann-Sieber (1),
C.-H. Nagel (1), A. Bunk (1), B. Weseloh (1), N. Beschorner (1), M.
Krepstakies (1), A. Schambach (3), J. Chusainow (2), C. Baum (3),
M. Manz (4), F. Buchholz (2), J. Hauber (1)
(2) Max-Planck-Institute for Molecular Cell Biology and Genetics,
Dresden, Germany
(3) Hannover Medical School, Hannover, Germany
(4) Institute for Research in Biomedicine, Bellinzona, Switzerland
Human immunodeficiency virus type 1 (HIV-1), the causative agent of the
aquired immunodeficiency syndrome (AIDS), integrates its proviral genome
permanently into the chromosome of the host cell. Thus, HIV-1 establishes a
persistent lifelong infection.
Current highly active antiretroviral therapy (HAART), which is
based primarily on virus enzyme
inhibitors and molecules that
inhibit virus attachement or viruscell fusion does not target the
integrated provirus and can therefore not eliminate HIV-1 from the
body.
(1) After entry into the cytoplasm, the RNA genome of retroviruses
is reverse transcribed into DNA. Subsequently, this proviral DNA
is integrated into the host genome and serves as template for
the production of progeny viruses during the host cell’s entire
life-span. (2) Reverse transcription results in sequence-identical
long terminal repeats (LTR; indicated by grey boxes). Engineered
LTR-specific recombinases (Tre-recombinase) recognize and recombine
LTR sequences of the integrated proviral DNA. (3) Tre-recombinase
activity results in the excision of the main body of the integrated
proviral DNA, thereby eradicating retrovirus infection.
In collaboration with Dr. Frank
Buchholz (MPI-CBG, Dresden)
we developed a novel strategy to
combat and possibly cure HIV-1
infection through expression of in
vitro taylored recombinases (Trerecombinases). Tre-recombinases
recognize asymmetric target
sequences within the native Long
Terminal Repeats (LTR) of the
integrated provirus and excise the
proviral HIV-1 DNA from the
genome of infected cells.
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In a proof-of-concept study we
demonstrated that Tre-recombinase quantitatively eliminates
the provirus from a human cell
culture infected with an HIV-1
Env-mutant in absence of cellular
toxicity. To advance this novel
therapy strategy towards clinical
development we subsequently
established an interdisciplinary
project team.
Tre-recombinase therapy will
most likely involve the introduction of Tre-encoding sequences
into the patient’s target cells by
somatic gene transfer. We therefore generated and analyzed in
collaboration
with
Prof.
Christopher Baum’s group (MHH)
replication-incompetent and selfinfactivating (SIN) lentiviral vectors of the newest generation.
A potential future Tre-therapy
may be envisaged as follows: first,
HIV-1 LTR sequences isolated
from the blood of the infected
subjects are determined and, subsequently, an adequate Trerecombinase is generated in vitro
by substrate linked protein evolution. The respective Tre-encoding
sequence is then be introduced ex
vivo into the patient’s hematopoietic progenitor/stem cells (HSC)
using lentiviral vectors. Following
quality control, these genetically
engineered HSC are then infused
into the respective patient. It is
expected, that the immune system
of this patient will be functionally
reconstituted over time, allowing
the elimination of long-living
latently-infected cells.
To further validate this therapy
approach Tre-recombinase has been
The delivery of Tre-recombinase into infected patients most likely
requires a gene therapy strategy. Current clinical protocols include,
after G-CSF mobilization, the harvest of hematopoietic stem cells
(HSC) by leukapheresis. Subsequently, ex vivo Tre-recombinase
gene transfer using replication-incompetent and self-inactivating
(SIN) lentiviral vectors and the subsequent autologous transplantation/infusion of the gene-modified cells would be performed.
In consequence it is expected that HIV-resistant cells will increase in
number. Remaining latently infected cells may now be targeted by
the partially reconstituted immune system of the patient.
(A) A replication-incompetent and self-inactivating (SIN) lentiviral
vector expressing Tre-recombinase and the eGFP reporter is
depicted. (B) The gene encoding Tre-recombinase was delivered
by the lentiviral SIN vector shown in panel A into PM1 cells. For
control, PM1 cells were also infected with the parental SIN vector
lacking Tre sequences. Subsequently, the respective cultures were
infected with wildtype HIV-1 (BaL). HIV-1 replication was determined
over a period of 10 weeks (w) in the Tre-expressing culture (Tre)
and in the negative control culture (NC).
successfully analyzed in cell cultures infected with fully replication-competent HIV-1
strain BaL. Furthermore, the target sequence specificity of Tre-recombinase has been
evaluated in detail and potential Tre-induced toxicities were studied in various cellular systems.
To monitor Tre-activity in an infected organism we also initiated experiments in
collaboration with Dr. Markus Manz (IRB, Bellinzona) employing Rag2-/-γc-/mice. Transplantation of human cord blood-derived CD34+ HSC into newborns
of these animals leads to de novo development of B, T, and dendritic cells; formation of structured primary and secondary lymphoid organs; and production of
functional immune responses. Consequently, these humanized animals can be
infected with HIV-1. Therefore, we are currently transplanting Rag2-/-γc-/- animals
with lentivirus-transduced human Tre-expressing HSC to evaluate in vivo Trerecombinase activity.
*Supported by Bundesministerium für Bildung und Forschung (BMBF)
Rapid response kinetics to HLA-B57/58 Gag-restricted CTL
epitopes is associated with slow disease progression in chronic
HIV-1 infection
P. Hartjen (1), I. Stahmer (1), J. Schulze zur Wiesch (1), A. Thomssen (1),
J.Hauber (2), J. van Lunzen (1,2)
Infectious Diseases Unit, Hamburg, Germany
Correlations between a person’s HLA composition and the ability of cytotoxic
T lymphocytes (CTL) to control HIV-1 replication are well documented.
Especially B27 and B57/58 restricted CTL responses directed against Gagderived epitopes are associated with slow disease progression. We were interested in defining the underlying mechanisms that may account for the beneficial role of these particular immune responses. Therefore, we assessed the
response kinetics of CTL from HIV infected subjects with slow disease progression in an IFN-γ-ELISPOT assay and phenotypically characterized immunodominant CTL by tetramer staining.
So far, cyropreserved PBL derived from 21 HIV-1 infected subjects were analyzed
with different characteristics in disease progression. Patients with non-progressive disease or slow progression and good viral control in the absence of HAART
were compared to a HLA-matched control group with progressive disease with or
without HAART according to current treatment guidelines. Patients were selected
on the basis of their HLA haplotype to ensure the same frequencies of “beneficial”
B57/58 and B27 haplotypes in both groups. Multiple samples were analyzed longitudinally (range 1-6 years) using a specialized IFN-γ-ELISPOT assay using
optimal B clade consensus peptides for HLA-A and -B haplotypes derived from
Gag, Pol, Nef, and/or Env regions.
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HLA-B57/58 and B27 haplotypes were evenly distributed in LTNP and controls
(8/10 vs. 8/11). The B57-and B58-restricted CTL-responses against Gag-epitopes
were detected to be the immunodominant response in these patients. Less frequently, B44-Gag, B44–Env and B27-Gag
restricted responses were detected
in descending order. These strong
CTL responses were detected in
LTNP as early as 30 min after peptide exposure in vitro and gradually
increased thereafter. This rapid
response pattern was conserved in
LTNP for more than 2 years and
lasted up to 6 years of follow up. In
contrast, most of the HLA-Arestricted responses were only
detectable after 24 hours of in vitro
stimulation, suggesting a comparatively delayed response kinetic for
non-B57/58 restricted epitopes in
the control group. Interestingly,
HLA-matched patients with progressive disease displayed considerably slower and weaker Gagrestricted responses, even in patients
CD8+ T cells are schematically assigned to distinct functional
with B57/58 haplotype.
subsets. A hypothetical model of linear CD8+ T-cell differentiation is
depicted. Four main phenotypic subsets as well as their respective
We further characterized immuno- enrichment into memory virus-specific CD8+ T cells are presented.
dominant CTL by tetramer stain- L and H indicates low expression and high expression, respecPhenotypes in brackets represent intermediate or minor
ing. In order to distinguish between tively.
populations. Cartoon adapted from Appay et al. 2002 Nature
the differentiated subsets “early”, Medicine 8:379-85.
“intermediate”
and
“late”
(CD28+CD27+, CD28-CD27+ and CD28-CD27-, respectively) we determine the
expression level of the costimulatory receptors CD27 and CD28 in tetramer-positive
CTL. This clearly revealed an enrichment of CTL presenting “early” memory phenotype in individuals with slow disease progression, which is associated with controlled
acute viral infection (e.g. EBV, CMV, chronic HIV). Interestingly, this phenotype
shifted towards “intermediate” and “late” phenotypes resembling uncontrolled
chronic infection in patients with progressive disease (e.g. chronic HIV/HCV).
We conclude, that the dominance of B57/58 haplotypes in slowly progressing HIV-1
infection is not only explained by the breadth and magnitude of CTL-responses but
also by the rapid response kinetics of specific CTL in patients with slow disease progression. These Gag-specific CTL may help preventing establishment of HIV-1
latency via targeting the virus life cycle very early at the pre-integration level.
Determinants of successful antiviral treatment of concomitant
acute HCV/HIV coinfection.
J. Schulze zur Wiesch (1), D. Pieper (2), A. Thomssen(1), P. Hartjen (1),
J. Hauber (2), J. van Lunzen (1,2)
(1) University Medical Center Hamburg-Eppendorf – Infectious Diseases
Unit, Hamburg, Germany
HIV and HCV share similar routes of infection and chronic HCV coinfection with all
its complications has become a major health threat to HIV-infected individuals
worldwide. Lately, outbreaks of sexually transmitted acute HCV infection in chronically HIV-infected patients have been reported from different parts of Europe. So far
only limited data exist concerning the clinical outcome and the immunologic
responses which are primed during simultaneously acquired acute HCV/HIV coinfection. In a pilot study we conducted a detailed clinical and immunologic analysis
of three individuals following concomitant acute HCV/primary HIV infection.
Additionally to longitudinal clinical parameters, virus-specific CD8+ and CD4+ T cell
responses were assessed using ELISPOT, standard proliferative (CSFE) and further in
vitro CD4+ T cell assays. In all patients anti-HCV treatment was started and HIV
antiretroviral therapy (ART) was co-administered early during primary infection.
HCV viremia was cleared under therapy with pegylated IFN-alpha in all three cases.
In two patients HIV replication was contained even after ART was interrupted, which
was associated with strong HIV-specific CD8+ and CD4+ T cell responses. In these two
patients multi-specific HCV CD4+ responses could also be detected. No HCV specific CD4+ responses were detected
in the third patient who also had the
lowest CD4+ nadir during PHI
(<200 cells/l). Our data indicate that
HCV antiviral therapy should be
considered early in the setting of
concomitant acute HCV/HIV coinfection and that a sustained virological response is possible even
during primary HIV infection.
Clearly, larger prospective trials have
to determine the best anti-HCV
treatment regimen and whether
early adjuvant antiretroviral treatment is needed to achieve the favorable clinical and immunological
Immunologic control of HCV pathogenesis. In a normal HCV monoinfection, HCV-specific CD4 and CD8 T cells are mobilized to contain outcome described here. A better
infection. In case infection is not cleared, such T cells contribute to understanding of immune responsantiviral, proinflammatory, and regulatory effects. There is emerging
evidence that the high viral loads of HCV during coinfection with HIV es following acquisition of HCV
are associated with profound defects in HCV-specific CD4 and also may shed insight into both, HCV
CD8 T cell responses. Moreover, some preliminary experimental data
pathogenesis in HIV-infected indisuggest that the cytokine environment in the liver is altered, which
could affect disease progression independently from any influence viduals and prospect for immunoof virus load (graphic courtesy of A. Kim, Boston).
therapies in this population.
53
Crossing the Species Barrier: Role of Env-Receptor Interactions in
Gammaretrovirus Infections
54
U. Bergholz (1), M. Ziegler (1), K. Stieler (2), A.D. Miller (3), M. Eiden (4),
N. Fischer (2), V. Prassolov (5), C. Stocking (1),
(1) Heinrich-Pette-Institute, Germany
(2) University Medical Center Hamburg-Eppendorf, Hamburg, Germany
(3) Fred Hutchison Cancer Center, Seattle, WA USA
(4) National Institute of Health, Bethesda, MD, USA
(5) Engelhardt-Institute of Molecular Biology, Moscow, Russia
The gammaretroviruses compromise a genus of exogenous and endogenous viruses
that are widely spread in vertebrates. Analysis of endogenous retrovirus (ERVs) in
the genomes of humans, mice, and other species indicates a longstanding association, probably dating back several hundred million years, during which retroviruses
have repeatedly colonized host genomes. Phylogenetic studies of class I (gamma and
epsilon) and class II (alpha, beta, delta and lenti) ERVs suggest that horizontal transfer of infectious virus between vertebrate classes occurs only rarely, although several
important examples have occurred. In addition to the well-known jumping of a
lentivirus from chimpanzees (SIVcpz) to humans (HIV-1), a recent report has demonstrated the first bonafide human infection with a xenotropic MLV-related gammaretrovirus (XMRV). Using retroviral pseudotype assays and receptor binding
assays, we have demonstrate that XMRV possesses a wide host range and efficiently
infects feral mouse cells, as well as established human cell lines and primary cells.
Interference assays confirm the classification of XMRV as a xenotropic MLV-like
virus, which uses the transmembrane protein Xpr1 as a receptor. Interestingly,
XMRV preferentially uses the human versus the murine Xpr1 variant, arguing that
XMRV already persists for a longer period in the human population.
Another example of cross-species transfer is the recent characterization of a
Koala gammaretrovirus (KoLV), which is currently "invading" the host genome.
This virus is closely related to the gibbon ape leukemia virus (GALV), although
the vector responsible for transmission of the virus between these two species
with distinct habitats has not been conclusively identified. We have recently isolated an ERV from Mus cervicolor, which we have dubbed McERV. McERV is
closely related to the GALV and KoLV isolates and thus may share a common
ancestor (Fig. X). Host and tissue spectrum analysis has shown that in contrast to
most other gammaretrovirus isolates that use ubiquitously expressed membrane
transporters as a receptor, McERV uses the myelin protein plasmolipin (PLLP) as
a cellular receptor. PLLP expression pattern is restricted to cells of nervous and
secretory systems (e.g. brain, spinal cord, kidney, and lungs) and thus retroviral
vectors pseudotyped with McERV show tissue expressions. We are currently
screening the mouse genome for other uncharacterized ERVs that may shed light
on the evolution and spread of gammaretrovirus, but also provide attractive tools
for cellular and molecular biology, as well gene therapy.
Requirement of Activation for Hepatitis B Virus Infection.
M. Bruns (1), C. Maenz (1)
Although in vitro models of human hepatitis B virus replication are established,
so far none could approximate infection efficiency as expected from in vivo
observations. Susceptibility for HBV infections has only been reported for primary hepatocytes of human, chimpanzee or Tupaia belangeri and the cell line
HepaRG. We showed that the insusceptible human hepatoma cell line HepG2 can
be infected, when the virus was beforehand activated by passage over whole duck
liver cell cultures. That suggests an activation step to be performed by specialized
liver cells.
*Supported by Deutsche Forschungsgemeinschaft
Enzymatic treatment of duck hepatitis B virus: topology of the
surface proteins for virions and noninfectious subviral particles
M. Bruns (1), C. Franke (1), U. Matschl (1)
The large surface antigen L of duck hepatitis B virus exhibits a mixed topology
with the preS domains of the protein alternatively exposed to the particles’ interior or exterior. After separating virions from subviral particles (SVPs), we compared their L topologies and showed that both particle types exhibit the same
amount of L with the following differences: first, preS of intact virions was enzymatically digested with chymotrypsin, whereas in SVPs only half of preS was
accessible; second, phosphorylation of L at S118 was completely removed by
phosphatase treatment only in virions; third, iodine-125 labeling disclosed a
higher ratio of exposed preS to S domains in virions compared to SVPs. These
data point towards different surface architectures of virions and SVPs. Because
the preS domain acts in binding to a cellular receptor of hepatocytes, our findings
implicate the exclusion of SVPs as competitors for the receptor binding and entry
of virions.
*Supported by the Deutsche Forschungsgemeinschaft
Entry of Duck Hepatitis B Virus into Primary Duck Liver and Kidney
Cells after Uncovery of a Fusogenic Region within the Large Surface
Protein
C. Maenz (1), S.F. Chang (1,2), A. Iwanski (1), M. Bruns (1)
(2) Biochemical Engineering Center, Chutung Hsinchu, Taiwan
Hepatitis B viruses exhibit a narrow host range specificity that is believed to
be mediated by a domain of the large surface protein, designated L. For duck
hepatitis B virus it has been shown that the preS domain of L binds to carboxy-
55
56
peptidase D, a cellular receptor present in many species on a wide variety of cell
types. Nonetheless only hepatocytes become infected. It so far remained vague, which
viral features procure host range specificity and organotropicity. Using chymotrypsin to treat duck hepatitis B virus we addressed the question, whether a putative fusogenic region within the amino-terminal end of the small surface protein
may participate in viral entry and possibly constitute one of the requirements for
the host range of the virus. The addition of the enzyme to virions resulted in an
increased infectivity. Remarkably, even remnants of enzyme-treated subviral particles
proved to be inhibitory for infection. A noninfectious deletion mutant, devoid of
the binding region for carboxypeptidase D, could be rendered infectious for
primary duck hepatocytes by treatment with chymotrypsin. Although due to the
protease treatment mutant and wild-type viruses may have become infectious in
an unspecific and receptor-independent manner, the host range specificity was
not affected as shown by the inability of viral replication in different hepatoma
cell-lines as well as primary chicken hepatocytes. Instead, the organotropicity could
be reduced, which was demonstrated by infection of primary duck kidney cells.
Elucidation of the SV40-induced DNA damage signaling cascades
and their components utilized by SV40
G. Rohaly (1), K. Korf (1), S. Dehde (1), A. Mena-Nunez (1), H. Will (1),
I. Dornreiter (1)
Simian Virus 40 (SV40) infection of permissive wildtype p53 (wtp53) CV-1 cells
activates the phosphoinositide 3-kinase-related kinase (PIKK) ATM (Ataxia
telangiectasia mutated) leading to ATM-catalyzed phosphorylation of cellular
down-stream substrates Chk2, H2AX, SMC1, p53 and viral T-Ag. Inactivation of
ATM by dominant-negative effects shortly after viral infection prevented phosphorylation of the aforementioned down-stream substrates, and unexpectedly
impaired T-Ag expression. Thus, data strongly indicate that SV40 utilizes ATM to
transactivate its early region, which encodes for the replication/transcription factor T-Ag. Accordingly, in SV40-infected, ATMkd expressing cells viral amplification and T-Ag-mediated transactivation of the late region, which encodes for the
capsid proteins, were completely abrogated. Another PIK kinase, which is activated upon viral infection, is ATR (ATM and Rad3-related protein). Inactivation
of ATR by Dox-induced expression of ectopic ATRkd shortly after viral infection
did not interfere with T-Ag expression and ATM activation, as corroborated by
the appearance of phosphorylated Chk2, H2AX, SMC1, p53 and T-Ag. However,
SV40-infected TO-CV-1 cells expressing dominant-negative ATRkd failed to activate the transcriptional activity of ∆p53, an activity that leads to up-regulation of
Cdk-inhibitor p21 and concomitantly inactivation of the S-phase promoting
cyclin A-Cdk2 complex. Furthermore, our data show that viral amplification was
significantly reduced (90%) in ATRkd expressing cells, indicating that ATR-∆p53p21-mediated inactivation of cyclin A-Cdk2 is essential for maximal viral ampli-
57
Formation of active SV40 replication centers depends on functional ATR.
fication. In SV40-infected CV-1 cells, about ten active viral replication centers are
detectable 24 hours post infection, which can be identified by EdU-incorporating
large T-Ag foci. In contrast, in SV40-infected, TO-CV-1-Flag-ATRkd cells formation of active viral replication centers was considerably impaired, despite cellular
replication, as evidenced by cellular EdU-incorporation and EdU-negative T-Ag
foci. In addition, inactivation of the ATR down-stream target ∆p53 by expressing
mut∆p53 or preventing up-regulation of ∆p53-transactivated p21 via expression
of shRNAp21 also impaired the formation of active viral replication centers,
although to a lesser extend. Thus, the ATR-∆p53-21-mediated inactivation of
cyclin A-Cdk2 is indispensable for facilitating viral amplification. Taken together,
our data demonstrate that the ATM- and the ATR-mediated DNA damage
response signaling pathways are purposefully activated in SV40-infected permissive cells, to enable adaptation of the host cell’s milieu for optimal viral progeny.
Role of the host’s p53 status in SV40 life cycle
K. Korf I (1), G. Rohaly (1), S. Dehde (1), H. Will (1), I. Dornreiter (1)
Although the transcriptional activity of p53 is inactivated in SV40-infected cells,
we propose that p53 plays a role in the SV40 life cycle. The assumption is supported by the finding that for the human adeno-associated virus type 2 (AAV2)
it was shown that intact p53 is required to prevent premature death of the host
cell and thus contributes to enhanced viral progeny. Therefore, we tested whether
the p53 status of the host influences the infection process and accordingly viral
amplification. Evaluation of viral replication efficiency in SV40-infected wtp53
CV-1 and mutp53-175D expressing wtp53 TO-CV-1 cells revealed that viral
amplification was, in comparison to the parental cells, significantly reduced
58
(70%) in TO-CV-1-Flag-mutp53 cells. Analysis of the cell cycle distribution did
not display any differences between wtp53 and mutp53 expressing CV-1 cells. In
either cell lines, the ATM-Chk2- and the ATR-∆p53-p21-mediated DNA damage
pathways were fully activated, leading to attenuation of S-phase progression,
thereby providing an optimal environment for viral amplification. In addition,
expression of the viral proteins VP1 and T-Ag as well as ATM-catalyzed phosphorylation of T-Ag were not impaired. Thus, at first sight no obvious reason was
found, which would explain the negative impact of mutp53 on viral progeny.
Since T-Ag interacts with wtp53 and the tumor suppressor is recruited into viral
replication centers one possible explanation is that the p53 hot spot mutant 175D
interferes with the viral replication process. Indeed, complex formation of
mutp53 with T-Ag as well as recruitment of mutp53 to viral replication centers,
which displayed diminished replication activity, was observed. Data strongly
indicate that the p53 status of the host cell plays an important part in SV40 DNA
replication. However, the molecular mechanisms involving transcriptional inactive wtp53 in viral amplification remain to be elucidated.
Elucidation of the molecular mechanisms that underlie SV40induced polyploidization
G. Rohaly (1), S. Dehde (1), K. Korf I (1), A. Mena-Nunez (1),
H. Will (1), I. Dornreiter (1)
The current model of origin licensing proposes a sequential assembly of proteins to form the pre-replication complex (pre-RC) and describes the basic
mechanisms responsible for preventing re-initiation of DNA synthesis within a
single cell cycle. In the first step, Cdc6, Cdt1 and cyclin E collaborate with the
origin recognition complex to load the replication helicase (Mcm2-7) into preRCs at replication origins. In the second step, two protein kinases, Cdc7-Dbf4
and the cyclin E-Cdk2, convert pre-RCs into bidirectional replisomes at each
origin. Re-initiation at replication origins within a cell cycle is prevented by the
inactivation of pre-replication components in S- and G2-phase. The Cdks play
a central role in establishing a block to re-replication through phosphorylation
of each of the components. At present several proteins of the mammalian preRC have been described to be phosphorylated by cyclin A-Cdk and as a result
degraded or inactivated. First, it was demonstrated that cyclin A-Cdk2dependent Cdt1 phosphorylation in S-phase induces its degradation. Second,
the licensing factor Cdc6 remains associated with chromatin throughout
S-phase, but is inactivated by cyclin A-Cdk2 catalyzed phosphorylation. Third,
cyclin A-Cdk2 phosphorylation decreases the helicase activity of the Mcm complex, reduces the complex’s binding to chromatin and may be at least partially
responsible for the gradual release of chromatin-bound Mcm proteins. Forth,
cyclin A-Cdk2 phosphorylation of origin bound DNA polymerase α-primase
complex (Pol α) immediately after origin firing prevents re-loading of the
replicase and accordingly re-initiation of bidirectional DNA replication. Last,
cyclin A-Cdk1 phosphorylation of the tetrameric Pol α complex prevents overall
loading of the replicase onto chromatin.
In SV40-infected permissive wtp53 cells, the continued replication of host cell
chromosomal DNA beyond 4N implies alteration of the normal controls acting
to limit DNA replication to once per cell cycle. Considering that cellular mechanisms blocking re-replication of DNA act by cyclin A-dependent phopshorylation of the aforementioned replication factors it is feasible to suggest that dysregulation of replication control in SV40-infected cells is altered at the Cdk
activity and cyclin expression level. Indeed, our results demonstrated that SV40
infection leads to inhibition of cyclin A-Cdk activity and accordingly S-phase
arrest, which is accomplished by the ATR-∆p53-p21-mediated DNA damage
pathway. Moreover, in SV40 infected cells degradation of the licensing factor
Cdt1 was not observed; instead, the factor remained chromatin associated until
the cells displayed a cytopathic effect. Another factor, which is an essential component of the origin-licensing complex and in concert with Cdk2 converts the
pre-RC into an active replication complex is cyclin E. In normal cycling cells
cyclin E is loaded onto chromatin during G1-phase and promotes loading of
the Mcm helicase into pre-RCs. Accordingly, expression of cyclin E is tightly
regulated so that peak levels occur at the G1/S boundary followed by a decline
and loss of expression during S-phase. Thus, degradation of cyclin E prevents
re-loading of the Mcm helicase and assembly of pre-replication complexes in
S-phase. However, in SV40-infected permissive cells cell cycle-dependent turnover of cyclin E is impaired due to sequestration of the Cul4 E3 ubiquitin ligase
by T-Ag, leading to accumulation of cyclin E as well as high cyclin E-Cdk2
activity. We found that in SV40-infected cells proteasome-resistant cyclin E
remains chromatin associated throughout S-phase, and together with Cdt1
facilitates loading of the Mcm helicase onto chromatin, thereby enabling relicensing of RCs. In addition, cyclin E-Cdk2 activity is essential to promote
loading of replication factors Cdc45 and Pol α into the licensed RCs. Since
SV40-infected cells display high cyclin E-Cdk2 activity and are devoid of the
replication antagonist cyclin A-Cdk2, loading of Cdc45 and Pol α onto the relicensed origins of replication is permitted. Taken together, our investigations
demonstrate that SV40 overrides DNA replication control through Cdk/cyclinderegulation, which is facilitated by the ATR-∆p53-p21- and the ATM-mediated
DNA damage response pathways.
Inhibition of duck hepatitis B virus infection of liver cells by combined treatment with viral e antigen and carbohydrates
C. Maenz (1), C. Loscher (1), A. Iwanski (1), M. Bruns (1)
The e antigen (eAg) of duck hepatitis B virus (DHBV) is a glycosylated secretory
protein with a currently unknown function. We concentrated this antigen from
the supernatants of persistently infected primary duck liver cell cultures by
ammonium sulphate precipitation, adsorption chromatography over concanava-
59
60
lin A Sepharose, preparative isoelectric focusing and molecular
sieve chromatography. The combined treatment of duck liver cells
with DHBV eAg (DHBe) concentrate and alpha-methyl-d-mannopyranoside strongly inhibited
DHBV replication at de novo
infection. When DHBe was added
to non-infected primary duck liver
cells, it was found to be associated
with liver sinusoidal endothelial
cells. This binding could be inhibited by the addition of alphamethyl-d-mannopyranoside and
other sugar molecules. The inhibitory effect of DHBe on infection
could play a role in maintaining
viral persistence.
*Supported by Deutsche
Forschungsgemeinschaft and
Bundesministerium für Bildung
Purification of DHBe. (a) Elution profile for DHBe over a Concanavalin
A-Sepharose column; (b) Isoelectric focusing of DHBe within a pH
gradient of 3.5-10; (c) Molecular sieve chromatography of DHBe in
Sephadex G-75; (d) Analysis of purfication steps by electrophoresis:
staining with Coomassie blue (left) or Western blotting with antiDHBc antiserum (right).
Solution structure of stem-loop alpha of the hepatitis B virus posttranscriptional regulatory element
M. Schwalbe (1), O. Ohlenschläger (1), A. Marchanka (1),
R. Ramachandran (1), S. Häfner (1), T. Heise (2), M. Görlach (1)
(1) Leibniz-Institut für Altersforschung/Fritz-Lipmann-Institut, Jena, Germany
Chronic hepatitis B virus (HBV) infections may lead to severe diseases like liver
cirrhosis or hepatocellular carcinoma (HCC). The HBV post-transcriptional regulatory element (HPRE) facilitates the nuclear export of unspliced viral mRNAs,
contains a splicing regulatory element and resides in the 3′-region of all viral transcripts. The HPRE consists of three sub-elements α (nucleotides 1151–1346), β1
(nucleotides 1347–1457) and β2 (nucleotides 1458–1582), which confer together
full export competence. Here, we present the NMR solution structure (pdb 2JYM)
of the stem-loop α (SLα, nucleotides 1292–1321) located in the sub-element α. The
SLα contains a CAGGC pentaloop highly conserved in hepadnaviruses, which
essentially adopts a CUNG-like tetraloop conformation. Furthermore, the SLα
harbours a single bulged G residue flanked by A-helical regions. The structure is
highly suggestive of serving two functions in the context of export of unspliced viral
RNA: binding sterile alpha motif (SAM-) domain containing proteins and/or
preventing the utilization of a 3′-splice site contained within SLα.
Analysis of Duck Hepatitis B Virus core protein phosphorylation
and dephosphorylation
C. Maenz (1), M. Bruns (1), H. Will (1)
The core proteins of hepatitis B viruses are phosphorylated at serin/threonine
sites during virus entry, shortly after or concomitantly during core protein
neosynthesis, and are dephosphorylated during viral DNA maturation shortly after core assembly. It is neither known which of the many ser/thr sites are
(de)modified at which stage of infection, nor which enzymes are involved,
nor whether the dynamic on/off of phospho groups is functionally relevant.
In order to address some of these questions we have overexpressed two known
cellular phosphatases (phosphatase 2B (calcineurin) and phosphatase 2A),
which we considered candidates potentially involved in core dephosphorylation, and tested their effects. Both enzymes induced hypophosphorylation of
the core protein compared to non-transfected cells but had no (with calcineurin) or only little effect (with PP2A) on steady state levels of viral protein expression, viral DNA synthesis, formation and secretion of viral particles as well as on infectivity of the secreted viral particles for primary duck
liver cells. Studies with several duck hepatitis B virus (DHBV) core protein
phosphosite specific antibodies which we produced indicated differential
effects of each enzyme for specific DHBV core phosphosite. Overexpression
of a dominant negative mutant of PP2A increased the level of intrahepatic
DHBV core protein and slightly increased viral particle secretion. Taken
Hypophosphorylation of DHBc by Calcineurin. Detection of DHBV core protein and calcineurin in transfected
hepatoma cells by indirect immunofluorescence staining (left panel) and calcineurin-mediated dephosphorylation
of DHBc but not of the envelope protein PreS as evidenced by immunoblotting (right panel).
61
together, these data argue against an essential role of phosphorylation at
any stage of DHBV core phosphorylation and suggest a stimulatory role of
phosporylation of unknown cellular and/or viral proteins for DHBV progeny
production.
62
Transactivation activity of the X and PreS protein of duck hepatitis
B virus are dispensible for DHBV infection in vitro and in vivo
C. Kranig (1), K. Dallmeier (2), C. Maenz (1), M. Bruns (1), H. Will (1)
(1) Heinrich-Pette Institute, Hamburg, Germany
(2) Institute of Virology, University Freiburg, Germany
Both human and duck hepatitis B viruses (HBV and DHBV) code for two proteins with transcriptional transactivation activity, the X proteins and the large
envelope proteins preS in phosphorylated forms. HBV mutants lacking both
activities cannot produce virions in hepatoma cells, whereas HBV deficient in
only one of both are viable. We have investigated whether the same applied to
DHBV. Viral DHBV DNA genomes unable to produce DHBx and phospho-preS
or both were created by mutagenesis. These mutant genomes were functionally
tested both in chicken hepatoma cells (LMH) by transfection, in primary duck
hepatocytes as well as in ducklings. All three mutant DHBV genomes were as
efficient in viral protein synthesis, viral replication and production of progeny as
the wildtype genome in LMH cells. Unexpectedly, there was also no detectable
difference in these parameters between the mutants and wildtype DHBV in infectivity and production of progeny when analysed in primary duck hepatocytes and
in ducklings. These data indicate that the transactivation activity of DHBx and
phospho-preS of DHBV are dispensible for the infectious life cycle of DHBV in
vitro and in vivo.
*Supported by the Bundesministerium für Bildung und Forschung
Host-range and pathogenicity of hepatitis B viruses
H.-J. Netter (1), S.-F. Chang (2), M. Bruns (3)
(1) Monash University, Department of Microbiology, Australia
(2) Industrial Technology Research Institute, Chutung Hsinchu, Taiwan
Hepatitis B viruses are small enveloped DNA viruses referred to as
Hepadnaviridae that cause transient or persistent (chronic) infections of the
liver. This family is divided into two genera, orthohepdnavirus and avihepadnavirus, which infect mammals or birds as natural hosts, respectively. They
possess a narrow host range determined by the initial steps of viral attachment and entry. Hepatitis B virus is the focus of biomedical research owing
to its medical significance. Approximately 2 billion people have serological
evidence of hepatitis B, and of these approximately 350 million people have
chronic infections (World Health Organisation, Fact Sheet WHO/204,
October 2000). Depending on viral and host factors, the outcomes of infection with hepatitis B virus vary between are acute hepatitis, mild or severe
chronic hepatitis or cirrhosis. Chronic infections are associated with an
increased risk for the development of hepatocellular carcinoma. We have
reviewed specific aspects of the host range and pathogenicity of hepatitis B
viruses which became recently apparent.
Polyomavirus-encoded miRNAs
N. Fischer (1), N. Walz (2), T. Christalla (2), T. Günther (2), U. Tessmer (2),
A. Grundhoff (2)
Institute of Med. Microbiology, Hamburg, Germany
Previously, we have developed a computational algorithm (VMir) for the ab
initio prediction of viral miRNAs, and sucessfully applied the method to identify miRNAs in several viruses, including the Polyomavirus SV40. SV40 encodes
a single pre-miRNA hairpin, which produces two mature miRNAs late in viral
infection. These miRNAs targets the early transcripts which encode large
T-antigen (LT-Ag), thus preventing accumulation of superfluous amounts of
A miRNA encoded by Merkel Cell Polyomavirus (MCPyV). (A) VMir Prediction of putative miRNAs encoded by
MCPyV. miRNA candidates are shown by genomic position (x-axis) and score (y-axis). The experimentally verified,
high scoring candidate is shown in red and marked with an arrow. Predictions with a score of less than 115 (dotted
line) are deemed insignificant. (B) Experimental validation (small RNA northern blot) of the miRNA candidate
predicted by VMir in 293T (left) and 293 (right) cells transfected with two different constructs (MCPyV I + II) which
express subgenomic regions harboring the miRNA candidate. The position of the mature miRNA is marked with
an arrow. (C) Predicted structure of the MCPyV miRNA precursor stem loop. The position of the mature miRNA
is shown in red.
63
64
the highly immunogenic LT-Ag protein. The pre-miRNA hairpin is located just
distal of the open reading frame encoding the major capsid antigen. It is fully
complementary to the LT-Ag transcripts which are being transcribed from the
other strand, and thus exerts its negative regulatory effect via cleavage of these
transcripts in an siRNA like manner. As predicted by VMir (and according to
subsequent experimental confirmation), the closely related polyomaviruses
JVC and BKV also encode a single pre-miRNA. These pre-miRNAs are located
at a similar genomic location as the SV40 miRNA, but are not related in
sequence. Nevertheless, as they are located antisense to the LT-Ag gene, they
retain their ability to negatively regulate LT-Ag transcripts via cleavage.
We have analyzed the recently discovered Merkel cell polyomavirus (MCPyV), a
novel polyomavirus which is thought to play a causative role in the pathogenesis
of Merkel cell carcinoma (MCC). A high scoring miRNA candidate predicted by
VMir was experimentally tested and found to encode a bona fide miRNA.
Interestingly, the MCPyV-encoded miRNA is encoded opposite to sequences
forming the proximal portion of the LT-Ag ORF, at a fundamentally different
genomic position than the miRNAs of SV40, JCV and BKV miRNAs. Phylogenetic
analysis shows that MCPyV is more closely related to the Mouse Polyomavirus
(MuPyV) branch of the Polyoma-virus family than to SV40, BKV and JCV.
Indeed, VMir analysis suggests that MuPyV, Hamster Polyomavirus (HPyV) and
Polyomavirus-encoded miRNAs. (A) Phylogenetic tree of the Polyomavirus family. The human members of
the family are shaded grey. (B) Alignment of selected Polyomavirus genomes and position of experimentally
verified (red) or VMir-predicted (blue) miRNAs, indicated by block arrows. Sequences encoding small, middle
and large T-Ag or capsid antigens (VP1, VP2 and VP3) are shown as yellow or green block arrows, respectively.
Aligned genomic regions are symbolized by the intermittent black line underneath. Gaps in the line indicate
the absence of sequences present in other genomes. (C) Alignment of sequences of mature 5p and/or 3p
miRNAs encoded by SV40, BKV, JCV, Merkel Cell Polyomavirus (MCPyV) and Mouse Polyomavirus (MoPyV),
showing the absence of significant sequence conservation. The SV40, BKV and JCV pre-miRNA hairpins produce mature miRNA molecules from both arms.
the lymphotropic Polyomavirus (LPV) all harbor miRNAs at a similar location as
MCPyV.
While Polyomavirus-encoded miRNAs thus are overall neither related in sequence
nor genomic location, they are nevertheless predicted to retain complementarity
to LT-Ag transcripts, suggesting the negative regulation of LT-Ag is functionally
important in vivo.
In addition to the experimental validation of HPyV and LPV miRNAs, we are
currrently investigating the molecular mechanisms governing polyomavirus
miRNA expression as well as the role of the MCPyV miRNA in the viral lifecycle
and its possible contribution to cellular transformation by MCPyV.
*Supported by Bundesministerium für Gesundheit (BMG)
A Global Analysis of Evolutionary Conservation Among Known and
Predicted Gammaherpesvirus microRNAs
N. Walz (1), T. Christalla (2), U. Tessmer (2), A. Grundhoff (2)
MicroRNAs (miRNAs) are small, non-coding RNAs which post-transcriptionally regulate mRNA expression. The current release of the miRNA registry lists
a total of 140 viral miRNAs. Strikingly, the vast majority (137) of these are
encoded by members of the herpesvirus family. Whereas the propensity to
Conservation of EBV and rLCV miRNAs. (A) Schematic depiction of the genomic location of EBV- and rLCVmiRNAs and their conservation. Open reading frames are shown as block arrows, the location of pre-miRNAs is
indicated by triangles underneath or above the EBV and rLCV backbones. pre-miRNA hairpins are depicted in
higher detail in the center: Light gray lollipops drawn proximal to the backbone symbolize non-conserved premiRNAs, grey or black lollipops depict conserved hairpins. Solid or dotted lines connect homologous hairpins
and indicate whether conservation includes the seed region of mature miRNAs or is limited to the pre-miRNA
(dotted line). (B) Experimental confirmation of novel rLCV and EBV miRNAs by small RNA northern blotting.
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encode miRNAs thus appears to be a conserved feature of herpesviruses, the
miRNAs themselves show little conservation: with the exception of 7 miRNAs
known to be shared between Epstein-Barr Virus (EBV) and its close relative
Rhesus Lymphocryptovirus (rLCV), all other herpesvirus miRNAs appear
unrelated in sequence. We have recently employed VMir, a computational ab
initio prediction method, to identify miRNAs in several viral genomes,
including 12 miRNAs encoded by Kaposi's sarcoma-associated herpesvirus
and 23 miRNAs encoded by EBV. We have modified the algorithm to allow
consideration of evolutionary conservation and conducted a global analysis
of all fully sequenced gammaherpesvirus genomes currently listed in the
NCBI database.
Our analysis confirmed the conservation of miRNAs between EBV and rLCV, but
predicted additional conserved miRNAs; indeed, subsequent experimental
validation confirmed the existence of 2 novel EBV- and 16 novel rLCV-encoded
miRNAs, with a total of 14 miRNAs sharing recognizable sequence homology. In
contrast, with the exception of viral genomes with a very high degree of overall
sequence conservation (i.e., different viral strains such as KSHV type M and P, as
well as the closely related rhadinoviruses Macaca Fuscata Rhadinovirus and
Conservation of Gammaherpesvirus miRNAs. (A) All complete gammaherpesvirus genomes deposited in the
Refseq database were blasted against each other. The upper panel the percentage of overall sequence identity
between individually aligned genomes. The lower panel shows the number of confirmed pre-miRNAs or premiRNAs predicted by VMir which were found to be conserved between genome pairs (Self-aligned genomes
show the total number of predicted pre-miRNAs). (B) VMir Prediction of Equid herpesvirus 2 (EqdHV2), Ovine
herpesvirus 2 (OvHV2), Alcelaphine herpesvirus 1 (AlHV1) miRNAs. These viruses are predicted to express large
numbers of miRNAs unrelated in sequence, but encoded at similiar genomic locations.
Rhesus Rhadinovirus), we have found little evidence for the existence of hitherto
unknown conserved miRNAs shared by other herpesviruses, although many
genomes are predicted to harbor large numbers of miRNAs at similar genomic
locations.
While EBV and rLCV thus share even more miRNAs than previously thought,
our results underline the notion that, in contrast to their cellular counterparts,
viral miRNAs show an almost total lack of sequence conservation.
*Supported (in part) by Deutsche Forschungsgemeinschaft (DFG) and
Hamburger Stiftung zur Förderung der Krebsbekämpfung
Monitoring the Epigenetic Fate of Gammaherpesvirus Genomes
T. Günther (1), U. Tessmer (1), A. Grundhoff (1)
Latent Infection with Kaposi´s Sarcoma associated Herpesvirus (KSHV) is etiologically linked to the development of several tumors, including Kaposi`s Sarcoma
(KS) and primary effusion lymphoma (PEL). Latency is a phase of the viral lifecycle during which viral gene expression is restricted to a small number of genes,
and although epigenetic modifications are very likely to be of fundamental
importance for latency establishment, their formation in the context of a de novo
infection has hitherto not been analyzed.
We have developed a system to globally monitor the epigenetic status of KSHV
genomes using a custom-designed, high resolution microarray. We have also devel-
Experimental Outline and Analysis of MeDIP Experiments. (A) Flow chart depicting the outline of MeDIP experiments. Genomic DNA is isolated from KSHV-infected or mock-infected cells. DNA from mock-infected cells
is subsequently mixed with either unmethylated KSHV-bacmid DNA (neg. control) or bacmid DNA which has
been fully methylated in vitro (pos. control). The samples are sheared, subjected to methylated DNA immunprecipitation, differentially labeled with Cy5 and Cy3, and analyzed on two color custom microarrays which
carry the KSHV genome in high resolution. Data analysis, normalization and visualization is performed with
custom developed software. A screenshot of the software interface is shown in (B).
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opped software for the refinement and
normalization of the microarray data, as
well as to provide graphic visualization of
modified viral genomes. These tools were
employed to analyze the epigenetic status
of KSHV genomes at various timepoints
following a de novo infection of endothelial SLK cells, using immunoprecipation of
DNA via 5´-methylcytidine-specific antibodies (MeDIP) or antibodies specific for
modified histones (ChIP protocol) to
provide input for array analysis.
We find that de novo infecting episomes
indeed adopt a characteristic and highly The epigenetic status of KSHV episomes in PEL cell lines and
distinct DNA methylation profile with de novo infected SLK cells. (A) Schematic Representation
of the KSHV genome. Open reading frames in forward or
astonishing similarity to viral genomes in reverse orientation are depicted as yellow or blue block
latently tumor-derived PEL cells. This is arrows, respectively. (B) CpG frequency across the KSHV
(C) Global DNA methylation patterns of KSHV
also true for deposition of acetylated genome.
genomes in de novo infected SLK cells after establishhistones, which showed a genome-wide ment of stable latency. Note that the patterns are not a
pattern nearly complementary to DNA pure function of CpG content of individual loci. (D) DNA
methylation patterns of KSHV genomes isolated from the
methylation. Furthermore, DNA methyla- tumor-derived PEL cell line BCBL1. (E) Histone acetylation
tion appears to evolve over much longer patterns across the KSHV genome in BCBL1 cells. The
form a near perfect complement of the observed
time periods than histone modifications, patterns
DNA methylation patterns.
suggesting the latter serve as an initial
signal which subsequently triggers CpG methylation patterns to reinforce the
latency program. Our approach, for the first time, allowed the global analysis of the
'epigenome' of de novo infecting KSHV genomes in a temporal and spatial manner.
The data from our analysis will shed light on the molecular mechanisms underlying
latency establishment as well as reactivation and lytic cycle induction.
*Supported (in part) by Deutsche Forschungsgemeinschaft (DFG)
A requirement for Drosha in the Maintenance of Kaposi's sarcomaassociated Herpesvirus Latency
T. Christalla (1), U. Tessmer (1), A. Grundhoff (1)
Kaposi’s Sarcoma associated Herpesvirus (KSHV) is the etiological agent of several
tumors, including Kaposi`s Sarcoma (KS) and primary effusion lymphoma (PEL).
The cells in PEL tumors as well as in PEL-derived cell lines are latently infected with
KSHV. Latency is a phase of the viral lifecycle during which viral gene expression is
restricted to a small number of genes. In contrast, during the lytic cycle the full complement of KSHV genes is expressed, culminating in the production and release of
viral progeny and death of the host cell. The molecular mechanisms of latency establishment are unclear, but it has been hypothesized that viral miRNAs might contribute to the maintenance of latency via the silencing of lytic genes. We therefore have
analyzed the effects of inhibiting the expression of Drosha (a RNAse III like enzyme
which initiates maturation of miRNAs by excising pre-miRNA hairpins from their
precursor transcripts) in the PEL-derived cell line BCBL-1. Indeed, shRNA-mediated
knockdown of Drosha resulted in rapid and efficient induction of the lytic cycle, with
an efficiency that was comparable to treatment with chemical agents such as phorbol
esters or histone deacetylase inhibitors (which are commonly used to induce the lytic
cycle in PEL cells). Surprisingly, however, knockdown of Dicer did not result in a
similar induction of the lytic cycle. Like Drosha, Dicer is an RNAse III like enzyme,
which processes pre-miRNA hairpins and is required for the production of mature
miRNAs. During miRNA processsing, Dicer acts downstream of Drosha, and our
data therefore indicate that the role of Drosha in the maintenance of KSHV latency
is distinct from its function in the miRNA pathway. Recent data suggest that such a
role may be the direct negative regulation of host transcripts which harbor premiRNA like hairpins. These transcripts are cleaved and directly destabilized by
Drosha (hence a mode of regulation independent of Dicer), but the excised premiRNA like structures do not feed into the miRNA pathway. We are currently working to establish the identity of cellular transcripts which are destabilized by Drosha
and, upon stabilization by Drosha knockdown, induce lytic KSHV replication.
*Supported (in part) by Deutsche Forschungsgemeinschaft (DFG)
Drosha knockdown induces lytic KSHV replication. (A) phase contrast (PC, top panel) and immunofluorescence
staining for the viral lytic cycle specific DNA polymerase processivity factor (PF) encoded by ORF59 (lower
panel) in the PEL-derived cell line BCBL1 after shRNA-mediated knockdown of Dicer or Drosha. (B) Higher
magnification of Drosha knockdown cells with additional staining for the latency associated nuclear antigen
(LANA) which is expressed during latent as well as lytic infection. (C) and (D) quantitative RT-PCR of transcripts
encoding PF (C) or the immediate early protein ORF50 (D) at various timepoints post transduction with lentiviruses encoding shRNAs that target Drosha or Dicer, or with a control lentivirus (pSico), or in cells which had
been mock infected (control).
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A FACS-based FRET assay to analyze interactions between viral and
cellular proteins in living cells
70
C. Banning (1), A. Düsedau (1), M. Warmer (1), R. Reimer (1),
H. Hohenberg (1), F. Kirchhoff (2), M. Schindler (1)
(2) University of Ulm, Institute of Virology, Ulm, Germany
Most methods to study protein interactions in biological systems require protein
purification or lysis and destruction of the cellular milieu. One of the few noninvasive techniques for protein interaction studies in living cells is Försters resonance energy transfer (FRET). In general, FRET measurements are done via fluorescence microscopy, which has serious limitations. For example it is difficult to
quantify and compare results of
multiple measurements or to analyze large amounts of samples and
cells. Here, we present a robust
flow cytometry based FRET assay
that allows to quantify protein
interactions in a large number of
cells. We applied this system to
analyze the dynamic interactions
of the human and simian immunodeficiency virus (HIV and SIV)
Nef proteins as well as the HIV-1
Vpu protein with various cellular
host factors. Amongst others, our
data suggest that - in strict contrast
to HIV-1 Nefs (Na7, NL4-3) –
SIVmac Nef strongly interacts with
CD3. Furthermore we demonstrate direct binding of HIV-1 Vpu
(NL43) to CD4 and tetherin
(CD317), which has recently been
shown to inhibit the release of
progeny virions from infected cells.
Currently we further develop our
assay to screen for protein-protein
interactions in high-throughput to HIV-1 and SIV Nef both colocalize with CD3 in 293T cells transfected
pECFP-CD3 and pEYFP-Nef, respectively (upper panel). In
generally identify unknown pro- with
contrast to this, only SIV-Nef interacts with CD3 as revealed by
tein interactions in living cells.
quantitative FACS-based FRET analysis (lower panel).
Inhibition of Transferrin-Receptor Uptake by SIV and HIV Nef-alleles
M. Widder (1), S. Gundlach (1), F.X. Gobert (2), P. Benaroch (2),
F. Kirchhoff (2), M. Schindler (1)
(2) Institut-Curie, Paris, France
The HIV-1 Nef protein contributes to the damaging high levels of immune activation associated with T-cell loss and AIDS progression and is therefore commonly considered a virulence factor. However, we recently found that Nef-alleles
derived from asymptomatic naturally simian immunodeficiency virus (SIV)
infected monkeys and the less pathogenic HIV-2 block the responsiveness of
virally infected T-cells to activation and may protect the infected host against
damaging high levels of immune
activation and loss of CD4+ T
cells.. Further analysis revealed
that Transferrin receptor (TfR)
levels were elevated in virally
infected primary T-cells expressing Nef-alleles from SIV and
HIV-2 but not from HIV-1. This
effect was verified in 293T, THP-I
and Jurkat T cells, as well as in
infected primary lymphocytes and
macrophages. Nef-expression
alone was sufficient to induce
upregulation of TfR. Nef-mediated
inhibition of T-cell activation did
not correlate with TfR upmodulation. Mutational analyses conModel of differential manipulation of iron-uptake by HIV and SIV firmed that both represent indenef-alleles. SIV and HIV-2 Nef inhibit TfR-internalization and might
therefore decrease cellular iron-levels. In strict contrast, HIV-1 Nef pendent Nef functions and SIV
has been reported to downmodulate HfE what should lead to an Nef
upregulates TfR by a
increase in TfR-internalization and hence iron-uptake. AP-2: adaptorN-terminal
AP2-binding motif.
protein 2; CCV: clathrin-coated-vesicle.
Internalization
experiments
revealed that high TfR levels are a result of reduced receptor endocytosis following Nef expression. Possible consequences in vivo could be lower levels of intracellular iron and reduced virus production. Our results show that upregulation of
TfR is an independent Nef function that may contribute to the asymptomatic or
attenuated course of infection observed in naturally SIV-infected monkeys and
HIV-2 in humans.
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Inefficient Nef-Mediated Downmodulation of CD3 and MHC-I
Correlates with Loss of CD4+ T Cells in Natural SIV Infection
72
M. Schindler (1,6), J. Schmökel (1), A. Specht (1), H. Li (2), J. Münch (1),
M. Khalid (1), D. Sodora (3), B. Hahn (2), G. Silvestri (4,5), F. Kirchhoff (1)
(2) University of Alabama at Birmingham, Birmingham, Alabama, USA
(3) University of Texas Southwestern Medical Center, Dallas, Texas, USA
(4) Yerkes Regional Primate Research Center, Emory University,
Atlanta, Georgia, USA
(5) University of Pennsylvania, Philadelphia, Pennsylvania, USA
Recent data suggest that Nefmediated downmodulation of TCRCD3 may protect SIVsmm-infected
sooty mangabeys (SMs) against the
loss of CD4+ T cells. However, the
mechanisms underlying this protective effect remain unclear. To further
assess the role of Nef in nonpathogenic SIV infection, we cloned nef
alleles from 11 SIVsmm-infected
SMs with high (>500) and 15 animals with low (<500) CD4+ T-cells/
Correlation between Nef-mediated CD3 and MHC-I downmodulatiµl in bulk into proviral HIV-1 IRES/ on in virally infected PBMCs. Correlation between the percentages
eGFP constructs and analyzed their of CD4+ T-cells in SIVsmm-infected SMs and n-fold downmodulatiof CD3 and MHC-I. Assays were performed in PBMCs and statieffects on the phenotype, activation, on
stical analysis was performed using the PRISM 4.0 package.
and apoptosis of primary T cells. We
found that not only efficient Nef-mediated downmodulation of TCR-CD3 but also
of MHC-I correlated with preserved CD4+ T cell counts, as well as with high numbers
of Ki67+CD4+ and CD8+CD28+ T cells and reduced CD95 expression by CD4+ T cells.
Moreover, effective MHC-I downregulation correlated with low proportions of effector and high percentages of naïve and memory CD8+ T cells. We found that T cells
infected with viruses expressing Nef alleles from the CD4low SM group expressed
significantly higher levels of the CD69, interleukin (IL)-2 and programmed death
(PD)-1 receptors than those expressing Nefs from the CD4high group. SIVsmm Nef
alleles that were less active in downmodulating TCR-CD3 were also less potent in
suppressing the activation of virally infected T cells and subsequent cell death.
However, only nef alleles from a single animal with very low CD4+ T cell counts rendered T cells hyper-responsive to activation, similar to those of HIV-1. Our data suggest that Nef may protect the natural hosts of SIV against the loss of CD4+ T cells by
at least two mechanisms: (i) downmodulation of TCR-CD3 to prevent activationinduced cell death and to suppress the induction of PD-1 that may impair T cell
function and survival, and (ii) downmodulation of MHC-I to reduce CTL lysis of
virally infected CD4+ T cells and/or bystander CD8+ T cell activation.
Single Nef Proteins from HIV-1 Subtypes C and F Fail to Up-Regulate
Invariant Chain Cell Surface Expression but are Active for other
Functions.
G. Turk (1), S. Gundlach (2), M. Carobene (1),
M. Schindler (2), H. Salomon (1), P. Benaroch (3)
(1) National Reference Center for AIDS, Buenos Aires, Argentinia
The HIV-1 Nef protein plays a major role in viral immunopathogenesis, modulating surface expression of several immune receptors, altering signal transduction
pathways and enhancing viral infectivity, among other activities. Nef also exhibits
great intersubtype diversity but most studies have only been focused on Nef proteins from the subtype B. Thus, little is known about the functional capacities of
non-subtype B Nef proteins in host cells. We investigated cell surface regulation of
MHC-I, MHC-II, the MHC-II-associated chaperone invariant chain (Ii), CD4,
CD3 and CD28 in cells transfected or infected with five different Nef-alleles including one HIV-1 Subype C and F allele. No significant difference among the Nef
proteins regarding CD3, CD28 and MHC-II downregulation was observed. The
NefC showed a slightly, yet significant, diminished capacity to downregulate
MHC-I in all cells, as well as to down-regulate CD4 in Jurkat cells and PBMCs.
Strikingly, the two alleles from NefC and NefF were unable to up-regulate the Ii
chain both in transfected and infected cells. Moreover, internalization rate of surface Ii chain was slightly affected by NefC and NefF, whereas it was drastically
reduced by NefB. Nef domains known to be involved in Ii chain up-regulation were
conserved among the five alleles analyzed here. In summary, we identified two primary HIV-1 NefC and NefF-alleles that are selectively impaired for Ii-upregulation
and which may help to elucidate the mechanism of this Nef function in the future.
It will be important to determine whether the observed differences are HIV-1 subtype dependent and influence viral immunopathogenesis.
*Supported by Stiftung Neuroviraler Erkrankungen (NVE)
Human Immunodeficiency Virus Type 1 Nef Expression Prevents
AP-2-Mediated Internalization of the Major Histocompatibility
Complex Class II-Associated Invariant Chain
H. Toussaint (1), F-X. Gobert (1), M. Schindler (2), C. Banning (2),
P. Kozik (3), M. Jouve (1), F. Kirchhoff (4), P. Benaroch (1)
(3) University of Cambridge, Cambridge CB2 0XY, UK
The lentiviral Nef protein has been studied extensively for its ability to induce the
downregulation of several immunoreceptors on the surfaces of infected cells.
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Distribution of internalized anti-Ii chain MAb and Tf in the presence of Nef. HeLa-CIITA cells transfected with
the bicistronic plasmid Nef NL4-3 (expressing Nef and GFP) or Nef-STOP (negative control expressing GFP only)
were allowed to bind at 0°C with either Tf-Alexa Fluor 647 or the Ii chain-specific MAb. Cells were then shifted
to 37°C for 5 min and fixed, and the Ii chain-specific MAb was revealed with Cy3-labeled secondary antibodies.
Confocal sections of the indicated stainings are presented together with their overlays.
However, Nef expression is unique in inducing highly effective upregulation of
the major histocompatibility complex class II-associated chaperone invariant (Ii)
chain complexes in different cell types. Under normal conditions, endocytosis of
the Ii chain and other molecules, like the transferrin receptor and CD4, is rapid
and AP-2 dependent. Human immunodeficiency virus type 1 (HIV-1) Nef
expression strongly reduces the internalization of the Ii chain, enhances that of
CD4, and does not modify transferrin uptake. The mutation of AP-2 binding
motifs LL164 and DD174 in Nef leads to the inhibition of Ii chain upregulation.
In AP-2-depleted cells, surface levels of the Ii chain are high and remain unmodified by Nef expression, further indicating that Nef regulates Ii chain internalization via the AP-2 pathway. Immunoprecipitation experiments revealed that the Ii
chain can interact with Nef in a dileucine-dependent manner. Importantly, we
have shown that Nef-induced CD4 downregulation and Ii chain upregulation are
genetically distinguishable. We have identified natural nef alleles that have lost
one of the two functions but not the other one. Moreover, we have characterized
Nef mutant forms possessing a similar phenotype in the context of HIV-1 infection. Therefore, the Nef-induced accumulation of Ii chain complexes at the cell
surface probably results from a complex mechanism leading to the impairment
of AP-2-mediated endocytosis rather than from direct competition between Nef
and the Ii chain for binding AP-2.
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Siglec expression on chimpanzee T-cells might contribute to the
asymptomatic course of lentiviral infection in human primates
H. Ströver (1) and M. Schindler (1)
Siglecs (sialic acid-binding immunoglobulin-like lectins) belong to the Sialic acidrecognizing IG-superfamily lectins. They consist of an amino-terminal V-set
immunoglobulin domain, which binds to sialic acid, and a variable number of
C2-set immunoglobulin domains. Most Siglecs have an ITIM (immunoglobulin
receptor family tyrosine-based inhibitory motif) and/or ITIM-like motif with
which they are proprosed to block cellular activation. Siglecs are prominently
expressed on immune cells, mainly of the leukocyte and the macrophage lineage.
However, there are great species-specific differences which correlate with susceptibility to AIDS.
HIV-1 was transmitted into the human population from Chimpanzees which
carry a virus closely related to HIV-1 (called SIV, simian immunodeficiency
virus). Interestingly, in Chimpanzees experimentally infected with HIV-1 the
virus establishes infection and replicates, but there is no AIDS progression
observed. Thus, Chimpanzee are most likely genetically resistant towards HIV-1
infection. In contrast to humans, Chimpanzees express high levels of Siglecs on
their T-cells, the main target cells of HIV-1. Siglec expression on those cells
could dampen the cellular activation which is essential for efficient HIV-1 replication. Therefore, the Siglec receptor familiy could be an important host factor preventing CD4+ T-cell depletion and AIDS progression in the non-human
Chimpanzee host.
So far, we confirm that different members of the Siglec family are able to suppress
cellular activation of human Jurkat T-cells and primary T-cells. Mutation of the
ITIM resulted in increased cellular activation, which demonstrates that Siglecs
constitutively inhibit T-cell signalling via this motif. Currently, we investigate the
effects of Siglec-expression on productive HIV-1 infection and replication.
HIV-1 budding and persistence in macrophages
C. Banning (1), B. Holstermann (1), H. Hohenberg (1), M. Schindler (1)
HIV-1 infected macrophages accumulate viral particles in large intracellular
vacuoles that originate from the lysosomal pathway. Those particle accumulation
are infectious over weeks and represent viral reservoirs which can not be eliminated by the immune system. Strikingly, those vesicles do not fuse with lysosomes
76
because of lack of acidification. The mechanism underlying this defect in acidification are largely unknown. Additionally, it is matter of intense debate if those
viral particle accumulations originate from direct budding into intracellular
vesicles or are reinternalized virions which have previously assembled at the
plasma membrane. To address this question, we generated R5-tropic HIV-1 constructs carrying GFP in the GAG region (iGAG) and infected primary macrophages. Live-cell imaging is performed to track GAG and assembly and budding
of newly synthesized virions. Furthermore we generated iGAG viruses with
single knock-out mutations in nef, vpu, vpr and env. The latter constructs are
used to investigate if acidification of lysosomal virus containing vesicles is prevented by one of these viral protein and as a consequence allows HIV-1 to
persist in macrophages.
Upper panel: ultrastructural analysis of HIV-1 infected macrophages, showing large vesicles inside the cell
(left). Viral particles are present inside those vesicles (right). Lower panel: HIV-1 GAG localization in different
cell types visualized by GFP expression. Macrophages, primary T-cells and 293Ts were infected with viruses
expressing GFP in the GAG-gene. Representative confocal images are presented.
A serine phosphorylation site in HIV-1 Vpu is required for efficient
counteraction of tetherin and replication in macrophages but not in
ex vivo human lymphoid tissue
M. Schindler (1,2), D. Rajan (2) C. Banning (1), A. Iwanski (1),
P. Wimmer (1), A. Specht (2), D. Sauter (2), F. Kirchhoff (2)
The human immunodeficiency virus type 1 (HIV-1) Vpu protein degrades CD4
and enhances the release of progeny virions by counteracting a restriction factor
named tetherin (CD317). The latter effect is cell type dependent most likely due to
differential levels of endogenous tetherin. Here, we show that mutation of a serine
phosphorylation site at position 52 (S52A) disrupts Vpu-mediated degradation of
CD4 and impairs its ability to antagonize tetherin. Nevertheless, the S52A substitution did not reduce the ability of Vpu to enhance HIV-1 replication or CD4+ T-cell
depletion in human lymphoid tissue. In strict contrast, HIV-1 expressing the S52A
Vpu replicated only poorly in macrophages. The reason for these diverse effects of
the S52A mutation on HIV-1 replication might be that activated CD4+ T-cells, that
are the major target cells in lymphoid tissues, express substantially lower levels of
tetherin than macrophages. Thus, our data suggest that a reduced ability of Vpu to
counteract tetherin has differential effects on HIV-1 replication in CD4+ T cells and
macrophages, the two most important cell types for viral replication in vivo.
HIV-1 Vpu S52A replicates efficiently in lymphoid tissue but not in macrophages. Left panel: cummulative p24
production over 15 days in tissues from eight donors infected with the indicated HIV-1 variants. Values are given
as percentages compared to cultures infected with NL4-3 wild-type (100%). Shown are means +/- SEM. Right
panel: cummulative reverse transcriptase (RT) production by macrophages infected with the NL4-3 variants over
a 20 day period. Values give averages +/- SEM of macrophages from three different donors infected with two
independent virus stocks. Abbreviations: N-, Nef-defective; U-, Vpu-defective; S52A, VpuS52A; PSL, photonstimulated luminescence; HLT, human lymphoid tissue; MDM, monocyte-derived-macrophages.
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Viral determinants for AIDS-progression in pediatric HIV-1 subtype
B and C infections
78
C. Banning (1), H. Ströver (1), N. Eickel (1), A. Iwanski (1), P. Walker (2),
L. Morris (2), M. Sornsakrin (3), R. Ganschow (3), M. Schindler (1)
(2) National Institute for Communicable Diseases (NICD),
Johannesburg, South Africa
Ambulance Clinic of Immune defects, Hamburg, Germany
The course of pediatric AIDS progression is characterized by high viral titers
accompanied by increased levels of immune activation and immune cell dysfunction. However, the viral determinants which are responsible for these effects are
largely unknown. Accumulating data from in vitro experiments demonstrated
that the HIV-1 Nef and Tat proteins lead to hyperactivation of virally infected
T-cells. Furthermore, Nef was reported to cause B-cell dysfunction and hence
hypergammaimmunoglobulinemia. The goal of our project is to assess the relevance of this in vitro findings in two cohorts of pediatric HIV-1 subtype B and C
infections. In close collaboration with the UKE and the NICD we assembled
blood samples and viral isolates from >50 HIV-1 subtype B and C infected children. The course of disease progression and clinical parameters are available for
the majority of the patients enrolled in our study. HIV-1 nef and tat genes from
all patient viral isolates will be amplified and analyzed for their capability to
hyperactivate t-cells and to induce cytokines to stimulate uninfected bystander
cells. Additionally, we will analyze multiple parameters which have been previously suggested to be important for disease progression. The data from the functional analysis of pediatric Nef and Tat proteins will be correlated to various
clinical parameters from the patients, to elucidate viral determinants which are
important for AIDS-progression.
Replication of ICP0-Null Mutant Herpes Simplex Virus Type 1
Is Restricted by both PML and Sp100
R.D. Everett (2), C. Parada (2), P. Gripon (3), H. Sirma H (1), A. Orr (2)
(2) MRC Virology Unit, Glasgow, U.K.
(3) INSERM U522, Rennes, France
Herpes simplex virus type 1 (HSV-1) mutants that fail to express the viral
immediate-early protein ICP0 have a pronounced defect in viral gene expression and plaque formation in limited passage human fibroblasts. ICP0 is a
RING finger E3 ubiquitin ligase that induces the degradation of several cellular
proteins. PML, the organiser of cellular nuclear sub-structures known as PML
nuclear bodies or ND10, is one of the most notable proteins that is targeted by
ICP0. Depletion of PML from human fibroblasts increases ICP0-null mutant
HSV-1 gene expression, but not to wild type levels. In this study we report that
HSV DNA is very commonly associated with PML Nuclear Bodies.
depletion of Sp100, another
major ND10 protein, results in a
similar increase in ICP0-null
mutant gene expression, and that
simultaneous depletion of both
proteins complements the mutant
virus to a greater degree. Although
chromatin assembly and modification undoubtedly play major
roles in the regulation of HSV-1
infection, we found that pharmacological interference with histone deacetylase activity was unable to complement the defect of
ICP0 null mutant HSV-1 in either
normal or PML depleted human
fibroblasts. These data lend further weight to the hypothesis that
ND10 play an important role in
the regulation of HSV-1 gene
expression.
*Supported by Deutsche Forschungsgemeinschaft (DFG) and Stiftung für neurovirale Erkrankungen
Duck Hepatitis B Virus Requires Cholesterol for Endosomal Escape
during Virus Entry
A. Funk (1), M. Mhamdi (1), H. Hohenberg (1), J. Heeren (2),
R. Reimer (1), C. Lambert (3), R. Prange (3), H. Sirma (1)
(2) University Medical Center Hamburg-Eppendorf, Hamburg, Germany
(3) Johannes-Gutenberg-University, Mainz, Germany
The identity and functionality of biological membranes is determined by
cooperative interaction between their lipid and protein constituents.
Cholesterol is an important structural lipid that modulates fluidity of biological membranes favouring the formation of detergent-resistant microdomains. In the present study, we evaluated the functional role of cholesterol
and lipid rafts for entry of hepatitis B viruses into hepatocytes. We show that
the duck hepatitis B virus (DHBV) attaches predominantly to detergent-soluble domains on the plasma membrane. Cholesterol depletion from host
membranes and thus disruption of rafts does not affect DHBV infection. In
contrast, depletion of cholesterol from the envelope of both DHBV and
human HBV strongly reduces virus infectivity. Cholesterol depletion increases the density of viral particles and leads to changes in the ultrastructural
appearance of the virus envelope. However, the dual topology of the viral
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envelope protein L is not significantly impaired. Infectivity and
density of viral particles are partially restored upon cholesterol
replenishment. Binding and
entry of cholesterol-deficient
DHBV into hepatocytes is not
significantly impaired, in contrast to their release from endosomes. We therefore conclude
that viral, but not host cholesterol is required for endosomal
escape of DHBV.
Cholesterol dependent entry steps of DHBV.
Strong and selective inhibitors of hepatitis B virus replication among
novel N4-hydroxy- and 5-methyl-beta-L-deoxycytidine analogues
E. Matthes (2), A. Funk (1), I. Krahn (2), K. Gaertner (2),
M. von Janta-Lipinski (2), L. Lin (1), H. Will (1), H. Sirma (1)
(2) Max-Delbrück-Center of Molecular Medicine, Berlin, Germany
Novel N(4)-hydroxy- and 5-methyl-modified beta-L-deoxycytidine analogues
were synthesized and evaluated as anti-hepatitis B virus (HBV) agents. Their in
vitro efficiencies were investigated in HepG2.2.15 cells stably transfected with
HBV. beta-L-2',3'-Didehydro-2',3'-dideoxy-N(4)-hydroxycytidine (beta-LHyd4C) was most effective in reducing secreted HBV DNA (50% effective concentration [EC(50)], 0.03 microM), followed by beta-L-2',3'-dideoxy-3'-thiaN(4)-hydroxycytidine (EC(50), 0.51 microM), beta-L-2',3'-dideoxy-N(4)hydroxycytidine (EC(50), 0.55 microM), and beta-L-5-methyl-2'-deoxycytidine
(EC(50), 0.9 microM). The inhibition of the presumed target, the HBV DNA
polymerase, by the triphosphates of some of the beta-L-cytidine derivatives was
also assessed. In accordance with the cell culture data, beta-L-Hyd4C triphosphate was the most active inhibitor, with a 50% inhibitory concentration of 0.21
microM. The cytotoxicities of some of the 4-NHOH-modified beta-L-nucleosides were dramatically lower than those of the corresponding cytidine analogues
with the unmodified 4-NH(2) group. The 50% cytotoxic concentrations for betaL-Hyd4C in HepG2 and HL-60 cells were 2,500 microM and 3,500 microM,
respectively. In summary, our results demonstrate that at least beta-L-Hyd4C can
be recommended as a highly efficient and extremely selective inhibitor of HBV
replication for further investigations.
Assembly and budding of a hepatitis B virus is mediated by a novel
type of intracellular vesicles
M. Mhamdi (1), A. Funk (1), H. Hohenberg (1), H. Will (1), H. Sirma (1)
Formation of enveloped viruses
involves assembly and budding at
cellular membranes. Here we elucidated the morphogenesis of
hepadnaviruses on ultrastructural and biochemical level using
duck hepatitis B virus (DHBV) as
a model system. Formation of
virus progeny initiates at the
endoplasmic reticulum (ER), and
is conserved in vitro and in vivo.
The morphogenesis proceeds via
membrane-surrounded vesicles
containing both virions and subviral particles (virus particlescontaining vesicles, VCVs). VCVs
are generated and maintained by
reorganization of endomembranes accompanied by a striking
disorganisation of the rough ER.
Model of DHBV morphogenesis.
VCVs are novel organelles with
unique identity and properties of ER, intermediate compartment, endosomes,
and multivesicular bodies. VCVs are dynamic structures whose size and shape
are regulated by both fusion and fission. In conclusion, our data indicate a
reorganisation of endomembranes during DHBV infection resulting in the
biogenesis of novel organelles serving as multifunctional platforms for assembly and budding of virus progeny.
*Supported by Studienstiftung des deutschen Volkes and Stiftung neurovirale
Erkrankungen (NVE)
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Avian Hepatitis B Viruses: Molecular and Cellular Biology,
Phylogenesis and Host Tropism
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A. Funk (1), M. Mhamdi (1), H. Will (1), H. Sirma (1)
The human hepatitis B virus (HBV) and the duck hepatitis B virus (DHBV) share
several fundamental features: both viruses have a partially double-stranded DNA
genome which is replicated via an RNA intermediate. The coding open reading
frames (ORFs) overlap extensively. In addition, the genomic and structural organization as well as replication and biological characteristics are very similar in both
viruses. It is worth to mention that most of the key features of hepadnaviral infection
were first discovered in the DHBV model system and subsequently confirmed for
HBV. However, there are also several differences between human HBV and DHBV.
Life cycle of hepatitis B viruses.
Efficiency of DHBV infection is restricted by an abortive entry
pathway leading to degradation of the majority of incoming viral
particles in vitro
A. Funk (1), M. Mhamdi (1), H. Sirma (1)
After endocytic entry into host cells, viruses exploit various strategies to bypass
degradative compartments within the host cell. Hepadnaviruses do so by escaping from the early endosome presumably with the help of a translocation motif
in the surface protein. Surprisingly, this escape is quiet inefficient, most of viral
particles are not able to translocate into the cytosol. Here, we determined the
overall stability of viral particles during entry and show that the majority of
incoming viral particles is degraded. Kinetics of degradation differs for viral
surface protein L and DNA. L has a half life of ca 1 h after viral entry and degradation depends on activity of specific cellular proteases and proteasome. In
contrast, viral DNA is 24 h after entry about ten times reduced. Disruption of
microtubules preventing infection led to further increased instability of viral
DNA after this time period. Statistically, about 1 virion per cell ‘survives!’. Three
days after infection, this single viral genome is amplified about 10 times. Taking
into account that only a minority of hepatocytes is infected, the incoming
rcDNA molecule is productively at least 100-fold amplified. Infection experiments with a mix of genotypically different DHBV largely confirmed the data.
Taken together, our data show that the majority of viral particles is degraded
after entry into host cells before escape into the cytosol and infection establishment. Our study indicates that efficiency of DHBV infection is determined by
an abortive entry pathway leading to degradation of the majority of incoming
viral particles.
Secretion of duck hepatitis B virus occurs via exocytosis
M. Mhamdi (1), A. Funk (1), H. Hohenberg (1), R. Reimer (1), H. Sirma (1)
Duck hepatitis B virus (DHBV) assembles and buds into virus particles containing-vesicles (VCVs) generated by the reorganization of the rough endoplasmic reticulum (rER) and harbouring marker proteins of the ER-Golgi intermediate compartment (ERGIC), endosomes and multivesicular bodies (MVBs). In
this study, we have investigated the intracellular pathway followed by DHBV to
secrete progeny virus using three well known inhibitors of the secretory pathway: Brefeldin A (BFA), monensin, and 20°C block. We found that viral secretion was drastically inhibited by BFA which induced the accumulation of viral
particles within large VCVs near the nucleus and in close proximity to the cell
surface. The other two inhibitors only slightly inhibited virus release and during the 20°C no accumulation of virus particles within the TGN was observed.
We conclude that DHBV was transported to the cell surface by a Golgi-
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independent pathway. Using electron microscopy in conjunction with live confocal microscopy, we showed that VCVs serves as transport carriers that are
directed to the cell surface were they liberate 40-80 virions and 46.000 SVPs per
hepatocyte and per hour via exocytosis.Taken together these data together with
our previously published data indicate that VCVs are multifunctional platforms
for assembly, budding, intracellular transport, and secretion of newly formed
virus particles.
*Supported by Deutsche Forschungsgemeinschaft (DFG), Studienstiftung des
deutschen Volkes und Stiftung für neurovirale Erkrankungen
Model of DHBV secretion.
Naturally occurring adefovir resistance mutation rtI233V selected
under adefovir therapy
O. Schildgen (2), C. Olotu (1), A. Funk (1), B. Zöllner (3),
M. Helm (4), H. Sirma (1)
(2) University Bonn, Institute of Medical Microbiology, Immunology and
Parasitology, Bonn, Germany
(3) University Medical Center Hamburg-Eppendorf, Institute of Med.
Microbiology, Hamburg, Germany
(4) Praxis Abelein/Helm, Nürnberg, Germany
Recently, we reported on three clinical cases of chronic hepatitis B virus (HBV)
infection resistant to adefovir (ADF) due to the pre-existing mutation rtI233V.
Here, we further evaluated the clinical role of the rtI233V mutation. Serum
samples from patients before and after ADF therapy were investigated for viral
HBs antigen level and HBV DNA load. The RT region of the viral polymerase
was analysed by sequencing of several cloned PCR products. We followed two
of our previously reported cases of lamivudine and ADF resistant HBV infection. Under tenofovir (TNF) treatment, one of the patients now has viral titers
below the detection limit. The second patient developed renal failure under
TNF therapy and was consequently treated with entecavir. Subsequently, the
viremia increased from below the detection limit to 104 GE/ml. Interestingly,
the rtI233V mutation partially converted to wildtype during entecavir therapy.
In addition, two patients epidemiologically independent to the afore mentioned cases were identified with ADF resistance associated to the rtI233V
mutation that was selected during ADF therapy. The results strongly suggest
that the mutation rtI233V is indeed responsible for ADF resistance. It can be
selected during antiviral therapy but occurs also as natural variant prior to
therapy. Inversely, cessation of selection pressure by ADF resulted in conversion
of the mutation to the wildtype sequence. A therapy switch from TDF to
entecavir in patients pretreated with ADF and lamivudine may decrease
efficiency of entecavir treatment as compared to therapy naïve patients.
Cellular Determinants of HBV Replication
H. Boukari (1), H. Sirma (1)
Hepatitis B Virus is one of the most important human pathogens. These small
DNA virus have a distinct liver tropism and causes liver-cirrhoses and primary
liver-carcinoma. The HepaRG cell line. Upon their differentiation, HepaRG cells
become susceptible for HBV infection. We found, that the HBV replication
depends on the state of differentiation, in a positive manner. Furthermore, interaction with the PML nuclear bodies and its components,, which have an important role in intrinsic antiviral response, were in focus. We found, that both HBV
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replication and gene expression are increased in PML- and Sp100-depleted cells,
which are constitutive members of the PML-NB. Our results show that HBV
replication depends on the differentiation state of the host cell and that PML-NB
components PML and Sp100 are important factors modulating HB viral replication and gene expression.
High resolution electron microscopical (EM)-analysis of the
distribution of TNF receptor 1 and the inhibition its internalization
using immunogold stereo surface replica techniques
H. Dobner (1), H. Hohenberg (1), B. Holstermann (1), R. Reimer (1),
C. Schneider (1), W. Schneider-Brachert (2), M. Warmer (1)
(2) Institute for Medical Microbiology and Hygiene,
University Regensburg, Germany
The adenoviral protein E3-14.7K
is an inhibitor of TNF-induced
apoptosis, but the molecular and
structural mechanism underlying
this protective effect has not been
explained so far. TNF-mediated
apoptosis is initiated by ligandinduced recruitment of TNF
receptor–associated death domain
(TRADD), Fas-associated death
domain (FADD), and caspase-8 to
the death domain of TNF receptor
1 (TNFR1), thereby establishing
the death-inducing signaling complex (DISC). In the first step of
our EM-study we analyse the exact
distribution of TNF receptors at
the plasma membrane surface of
adenovirus–infected human cells Adenovirus-infected HeLa cells in a high resolution FESEM (overview).
applying the heavy metal immunogold surface replica technique. In a second step we will investigate by 3D
ultrathin section reconstruction techniques the more complex intracytoplasmatical mechanisms that allow the adenovirus 14.7K protein to inhibit ligandinduced TNFR1 internalization.
Cryotomography Ultrastructural analysis of cytomegalovirus tegument protein interactions
H. Hohenberg (1), B. Holstermann (1), R. Reimer (1), C. Schneider (1),
M. Warmer (1), M. Winkler (2)
(2) Institute for Infection Medicine, Kiel, Germany
Cytomegalovirus tegument proteins are critical constituents of
the virus particle that play a role
in particle assembly, virus entry
and exit, establishment of infection and immunomodulation.
Based on an established map of
protein interactions between tegument proteins, which provides a
scaffold for further definition of
virus structure assembly and function, the main focus of the investigations is the interaction between
ppUL82 (pp71) and ppUL35. Both
proteins localize to PML oncogenic domains and cooperatively
transactivate the immediate-early
enhancer of HCMV. In addition,
deletion of the UL35 gene affects
Tomographic reconstruction of the different intracellular maturation
virus assembly by modulating
steps of cytomegalovirus after cryoprocessing.
nucleocytoplasmic localization of
other tegument proteins. Thus, ppUL35 is part of a cell-biological switch critical
for immediate-early regulation and virus assembly. The use of Carbocell microreactors for sample preparation and ultrastructural analysis assures the perpetuation of virus components in situ during the whole investigation process.
Cryofixation by high pressure freezing and deep temperature substitution are
used to preserve the tegument proteins in their native state for further investigation by immunogold-labelling and high resolution transmission electron microscopy.
*Supported by German Academic Exchange Service (DAAD)
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Dynamic processes in organic and biological samples investigated
with pump-probe experiments. Insights into the process of virus
self-assembly in cells using a combination of microdiffraction and
imaging techniques
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H. Hohenberg (1), A. Meents (2), B. Reime (2), R. Reimer (1), T. Pakendorf (2)
(2) DESY, Hamburg, Germany
The bio-imaging and diffraction beamline P11 at DESY enables to investigate
the structure of crystalline and non-crystalline biological materials at two different end stations using an x-ray energy range between 3keV and 30keV with
x-ray foci ranging from 10 nm to 250 nm. The DESY bio-imaging source allows
the application of fluorescence tomography, phase contrast imaging and coherent diffraction imaging. In a collaboration project between the HPI-imaging
group and the DESY the ultra-structure of cryo-processed viral protein assemblies are investigated with a combination of x-ray imaging and diffraction
techniques and compared with high resolution cryo-electron tomography.
A: Experimental hutch layout for beamline P11: All components are mounted on a granite block on which an
intermediate focus, acting as a secondary source, is located. B: TEM image of cryoprocessed HIV capsid protein
aggregates in a transfected E.coli cell.
Nanoparticle-Loaded Magnetophoretic Vesicles
M. Krack (1), H. Hohenberg (2), A. Kornowski (3), P. Lindner (1),
H. Weller (1) and S. Förster (1)
(1) University Hamburg, Institute of Chemistry, Hamburg, Germany
For many biomedical applications, the controlled uptake and release of active
ingredients is of particular importance. In case of block copolymer vesicles,
external stimuli such as changes in pH, temperature, UV-light, and concentration of oxidizing agents can be used to trigger the release of substances in specific therapeutic windows. A further attractive external trigger for diagnostics
and therapeutic treatments are external magnetic fields. Magnetic nanoparticles have been employed for magnetic resonance imaging (MRI) and for magneto-thermal cancer therapy. The incorporation of magnetic nanoparticles into
vesicles would create a versatile diagnostic and therapeutic tool that would be
biocompatible, could be magnetically moved or targeted to specific cells or
organs, and could be used for controlled release stimulated by external magnetic fields. Block copolymer vesicles are particularly suited for this purpose
because of their increased stability and bilayer thickness compared to lipid
vesicles.
Size and Surface Effects on the MRI Relaxivity of Manganese Ferrite
Nanoparticle Contrast Agents
U. I. Tromsdorf (1), N. C. Bigall (1), Michael G. Kaul (2), O.T. Bruns (3),
M. S. Nikolic (1), B. Mollwitz (3), R. A. Sperling (4), R. Reimer (5),
H. Hohenberg (5), W. J. Parak (4), S. Förster (1), U. Beisiegel (3),
G. Adam (2) and H. Weller (1)
(1) Institute of Physical Chemistry, University of Hamburg
(2) Department of Diagnostic and Interventional Radiology,
University Medical Center Hamburg-Eppendorf
(3) Department of Biochemistry and Molecular Biology II:
Molecular Cell Biology, University Medical Center Hamburg-Eppendorf
(4) Ludwig-Maximilians-University, Center of NanoScience
Superparamagnetic MnFe2O4 nanocrystals of different sizes were synthesized in
high-boiling ether solvent and transferred into water using three different
approaches. First, we applied a ligand exchange in order to form a water soluble
polymer shell. Second, the particles were embedded into an amphiphilic polymer
shell. Third, the nanoparticles were embedded into large micelles formed by lipids. Although all approaches lead to effective negative contrast enhancement, we
observed significant differences concerning the magnitude of this effect. The
transverse relaxivity, in particular r2*, is greatly higher for the micellar system
compared to the polymer-coated particles using same-sized nanoparticles. We
also observed an increase in transverse relaxivities with increasing particle size for
the polymer-coated nanocrystals. The results are qualitatively compared with
theoretical models describing the dependence of relaxivity on the size of magnetic spheres.
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Real-time magnetic resonance imaging and quantification of
lipoprotein metabolism in vivo using nanocrystals
90
O.T. Bruns (1), H. Ittrich (2), K. Peldschus (2), M.G. Kaul (2),
U.I. Tromsdorf (3), J. Lauterwasser (1), M.S. Nikolic (3), B. Mollwitz (1),
M. Merkel (4), N.C. Bigall (5), S. Sapra (5), R. Reimer (6), H. Hohenberg (6),
H. Weller (3), A. Eychmüller (5), G. Adam (2), U. Beisiegel (1)
and J. Heeren (1)
(1) IBM II: Molecular Cell Biology,
University Medical Center Hamburg-Eppendorf, Hamburg, Germany
(2) Department of Diagnostic and Interventional Radiology,
(3) Institute of Physical Chemistry, University of Hamburg
(4) Asklepios Clinic St. Georg, 1. Department of Internal Medicine,
Hamburg, Germany
(5) Institute of Physical Chemistry and Electrochemistry, TU Dresden,
Dresden, Germany
Semiconductor quantum dots and superparamagnetic iron oxide nanocrystals
have physical properties that are well suited for biomedical imaging. Previously,
we have shown that iron oxide nanocrystals embedded within the lipid core of
micelles show optimized characteristics for quantitative imaging. Here, we
embed quantum dots and superparamagnetic iron oxide nanocrystals in the
core of lipoproteins - micelles that transport lipids and other hydrophobic substances in the blood - and show that it is possible to image and quantify the
kinetics of lipoprotein metabolism in vivo using fluorescence and dynamic
magnetic resonance imaging. The lipoproteins were taken up by liver cells in
wild-type mice and displayed defective clearance in knock-out mice lacking a
lipoprotein receptor or its ligand, indicating that the nanocrystals did not influence the specificity of the metabolic process. Using this strategy it is possible to
study the clearance of lipoproteins in metabolic disorders and to improve the
contrast in clinical imaging.
Programmbereich
„Zelluläre Dysregulation“
Research Program
“Cellular Dysregulation“
Head: Dr. Carol Stocking
Introduction
The multitude and complexity of the networks operating within and between
cells renders a cell vulnerable to dysregulation, either precipitated by exogenous
stimuli or endogenous to the system. Even minor cellular disruptions can threaten the fragile functional balance of a cell, leading to cellular damage at the level
of individual organelles and/or at the level of the genome. To cope with such
endogenous or exogenous stress situations, cells have developed a variety of protective pathways that lead to either repair of the damage incurred or elimination
of the damaged cell. Nevertheless, despite the elaborate protective arsenal of a cell,
situations do occur that result in either transient or permanent alterations in
cellular signaling characteristic of pathologic alterations. Important examples
are viral infections and tumor development. In both conditions, cellular
safeguards have failed, leading to the reprogramming of cellular pathways for the
needs of viral replication or for successful tumor evolution. To understand these
and other pathologic conditions, it is necessary to study in detail the basic
mechanisms of cellular signaling, as well as the safeguard pathways insuring their
proper function.
It is becoming increasingly evident that many pathological conditions lead to
alterations in common cellular pathways or violate similar protective actions of
the cell. In analyzing these pathways at the cellular level, the distinction between
pathways dysregulated by viral infections or tumor evolution become irrelevant.
Indeed, approaching the problems of cellular dysregulation from different angles
clearly has synergistic effects. Thus the broad spectrum of projects addressing
this topic serves the major goal of understanding the regulatory mechanism that
govern the appropriate functioning of a cell, with the hope that understanding
such mechanisms, and how they are deregulated under various pathologic conditions, will help us to identify handles for either preventing dysregulation or for
curing the effects of cellular dysregulation at the level of the organism.
The Cellular Dysregulation Program has three core themes that are connected by
this common research: 1) Cellular defense mechanism and tumor suppression;
2) Cellular controls of self-renewal, proliferation, and differentiation; and
3) Molecular mechanisms for “fine tuning” normal cell processes. All themes are
also connected by the fact that both viral infections and tumor evolution impinge
on one or, more often, many of these cellular control elements. This fact is also
reflected in the different experimental approaches used within our Program to
dissect the underlying mechanisms behind normal and abnormal cellular functions. Although traditionally the emphasis has and continues to be the prominent
use of various viruses (e.g. adenovirus, polyoma virus, SV40, hepatitis virus and
retrovirus) to identify and characterize cellular control mechanisms, there is also
a strong deployment of animal models to mimic tumor growth in vivo, in which
either viral elements or mutated cellular genes identified in patient samples are
ectopically expressed. Mouse models hold the promise of yielding results that can
be applied to tumor diagnosis and treatment and are highly adaptable to genetic
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Cellular Dysregulation
92
analysis for identifying causal relationships (within the normal environment).
However, the success of many of the studies carried out within the Cellular
Dysregulation program rely on new technical developments that extend the
strength of cell biological experimentation in vitro. Here, the outstanding imaging
resources available at our institute (fluorescent confocal microscopy, transmission
electron microcscopy tomography, and correlative light / electron microscopy
methods), as well several flow cytometry instruments (including a fluorescenceactivated cell sorter) must also be mentioned.
In this biannual report, we have documented in form of brief abstracts many of
the ongoing research projects, as well as successfully completed projects, in 2007
and 2008 within the program area “Cellular Dysregulation.” These abstracts have
been grouped together on basis of the three core themes, which are briefly outlined below.
A. Cellular defense mechanism and tumor suppression
The mammalian cell has evolved many mechanisms of protection against external assault (e.g. radiation, chemicals, infections, nutrient deficiencies, etc.) or
inherent or sponataneous mutations in the cellular machinery. These mechanisms are part of a genetic program that is normally latent but is activated by
poorly understood stress sensors. Activation of the defense program generally
leads to cell-cycle arrest and DNA repair, however, in severely damaged cells the
program triggers cell death through apoptosis and/or autophagy or in the execution of a program that leads to a permanent growth arrest known as senescence.
These responses are considered crucial tumor suppressive mechanisms, and
thus are the targets of transforming viruses or spontaneous genetic mutations
leading to cancer. Study of these defense mechanisms remains a major focus
of the Cellular Dysregulation Program. These studies can be divided into three
sections:
Historically, viruses have been essential for the identification of cellular proteins
and circuitries that are targeted during cellular transformation. Thus it was the
analysis of DNA viruses that first led to the identification of p53, a central player
in coordinating and executing cellular defense and protection mechanisms
against endogenous and exogenous cellular stress situations. These types of
studies are still very relevant, as demonstrated by ongoing work to dissect the
transformation potential and cellular counterparts of gene products encoded by
the E1B and E4 transcription units of the human adenoviruses or by the newly
discovered Merkel cell polyomavirus (MCPyV), which is associated with a highly
aggressive skin carcinoma.
When damage accumulates irreversibly, mitotic cells rely on either of two mechanisms to avoid replication: permanent cell cycle arrest (cellular senescence) or cell
death programs. Although apoptosis is the best-described form of programmed
cell death, autophagy, which is a lysosomal degradation pathway essential for
homeostasis, also contributes to cell death. Studies within our program address
the important players in both apoptosis and senescence, as well as to identify cellular genes that counteract the activation of these programs in leukemia or other
tumors, and thus important targets for therapy. Collaborative studies within our
program also address the mechanisms in which bacteria subvert activation of the
autophagy pathway as part of their virulence strategy.
A3. DNA repair mechanisms
Mechanisms by which defense strategies promote DNA repair remain an important focus of our work. A coordinated response to DNA damage is required to
maintain cellular viability and prevent disease. The ATM and ATR protein
kinases act as important regulators of the DNA-damage response by signaling to
control cell-cycle transitions, DNA replication, DNA repair and apoptosis. Our
recent studies have provided new insights into the mechanisms by which the
ATR-activated pathway delays cell cycle progression and irreversible inhibition of
DNA replication involving the alternatively spliced p53 protein, termed ∆p53.
Recent work has also implicated the SPOC1 protein in the ATM-dependent
DNA-repair process. In addition to cellular protein, viral proteins (e.g. adenovirus
E1B-AP5) also regulate ATR-induced DNA damage signaling pathways during
infection, thereby promoting viral replication.
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B. Cellular controls of self-renewal, proliferation, and differentiation
There is increasing evidence that tumors are maintained by a subset of cancers
cells that have the ability to self-renew and differentiate into the morphologically
heterogeneous populations that are often observed in tumors, a concept first
established for human myeloid leukemia. Thus understanding the complex circuitry that regulates self-renewal, proliferation, and differentiation in tissue stem
cells is important for deciphering the molecular events that occur in the generation of the cancer stem cell. Our recent findings are presented in two subsections:
Characterization of the genetic lesions in acute leukemia samples have demonstrated the importance of deregulated transcription factors in initiating neoplastic transformation by modulating the balance between self-renewal and differentiation. More recently, the growing importance of epigenetic changes in DNA and
chromatin structure, which allows the accessibility of transcriptional factors at
specific DNA-binding sites, has also been recognized. Several studies within the
Cellular Dysregulation Program address the impact of hematopoietic transcription factors on transformation and differentiation. In addition, more recent
studies assess the impact of mutant p53 proteins on transcription regulation to
understand their gain-of-function role in tumorigenesis. Similarly, several findings support the role of SPOC1, a protein associated with aggressive ovarian
cancer, as a pivotal regulator of chromatin structure during mitosis.
Another main focus of the research in this Program is the characterization of cancer
stem cells and identifying the genetic lesions that are necessary to maintain them.
In these studies both in vivo models of leukemia and mammary carcinoma are
being used, as well as an in vitro model of embryonic carcinoma. These important
studies have revealed a role for p53 and the MADS transcription factor Mef2c in
regulating decisions of cell fate, which may have critical consequences for tumor/
leukemia induction. Studies are also underway to assess the importance of TERT
(encoding telomerase), which is normally down-regulated during differentiation
and is postulated to be in important in maintaining the stem cell self-renewal
potential. In addition to transcription factors, the role of activated receptor tyrosine
kinase and their downstream signaling pathways are being investigated for their
role in disrupting signals regulating proliferation and differentiation in leukemia.
Proteins are the workhorse of several cellular processes and it is becoming increasingly clear from studies of viral and cellular proteins that their functional activation
is regulated at several post-transcriptional levels that modulate their amount, localization, activity, and stability. These controls can occur both at the RNA and protein
levels, and are the subject of several research endeavors within this program.
Several cellular mechanisms by which protein amounts are controlled at the RNA
level have been revealed by analysis of viral control elements and/or mechanisms of
disease. This includes the CRM1-mediated nucleoytoplasmic transport of RNA,
first identified by analysis of HIV-1, and the RNA chaperone La, an essential transacting factor in IRES-dependent translation initiation of picornaviruses. Our
studies show that these mechanisms to control translation and elongation efficiencies
are not restricted to viral RNA but are also used by a subset of cellular mRNAs.
Similarly, novel insight into mechanisms that control mRNA maturation and
stability has been gained by analysis of the consequence of point mutations or
chromosomal translocations associated with disease. This includes nonsense
mediated decay (NMD), a cellular mechanism of mRNA surveillance to detect
nonsense mutations and prevent the expression of truncated or erroneous proteins,
and R-loop formation during transcription, which can disrupt normal splicing and
increase genomic instability, and eventually malignant cell transformation.
Post-translational modifications are key modulators of protein function. Several
studies within our research program address the consequence of phosphorylation, ubiquitination, and SUMOylation on the activity of viral and cellular proteins, and aim to identify the modifier proteins that regulate these processes.
Importantly, the ability of p53 to bind to enzymes that mediate either phosphorylation or ubiquitination is an important mechanism by which the specific
activity of p53 as a transcription factor is controlled. Similarly, the adenovirus
E1B-55K protein also exploits both ubiquitin and ubiquitin-like systems to target
host cell proteins in transformation (e.g. SUMOylation of p53).
The following short reports document the high standard of research in this program area and the significant progress made during the last two years in the
individual projects. The large number of publication in scientific journals with a
high impact factor best supports this statement.
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Research Projects
A. Cellular defense mechanisms
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Cell transformation by human adenoviruses
B. Härtl (1), T. Sieber (1), P. Blanchette (3), P. Wimmer (1),
E. Kremmer (4), T. Dobner (1)
(4) Institute for Molecular Immunology, Helmholzzentrum München,
Germany
Human adenoviruses (Ads) are classified as DNA tumor viruses by virtue of their
ability to cause tumors when inoculated into rodents. Two classes of oncogenic
human Ads can be distinguished based on the types of tumor they elicit in animals and the viral oncoproteins responsible for their tumorigenic potential. All
members of the oncogenic subgroups A and B cause mostly undifferentiated
sarcomas at the site of injection, while two members of the otherwise non-oncogenic subgroup D (Ad9 and Ad10) elicit exclusively estrogen-dependent mammary tumors following subcutaneous or intraperitoneal injection in female rats.
Following the initial discovery of their oncogenic potential in animals it soon
became apparent that human Ads provide an excellent experimental tool to analyze fundamental events of normal and malignant cell growth. In particular, their
ability to oncogenically transform primary mammalian cells in culture, which are
then capable of initiating tumor growth in a susceptible host animal, has been of
immense value in elucidating events in cellular and viral growth control. Much of
our current understanding of the molecular mechanisms underlying virus-mediated oncogenic transformation has derived from the study of human Ads and the
viral gene products involved in transformation. These studies led to the identification of the adenovirus oncoproteins, which in turn have served as molecular
probes to resolve many fundamental aspects of cellular gene expression, cell
cycle control and programmed
cell death.
Today, it is well established that
cell transformation by subgroup A
and C Ads is a multistep process
involving the cooperation of several gene products encoded in
transcription units E1A, E1B and The genomic organization of Ad5. The linear double-stranded
genome is depicted in the center as a thin line, with the inverted
E4, as well as epigenetic changes terminal repeats (ITR) at each end: lengths are marked in map units.
resulting from viral DNA integra- Transcription units are shown relative to their position and orienin the Ad5 genome. Early genes (E1A, E1B, E2A, E2B, E3 and
tion. While E1A is clearly the key tation
E4) are indicated by black bars. Genes expressed at intermediate
player in subgroup A and C times of infection (IX and IVa2) are indicated by gray bars. Open
Ad-mediated cell transformation rectangles denote introns. Late genes (L1 - L5) produced from the
late promoter (MLP) all contain the tripartite leader (TPL) at
and oncogenesis, it is becoming major
their 5’ ends and are shown at the top of the figure. Small arrows
increasingly clear that other gene denote virus-associated RNAs I and II (VA RNAs).
products substantially affect the tumorigenic phenotype of the transformed cell.
In particular, work in our group on the oncogenic potential of E1B and E4 gene
products have now revealed new pathways that point to novel, general mechanisms of virus-mediated oncogenesis.
Products of early region 1B (E1B) from human Ads are capable of cooperating
with Ad E1A to transform primary mammalian cells in culture. It is generally
considered that the large E1B-55K products contribute to complete cell transformation by antagonizing apoptosis and growth arrest, which primarily result from
the induction and metabolic stabilization of the tumor suppressor protein p53 by
Ad E1A. These growth-promoting activities correlate with their ability to act as
a direct transcriptional repressor that is targeted to p53-responsive promoters by
Protein interaction domains and functional motifs in the Ad5 E1B-55K protein. Domains involved in nucleocytoplasmic shuttling, RNA-binding, transcriptional repression and nuclear localization (NLS) are indicated on
top. Conserved amino acid residues (cysteine histidine, serine and threonine) are indicated below. Mutations
that affect various processes shown on the right are indicated by boxes and triangles, and numbers refer
to their positions in the E1B amino acid sequence. Arrows indicate the high conservation of the USP7- and
CRM1-binding motif and the SUMO-conjugation site, as illustrated by the absolute complexity diagram
(bottom).
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binding to p53. In addition it has been hypothesized that the mode of action of
Ad E1B-55K products during transformation may involve additional functions
and other protein interactions.
Over the past years we performed a comprehensive mutational analysis to assign
further transforming functions of Ad5 E1B-55K to distinct domains within the viral
polypeptide. Results from these studies show that the functions required for transformation are encoded within several patches of the 55K primary sequence including
several clustered cysteine and histidine residues, some of which match the consensus
for zinc fingers. In addition, two amino acid substitutions (C454S/C456S) created
a 55K mutant protein, which was substantially reduced in transforming activity.
Interestingly, the same mutations affected neither binding to p53 nor inhibition of
p53-mediated transactivation. Therefore, an activity necessary for efficient transformation of primary rat cells can be separated from functions required for inhibition
of p53-stimulated transcription. Our data indicate that this activity is linked to the
ability of the Ad5 protein to bind to components of the Mre11/Rad50/NBS1 DNA
double-strand break repair complex and/or its ability to assemble multiprotein aggregates in the cytoplasm and nucleus of transformed rat cells. These results introduce
a new function for Ad5 E1B-55K and suggest that the viral protein contributes to cell
transformation through p53 transcription-dependent and -independent pathways.
Further support for this model originates from genetic and biochemical analyses to
assess role of E1B-55K related proteins E1B-156R, E1B-93R and E1B-84R in the
transformation process. Results from these assays show that E1B-156R, which comprises the 79 amino-terminal and 77 carboxy-terminal amino acids of E1B-55K,
also enhances focal transformation of primary rat cells in cooperation with E1A.
Since E1B-156R seemed unable to relocalize p53 and inhibit its transactivating
function, it must be assumed that it contributes to transformation independently
of repression of p53-stimulated transcription. Furthermore, we found that E1B156R contains a functional transcriptional repression domain and binds the cellular
apoptosis regulator Daxx. Taken together, these analyses introduce E1B-156R as a
viral oncoprotein that contributes to cell transformation by a novel mechanism,
which likely involves binding to and modulation of the cellular Daxx protein.
*Supported by Deutsche Forschungsgemeinschaft and in part
by Wilhelm Sander-Stiftung
RNA interference as a tool to study adenovirus E1A/ E1B-mediated
cell transformation
E. Koyuncu and T. Dobner
In this study we evaluated the use of RNA interference (RNAi) as an experimental
tool to assess the role of known and potential E1B-55K interaction partners on
E1A- plus E1B-mediated transformation of primary baby rat kidney (BRK) cells.
Results from these studies demonstrate that coexpression of E1A with p53-specific
short hairpin RNAs (shRNAs) efficiently induced the formation of transformed
clones which could be established into permanent cell lines. Surprisingly, shRNA-
p53 appeared to be more efficient to promote E1A-induced focus formation than
E1B-55K. More importantly, the total number of transformed cells was further
greatly increased when both plasmids were simultaneously cotransfected with
E1A, suggesting that E1B-55K and shRNA-p53 can synergistically promote focusformation in the presence of E1A. These data demonstrate that silencing of cellular
targets of E1B-55K through RNAi can be used to evaluate their role in cell transformation and provide further support for the idea that the viral oncoprotein
contributes to oncogenesis through p53-dependent and -independent pathways.
*Supported by Erich und Gertrud Roggenbuck-Stiftung
Merkel cell polyomavirus (MCPyV) in Merkel cell carcinoma cell lines
N. Fischer (1), J. Brandner (2), F. Fuchs (2) , I. Moll (2), A. Grundhoff (3)
Institute of Dermatology, Hamburg, Germany
MCPyV in Merkel cell carcinoma (MCC) cell lines. (A) MCPyV copy numbers in MCC cell lines relative to GAPDH
copy numbers, as determined by quantitative PCR. All values are depicted as mean ± standard deviation of
three independent experiments. (B) Detection of MCPyV genomes in three MCC lines by southern blotting.
(C) Schematic representation of the MCPyV LT-Ag and the position of point mutations (oval shapes) or deletions
(grey boxes) which leads to premature termination of translation.
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Merkel cell carcinoma (MCC) is a highly aggressive tumor of the skin which
predominantly afflicts elderly and immunocompromised patients. The recently
discovered Merkel cell polyomavirus (MCPyV) is believed to play a causative
role in the pathogenesis of MCC. This hypothesis is supported by several lines
of evidence: (i) MCPyV is detected with a frequency of ~80 % in MCC tissues,
(ii) the tumors harbor clonally integrated MCPyV genomes, indicating that
integration preceeds tumor formation and (iii) the gene encoding large
T-antigen (LT-Ag) gene of integrated MCPyV genomes unequivocally harbor
mutations which selectively abrogate its ability to support viral replication while
preserving its ability to target the cellular tumor supressor Rb.
While tumor derived cell lines represent valuable tools for the in vitro study of
MC carcinomas in general and the possible contribution of MCPyV in particular, only a few MCC cell lines have hitherto been established and only one of
these has been reported to be infected with MCPyV. We have analyzed 3 existing
and 4 newly established MCC cell lines for the presence, integration pattern and
copy number of MCPyV. In five cell lines, MCPyV specific sequences were
detected. In three of these lines, Southern Blotting and/or quantitative PCR suggest clonal integration of concatemeres of MCPyV genome. All three lines contained the LT-Ag truncating mutations characteristic for MCPyV genomes
found in primary MCC tumors. The premature stop codons were localized in
the region between the regions coding for the Rb binding and origin binding
domains of LT-Ag.
In contrast, the remaining two lines had average copy numbers equal to or less
than 1/50 per cell, indicating that the cultures consist of a mixture of infected
and uninfected cells. This may reflect outgrowth of original MCPyV-positive
Classification of Merkel cell carcinoma (MCC) cell lines. (A-B) Immunofluorescence staining of CgA in the variant
MCC cell line MCCL-6 (A, A´) and the classic line MCCL-3 (B, B´). (A, B) epifluroescence picture of CgA staining.
(A´, B´) overlay of epifluorescence pictures of CgA (red) and DAPI (blue) staining. (C-F) Morphological subtypes
of MCC cell lines: (C) MCCL-5 representing type I, (D) MCCL-7 representing type II, (E) MCCL-11 representing
type III and (F) MCCL-12 representing type IV. Note that all cell lines are grown on feeder layer which is also
seen in (C-F). Bars: (A-C): 20 µm, (C-F): 100 µm.
tumor cells along with uninfected bystander cells during cell line establishment,
or elimination of viral genomes upon cultivation in vitro. The latter would argue
for the presence of a selectional pressure against MCPyV in some, but not all
lines, a hypothesis which is currently under investigation.
MCC cell lines are conventionally categorized as “classic” or “variant” and further
divided into four subtypes, based on expression of neuroendocrine markers such
as chromogranin A (CgA) and cell as well as colony morphology. While it has
been suggested that the presence of MCPyV might promote a classic phenotype,
we identified MCPyV-positive as well as -negative lines of the classic variety, indicating that the distinguishing features are either inherently independent of viral
infection or have become so in the course of tumorigenesis and/or cell line establishment. We are currently investigating the contribution of MCPyV infection to
the maintenance of the transformed status of the various MCC cell lines, as well
as the transforming potential of the shortened large T-Ag proteins in heterologous systems.
*Supported by Bundesministerium für Gesundheit (BMG)
p53-Rb2/p130 crosstalk in cellular senescence
W. Bohn (1), H. Helmbold (1), N. Kömm (1), U. Galderisi (2)
(2) University of Naples, Italy
Cellular senescence is a basic response of diploid mammalian cells to genotoxic stress. It
is activated to withdraw DNA damaged cells irreversibly from the cell cycle in order to
prevent uncontrolled proliferative activity. The response is induced by intrinsic stress
signals, arising from shortening of telomeres or from the activity of viral and cellular
oncogenes. The response can also be induced artificially by extrinsic signals, such as
irradiation or the application of chemotherapeutic drugs. The appearance of senescent
cells specifically in premalignant tumor lesions suggests that cellular senescence acts to
suppress the transition from the benign to the malignant stage in tumor development.
We aim at identifying signaling pathways that determine induction and maintenance of the senescent growth arrest, and are of prognostic relevance in tumor
treatment. It is known that p53 and retinoblastoma (Rb) proteins play the most
important role in the senescence response. Functional inactivation of these proteins
or their signaling pathways abrogates the senescent growth arrest. Using rat glioma
cells as an initial experimental cell system we identified Rb2/p130 as the retinoblastoma protein specifically interacting with p53 in this response. The cells are precisely arrested between the cyclin-E to cyclin- A mediated steps in the cell cycle.
While remaining arrested, the cells gradually adopt a senescence phenotype, which
is indicative of reaching an irreversible stage of growth arrest. Irreversibility is based
on epigenetic changes leading to irreversible chromatin silencing. We extended the
studies to human tumor cells subjected to DNA damaging treatment. We obtained
evidence that the Rb2/p130 - p53 crosstalk is of general relevance for activation of
the senescence response. siRNA mediated knock-down of Rb2/p130 but not of Rb1
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impaired arrest activation and led to progression of cells into S-phase of the cell
cycle. The same outcome was obtained when p53 or its downstream target CDKinhibitor p21WAF1 were inactivated. Thus human tumor cells can activate cellular
senescence in response to DNA damaging treatment even when they lack a functional Rb1 signaling pathway, provided they have a wild-type p53 gene status.
Activation of senescence pathways by unfavorable culture conditions could also be
responsible for the limited life span of mesenchymal stem cells (MSC) in vitro, a
major obstacle for their use in basic molecular and cell therapy studies. Using rat
mesenchymal cells as a model system, we have determined the relationship between
adopting a senescent phenotype and changes in stemness markers and DNA repair
mechanisms. We found a strong and general reduction in the expression of all genes
involved in DNA repair, and a loss of stemness markers. This suggests that DNA damage sustains the senescence process in MSCs in an auto-loop-like pathway. Entry into
senescence occurred with characteristic changes in Rb expression patterns. Rb1 and
p107 gene expression decreased during in vitro cultivation. By contrast, pRb2/p130
became the prominent Rb protein. This suggests that Rb2/p130 could be a marker of
senescence or that it even plays a role in triggering the process in MSCs.
*Supported by Deutsche Krebshilfe
Dissecting the mechanism of apoptosis-resistance in hematopoietic
malignancies expressing wild-type p53
A. Engelmann (1), K. Schulz (1), D. Speidel (1), G. Bornkamm (2)
W. Deppert (1), C. Stocking (1)
(2) GSF, Munich, Germany
Tumors that have acquired resistance against death stimuli constitute a severe
problem in the context of cancer therapy. To determine genetic alterations that
favor the development of stress resistant tumors in vivo, we took advantage of
polyclonal tumors created by retroviral infection of newborn Eλ-myc mice, in
which the retroviral integration acts as a gene mutagen to enhance tumor progression. Tumor cells were cultivated ex vivo and exposed to gamma-irradiation
prior to their re-transplantation into syngenic recipients, thereby providing a
strong selective pressure for pro-survival mutants. Secondary tumors developing from treatment resistant cell clones were analyzed for retroviral integration
sites to reveal potential candidate genes whose dysregulation confer the selection advantage. In addition to the anti-apoptotic gene Bcl-xL, we identified the
Gadd45β locus to be a novel common integration site in these stress-resistant
tumors. Enhanced expression of Gadd45β was observed in secondary tumors,
but not in cells from primary non-stressed parental tumors. In accord with a
thus far undocumented supportive role of Gadd45β in tumorigenesis, we
showed that Gadd45β over-expressing NIH3T3 cells form tumors in NOD/
SCID mice. Interestingly, and in contrast to “classical” anti-apoptotic factors,
high Gadd45β levels did not protect against c-myc, UV or gamma-irradiation
induced apoptosis, but conferred a strong and specific survival advantage
against serum withdrawal.
To identify other candidate genes that protected tumors from stress-induced
apoptosis, several tumor pairs (radiation sensitive vs. resistant tumors derived
from the same parental tumor) were screened by profiling expression arrays.
Three candidate genes were identified, whose expression was upregulated in
radiation resistant tumors by fivefold. Analysis demonstrated that these were all
normally upregulated after apoptosis stimulation. Complementary DNA were
isolated and expressed in hematopoietic cells and their ectopic expression is currently being tested for their ability to protect from radiation or other stress
response.
FLASH meets nuclear bodies: CD95 receptor signals via a nuclear
pathway
E. Krieghoff (1), K. Milovic-Holm, (2) T.G. Hofmann (1)
(1) German Cancer Research Center, Heidelberg, Germany
The CD95 receptor signals via assembly of a multi-protein complex termed
death-inducing signaling complex (DISC) which triggers activation of receptorbound caspase-8/FLICE molecules. Most cells (type II cells) depend on a mitochondrial amplification pathway to commit apoptosis upon CD95 activation.
The caspase-8-binding protein FLICE-associated huge protein (FLASH) has
been previously implicated in the regulation of caspase-8 activation at the DISC.
Model of the apoptotic and non-apoptotic functions of FLASH. (A) Nuclear body associated FLASH is shuttled
into the cytoplasm upon activation of CD95, dependent on caspase activity and CRM-1. Cytoplasmic FLASH
then associates with mitochondrial caspase-8 thereby promoting apoptosis. This function can be inhibited by
adenoviral protein E1B19K that associates with both caspase 8 and FLASH at mitochondria. (B) Non-apoptotic
functions of FLASH. After TNFalpha treatment FLASH may translocate into the nucleus and interacts there
with hormone receptors and receptor coactivators to modulate transcription. FLASH is important for Cajal
body architecture, interacts with NPAT and enhances histone gene transcription and S-phase progression;
right: FLASH promotes TNFalpha-mediated activation of NFkappaB signalling by forming a complex with TRAF2,
leading to degradation of IkapppaB, nuclear accumulation of NFkappaB and expression of its target genes.
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However, recent findings demonstrated that FLASH is a Cajal body component
and regulates progression through S-phase of the cell cycle in the nucleus. Our
recent work identified FLASH as binding partner of the PML nuclear body
(PML NB) constituent Sp100 and demonstrated that FLASH partially localizes
to PML NBs. Upon CD95 activation FLASH exits the nucleus and translocates
to mitochondria where it meets caspase-8 to promote its activation. Our findings reconcile conflicting views on FLASH localization and its role in apoptosis
regulation, and suggest that CD95 signals via a nuclear pathway. These findings
have several implications for understanding FLASH function.
* Supported by Deutsche Forschungsgemeinschaft and Deutsche Krebshilfe
Functional p53 is required for effective telomerase inhibition in
BCR-ABL-positive CML cells in vitro
U. Brassat (1), S. Balabanov (1), H. Sirma (2), C. Günes (2), M. Braig (1),
D. Bali (3), K. Borgmann (3), T.H. Brümmendorf (1)
Hubertus Wald University Cancer Research Center, Hamburg, Germany
Radiobiology and exp. Radiooncology, Hamburg, Germany
Telomeres consist of repeat structures such as (TTAGGG)n in vertebrates and are
localized at the end of chromosomes. Replication-dependent telomere shortening
due to the end-replication problem can be counteracted by upregulation of an
endogenous reverse-transcriptase called telomerase. Increasing evidence suggests
that critical telomere shortening results in genetic instability which may promote
tumour evolution and telomerase activation during which critically short telomeres are stabilised and ongoing tumour growth is facilitated. In Chronic myeloid
leukemia (CML) the high turnover of the malignant clone is driven by the oncogene BCR-ABL and leads to accelerated telomere shortening in chronic phase (CP)
compared to telomere length in healthy individuals. Telomere shortening has been
demonstrated to be correlated with disease stage, duration, prognosis and response
to molecular targeted treatment. Despite of the accelerated telomere shortening
observed, telomerase activity is increased in CP CML and further upregulated with
progression of the disease to accelerated phase or blast crisis (AP/BC). To investigate the effect of telomerase inhibition on BCR-ABL-positive cells, we expressed a
dominant-negative mutant of hTERT (vector pOS DNhTERT-IRES-GFP) in K562
cells. The cells were single sorted and clones in addition to bulk cultures were long
term expanded in vitro. The expression of the transgene DNhTERT was monitored
by the expression of GFP and function of DNhTERT was analyzed by measurement of telomere length (by flow-FISH) and telomerase activity (TRAP assay).
Evaluation of these parameters showed the following patterns of growth kinetics
and telomere biology in individual clones: Two clones lost telomere repeats and
were transiently delayed in growth kinetics but eventually escaped from crisis
without loss of GFP expression (indicated by a re-increase in telomere length and
growth rate, group A) Three other clones lost GFP expression after initial and sig-
nificant telomere reduction indicating loss of the transgene (group B). Finally,
telomere length and growt0h kinetics of two remaining clones and of the bulk
culture cells remained unaffected by expression of DN-hTERT (group C). Of note,
none of the clones analyzed either died or entered cell cycle arrest. Further analyses
of one clone of group A revealed impaired DNA damage response indicated by two
fold increase in number of γH2AX foci in comparison to control cells. Moreover,
the expression pattern of genes involved in DNA repair was significantly altered
(Dual chip®). Network analysis of the altered genes using MetaCore® software
confirmed p53 as a key regulator in signaling of DNA damage in these cells. CML
blast crisis cell lines such as K562 are typically negative for functional p53 and
p16INK4A. Induction of p53 in cells with critically short telomeres (telomere length
4-5 kb) leads to immediate induction of apoptosis while vector control cells
continued to escape from crisis. These results suggest that the success of strategies
aimed at telomerase inhibition in CML is dependent on the presence of
functional p53 in BCR-ABL-positive cells, which argues in favor of applying these
strategies preferentially in CP as opposed to BC.
Invasin- and β1-Integrin-dependent uptake of bacteria is coupled to
the activation of autophagy and suppressed by Yersinia enterocolitica
A. Deuretzbacher (1), N. Czymmeck (1), R. Reimer (3), H. Hohenberg (3),
K. Trülzsch (2), J. Heesemann (2), M. Aepfelbacher (1), and K. Ruckdeschel (1)
(1) Institute for Medical Microbiology, Virology and Hygiene,
University Medical Center Eppendorf, Hamburg, Germany
(2) Max von Pettenkofer-Institute for Hygiene and
Medical Microbiology, Munich
Autophagy is a central lysosomal degradation process that is essential for the
maintenance of cellular homeostasis. Autophagy-related processes also promote separation and degradation of intracellular microorganisms, which contributes to the development of innate and adaptive immunity. Here, we investigated the impact of the enteropathogenic gram-negative bacterium Yersinia
enterocolitica on autophagy in infected macrophages. Autophagy was analyzed
by monitoring processing of the marker protein LC3 and by visualizing
autophagosome formation. Our results show that Yersinia virulence factors
differentially target the autophagic response in macrophages. Avirulent
yersiniae that were defective in virulence plasmid-associated type III protein
secretion efficiently activated autophagy. The autophagy-related events included the formation of autophagosomes, processing of LC3, redistribution of
GFP-LC3 to bacteria-containg vacuoles, and the segregation of intracellular
bacteria by autophagosomal compartments. Autophagy followed engulfment
of the bacteria by macrophages and specifically involved the Yersinia outer
membrane protein invasin. The autophagic events depended on engagement
of β1-integrin receptors. This delineates a novel pathway through which the
sensing of a bacterial microbe triggers autophagy. Wild type Y. enterocolitica on the
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contrary inhibited bacterial
uptake and the activation of
autophagy in a type III protein
secretion dependent manner. The
autophagy responses elicited by
rapamycin and chloroquine were
also suppressed by wild type
Yersinia infection. This indicates
global targeting of the autophagic
system by Yersinia-dependent
immunomodulation
which
involves several Yersinia outer
proteins (Yops) mobilized by
type III secretion. We propose
that the subversion of autophagy
is part of the Y. enterocolitica
virulence strategy that supports
bacterial
survival
when
β1-integrin-dependent internalization into macrophages is deleterious for the pathogen and
conducive for host immunity.
Internalized Yersiniae in a murine J774A.1 macrophage cell in TEM.
A3. Mechanisms DNA repair
Regulation and function of the stress-induced polymerase α-primasewtp53 complex in the ATR-mediated intra-S-phase checkpoint
G. Rohaly, A. Mena-Nunez, S. Dehde, H. Will, I. Dornreiter
In UV-damaged S-phase cells, it is inopportune to initiate DNA synthesis when
template strands are damaged because mutations and chromosomal aberrations
are induced through the process of replication of damaged DNA. One important
response to replication stress is the activation of the ATR-mediated intra-S checkpoint, which signals through the combined action of the Chk1-Cdc25A-cyclin
E-Cdk2 and the ∆p53-p21-cyclin A-Cdk2 pathways. The damage pathways delay
further progression through the cell cycle, inhibit origin firing, and promote
lesion repair. Although the transcriptional activity of p53 is repressed in attenuated S-phase cells, complete inhibition of DNA replication is only observed in
wtp53, but not in mutp53 primate cells. Our investigations revealed that wtp53
interacts exclusively with origin-competent DNA polymerase α-primase (Pol α)
only during the damage induced intra-S checkpoint. The p53 interacting domain
is located in the N-terminus of polymerase α, containing the Cdk phosphorylation sites that are involved in initiation control. A prerequisite for Pol α-wtp53
complex formation is ATR-∆p53-p21-mediated down regulation of cyclin A-Cdk2
activity, while high cyclin A-Cdk2/1 activity leads to complex dissociation.
Moreover, in vitro data demonstrate that wtp53 inhibits the primase but not the
polymerase α activity of the replicase. Thus, data suggest that the intra-S checkpoint induced interaction of wtp53 with Pol α prevents origin firing to allow
unperturbed repair of genotoxic lesions before the onset of DNA replication.
Repair fidelity of UV-induced lesions depends on ATR and
the p53 status
V. Deing, G. Rohaly, A. Mena-Nunez, S. Dehde, H. Hildebrandt,
H. Will, I. Dornreiter
In UV-damaged cells, the photoproducts [6-4]PP are regarded as the principal
UV-induced lesion that jeopardize replication fidelity and thus should activate
the intra-S checkpoint response. The fact explains why the [6-4]PP lesions are
much more efficiently repaired than CPDs and >80 % of the [6-4]PPs are
removed from human fibroblast DNA within three hours after UV irradiation.
Therefore, with such rapid repair of damage, addition of an extra hour or two
of repair before replication should have a significant impact on outcome. In a
recent study of our own, we found that only in wtp53 cells onset of DNA replication is delayed for six hours after treating the cells at the G1/S transition with
a low dose of UV. Our data demonstrate that during the six-hour replication
arrest, high repair activity is observed. Thus, it seems reasonable to assume that
inhibition of origin firing is a mechanism needed to allow complete repair of
[6-4]PP lesions and accordingly to prevent error prone bypass replication.
Accordingly, we assumed that the ATR-∆p53-p21-cyclin A-Cdk2-mediated
attenuation of S-phase progression and -mediated inhibition of origin-dependent initiation serves the purpose to enable repair of the genotoxic photoproducts [6-4]PP prior to the onset of replication. The assumption was investigated
by measuring the repair efficiency in UV-irradiated G1/S wtp53 CV1 and
TO-CV-1-mut∆p53 cells, where Dox addition leads to inactivation of endogenous ∆p53. Data demonstrated that in UV-irradiated G1/S CV-1 cells [6-4]PPs
were completely removed after 6 h. In contrast, in damaged TO-CV-1-mut∆p53
cells, where attenuation of S-phase progression and extended inhibition of replication is abrogated, the [6-4]PP lesions were removed in less than 2 h. Although
the [6-4]PP lesions were removed just in time before replication was initiated,
irradiated TO-CV-1-mut∆p53 cells responded with checkpoint activation in
G2-phase. During attenuation of G2-phase progression, high repair activity was
observed indicating that the accelerated [6-4]PP-repair process was executed at
the expense of repair fidelity, leading to secondary DNA damage. The same
results were obtained when ∆p53-transactivated p21 was knocked down by
Dox-induced shRNAp21 expression. It is noteworthy that the CPD repair kinetic was not altered in UV-irradiated G1/S cells devoid of functional ∆p53 or p21.
However, inactivation of endogenous ATR by dominant-negative effects severely impaired repair of the photoproducts CPD and [6-4]PP in UV-irradiated
G1/S TO-CV-1-ATRkd cells. Data strongly indicate that besides the ATR-activated
∆p53-p21-cyclin A-Cdk2 pathway, which delays S-phase progression and initia-
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tion of replication in favor of high fidelity [6-4]PP lesion repair, ATR is involved
in enabling nucleotide excision repair in damaged S-phase cells in general by an
yet unknown mechanism.
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In UV-damaged S-phase cells, delayed origin loading of
Pol α depends on the ATR-∆p53-p21-cyclin A-Cdk2 pathway
G. Rohaly, V. Deing, H. Will, I. Dornreiter
Our investigations demonstrated that the UV-induced ATR/Chk1 signaling pathway
causes transient delays in S-phase progression and reversible inhibition of DNA replication for 6 h. The key players of this pathway, which allow extended S-phase attenuation
and inhibition of replication, are transcriptional active ∆p53 and transcriptional
impaired wtp53. The transcriptional activity of ∆p53 leads to p21-mediated down regulation of cyclin A-Cdk2 activity, an activity that is required for S-phase progression and
DNA synthesis. Down regulation of cyclin A-Cdk2 activity is a prerequisite for facilitating interaction of the tumor suppressor wtp53 with hypophosphorylated, origin-competent Pol α, an event that correlates with inhibition of origin firing. However, it is not
known if p53 binds to origin-bound Pol α or sequestrates Pol α in order to prevent
origin loading. The question was addressed by using the ChIP-PCR method to determine the time point of origin-associated hypophosphorylated Pol α in sham- and
UV-treated CV-1 cells at specific time points post treatment. In order to establish ChIPPCR for origin-associated proteins, the well-characterized component of the originrecognition complex Orc2 was immunoprecipitated with an anti-Orc2 antibody from
CV-1 cell lysates. Association of Orc2 with ors12 origin and non-origin was analyzed by
PCR amplification. Results show that immunoprecipitated Orc2 contained only origin,
but not non-origin DNA, indicating that the ors12 locus is suitable for analyzing origin
interacting factors by ChIP-PCR in monkey CV-1 cells. Accordingly, investigations were
performed to evaluate whether hypophosphorylated Pol α interacts with the ors12 origin
during the UV-induced six-hour S-phase attenuation. For the immunoprecipitation,
two monoclonal anti-Pol α antibodies were used: HP180-12, which recognizes hypophosphorylated, origin-competent Pol α and SJK132-20, which is specific for the cyclin
A-Cdk-modified, origin-incompetent replicase. Results demonstrated that in mock
treated CV-1 cells HP180-12-reactive Pol α was associated with the ors12 origin only in
early S- (3 h post G1/S) but not in late S-phase (7 h post G1/S), confirming that all origins
had fired at the end of the replicative S-phase. In contrast, in UV-irradiated CV-1 cells
origin DNA was only recovered from HP180-12 immunoprecipitates at 7 h but not 3 h
post irradiation, indicating that in attenuated S-phase cells origin loading of Pol α is
prevented. In contrast, ChIP-PCR performed with sham-or UV-treated mut∆p53
expressing TO-CV-1 cells demonstrated that origin loading of HP180-12-reactive Pol α
was not delayed, thus resembling the pattern that was derived from non-irradiated cells.
In addition, data showed that in mock as well as in UV-irradiated CV-1 or TO-CV-1Flag-mut∆p53 cells SJK132-20-reactive Pol α was never associated with the ors12 origin.
The fact that origin loading of hypophosphorylated Pol α is prevented during the sixhour continuing UV-induced ATR-∆p53-p21-cyclin A-Cdk2-mediated S-phase attenu-
109
Loading of HP180-12-reactive, origin-competent Pol α onto the ors12 origin in mock- or UV-irradiated CV-1 and
TO-CV-1-Flag-mut∆p53 cells at specific times post irradiation.
ation suggests that p53 sequestrates and accordingly abrogates origin loading of the
replicase. In addition, confocal microscopy was performed to examine chromatin association of the HP180-12- and SJK132-20-reactive Pol α in mock- and UV-irradiated
cells, which were pre-extracted at specific time points post treatment. Data demonstrated
that loading of HP180-12-reactive Pol α onto chromatin is significantly delayed in
UV-irradiated CV-1 but not in TO-CV-1-Flag-mut∆p53 cells and never co-localized
with p53. However, it remains to be elucidated if in mutp53 expressing CV-1 cells origin
loading of Pol α is also delayed during S-phase attenuation.
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Dissection of the role of SPOC1 in DNA repair
A. Mund, G. Rohaly, I. Dornreiter, S. Schönfeldt, A. Mena-Nunez,
S. Dehde, K. Reumann, H. Will
Heinrich-Pette Institute, Hamburg, Germany
Chromatin modifications are important for all cellular processes that involve
DNA, including transcription, DNA replication and repair to maintain genomic
stability. One crucial event is the phosphorylation of histone H2AX (γH2AX) in
damage-induced modification of chromatin structure, which links the recruitment
of chromatin remodeling and repair complexes to sites of damaged DNA. Recently
our investigations revealed that our newly discovered protein SPOC1 (Survival
time associated PHD finger in Ovarian Cancer 1), a tightly cell cycle regulated
chromatin associated protein, co-localizes with DNA repair proteins in response
to DNA double strand breaks. Laser scanning microscopy demonstrated that
SPOC1 co-localizes with γH2AX at nuclear foci shortly before the onset of DNA
replication (G1/S-transiton). Additionally, we could show that the damageinduced SPOC1 foci formation depends on functional ATM. ATM is a member of
the phosphoinositide 3-kinase-related kinases (PIKK), which activate a complex
Formation of ATM-dependent SPOC1 (green) and gamma-H2AX (red) foci in human primary fibroblasts after
gamma irradiation. A/B: ATM mutants show no SPOC1 foci after 30min of 2Gy irradiation but still disperse
g-H2AX foci formation. C/D: Wt cells show distinct SPOC1 foci that co-localize with gamma-H2AX foci.
network involved in the DNA damage response pathways. Thus, our results
strongly indicate that SPOC1 is a new component of the ATM-mediated DNAdamage response pathway that operates in the G1/S-checkpoint.
*Supported by Deutsche Krebshilfe and Müggenburg Stiftung
A role for E1B-AP5 in ATR signalling pathways during adenovirus
infection
A. Blackford (1), R. Bruton (1), O. Dirlik (2), G. Stewart (1), A. Malcolm (1),
R. Taylor (1), T. Dobner (2), R. Grand (1), A. Turnell (1)
(1) CRC Institute for Cancer Studies, The University of Birmingham, UK
E1B-55K-Associated Protein 5 (E1B-AP5), is a cellular, heterogeneous nuclear
ribonucleoprotein that is targeted by adenovirus E1B-55K during infection. The
function of E1B-AP5 during infection, however, remains largely unknown. Given
the role of E1B-55K targets in the DNA damage response, we examined whether
E1B-AP5 function was integral to these pathways. Here we reveal a novel role for
E1B-AP5 as a key regulator of ATR signaling pathways activated during adenovirus infection. Specifically, we show that E1B-AP5 is recruited to viral replication
centers during infection where it co-localizes with ATR-Interacting Protein
(ATRIP) and the ATR substrate, RPA32. Indeed, E1B-AP5 associates with ATRIP
and RPA complex component, RPA70, in both uninfected and Ad-infected cells.
Additionally, GST pull-downs show that E1B-AP5 associates with RPA components, RPA70 and RPA32, directly in vitro. Significantly, we have determined that
E1B-AP5 is required for the ATR-dependent phosphorylation of RPA32 during
infection and contributes to the Ad-induced phosphorylation of Smc1 and H2AX.
In this regard, it is interesting to note that Ad5 and Ad12 differentially promote the
phosphorylation of RPA32, Rad9 and Smc1 during infection, such that Ad12 promotes significant phosphorylation of RPA32 and Rad9, whereas Ad5 only weakly
promotes RPA32 phosphorylation, and does not induce Rad9 phosphorylation.
These data suggest that Ad5 and Ad12 have evolved to utilize different strategies
to regulate DNA damage signaling pathways during infection in order to promote
viral replication. Taken together, our results define a role for E1B-AP5 in ATR
signaling pathways activated during infection. This might have broader implications for regulation of ATR activity during the cellular DNA replication cycle or in
response to DNA damage.
*Supported by Cancer Research UK
111
B. Cellular controls of self-renewal, proliferation,
and differentiation
112
Modulation of gene expression in U251 glioblastoma cells by
binding of mutant p53 R273H to intronic and intergenic sequences
M. Brázdová (1), T. Quante (1), L. Tögel (1), K. Walter (1), C. Loscher (1),
V. Tichý (2), L. Činčárová (2), W. Deppert (1), G.V. Tolstonog (1)
(2) Institute of Biophysics, Brno, Czech Republic
Missense point mutations in the TP53 gene are frequent genetic alterations in
human tumor tissue and cell lines derived thereof. Mutant p53 (mutp53) proteins
have lost sequence-specific DNA binding, but have retained the ability to interact
in a structure-selective manner with non-B DNA and to act as regulators of transcription. To identify functional binding sites of mutp53, we established a small
library of genomic sequences bound by p53R273H in U251 human glioblastoma cells
using chromatin immunoprecipitation (ChIP). Mutp53 binding to isolated DNA
fragments confirmed the specificity of the ChIP. The mutp53 bound DNA sequences are rich in repetitive DNA elements, which are dispersed over non-coding DNA
regions. Stable down-regulation of mutp53 expression strongly suggested that
mutp53 binding to genomic DNA is functional. We identified the PPARGC1A and
FRMD5 genes as p53R273H targets regulated by binding to intronic and intra-genic
sequences. We propose a model that attributes the oncogenic functions of mutp53
to its ability to interact with intronic and intergenic non-B DNA sequences and
modulate gene transcription via re-organization of chromatin.
*Supported by the Deutsche Forschungsgemeinschaft (DFG), by EU FP6 and by
the German-Israeli Foundation (G.I.F.)
Mutant p53R273H is targeted to the nucleus in U251 glioblastoma cells and associates with transcription factories.
Mutant and wild-type p53 as modulator of global chromatin
organization
T. Quante (1), A. März (1), L. Tögel, S. Hess (2), D.P.F. Möller (2),
G. Warnecke (1), M. Kühl (1), C. Loscher (1), W. Deppert (1),
G.V. Tolstonog (1)
(2) Department of Informatics, University of Hamburg, Germany
Missense mutations in the TP53 gene are frequent genetic alterations in human tumor
tissue and cell lines. In contrast to its wild-type counterpart, the mutant p53 (mutp53)
protein is long-lived and has lost the transcriptional activity towards pro-apoptotic and
growth arrest genes, but retained the propensity for targeting to chromatin. Expression
of mutp53 is advantageous for tumor cells, however the molecular mechanism of
mutp53 action is still not known. We used tumor cell lines expressing endogenous and
inducible mutp53 proteins as models to study the role of mutp53 in transcriptional
regulation. Mutp53 has lost sequence specificity in DNA binding activity but retained the
property to recognize DNA secondary structures, and based on our ChIP-Seq data is
prone to interact preferentially with repetitive sequences possessing conformational flexibility. Therefore we assumed that mutp53 operates on the level of global chromatin
organization rather than on modulating the expression of individual genes. In support,
we found that differentially regulated genes frequently map to the same chromosomal
locations or even are organized as physically-linked gene clusters. This implies that
mutp53 might be a factor involved in regulating the recruitment of gene promoters to
immobile transcription factories containing active RNA polymerases. To test this
hypothesis, physical DNA contacts in several genomic loci encompassing co-regulated
gene clusters were profiled using the chromosome conformation capture (3C) method.
Preliminary results strongly support the proposed function of mutp53 in modulating
the gene expression program at a higher level.
*Supported by Deutsche Forschungsgemeinschaft (DFG), by EU FP6, and by the
German-Israeli Foundation (G.I.F.)
P53 as a potential epigenetic modulator of gene expression
J. Hildebrand (1), A. März (1), L. Tögel (1), T. Quante (1), S. Hess (2),
D.P.F. Möller (2), G. Warnecke (1), M. Kühl (1), C. Loscher (1),
W. Deppert (1), G. V. Tolstonog (1)
(2) Department of Informatics, University of Hamburg, Germany
Transcription factors may act as dual-purpose regulators, controlling gene expression
either via direct targeting of a gene or via (re)organizing higher-order chromatin structure. In this regard, recent genome-wide studies of the p53 binding to DNA demonstrated that p53 binding not only is restricted to regulatory sequences around transcription start sites, but rather that binding events are widely distributed over the whole
genome. Extending the term “epigenetics” to (i) transcription events outside of coding
genes and (ii) regulatory reactions primarily based on nuclear (re)organization, we want
to propose that the function of p53 in regulating the gene expression program includes
also a role in epigenetic processes. In the frame of this hypothesis we tested the possibility that p53 may regulate transcription of long interspersed nucleotide elements (LINEs),
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114
a family of transcriptionally potent repetitive sequences, via binding to their promoters.
Around 12.000 full-length LINEs are dispersed throughout the human genome and can
be potentially engaged by the transcription machinery. Most of the full-length LINEs are
non-coding, whereas only 1 % has retained a coding potential for two proteins required
for retrotransposition. LINE transcription is developmentally regulated, whereby the
highest expression is observed during embryogenesis, in germ line cells and tumors. To
test the putative transactivation function of p53 on the LINE transcription, we constructed an episomal eGFP-based reporter gene, which allowed us an accurate measurement of
LINE promoter activity via eGFP fluorescence. In H1299 cells engineered for inducible
expression of p53, we observed significant stimulation of the episomal LINE promoter,
accompanied by a p53-dependent increase of endogenous LINE transcription. The latter
observation was confirmed in 5-FU treated HCT116 (p53+/+) cells and the teratocarcinoma cell lines 2102Ep (human) and F9 (mouse), and could be reversed by p53-specific
siRNA. Binding of p53 to endogenous LINE promoters was confirmed by ChIP and correlated with the occupation of the respective promoters by RNA polymerase II.
Analyzing the transcriptome profiles of H1299 cells expressing Tet-inducible p53 we
noted that many regulated genes frequently map to the same chromosomal locations or
even are organized in physically-linked functional gene clusters. The number of genes in
such co-regulated clusters correlated well with the time-dependent increase in p53 concentration after beginning of Tet-induced ectopic p53 expression. To allow for a systematic analysis of gene co-regulation, we programmed a script, which can match data from
the most common commercial platforms to current genome annotation, search for the
co-regulated genes based on variable criteria, and do cross-analysis between different
samples to find groups of common co-regulated genes. By genome-wide ChIP-on-chip
analysis we observed that significant number of co-regulated gene clusters was associated
with the single p53 binding events, supporting our assumption that p53 is additionally
involved in global epigenetic processes leading to modulation of the transcriptional
activity of the whole chromatin domain.
*Supported by Deutsche Forschungsgemeinschaft (DFG), and EU FP6
Oncogenicity and expression profiling of SPOC1 in normal and
tumor tissues
H. Staege (1), J. Weise (1), J. Hengstler (2), K. Milde-Langosch (3),
C. Günes (1), S. Kinkley (1), U. Matschl (1), G. Mohrmann (1), H. Will (1)
(2) IfADo, Dortmund, Germany
Institute of Gynpathology, Hamburg, Germany
We have analyzed the expression levels of the novel SPOC1 protein in different tissues of
human and mouse origin. Highest expression of SPOC1 RNA and protein was detected
in the testis and lower levels were found in all other tissues examined. In general, the level
of SPOC1 expression correlated with the proliferative state of the tissue, indicating a possible role for SPOC1 in normal cellular proliferation. Prompted by our initial observation that high levels of SPOC1 RNA are correlated with poor survival and residual disease in a subset of ovarian cancer patients, we extended our studies to investigate SPOC1
protein expression in ovarian cancer.
Consistent with the RNA studies, we
found that SPOC1 protein levels were
significantly increased in a subset of
ovarian tumors. These findings suggest that SPOC1 may have oncogenic
potential and could be a useful diagnostic/prognostic marker for ovarian
cancers. Studies to investigate SPOC1
expression in other types of cancer
and to correlate its expression with
other described tumour markers are
ongoing.
Furthermore, preliminary evidence
shows that cell lines with elevated
SPOC1 protein expression gives rise
Increased SPOC1 levels in a subset of ovarian carcinomas. SPOC1
protein levels were determined in lysates from normal ovary and to faster growing tumors than the
from ovarian carcinoma tissues by immunoblotting. SPOC1 was parental cell lines alone in nude mice.
detected with a rat monoclonal anti-SPOC1 antibody and β-actin
A more extensive study evaluating
served as loading control.
SPOC1 overexpressing cell lines in
comparison to the parental cell lines with lower steady state levels of SPOC1 protein in
nude mice are underway. These studies should provide conclusive evidence as to the
oncogenicity of enhanced SPOC1 protein expression.
*Supported by Deutsche Krebshilfe and the Müggenburg Stiftung
SPOC1: A novel PHD containing protein modulating chromatin
structure and mitotic chromosome condensation
S. Kinkley (1), H. Staege (1), G. Mohrmann (1), G. Rohaly (1),
T. Schaub (1), E. Kremmer (2), A. Winterpacht (3), and H. Will (1)
(2) GSF-Research Center, München, Germany
(3) University of Erlangen, Institute of Human Genetics, Erlangen, Germany
In this study, we characterize the molecular and functional features of a novel protein
called SPOC1. SPOC1 RNA expression was previously reported to be highest in highly
proliferating tissues and increased in a subset of ovarian carcinoma patients which statistically correlated with poor prognosis and residual disease. These observations implied
that SPOC1 may play a role in cellular proliferation and oncogenesis. Here we show that
the endogenous SPOC1 protein is labile, primarily chromatin associated and its expression as well as localization are regulated throughout the cell cycle. SPOC1 demonstrates a
dynamic regulation during mitosis with increased expression levels and biphasic localization to mitotic chromosomes, arguing for a functional role of SPOC1 in mitotic processes. Consistent with this postulate, SPOC1 siRNA knockdown experiments resulted in
defects in mitotic chromosome condensation, alignment and aberrant sister chromatid
segregation. Finally, we have been able to show via micrococcal nuclease (MNase) chromatin digestion assays that SPOC1 expression levels proportionally influence the degree
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116
Overexpression of SPOC1 induces global chromosome condensation and this depends on its plant homeo
domain (PHD).
of chromatin compaction. Collectively, our findings show that SPOC1 modulates chromatin structure and that tight regulation of its expression levels and subcellular localization during mitosis are crucial for proper chromosome condensation and cell division.
Enhanced SPOC1 expression induces chromatin instability
S. Kinkley (1), H. Staege (1), K. Reumann (1), M. Cremer (2),
A. Jauch (3), and H.Will (1)
(2) LMU Biozentrum, Department Biology II, Martinisried, Germany
(3) Institute of Human Genetics, University Hospital Heidelberg,
Heidelberg, Germany
SPOC1 is a novel chromatin associated protein that shows elevated expression levels
and a dynamic regulation during mitosis. Recent results suggest that SPOC1 is
important for proper chromatin architecture and condensation of mitotic chromosomes arguing indirectly that it may also be important for genomic stability.
Consistent with this possibility, several doxycyclin-inducible U2OS SPOC1
over-expressing cell lines became 100 % polyploid in the absence of doxycyclin after
multiple passages in culture, due to a low level of leaky SPOC1 over-expression
(2-fold) in these cell lines. The polyploidization of these cell lines were charted by
FACS analysis and later confirmed by M-FISH. Furthermore, doxycyclin induction
of SPOC1 over-expression (~30-fold) in these polyploid cell lines resulted in
chromosome losses, inducing an aneuploidy-like state from a tetraploid intermediate. These observations were reproducible also in Dld-1 cells, which had originally a
117
Low level of SPOC1 overexpression results in 100 % polyploidization of cell lines. A. Immunoblotting of SPOC1
expression levels in wild-type U2OS cells and in two U2OS inducible SPOC1 clones (Cl. 5 and Cl.14) in their
uninduced (UI) and induced (Ind.) states. Actin served as a loading control. B. FACS profiling of these cell lines
shows that both U2OS SPOC clone 5 and clone 14 are 100 % polyploidy in comparison to the U2OS cell line. C.
M-FISH analysis confirms that the U2OS SPOC1 clones are polyploidy in comparison to the parental U2OS and
shows that chromosome losses occur when the SPOC1 clones are induced for SPOC1 over expression.
normal diploid chromosome count and a single stable chromosome translocation,
demonstrating a global penetrance and potency of SPOC1. The underlying
mechanism of SPOC1 induced polyploidization and chromatin instability is not yet
clear, however it appears to involve the deregulation of key cyclins/cdk complexes
and some of their downstream targets. Elucidating this mechanism is a main focus
of our research program and is currently being actively pursued. Together these
findings indicate that a tight regulation of SPOC1 expression levels is crucial for the
maintenance of a normal diploid state in cells and therefore for genomic stability.
SPOC1: A novel chromatin remodeling protein?
S.Kinkley (1), H. Staege (1), N. Lohrengel (1), G. Mohrmann (1),
K. Reumann (1), A. Eberharter (2), and H. Will (1)
(2) Adolf-Butenandt Institute of Molecular Biology,
University of Munich, Munich, Germany
SPOC1 is a chromatin-associated protein with a described function in the regulation of
mitotic chromatin architecture. A more detailed analysis of SPOC1’s relationship with
chromatin revealed that it is capable of specifically interacting with DNA, histones and
nucleosomes, arguing strongly for chromatin related function of SPOC1. To further
clarify the specificity of SPOC1’s interaction with chromatin and to gain some insight
into the potential role it plays there, we systematically evaluated its ability to bind dif-
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Mitotic Changes in SPOC 1 Localisation and Expression. SPOC1 changes its localization during the mitotic cell
cycle as shown by immuno-fluorescence staining and by immuno-blotting using synchronized cells.
ferentially modified histones. Recently there have been several publications describing
PHD domains as the molecular reader of H3K4 di- and tri- methylation marks.
Consistent with the literature, SPOC1 was found to specifically associate with di- and
tri-methylated H3K4 in a PHD domain dependent manner. SPOC1 is also capable of
binding to other modified histones however, in a PHD-domain independent manner,
implicating that another as of yet undefined region in the SPOC1 protein is also capable
of interacting with chromatin. Interestingly, transient over expression of the SPOC1
protein in multiple cell lines results in massive global hyper-condensation of chromatin
in a PHD-domain dependent manner. This dramatic phenomenon results in the condensation and global remodeling of the entire genome, similar to what is observed
during mitosis. It is our current hypothesis, that SPOC1 may act either directly or indirectly to recruit condensing remodeling complexes to the chromatin and may therefore
act as a molecular switch between a euchromatin state to a more heterochromatic state.
Consistent with this possibility, SPOC1 is found to co-elute by column chromatography
in three high molecular weight fractions of ~400 kDa, 800 kDa and 2000 kDa, arguing
that SPOC1 is part of a greater protein complex. Elucidation of these complex members
should help to clarify the biological functional roles of SPOC1 as well as potentially the
mechanism of SPOC1 induced chromosome condensation. Therefore in this study we
aim to understand the molecular context and consequences of SPOC1 on chromatin.
The RUNX1 transcription factor: A gatekeeper in acute leukaemia
J. Cammenga (1,2), M. Fischer (1),), K. Weber (1,3), U. Bergholz (1),
M. Ziegler (1), S. Roscher (1), U. Müller (1), C. Stocking (1)
(2) Lund Strategic Research Center for Stem Cell Biology and Cell Therapy,
University of Lund, Sweden
Center of BM-Transplantation, Hamburg, Germany
RUNX1 is one of the most frequently mutated genes in human acute leukemias
and hematopoietic disorders. Located on chromosome 21, it is directly targeted by
several chromosomal translocations in AML, with the t(8;21) being the most
common, occurring at a frequency of approximately 15 %. Moreover, 20 % of pediatric
B-cell acute lymphoblastic leukemia (ALL) carry the t(12;21), juxtaposing the
RUNX1 gene with the gene encoding the ETS transcription factor ETV6. In addition
to translocations, inactivating or dominant-negative mutations in the RUNX1
gene have been identified in 15 to 25 % of the relatively rare, minimally differentiated
M0 AMLs, up to 25 % of myelodysplastic syndromes associated with AML
The RUNX1 gene is a frequent target of genetic disruption in acute leukemia. Specific chromosomal translocations or point mutations involving the RUNX1 gene are found in acute myeloid or lymphoid leukemia with a
distinctive phenotype. Depicted is the lineage and differentiation stage in which the bulk of the leukemia cells
with a characteristic genetic mutation accumulate. The genetic mutations result in either fusion or mutated
RUNX1 proteins, as depicted.
119
development, and in pedigrees of familial platelet disorder with a propensity to
develop AML. RUNX1 gene product (RUNX1 or often called AML1) is one of
three members of the RUNX family of transcription factors that play pivotal roles
in normal development. RUNX proteins bind DNA at sequence-specific motifs
through a conserved Runt homology domain, which is also the interacting domain
of a shared β-subunit that modulates nuclear localization and DNA binding.
Significantly, disruption of the gene encoding the β-subunit is also observed in
5 % of AMLs.
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Over the last few years, we have established mouse models for translocations
t(8;21) and t(12;21) and, more recently, for mutated RUNX1 genes, using a retroviralvector transduction protocol coupled with bone marrow transplantation. The
coexpression of GFP in this model has allowed the evaluation of changes in the
hematopoietic system before the onset of an overt leukemia, providing an excellent
model to study the multiple steps of leukemogenesis. The evaluation of these
mice have lead to the following conclusions: 1) intrinsic functions of the fusion
protein or mutated protein play a determining role in the phenotype (myeloid vs
lymphoid; early or late; T- or B-cell) of the ensuing leukemia; 2) whereas repression
of direct target genes of RUNX1 is a common functional denominator of RUNX1
mutations, DNA-binding-independent functions of RUNX1 play an important
role in leukemogenesis; 3) RUNX1 gene alterations alone are insufficient to
induce a leukemia, but probably act as gatekeeper, permitting the outgrowth of a
preleukemic clone, which is the target of secondary events.
More recent data have demonstrated that Runx1 forms large multiprotein
complexes with several classes of chromatin modulators and plays a pivotal role
in the initial chromatin remodeling during lineage speciation. We favor the
hypothesis that Runx1 regulates B-cell specification by chromatin modulation of
the PU.1 locus, in contrast myeloid differentiation per se does not require continued
Runx1 expression, but Runx1 is required to modulate proliferation or anti-apoptotic
stimuli. We are currently testing this hypothesis in Runx1 conditional mice. We
are also extending our work to human hematopoietic cells, using a xenograft
transplantation model (NOD/scid mice). Differences in the transcriptional
control of the hematopoietic system between mouse and man are known, and
may greatly influence the action of an altered transcription factor. For these studies
we have developed lentiviral vectors that allow the efficient expression of a
fluorescent marker gene (e.g. VENUS, tdTomato, EGFP, or Cyan), a potential
oncogene, and a cassette to express short-hairpin RNA, suppressing gene expression
Runx1 to determine its function in human hematopoiesis.
*Supported by Fritz-Thyssen-Stiftung, Deutsche José Carrera's LeukämieStiftung, and Deutsche Krebshilfe.
The MADS transcription factor MEF2C is an important regulator
of monopoiesis and the leukemic stem cell compartment in
MLL-associated leukemia
A. Schüler (1), M. Täger (1), M. Forster (1), J. Löhler (1), M. Ziegler (1),
S. Roscher (1), R. Delwel (2), E. Olson (3), R. Slany (4), C. Stocking (1)
(2) Dept of Hematology, Erasmus University, Rotterdam, The Netherlands
(3) University of Texas Southwestern Medical Center, Dallas TX, USA
(4) Friedrich-Alexander University Erlangen-Nürnberg, Germany
Multiple lines of evidence indicate that tumorigenesis is a multistep process originating
from a single cell. In myeloid leukemia, the concept that a hematopoietic stem cell
(HSC) is the first normal cell that becomes subverted in the leukemogenic process
is gaining wide acceptance. Importantly, several recent in vivo studies have also
demonstrated that a small number of the immediate progeny of this cell, which still
share many of characteristics of the HSC compartment, are responsible for maintaining
the leukemia and thus are denoted the leukemic stem cell (LSC) compartment.
Understanding how a HSC is converted to a LSC is critical for establishing effective
therapeutic intervention of acute myeloid leukemia (AML). We have used retroviral
insertional mutagenesis to identify novel genes that regulate myeloid differentiation
– and thus are potential targets of gene disruptions that establish the LSC. Two
members of the MEF2 gene family were identified in this screen.
The MEF2 proteins are members of the MADS (MCM1-agamous-deficiens-serum
response factor) family of transcription factors, which play key roles in pattern development in plants and coordinate diverse and important biological functions, including
proliferation, differentiation, and apoptosis, in the animal. Importantly, the activity of
MADS-box proteins is profoundly
influenced by developmental cues
and signals from extracellular
environment through several transcriptional and post-translational
controls. The MEF2 subfamily,
which consists of four members
(A-D) and distinguishes itself with
the presence of both a MADS
domain and a conserved MEF2
domain, important for homo/and
heterodimerization. In contrast to
the accumulating evidence supporting
Mef2c is an important link between extracellular stimuli and intrathe
importance of MEF2 in neuronal
cellular controls. Our work has shown that cell fate decisions between the monocytic and granulocytic lineages are modulated by and myogenic development and
the Mef2c transcription factor upon activation through extracelludespite the fact that several MEF2
lar signals (e.g. growth factors or bacterial infections). Activation of
Mef2c upregulates expression of the bZIP transcription factor cJun. proteins are highly expressed in
We postulate that cJun forms heterodimers with C/EBP proteins, hematopoietic tissue, little is known
thereby crippling their ability to regulate transcription of important
granulocytic genes and thus favouring monocytic differentiation about their role in myeloid and
driven by the ets transcription factor PU.1.
lymphoid development.
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122
Using a retroviral gene transduction – bone marrow transplantation model we have
been able to demonstrate the importance of MEF2C in AML development in
susceptible mice. This analysis has also shown the importance of MEF2C in lineage
commitment to the monocytic lineage, which has been corroborated by analysis of
conditional Mef2c knock-out mice. cJun was determined to be an important downstream target of MEF2C for this activity. Analysis of AML patient samples provided
the insight that high MEF2C expression is found in myelomonocytic leukemia samples
characterized by alterations of the bithorax orthologue MLL. Deletion of the Mef2c
gene in MLL-transformed cells cripples their ability to induce leukemia in vivo and
thus may play an important role in maintaining the LSC compartment.
*Supported by Deutsche José Carrera's Leukämie-Stfitung
Aldehyde dehydrogenase 1 (ALDH1) as a functional marker of cancer
stem cells in a mammary carcinoma mouse model
F. Wegwitz (1), C. Maenz (1), J. Oehlmann (1), A. Diesterbeck (1),
A. Preuß (1), G. Pilnitz-Stolze (1), A. Düsedau (1), A.-M. Kluth (1),
U. Schumacher (2), G. Tolstonog (1), W. Deppert (1)
Dep. of Anatomy, Hamburg, Germany
Since the discovery that a small subset of self-renewing leukemia cells drive cancer
growth and propagation about 10 years ago, the cancer stem cell theory has been
extended to solid tumours, including breast cancer. However, despite the attractiveness
of the cancer stem cell theory, most “cancer stem cells” are still only operationally
defined and characterized by empirically established cell surface markers.
Several studies describe the expression of ALDH1, which can be easily detected by
its fluorescent substrate Aldefluor®, as a prospective marker of mammary carcinoma
stem cells (MCSC). Our WAP-T mouse model for oncogene-induced mammary
carcinoma appeared to be suitable to test the value of ALDH1 expression as a
marker for MCSC derived from WAP-T tumors. Orthotopic transplantation of
isolated primary tumor cells into fat pads of syngeneic mice leads to the development
of tumors of similar morphology like the primary tumor. To isolate populations
enriched for MCSC we performed fluorescence activated cell sorting prior to
transplantation of different amounts of ALDH1+ and ALDH1- populations. We
observed tumor growth from as few as 100 cells. There was no significant difference
in the morphology or growth characteristics between ALDH1+ and ALDH1tumor cells. Similar results we obtained with a mammary carcinoma cell line
established from a primary WAP-T tumor (GeTo). GeTo cells exhibit a bilinear
differentiation potential with characteristics of both basal and luminal mammary
epithelial cells. Upon transplantation of as few as 10 unsorted cells tumor growth
can be observed. Proliferation of transplanted ALDH1- GeTo cells is only marginally
delayed compared to that of transplanted ALDH+ GeTo cells.
We conclude that ALDH1 is unlikely to be a marker for a MCSC enriched population
in our WAP-T mouse model. Furthermore, in this model MCSC are present at a
much higher frequency than formerly assumed. These observations are in line with
the recent publications suggesting that tumor stem cells are not necessarily a rare
population.
*Supported by Deutsche Krebshilfe and by Deutsche Forschungsgemeinschaft
(DFG)
Hierarchy and p53-dependent differentiation of embryonal
stem/progenitor cells in mouse F9 teratocarcinoma cell line
M.-A. Kluth, R. Reimer, H. Hohenberg, W. Deppert, G.V. Tolstonog
Differentiation leads to gradual specialization (commitment) of a dividing stem/
progenitor cell during embryonic development and adult tissue homeostasis.
Commitment accompanies the division of stem/progenitor cells and results in equal
(symmetric) or unequal (asymmetrical) separation of epigenetically-determined
instructions between two genetically identical daughter cells. The whole process is
precisely orchestrated by regulatory factors, controlling the self-renewal and
expansion of stem and progenitors on each level of commitment. Using mouse F9
teratocarcinoma cell line, a well-characterized cell differentiation model, we applied
FACS-based analytical techniques to isolate and characterize the subfractions of F9
cells exhibiting properties that fit the criteria of stem/progenitor cells, namely
self-renewal and potency. The “Aldefluor®”-reagent, commonly used in the stem cell
research, is metabolized into a fluorescent derivate by cells expressing aldehyde
dehydrogenases (ALDH), thereby allowing us to fractionate F9 cells into the ALDE+
and ALDE- populations. The microarray-based comparison of the gene expression
profiles of these subpopulations with those of F9 cells treated with retinoic acid (RA)
and dibutryl cAMP (dbcAMP) for differentiation and control cells revealed that
ALDE+ cells are related to control, undifferentiated cells, whereas the transcriptome
of ALDE- cells is similar to that of differentiated cells. As no differences in the
expression of ALDH isoforms was found between ALDE+ and ALDE- populations,
we analyzed the morphology and membrane potential of mitochondria, where most
of the ALDH isoforms are located. Applying the fluorescent dye JC1 as a vital sensor
of the mitochondrial membrane potential, we observed comparable morphology
but dramatic difference in the mitochondrial activity between ALDE+ and ALDEcells. The low membrane potential in ALDE+ cells explains the high metabolization
of the “Aldefluor®”-reagent, which can passively traverse the uncharged mitochondrial
membrane. ALDE+ F9 cells exhibit properties of stem/progenitor cells, as they
regenerate the original F9 cell population, which retains the differentiation potential
both under 2D (plastic dishes) and 3D (CarboCellTM micro-tubes) cell culture
conditions. In summary, we provided evidence that the F9 cell culture comprises
a heterogeneous population of stem and committed cells, which differentiate upon
inductor treatment.
In order to find the regulator(s) governing the composition of F9 cell population, we
paid attention to the p53 protein, which is highly expressed in ALDE+ cells, but
down-regulated in ALDE- cells and upon RA/dbcAMP treatment. The p53 gene in
F9 cells is wild-type and expressed at detectable levels mainly in the nucleus.
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Therefore, we performed ChIP experiments to analyze the DNA binding activity of
p53 and correlate it with the activity of the respective genes. Beyond binding to
promoters of known p53 target genes, like Mdm2 and Cdkn1a, we identified a new
target gene transactivated by p53, Utf1, which encodes a protein known as a transcriptional regulator of embryonic cell differentiation. shRNA mediated knockdown
of p53 resulted in an inductor-independent cell differentiation, whereas overexpression
of p53 reversed this effect and protected cells against the effects of differentiation
inductors. The data indicate that p53 may control the fate of stem and committed
cells and regulate their asymmetric division. In support of this hypothesis, we
demonstrated by BrdU pulse/chase labeling and BrdU-Hoechst 33342 quenching
assays that ALDE+ cells are dividing both symmetrically and asymmetrically.
p53 as a regulator of asymmetric cell division.
Asymmetrically dividing cells retain the BrdU label and express higher amounts of
p53 than the rest of the population, providing phenomenological evidence for a role
of p53 in the regulation of asymmetric division. The finding opens new perspectives
for the study of p53 function in self-renewal and commitment of stem cells. Our
data establish a link between loss of p53 function and initiation of tumorigenic
conversion of normal stem/progenitor cell to cancer initiating cell, which lose the
tight control mechanisms over cell fate regulation and expand via symmetric
divisions.
Spontaneous F9 cell differentiation in a capillary-based 3D cell culture
M.-A. Kluth (1), R. Reimer (1), H. Hohenberg (1), S. Beck (2),
M.W. Linscheid (2), W. Deppert (1), G.V. Tolstonog (1)
(2) Institute of Chemistry, Humboldt-University Berlin, Germany
Cell differentiation is a fundamental process of gradual specialization
(commitment) of a dividing stem/progenitor cell during embryonic development
and adult tissue homeostasis. Single steps of differentiation pathway can be followed in vitro under cell culture conditions on a limited number of established cell
lines which retained differentiation potential. Mouse F9 teratocarcinoma cells are
widely used as a model for the analysis of the molecular mechanisms of cell
differentiation and for studying steps which occur during early embryogenesis.
This cell line is characterized by the inability to differentiate spontaneously
(nullipotency), but upon treatment with retinoic acid (RA) alone or in
combination with dibutyryl cAMP (dbcAMP), the F9 cell population
differentiates towards extraembryonal parietal or visceral endoderm. Treatment
with inductors induces cell arrest and partial or complete differentiation of the
majority of cells, whereas a small population of cells remains in the proliferative
phase. The differentiation of F9 cells is a rapid process, running into completion
within 3 days of treatment. The differentiated cells are characterized by a migratory
phenotype (parietal endoderm) and enhanced expression of extracellular matrix
proteins, collagen IV and laminins (visceral endoderm). Inductor withdrawal by
change of culture media induces rapid expansion of the proliferation-active
subpopulation and the generation of differentiation-competent cells.
In order to study cell differentiation under conditions which closely mimic the
natural settings, we used a unique 3D-cell culture model established in the HPI,
the CarboCell TM micro-tubes. Compared to other 3D-cell culture techniques, the
growth of seeded cells and the diffusion of secreted auto- and paracrine factors are
restricted by the space of the capillary tube and their permeability. The transfer of
cells from 2D culture into CarboCell TM micro-tubes dramatically changed the
behavior of F9 cells by: (i) shortening of the dividing time from 12 hours to 3.6
hours; and (ii) spontaneous inductor-independent differentiation of F9 cells into
visceral endoderm. The differentiation status of F9 cells in CarboCell TM
micro-tubes was confirmed by immunohistochemical and electron microscopical
detection of the components of extracellular matrix (collagen IV and α-laminin).
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Microarray-based transcriptome analysis of RA/dbcAMP-treated cells cultured
under 2D conditions and cells from CarboCell TM micro-tubes revealed that around
2000 genes were similarly regulated under both cell culture conditions. Considering
that RA-signaling pathway plays a key role in the differentiation of embryonic cells
we tested the hypothesis that spontaneous, inductor-independent differentiation
of F9 cells is triggered by retinoic acid which had been intracellularly deposited
while the cells were passaged under 2D conditions in fetal calf serum (FCS)
containing medium. In support of this hypothesis, we observed that pre-culturing
of F9 cells in nutrient-rich, but FCS-free medium completely abolished the differentiation under 3D conditions, but did not prevent their growth. An HPLCbased analysis of the lipid fractions extracted from FCS and F9 cells cultured
in the CarboCell TM micro-tubes demonstrated the presence of retinoic acid,
thereby providing ultimate support for our hypothesis. In conclusion, applying
CarboCell TM micro-tubes we discovered a new mechanism of embryonic cell
differentiation, which is governed by storage and release of the differentiation
inductor retinoic acid.
Differentiation of F9 cells under 2D and 3D cell culture.
SPOC1 is essential for spermatogonial stem cells
H. Staege (1), S. Ficke (1,2), S. Kinkley (1), U. Matschl (1), T. Schaub (1),
U. Schumacher (2), H. Will (1)
Dep. of Anatomy, Hamburg, Germany
We have previously reported that
SPOC1-RNA levels are highest in
spermatogonia, mitotic germ cells
in adult testis which give rise to
sperm during spermatogenesis.
Using self-made mono- and
polyclonal anti-SPOC1 antibodies,
SPOC1 protein expression was
only detected in spermatogonia,
but not in other cells of the testis.
The expression pattern largely
overlapped with that of the PLZF
protein, which is known to be
essential for germ cell maintenance.
The data prompted us to test
whether SPOC1 plays a role in the
maintenance of spermatogonia
and spermatogenesis. For these
studies we produced mice with
doxycyclin-inducible shRNAmediated knock-down of SPOC1
protein expression. Histological
analysis of the testis obtained from
doxycyclin-treated mice showed
SPOC1 and PLZF are co-expressed in spermatogonia. Sections from the absence of spermatogonia in
adult mouse testis were co-stained with rat monoclonal anti- some tubules and in other tubules
SPOC1 (red) and mouse monoclonal anti-PLZF (green). DNA was
only sertoli cells. Sertoli cells are
stained with DRAQ5.
the only cells not derived from
spermatogonia. Taken together, these data strongly suggest that the SPOC1
protein is essential and a novel marker for male germ cell generation and/or
survival. These findings are consistent with additional evidences we have indicating
that SPOC1 is important for cell proliferation.
*Supported by Deutsche Krebshilfe and the Müggenburg Stiftung
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Activated receptor tyrosine kinases and their downstream
pathways in acute myelogenous leukemia: Important targets
for therapeutic intervention
S. Horn (1,2), J. Bäsecke (2), K. Schulz (1), M. Jücker (3), U. Bergholz (1),
L. Trumper (2), C. Stocking (1)
(2) Department of Hematology and Oncology, University of Göttingen
(3) University Medical Center Hamburg-Eppendorf, Hamburg
It is now well established that leukemia, like other cancers, is the consequence of multiple
genetic mutations. A popular notion is the two-hit model, in which gene mutations
blocking differentiation [e.g. dysregulation of transcription factors (TF)] together with
activation of a proliferation and/or anti-apoptoic signal [e.g. activated receptor tyrosine
kinase (RTK)] is sufficient to induce leuekmia – and thus may represent the key events
in leukemogenesis. As the latter are attractive targets for therapy, much emphasis has
been placed on defining RTK mutations and their signalling pathways in leukemia. Two
members of the class III RTK are frequent targets of mutations in acute myelogenous
leukemia (AML). FLT3 mutations, involving either the juxtamembrane region or the
tyrosine kinase domain II, are found in circa 30 % of AML samples and are not limited
to a specific AML subtype. In contrast, mutations in the extracellular domain of the KIT
Activation of RTK, either by ligand or by mutation, lead to the activation of several well-characterized signaling
pathways, which leads to proliferation, differentiation, and/or survival. Depicted are the major players in the
PI3K-AKT pathway (orange), the JAK-STAT pathway (green), or the RAS-MAPK pathway (blue). Constitutive
activation of the PI3K-AKT pathway is observed in approximately 70 % of AML samples.
receptor are almost exclusively found in AML patients carrying translocations or
inversions affecting the RUNX1 transcription or its cofactor CBFβ. Importantly, the
presence of RTK mutations correlates with poor survival and/or high risk of relapse. We
are interested in determining the relevant downstream signaling pathways of these
mutated RTK, as well as determining if the leukemia stem cell is also “addicted” to the
activated RTK for maintenance.
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Mutations in RTK or RAS proteins are frequently observed in leukemia, resulting in a high frequency of constitutive
activation of the PIK3K-AKT pathway. To determine if this activated pathway is sufficient to induce leukemia
in mice, constitutive activated forms of the catalytic domain or localization mutants (as depicted) were tested
for the tumorigenic potential by introduction via retroviral vectors into hematopoietic cells and injection into
mice. As controls, empty GFP vectors, or vectors expressing constitutive activated forms of cKIT RTK are shown.
Strikingly, although activated PI3K subunits are able to induce tumorigenic growth, these tumors are not as
aggressive as those receiving activated KIT, which signals through multiple pathways.
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Constitutive activation of the PI3K-AKT pathway is observed in up to 70 % of
acute myelogenous leukemia, primarily due to activating mutations in either
receptor tyrosine kinase or RAS proteins that signal through this pathway. To
investigate the relevance of an activated PI3K-AKT pathway in hematopoietic
malignancies, we analyzed the effect of point mutations in the catalytic subunit
of class IA PI3K (p110α), which are associated with several human neoplasms.
We demonstrate that mutations in the helical (E542K, E545A) and kinase domain
(H1047R) of p110α constitutively activate the PI3K-AKT pathway and lead to
factor-independent growth of early hematopoietic cells. Proliferation and
survival of the cells were inhibited in a time- and dose-dependent manner using
inhibitors of either PI3K or AKT. The mammalian target of rapamycin (mTOR)
was demonstrated to be important for mitogenic, but not anti-apoptotic
signaling of mutant p110α. In a syngenic mouse model, hematopoietic cells
expressing p110α point mutations induced a leukemia-like disease characterized
by anemia, neoplastic infiltration of hematopoietic organs, and 90 % mortality
within 5 weeks. Our data show that an activated PI3K-AKT pathway confers
factor-independency to hematopoietic cells in vitro and induces oncogenic
activity in vivo, providing a useful model to evaluate pharmacological inhibitors.
The development and testing of tyrosine kinase inhibitors (TKI) specific for activated
RTKs has been pushed forward during the last few years. Unfortunately, clinical
trials of different TKIs have been disappointing – due to either a minimal initial
response or the outgrowth of drug-resistant tumor cells. We have developed two
mouse models to characterize the molecular mechanisms behind this problem. In
the first mouse model, the importance of the two separate components leading to
leukemia (mutated TF and activated RTK) can be independently tested. In this
model, expression of the dysregulated TF (e.g. the fusion protein RUNX1/ETO)
can be “turned off ” by an inducible retroviral vector system, which incorporates
components of the loxP-CRE recombinase system and either interferon- or tetracycline-responsive promoters. The activated RTK can be independently inhibited
by administration of TKI. With this system, we are currently testing if the
inactivation of one pathway is sufficient to reverse the leukemia. These models
should provide invaluable insight into using TKI in the clinic.
*Supported by Deutsche Krebshilfe und AstraZeneca
Identification of genetic mutations that cooperate with constitutively
active FLT3 in the induction of acute lymphoblastic leukemia (ALL)
A.N. Lehmann, B. Niebuhr, K. Schulz, M. Ziegler, J. Meyer, S. Horn,
and C. Stocking
Acute lymphoblastic leukemia (ALL) is the most common form of childhood
malignancy and is a heterogeneous disease with subtypes that differ markedly in
their cellular and molecular characteristics, as well as their response to therapy and
subsequent risk of relapse. The FMS-like receptor tyrosine kinase FLT3 contributes
to normal differentiation, proliferation and apoptosis of primitive hematopoietic
cells in both early myeloid and lymphoid lineages. Mutations constitutively activating
the FLT3 receptor are commonly found in acute leukemias of both lineages, making
FLT3 one of the most frequently mutated genes in hematological malignancies. The
availability of FLT3 inhibitors provides an attractive therapeutic strategy for patients
harboring these mutations. In ALL, FLT3 mutations are associated with either MLL
or hyperdiploid subtypes, raising the question if specific genetic events synergize
with FLT3 mutations in ALL induction and if inhibiting FLT3 activation alone will
be effective in leukemia treatment. To better understand the role of FLT3 activating
mutations in ALL, we have established an allograft mouse model, in which bone
marrow (BM) progenitors were transduced with a retroviral vector expressing FLT3
with an internal tandem duplication (FLT3-ITD), followed by transplantation into
syngenic recipients. Mice receiving murine FLT3-ITD bone marrow succumbed to
an aggressive leukemia with a mean latency of 45 days. Based on surface marker
analysis and immunoglubulin (Ig) gene rearrangements, these tumors could be
classified as pre-B-cell tumors. The disease was transplantable down to a cell number
<200 and clonal (based on retroviral integration sites). Strikingly, the FLT3 inhibitor
PKC412, as well inhibitors of the PI3K and MAPK pathways or dominant-negative
forms of STAT5, profoundly inhibited proliferation of leukemic cells in vitro but did
not induce apoptosis. However, anti-apoptotic stimulus provided in methylcellulose
cultures was necessary to maintain leukemic cells in culture, which coincided with
upregulation of surface expression of the Ig heavy chain of the preB-cell receptor
(preBCR). These results suggest that the preBCR is active in these tumors but
progression to the B-cell stage is blocked, either through FLT3 signaling and/or
through independent lesions. Strikingly, each leukemia contained up to 6 to 8
independent retroviral integrations sites, a much higher number than would be
expected given the circa 15-20 % infection frequencies of BM cells before
transplantation. The high number of integrations could reflect two events: 1) preferential infection of a subset of cells more permissive to infection; and/or 2) the
preferential outgrowth of these infected cells due to the increased likelihood that
cooperating genes were activated through the integration site. To determine if
integration of the retroviral vector near putative oncogenes or tumor suppressors
contributed to the induction of leukemia, retroviral integration sites were analyzed.
Consistent with this hypothesis, integrations were found near genes that have been
identified as “common integration sites” (CIS) or loci that are targeted in independent
tumors – an event highly unlikely to occur merely by chance. Based on the function
of gene products deregulated by the retroviral integration, we predict that disruption
of the preBCR signaling pathway is necessary for FLT3-induced preB-cell leukemia.
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Differential regulation of human TERT gene expression and telomerase
activity during liver regeneration and liver cell differentiation
H. Sirma (1), M. Kumar (1), B. Wittek (1), J. Weise (1), P. Gripon (2),
S. Ande (3), K.-L. Rudolph (3), C. Günes (1).
(2) INSERM U522, Rennes, France
(3) MPI-Group, University of Ulm, Germany
Telomerase activity is non-detectable in human liver biopsies, whereas telomerase
activity is upregulated during hepatocarcinogenesis, indicating that telomerase
activity is repressed during differentiation of human liver and reactivation must
occur during carcinogenesis. Here we analyzed the regulation of telomerase activity
during liver cell proliferation and differentiation in both in vitro and in vivo models.
As an in vivo model we investigated the regulation of human TERT gene promoter
activity during regeneration induced by partial hepatectomy in hTERTp-lacZ transgenic mice which contain an 8.0 kbp fragment of the human TERT gene promoter.
As an in vitro model we used HepaRG cell line which recently was established as a
new system for human liver cell differentiation. We found a sharp increase of human
TERT promoter activity at day 3 after partial hepatectomy whereas murine TERT
mRNA expression was only weakly induced at the same time. A weak but significant
increase in telomerase activity was observed at day 2 after PH which did not correlate with TERT mRNA levels during regeneration. On the other hand, telomerase
activity and TERT gene expression is detectable in proliferating HepaRG cells and is
markedly down-regulated as cells reach confluency and cease proliferation. Luciferase
reporter gene analyses indicated that the 0.3 kbp TERT promoter fragment posseses
all necessary cis regulatory elements for TERT gene repression in HepaRG cells. We
established a suitable in vivo and in vitro model to dissect the molecular mechanisms underlying the regulation of telomerase activity in liver cells.
Regulation of human and mouse TERT promoter activity during
progression of mammary carcinogenesis in WAP-T x hTERTp-lacZ
bitransgenic mice
B. Wittek, M. Kumar, J. Schröder, J.M. Weise, W. Deppert, C. Günes
Telomerase activity is repressed in most human somatic tissues during differentiation processes, but strongly up-regulated in the vast majority of human tumors.
Therefore, one major focus of our research is the analysis of TERT gene expression
during tumorigenesis. We have generated transgenic mice with an 8.0 kbp human
TERT promoter fragment driving expression of the bacterial lacZ gene (hTERTplacZ transgenic mice). We could show that this transgenic mouse model provides a
suitable in vivo system to analyze the expression of the human TERT gene under
physiological conditions and during tumorigenesis. One model system for tumor
induction and progression is the SV40 Early Region (SV40-ER) antigen induced
tumor formation in the mammary tissue. A transgenic mouse model where the
(SV40-ER) gene is under the control of the whey acidic promoter (WAP-T mice)
exists in our laboratory. Here, ductal carcinomas in situ (DCIS) develop after lactation in this transgenic mouse which results in invasive tumors in a defined time
period. The experimentally induced DCIS is similar to the human DCIS which further develops to mammary carcinomas. Human mammary tissues, as is the case for
most of the human somatic cells, are telomerase negative and reactivation of telomerase is thought to be one of the key events during carcinogenesis, also for breast
carcinomas. By back-crossing the TERT-promoter transgenic mouse with the SV40LT transgenic mice we analyze the time point of telomerase reactivation. Our current results indicate that activation of telomerase is only detectable in invasive carcinoma but not at early time-points during tumor development. We also find that
C/EBPα is a crucial repressor of TERT gene expression in normal breast and that its
loss during tumorigenesis is one of the genetic events responsible for telomerase
reactivation in breast tissue.
*Supported by Deutsche Krebshilfe and Erich und Gertrud Roggenbuck-Stiftung
Physiological role of cellular prion protein in insulin producing β-cells
D. J. Picketts (1), R. Reimer (2), F.W. Scott (1), A. Strom (1),
A.W. Stuke (3), G.S. Wang (1)
(1) Chronic Disease Program, Ottawa Health Research Institute
(3) German Primate Centre, Göttingen
Cellular prion protein (PrPC) is ubiquitously expressed within the nervous system
and is also present in several other organs and tissues. In the pancreas, PrPC is specifically and abundantly expressed in the glucagon, insulin, pancreatic polypeptide,
and somatostatin secreting endocrine cells which are localized in the islets of
Langerhans. To date, PrPC is suggested to be involved in a number of cellular functions. However, the function of PrPC in the islets and specifically in insulin producing ß-cells is not known. We recently reported strong PrPC expression in all hormone
secreting cells in the islets. Of particular note, cytosolic PrPC aggregates were
observed exclusively in insulin producing ß-cells in rats. The aim of the present
study was to characterize PrPC function in insulin producing ß-cells. In order to
detect PrPC interacting proteins we performed a protein overlay assay (far-Western
immunoblot) using protein extracts from INS-1E rat insulinoma cells as bait and
recombinant bovine PrP (rbPrP) as a probe. We detected several putative PrPC
binding proteins. To date, we identified two candidate PrPC interacting molecules
which were of nuclear origin. We confirmed the in vitro interaction of the rbPrP
with the candidate molecules using purified recombinant proteins. Under specific
immunohistochemical conditions we also detected in vivo the localization of PrPC
to the nuclear lamina in endocrine and neuronal cells. Furthermore, intraperitoneal
glucose tolerance tests showed that PrP0/0 mice have impaired glucoregulation.
These results suggest the involvement of PrPC in epigenetic control of ß-cell function
associated with glucoregulation.
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Dissecting nucleocytoplasmic transport of CD83 mRNA
J. Chemnitz (1), D. Pieper (1), B. Fries (1), C. Grüttner (1),
A. Steinkasserer (2), R.H. Kehlenbach (3), J. Hauber (1)
(2) University Hospital Erlangen – Department of Dermatology,
Erlangen, Germany
(3) University of Göttingen – Centre of Biochemistry and Molecular Cell
Biology, Göttingen, Germany
Mature dendritic cells (DC) are capable of sensitizing even naïve CD4+ and
CD8+ T cells, and are therefore frequently termed “nature’s adjuvant”. Immature
DC reside in the peripheral tissues and upon uptake of antigen and exposure to
inflammatory stimuli, they migrate to the peripheral lymph nodes, where now
fully matured, they induce antigen-specific T cell responses. The DC maturation
process includes the modulation of chemokine and chemokine receptors, as
well as the up-regulation of several cytokines, costimulatory and adhesion
molecules.
Human CD83 serves as a major marker molecule for mature DC that belongs to
the immunoglobulin super family. What is less known is that CD83 is also
expressed, although to a lesser extent, by activated T and B cells and appears to be
important for efficient T-cell activation.
The vast majority of cellular mRNAs are transported from the site of transcription,
the cell nucleus, to the cytoplasmic site of protein synthesis via the NXF1/Tap
pathway. In contrast, recently we
demonstrated that the nucleocytoplasmic transport of a small subset
of cellular messages, including the
CD83 mRNA, is mediated in
activated T cells by the unrelated
CRM1 pathway. The subsequent
analysis of the CD83 transcript
revealed that this mRNA contains
in its coding region a highly
structured
cis-active
RNA
sequence, termed the posttranscriptional regulatory element
(PRE), and that this RNA element
is directly recognized by the cellular RNA-binding shuttle protein
of CD83 mRNA export. In the nucleus, the shuttle
HuR, a member of the ELAV family Regulation
protein HuR binds to the PRE sequence in CD83 mRNA. Subsequently,
of AU-rich element RNA-binding HuR recruits its protein ligand APRIL (ANP32B) that connects the
proteins. Interestingly, PRE:HuR CD83 mRNA:HuR ribonucleoprotein complex and the nuclear
receptor CRM1. Nuclear export via the CRM1 pathway
interaction does not affect the export
occurs upon phoshorylation of APRIL on residue Thr244 by casein
stability of this transcript. Instead, kinase 2 (CK2).
this interaction induces the CRM1-dependent cytoplasmic accumulation of
CD83 mRNA. We also provided evidence demonstrating that the interaction of
HuR and CRM1 is indirect and is accomplished by the HuR protein ligand APRIL
(ANP32B).
APRIL belongs to the growing family of ANP32 proteins (ANP32a-h), a group of
leucine-rich phosphoproteins, which have been linked to various cellular activities
such as, for example, gene expression, signaling, adhesion and apoptosis. Our
further analyses identified Thr244 to be an as yet unrecognized phosphate acceptor
site in APRIL and demonstrated that phosphorylation of this site affects APRIL’s
subcellular distribution. Subsequently, we identified casein kinase 2 (CK2) to be
the cellular protein kinase that phosphorylates Thr244 in APRIL. We were able to
show that inhibition of CK2 by a small-molecular weight inhibitor specifically
abrogates CD83 de novo expression. Interestingly, this effect was caused by
pronounced inhibition of the nucleocytoplasmic transport of CD83 mRNA.
Thus, the pharmaceutical interference with CK2 activity may provide a novel
strategy to inhibit CD83 expression, thereby blocking DC-mediated T cell activation.
*Supported in part by Wilhelm Sander-Stiftung and in part by Deutsche
Forschungsgemeinschaft (DFG)
Qualitative and quantitative image analysis of ultra structural changes
in sarcomere assembly of cMyBP-C knock-in mice
L. Carrier (1,2,3), T. Eschenhagen (1), B. Fraysse (2,3), J. Guiard (4),
H. Hohenberg (5), B. Holstermann (5), D. Juhr (1), E. Krämer (1),
G. Mearini (1), N. Mougenot (3), H. Pointu (4), R. Reimer (5),
S. Schlossarek (1), K. Schwartz (2,3), M. Vernet (4), N. Vignier (2,3)
(1) Institute of Experimental and Clinical Pharmacology and Toxicology,
(2) Institut de Myologie, Paris, France
(3) University Pierre et Marie Curie, Paris, France
(4) CEA-Grenoble, Grenoble, France
Mutations in the MYBPC3 gene encoding cardiac myosin-binding protein C
(cMyBPC) are frequent causes of familial hypertrophic cardiomyopathy (FHC),
but the mechanisms leading from mutations to disease remain elusive. The recently
developed first targeted cMyBP-C knock-in mouse carries a human MYBPC3 point
mutation. It is a G>A transition on the last nucleotide of exon 6, which is expected
to result either in exon skipping and premature stop or production of a full-length,
missense protein or both. The homozygotes exhibited myocyte and left ventricular
hypertrophy with reduced fractional shortening and interstitial fibrosis, the
heterozygotes developed only mild myocyte hypertrophy. In the homozygotes, the
amount of missense cMyBP-C was 90 % lower and the expected truncated protein
was undetectable, even after inhibiting the proteasome with MG132. Both missense
and nonsense mRNAs were present, but the level of total cMyBP-C mRNA was
markedly lower. Inhibition of nonsense-mediated mRNA decay in cultured cardiac
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myocytes or in vivo with cycloheximide or emetine increased the
level of nonsense mRNA severalfold. Electron microscopy analysis
has shown a correct sarcomere
ultrastructure but slightly different
distances between the sarcomere
bands in the homozygotes. In conclusion, these data indicate that the
expression of the point mutation in
the whole animal is regulated
mainly at the mRNA level, involving nonsense-mediated mRNA
decay. These data contribute to an
improved understanding of the
molecular mechanisms of FHC.
Ultra-structure of sarcomere assembly of cMyBP-C knock-in mice
in TEM.
Translational Elongation of Cyclin D1 requires an RNA Chaperone
J. Dittmann (1,2), G. Sommer (2), K. Reumann (1), J. Kuehnert (2),
O. Mauermann (1), H. Will (1), T. Heise (1, 2)
(2) Medical University of South Carolina (MUSC), Charleston, USA
We report that the human RNA chaperone La stimulates translation elongation of
cyclin D1 by interacting specifically with the La response element (LaRE), which
is located within the cyclin D1 open reading frame. By using a cell-free translation
system, we showed that the presence of the LaRE hinders translation elongation
and induces accumulation of a translation intermediate. Strikingly, if La protein
is added to the translation reaction, the abundance of the intermediate is strongly
decreased in concert with an increase in the full-length translation product. We
propose a model for translational regulation of the cellular cyclin D1 mRNA pool
in which the human La protein binds the LaRE located within the coding region
of cyclin D1 mRNA and acts as an RNA chaperone which supports translation
elongation.
The fusion protein OTT-MAL induces R-loop formation and genomic
instability
G. Sommer (3), R. Fleischer (1), K. Reumann (1), M. Ruthardt (2),
C. Stocking (1), H. Will (1), T. Heise (1, 3)
(2) Johann Wolfgang Goethe-University, University Medical Center II,
Hematology, Frankfurt, Germany
(3) Medical University of South Carolina, Department of Biochemistry &
Molecular Biology, Charleston, USA
The chromosomal translocation t(1;22) is associated with a severe childhood
acute megakaryocate leukaemia (AMKL). Our study demonstrates that the
corresponding fusion protein OTT-MAL interacts with endogenous RNA
Polymerase II, induces transcriptional R-loop formation and phosphorylation of
H2A.X (γH2A.X), a marker for genomic double strand breaks. Interestingly, the
levels of spliced mRNAs are strongly reduced and OTT-MAL colocalizes with
RNA polymerase II and γH2A.X in distinct nuclear foci. Our data suggest a novel
oncogenic mechanism, in which a fusion protein disturbs cotranscriptional
mRNA maturation and thereby induces R-loop formation, genomic instability,
and eventually malignant cell transformation.
*Supported by Deutsche José Carreras Leukämie-Stiftung
Analysis of leukemia (AML-M7) associated OTT-MAL fusion protein.
137
138
Adenovirus type 5 early region 1 B 55-kDa oncoprotein is
modified by different isoforms of the small ubiquitin-related
modifier (SUMO).
P. Wimmer, S. Schreiner, P. Groitl, T. Dobner
We have previously shown that adenovirus type 5 (Ad5) E1B-55K is a substrate of
the SUMO conjugation system in intact cells as well as in vitro. Posttranslational
modification of E1B-55K by SUMO1 at lysine 104 (K104R) regulates the subcellular
distribution of the viral protein in plasmid transfected and productively infected
cells. Furthermore, this modification is required to completely transform primary
rat cells in cooperation with Ad5 E1A and to repress p53 tumor suppressor functions.
Here, we report that E1B-55K can be modified by SUMO isoforms 2 and 3
(SUMO2, SUMO3) at K104. As predicted, the molecular characteristics of SUMO2
and SUMO3 give rise to additive SUMOylation of E1B-55K generating multiple
modified forms of the viral protein. We also demonstrate that E1B-55K contains
three amino acid patches, which closely resemble the consensus of SUMOinteraction motifs (SIMs). This motif, V/I-X-V/I-V/I mediates non-covalent
interactions with SUMO or SUMOylated proteins, and exists in nearly all proteins
known to be involved in SUMO-dependent processes, such as most notably PML.
Using site-directed mutagenesis we show that these motifs regulate the cellular
distribution of the Ad protein and are required for efficient repression of
p53-mediated transactivation in transient reporter gene assays. Future studies are
directed towards elucidating the role of the SIMs in regulating protein interactions
of 55K with viral and cellular factor as well as investigating their importance for
E1B-55K functions in productive infection and cell transformation.
*Supported by Studienstiftung des Deutschen Volkes
The adenovirus E1B-55K oncoprotein induces SUMO modification of p53
S. Müller (1), T. Dobner (2)
(1) Max Planck Institute of Biochemistry, Martinsried, Germany
The ability of adenovirus to induce cell transformation depends on the E1A and
E1B-55K oncoproteins. While E1A functionally inactivates the retinoblastoma
tumour suppressor, E1B-55K primarily interferes with the function of p53. In
adenovirus transformed cells E1B-55K can directly affect p53-dependent
transactivation. In virus-infected cells E1B-55K additionally cooperates with the
viral E4orf6 protein to induce ubiquitin-dependent degradation of p53. Here we
unravel a novel activity of E1B-55K by demonstrating that it drastically stimulates
the post-translational modification of p53 by the ubiquitin-like SUMO modifier.
Consistent with this finding the extent of p53 SUMOylation is highly elevated in
adenovirus transformed cell lines. E1B-55Kmediated SUMOylation depends on
the direct interaction of E1B-55K with p53 and additionally requires SUMO
modification of E1B-55K. These data suggest that E1B-55K exploits both ubiqutin
and ubiquitin-like systems to target host cell proteins and thus shed new light on
the molecular mechanisms of E1B-55K function. Moreover, the data expand the
emerging concept of dual-specificity factors that act in both the SUMO and
ubiquitin pathway and identify E1B-55K as the first viral protein that shares this
dual activity.
Characterization of posttranslational modifications of SPOC1
T. Schaub, S. Kinkley, H. Staege, K. Reumann, H. Will
SPOC1 is a 300 amino-acid protein containing a single predicted PHD (plant
homeodomain) found in chromatin-associated proteins, a bipartite NLS (nuclear
localization sequence) and two PEST domains (potential protein degradation
motifs). Furthermore, the SPOC1 protein is predicted to be highly posttranslationally
modified, suggesting that it is a very tightly regulated protein. SPOC1 contains
several putative Serine and threonine phosphorylation motifs, potentially
phosphorylated by Glykogen-synthase-3 β (GSK3-β), Cyclin-dependent kinases,
Polo-like kinase, Casein kinase-1. In addition, there are predicted binding sites for
Proline-directed kinases and Cyclins. In addition, SPOC1 contains several
consensus motifs for sumoylation. To date, we have been able to show that SPOC1
is a cell-cycle regulated chromatin-associated protein and that its stability is regulated
at least in part via its PEST domains and by GSK3β. In these ongoing studied we
investigate the effects of the different posttranslational modifications of the
SPOC1 protein on the regulation of SPOC1 functions. To this end, we have generated
SPOC1 phosphorylation and sumoylation site mutants as well as phospho-specific
antibodies. Elucidation of the posttranslational modification of SPOC1 will be
important to understand how its subcellular localisation, interaction with cellular
SPOC1 domains and predicted posttranslational modification sites.
139
proteins and various functions are regulated. In light of the accumulating data
arguing that misregulated SPOC1 levels has a detrimental effect on genomic integrity,
clarification of these posttranslational modifications should prove to be very
informative.
140
The ubiquitin-specific protease USP7 is a target of the adenovirus
E1B-55K oncoprotein
E. Koyuncu, B. Härtl, T. Dobner
The 55K product from human adenovirus type 5 early region 1B (E1B-55K)
belongs to a group of adenoviral regulatory proteins required for maximal virus
production. In addition, E1B-55K provides functions necessary for oncogenic
transformation of primary mammalian cells in culture. It is generally considered
that these multifunctional properties involve a variety of physical interactions with
key regulators of viral and cell growth such as E4orf6, p53, components of the DNA
damage response and PML nuclear bodies as well as other still unknown factors.
The USP7 protein is relocalized to the viral transcription and replication centers during infection. A549 cells
were infected with wt Ad5 at a multiplicity of 20 FFU per cell. Cells were fixed at different times after infection
(h p.i.) and labeled in situ with antibodies specific for cellular PML, Ad5 E4orf3, Ad5 E1B-55K, Ad5 DBP and
cellular USP7 (bottom panel), which progressively colocalizes with the DBP-positive sites of viral transcription
and DNA replication during the course of the infection. Magnification x 7600.
In a yeast two-hybrid screen, using E1B-55K as a bait, we identified the cellular
protein USP7, also know as HAUSP (Herpesvirus-associated ubiquitin-specific
protease), as a novel interaction partner of the 55K product. USP7 belongs to
the large family of ubiquitin-specific proteases (USPs) and is a critical component of the p53-Mdm2 stress response pathway by acting as a deubiquitinase of
both p53 and Mdm2. The interaction between E1B-55K and USP7 was verified
by coimmunoprecipitation experiments. In vitro binding assays demonstrate
that the regions involved in complex formation map to two highly conserved
amino acid residues in E1B-55K and the TRAF-like domain of USP7,
previously shown to mediate the binding of p53, Mdm2 and the EBNA1 protein
from EBV. Furthermore, USP7 activity and binding to 55K is required for
efficient transformation of primary rat cells and to maintain high levels of the
viral protein indicating that USP7-mediated deubiquitination regulates
E1B-55K protein stability and transforming functions. Taken together, our data
identify the deubiquitinating enzyme USP7 as a novel cellular binding partner
of E1B-55K and suggest an important role of USP7 for 55K protein function in
virus replication and cell transformation. This together with the demonstration
that EBV EBNA1 and HSV-1 ICP0 also interact with the ubiquitin-specific
protease hint at the possibility that USP7 is a common target of key viral
regulatory proteins.
*Supported by Wilhelm Sander-Stiftung
The MDM2 ubiquitination signal in the DNA-binding domain of
p53 forms a docking site for calcium calmodulin kinase superfamily
members
J. Chrystal (1), A.L. Craig (1), I. Dornreiter (2), J. Fraser (1), B. Harrison (1),
T.R. Hupp (1), Y. Lin (1), M. Scott (1), N. Sphyris (1)
(1) University of Edinburgh, Edinburgh, UK
Genetic and biochemical studies have shown that Ser(20) phosphorylation in
the transactivation domain of p53 mediates p300 catalyzed DNA-dependent
p53 acetylation and B-cell tumour suppression. However, the protein kinases
that mediate this modification are not well-defined. A cell-free Ser(20)
phosphorylation site assay was used to identify a broad range of Calciumcalmodulin kinase superfamily members including CHK2, CHK1, DAPK-1,
DAPK-3, DRAK-1, and AMPK as Ser(20) kinases. Phosphorylation of a p53
transactivation domain fragment at Ser(20) by these enzymes in vitro can be
mediated in trans by a docking site peptide derived from the BOX-V domain
of p53, which also harbours the ubiquitin-signal for MDM2. Evaluation of
these Calcium-calmodulin kinase superfamily members as candidate Ser(20)
kinases in vivo has shown that only CHK1 or DAPK-1 can stimulate p53
transactivation and induce Ser(20) phosphorylation of p53. Using CHK1 as a
prototypical in vivo Ser(20) kinase, we demonstrate that (i) CHK1 protein
depletion using siRNA can attenuate p53 phosphorylation at Ser(20), (ii) an
EGFP-BOX-V fusion peptide can attenuate Ser(20) phosphorylation of p53 in
141
vivo, (iii) the EGFP-BOX-V fusion peptide can selectively bind to CHK1 in
vivo, and (iv) the Deltap53 spliced variant lacking the BOX-V motif is refractory
to Ser(20) phosphorylation by CHK1. These data indicate that the BOX-V
motif of p53 has evolved the capacity to bind to enzymes that mediate either
p53 phosphorylation or ubiquitination thus controlling the specific activity of
p53 as a transcription factor.
142
Homeodomain-interacting protein kinase 2 is the ionizing
radiation-activated p53 serine 46 kinase and is regulated by ATM
I. Dauth (1), J. Krüger (1,2), T.G. Hofmann (1,2)
(1) German Cancer Research Center, Heidelberg, Germany
Phosphorylation of p53 at Ser(46) is important to activate the apoptotic
program. The protein kinase that phosphorylates p53 Ser(46) in response to
DNA double-strand breaks is currently unknown. The identification of this
kinase is of particular interest because it may contribute to the outcome of
cancer therapy. Here, we report that ionizing radiation (IR) provokes homeodomaininteracting protein kinase 2 (HIPK2) accumulation, activation, and complex
formation with p53. IR-induced HIPK2 up-regulation strictly correlates with
p53 Ser(46) phosphorylation. Down-regulation of HIPK2 by RNA interference
specifically inhibits IR-induced phosphorylation of p53 at Ser(46). Moreover,
we show that HIPK2 activation after IR is regulated by the DNA damage
checkpoint kinase ataxia telangiectasia mutated (ATM). Cells from ataxia
telangiectasia patients show defects in HIPK2 accumulation. Concordantly,
IR-induced HIPK2 accumulation is blocked by pharmacologic inhibition of
ATM. Furthermore, ATM down-regulation by RNA interference inhibited
IR-induced HIPK2 accumulation, whereas checkpoint kinase 2 deficiency
showed no effect. Taken together, our findings indicate that HIPK2 is the
IR-activated p53 Ser(46) kinase and is regulated by ATM.
*Supported by Deutsche Krebshilfe and Deutsche Forschungsgemeinschaft
Leibniz Center for Infection (LCI)
Ultrastructural investigation of the in vitro life cycle of Schistosoma
mansoni, analysed by cryo-techniques and TEM-tomography,
inclusive the localisation of specific regulative proteins in situ
H. Haas (1), H. Hohenberg (1), B. Holstermann (1), R. Reimer (1),
C. Schneider (1), G. Schramm (2), M. Warmer (1)
(2) Research Center Borstel, Borstel, Germany
S. mansoni affects 200 million people worldwide, often with serious consequences.
This cooperation project between HPI and FZB will provide a transparent model
for studying host/parasite interaction and parasite development (a.o. drug testing)
and for obtaining parasite immunomodulatory key molecules. Moreover, it
represents an alternative to animal experiments. The various life cycle stages of
S. mansoni differ profoundly in their effects on the host. Since access to individual
stages for immunobiological studies is hampered during in vivo infection, in vitro
culture of the parasite is an
easy-access alternative. Moreover,
only our ultrafast cryo-fixation
and preparation methods can
arrest the different states of
infectious eggs. Preserving the
life-like state of the parasite we are
able to study the time course of
expression of individual immunomodulatory egg molecules by 3D
ultrastructural
investigations
combined with the pinpoint
localisation of these molecules by
immunocytological studies.
Ultrastructure of an Schistosoma mansoni egg after high pressure
freezing in situ and cryoprocessing in the transmission electron
microscope.
143
144
Electron microscopical dissection of the exoerythrocytic forms of
Plasmodium berghei in situ
V.T. Heussler (2), H. Hohenberg (1), B. Holstermann (1), R. Reimer (1),
A. Rennenberg (2) C. Schneider (1)
(2) Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany
The initiation of clinical malaria evolves after the schizogonic division of the
exoerythrocytic forms (EEFs) of the parasite and the formation of merozoites
inside host hepatocytes. However, the mechanism by which hepatic merozoites
reach blood vessels (sinusoids) in the liver and escape the host immune system
before invading erythrocytes still remains unknown. Recently it was shown that
parasites induce the death and the detachment of their host hepatocytes,
followed by the budding of parasite-filled vesicles (merosomes) into the sinusoid
lumen. Parasites simultaneously inhibit the exposure of phosphatidylserine on
the outer leaflet of host plasma membranes, which act as “eat me” signals to
phagocytes. Thus, the hepatocyte-derived merosomes appear to ensure both the
migration of parasites into the bloodstream and their protection from the host
immune system. With the help of specially adapted correlative light / electron
microscopy methods and novel pre-selection and micro-preparation techniques
we were able to localize and to make a pin-point dissection of EEF´s in situ in
the liver.
A: Hepatic merozoit of Plamodium berghei in liver tissue (arrow). B: Schematic drawing of the different budding
steps of parasite-filled vesicles (merosomes) into the sinusoid lumen (from Sturm et al., 2006)
Personelle Zusammensetzung der
Wissenschaftlichen Abteilungen,
Forschungs- und Nachwuchsgruppen
Personelle Zusammensetzung
Abteilung Molekulare Virologie
146
Abteilungsleiter
Thomas Dobner, Prof. Dr. rer. nat.
Wissenschaftler
Barbara Härtl, Dr. rer. nat.
Kathrin Kindsmüller, Dr. rer. nat.*
Timo Sieber, Dr . rer. nat.
Doktoranden
Willhelm Ching*, Dipl. Chem.
Orkide Dirlik-Koyuncu, M.Sc.
Natascha Kömm*, Dipl. Biol.
Emre Koyuncu, M.Sc.
Agnes Lesniewski*, Dipl. Biol.
Daniela Müller*, Dipl. Biol.
Marie-Sophie Sander*, Cand. med.
Timo Sieber*, Dipl. Biol.
Melanie Schmid*, Dipl. Biol.
Sabrina Schreiner, Dipl. Biol.
Thomas Speiseder, Dipl. Biol.
Peter Wimmer*, Dipl. Biol.
Diplomanden
Wilhelm Ching*
Franziska Kopp
Florian Leinenkugel
Andreas Malitzky
Christian Peters*
Melanie Schmid*
Peter Wimmer*
Bachelor Studenten
Julia Godau
Maria Sekutovicz
Timo Fischer
Praktikanten
Technische Assistenten
Gäste
Sekretariat
Assoziierte Klinische
Kooperationsgruppen
Karolin Berg*
Wilhelm Ching*
Anabelle von Davier*
Nicholas Dobner*
Christine Henning*
Florian Henkel*
Hannes Homfeld*
Linda Hüske*
Fabian Imse*
Franziska Kopp*
Denise Lehnhardt*
Agnes Lesniewski*
Kristina Metz
Sandra Postels*
Folusho Osuntuyi*
Kristina Ritter*
David Rohde*
Mareike Ruhnau
Peggy Synwoldt*
Anne Winkler*
Gabriele Dobner*
Peter Groitl, Dipl. Biol.
Anna Bresovitsky*
Peter Forster*
Martin Kruse
Timo Sieber*
Susan Watson
Peter Wimmer*
Tina Wunder*
Harald Wodrich
Edda Renz
Franz Rinninger, Prof. Dr. med.
May Brundert, PD Dr. med.
Udo Schumacher, Prof. Dr. med.
Olaf Pongs, Prof. Dr. med.
(*zeitweise beschäftigt)
147
Abteilung Allgemeine Virologie
148
Abteilungsleiter
Hans Will, Prof. Dr. rer. nat.
Wissenschaftler
Anneke Funk*, Dr. rer. nat.
Gabor Rohaly, Dr. rer. nat.
Gunhild Sommer*, Dr. rer. nat.
Hannah Staege, Dr. rer. nat.
Hüseyin Sirma, PD Dr. med.
Tilman Heise*, Dr. rer. nat.
Irena Dornreiter, Dr. rer. nat.
Mouna Mhamdi*, Dr. rer. nat.
Sarah Kinkley*, Dr. rer. nat.
Doktoranden
Alejandro Mena-Nunez*, Dipl. Biol.
Andreas Mund*, Dipl. Biol.
Cynthia Olotu*, cand. med.
Sarah Kinkley*, Dipl. biol.
Malte Kriegs*, Dipl. Biol.
Mouna Mhamdi*, Dipl. Biol.
Stefan Ficke*, cand med.
Diplomanden
Marc Bohlmann*
Katrin Gießelmann*
Christine Kranig*
Theres Schaub*
Diplomanden
Bachelor Studenten
Praktikanten
Gäste
Sekretariat
Katharina Korf*
Verena Deing*
Tobias Schubert*
Heithem Boukari*
Lutz Homann*
Agnes Hunger*
Daniela Ploen*
Heithem Boukari*
Katharina Korf*
Maren Ahlers*
Nancy Lachner*
Sandra Münch*
Tobias Schubert*
Theres Schaub*
Emily-Marie Latuske*
Margarethe Winnicki*
Katrin Wagner Prigge*
Heike Hildebrandt*
Kerstin Reumann
Nicole Lohrengel*
Oliver Mauermann*
Susann Schönefeldt*
Silke Dehde*
Urte Matschl*
Prof. U. Schumacher*
Ingrid Michalski*
Jacqueline Bukatz*
Annette Preuß*
Deborah Maus*
Lutz Homann*
Katja Kleinsteuber*
Melanie Piedavent*
Sabrina Köcher*
Tina Bohstedt*
Verena Deing *
Anneli Sagar*
Nora Pallmann*
Leah Eissing*
149
Abteilung Tumorvirologie
150
Abteilungsleiter
Wolfgang Deppert, Prof. Dr. rer. nat.
Wissenschaftler
Wolfgang Bohn, PD Dr. rer. nat.
Michael Bruns, PD Dr. med. vet.
Cagatay Günes, PD Dr. rer. nat.
Christina Heinlein, Dr. rer. nat.*
Heike Helmbold, Dr. rer. nat.*
Andrea Hermannstädter, Dr. rer. nat.*
Frauke Krepulat, Dr. rer. nat.*
Mukesh Kumar, Dr. rer. nat.*
Claudia Mänz, Dr. rer. nat.*
Annette März, Dr. rer. nat.*
Lars Tögel, Dr. rer. nat.*
Genrich Tolstonog, Dr. rer. nat.
Julia Weise, Dr. rer. nat.*
Britta Wittek, Dr. rer. nat.*
Doktoranden
Satish Babu, Master Sci.
Heidrun Gevensleben, cand. med.*
Janosch Hildebrand, Dipl. Biol.*
Mike Jahn, Dipl. Biol.*
Andreas Kluth, Master Sci.*
Tanja Knauer, Dipl. Biol.*
Eva Lenfert, Dipl. Biol.*
Timo Quante, Dipl. Biochem.
Martin Rütze, Dipl. Biotech.
Claudia Schlesiger, Dipl. Biol.*
Jara Wanger, cand. med. vet.*
Florian Wegwitz, Dipl. Biochem.
Diplomanden
Praktikanten
Technische
Assistenten
Gäste
Spülküche
Sekretariat
Katharina Gruner*
Norbert Günther*
Natascha Kömm*
Dorothea Pieper*
Jarob Saker*
Anneli Sagar*
Thomas Schulze*
Andrea Diesterbeck
Silvia Dolski*
Marion Kühl
Christine Loscher*
Jasmin Oehlmann*
Gundula Pilnitz-Stolze*
Annette Preuß*
Julia Schröder
Gabriele Warnecke
Doris Weidemann
Claudia Annan*
Marie Brazdova*, Dr. rer. nat.
Umberto Galderisi*, Prof. Dr.
Kamila Iwanski, cand. med.*
Ella Kim, PD Dr. rer. nat.
Katharina Möllmann*
Peter Pably, Dipl. Biochem.*
Klaus Wiegers, Dr. rer. nat.*
M. Cengiz Yakicier, Dr. rer. nat.*
Irmgard Haye*
Monika Müller*
Semsa Reus
Martina Hintz-Malchow
151
Abteilung Zellbiologie und Virologie
152
Abteilungsleiter
Joachim Hauber, Prof. Dr. rer. nat.
Wissenschaftler
Jan Chemnitz, Dr. rer. nat.
Janet Chusainow, Dr. rer. nat.*
Ilona Hauber, Dr. rer. nat.
Helga Hofmann-Sieber, Dr. rer. nat.*
Claus-Henning Nagel, Dr. rer. nat.*
Poornima Priyadarshini C.G., Dr. rer. nat.*
Klaus Wiegers, Dr. med.*
Doktoranden
Christina Ehlers, Dipl. Chem.*
Dirk Hoffmann, Dipl. Biochem.
Marcel Krepstakies, Dipl. Chem.
Dorothea Pieper, Dipl. Biol.*
Bachelor Studenten
Stephan Hofmann*
Praktikanten
Stephan Hofmann*
Susanne Scheu*
Technische
Assistenten
Gäste
Sekretariat
Assoziierte Klinische
Kooperationsgruppe
Bettina Abel
Niklas Beschorner, Dipl. Biotech.*
Andrea Bunk
Cordula Grüttner
Hendrik Plagmann*
Britta Weseloh
Basma Abdel Motaal*
Dr. Gerald Bacher*
Stephanie Frankenberger*
Nele Jensen*
Prof. Dr. Annette Kaiser*
Ute Neumann
Kristina Colberg *
Philip Hartjen, Dr. rer. nat.*
Nele Jensen*
Silke Kummer*
Janine Mohn*
Dorothea Pieper*
Julian Schulze zur Wiesch, Dr. med.*
Ingrid Stahmer, Dr. rer. nat.*
Adriana Thomssen*
Jan van Lunzen, PD Dr. med.
153
Forschungsgruppe
Elektronenmikroskopie und Mikrotechnologie
154
Forschungsgruppenleiter
Wissenschaftler
Diplomanden
Bachelor Studenten
Heinrich Hohenberg, Dr. rer. nat.
Rudolph Reimer, Dr. rer. nat.
Robert Getzieh*
Yonas Shaikh*
Praktikanten
Hendrick Herrmann*
Rasa Raithore*
Technische
Assistenten
Carola Schneider
Barbara Holstermann
Martin Warmer
Hendrick Herrmann*
Forschungsgruppe Molekulare Pathologie
155
Forschungsgruppenleiterin
Carol Stocking, Dr. phil.
Wissenschaftler
Stefan Horn, Dr. rer. nat.
Maike Täger, Dr. rer. nat.
Doktoranden
Folake Akinduro, Dipl. Biol.
Anneke Lehmann, Dipl. Biol.
Birte Niebuhr, Dipl. Agarbiol.
Andrea Schüler, Dipl. Biol.
Katrin Schulz, Dipl. Biochem.
Diplomanden
Kira Behrens*
Praktikanten
Technische
Assistenten
Gäste
Sabine Leisten*
Kira Behrens*
Frederik Tellkamp*
Ulla Bergholz
Ursula Müller, Dipl.-Ing.
Susanne Roscher
Marion Ziegler
Klaus Harbers, Dr. rer. nat.
Jürgen Löhler, Dr. med.
Vladimir Prassolov, Prof. Dr
Nachwuchsgruppe Zelluläre Virusabwehr
156
Nachwuchsgruppenleiter
Doktoranden
Gäste
Adam Grundhoff, Dr. rer. nat.
Thomas Christalla
Nicole Walz
Thomas Günther
Christine Henning
Uwe Tessmer, Dipl. Ing.
Dr. Nicole Fischer (UKE)
Nachwuchsgruppe Virus-Pathogenese
157
Nachwuchsgruppenleiter
Michael Schindler, Dr. biol. hum.
Doktoranden
Carina Banning
Swantje Gundlach
Miriam Widder
Diplomanden
Nina Eickel*
Heike Ströver*
Praktikanten
Technische
Assistenten
Karen Bayer*
Nina Eickel*
Konstantin Hoffer*
Heike Ströver*
Alicja Iwanski, Dipl. Biol.
Veröffentlichungen
2007
Peer-Reviewed Original Publications
Balabanov, S., Gontarewicz, A., Ziegler, P., Hartmann, U., Kammer, W., Copland,
M., Brassat, U., Priemer, M., Hauber, I., Wilhelm, T., Schwarz, G., Kanz, L.,
Bokemeyer, C., Hauber, J., Holyoake, T.L., Nordheim, A., and Brümmendorf,
T.H. (2007). Hypusination of eukaryotic initiation factor 5A (eIF5A): a novel
therapeutic target in BCR-ABL-positive leukemias identified by a proteomics
approach. Blood 109, 1701-1711.
158
Baschuk, N., Utermöhlen, O., Gugel, R., Warnecke, G., Karow, U., Paulsen, D.,
Brombacher, F., Krönke, M., and Deppert, W. (2007). Interleukin-4 Impairs
Granzyme-Mediated Cytotoxity of Simian Virus 40 Large Tumor AntigenSpecific CTL in BALB/c Mice. Cancer Immunol. Immunother. 56, 1625-1636.
Bruns, K., Studtrucker, N., Sharma, A., Fossen, T., Mitzner, D., Eissmann, A.,
Tessmer, U., Röder, R., Henklein, P., Wray, V., Schubert, U. (2007). Structural
characterization and oligomerization of PB1-F2, a proapoptotic influenza A
virus protein. J Biol. Chem. 5; 282: 353-363.
Cammenga, J., Niebuhr, B., Horn, S., Bergholz, U., Putz, G., Buchholz, F., Löhler,
J., and Stocking, C. (2007). RUNX1 DNA-binding mutants, associated with
minimally differentiated acute myeloid leukemia, disrupt myeloid differentiation. Cancer Res. 67, 537-545.
Craig, A.L., Chrystal, J., Fraser, J., Sphyris, N., Lin, Y., Harrison, B., Scott, M.,
Dornreiter, I., and Hupp, T.R. (2007). The MDM2 Ubiquitination Signal in the
DNA-Binding Domain of p53 Forms a Docking Site for Calcium Calmodulin
Kinase Superfamily Members. Mol. Cell. Biol. 27, 3542-55.
Dauth, I., Krüger, J., and Hofmann, T.G. (2007). Homeodomain-Interacting
Protein Kinase 2 Is the Ionizing Radiation-Activated p53 Serine 46 Kinase and
Is Regulated by ATM. Cancer Res. 67, 2274-2279.
Deppert, W. (2007). Mutant p53: From Guardian to Fallen Angel? Oncogene 26,
2142-2144. Review.
Franke, C., Matschl, U., and Bruns, M. (2007). Enzymatic treatment of duck
hepatitis B virus: Topology of the surface proteins for virions and noninfectious subviral particles. Virology 359, 126-136
Fries, B., Heukeshoven, J., Hauber, I., Grüttner, C., Stocking, C., Kehlenbach,
R.H., Hauber, J., and Chemnitz, J. (2007). Analysis of nucleocytoplasmic trafficking of the HuR ligand APRIL and its influence on CD83 expression. J. Biol.
Chem. 282, 4504-4515.
Funk, A., Mhamdi, M., Will, H., and Sirma, H. (2007). Avian hepatitis B viruses:
Molecular and cellular biology, phylogenesis, and host tropism. World J.
Gastroenterol 13, 91-103.
Veröffentlichungen
Kaiser, A., Hammels, I., Gottwald, A., Nassar, M., Zaghloul, M.S., Motaal, B.A.,
Hauber, J., and Hoerauf, A. (2007). Modification of eukaryotic initiation factor 5A from Plasmodium vivax by a truncated deoxyhypusine synthase from
Plasmodium falciparum: An enzyme with dual enzymatic properties. Bioorg.
Med. Chem. 15, 6200-6207.
Kim, E., and Deppert, W. (2007). Interactions of Mutant p53 with DNA: Guilt
by Association. Oncogene 26, 2185-2190. Review.
Kindsmuller, K., Groitl, P., Hartl, B., Blanchette, P., Hauber, J., and Dobner, T.
(2007). Intranuclear targeting and nuclear export of the adenovirus E1B-55K
protein are regulated by SUMO1 conjugation. Proc Natl Acad Sci USA 104,
6684-6689.
Krieghoff, E., Milovic-Holm, K., and Hofmann, T.G. (2007). FLASH meets nuclear
bodies: CD95 receptor signals via a nuclear pathway. Cell Cycle 6, 771-775.
Krummheuer, J., Johnson, A.T., Hauber, I., Kammler, S., Anderson, J.L., Hauber,
J., Purcell, D.F., and Schaal, H. (2007). A minimal uORF within the HIV-1
vpu leader allows efficient translation initiation at the downstream env AUG.
Virology 363, 261-271.
Maenz,C., Chang,S.-F., Iwanski,A., and Bruns,M. (2007). Entry of duck hepatitis
B virus into primary duck liver and kidney cells after discovery of a fusogenic
region within the large surface protein. J. Virol. 81, 5014-5023.
Matthes, E., Funk, A., Krahn, I., Gaertner, K., von Janta-Lipinski, M., Lin, L.,
Will, H., and Sirma, H. (2007). Strong and selective inhibitors of HBVreplication among novel N4-hydroxy-and 5-Methyl-β-L-deoxycytidine analogues. Antimicrobial Agents and Chemotherapy 51, 2523-2530.
Metzner, A. Horstmann, M.A, Fehse, B., Ortmeyer, G., Niemeyer, C.M., Stocking,
C., Mayr, G.W. and Jücker, M. (2007). Gene transfer of SHIP-1 inhibits proliferation of juvenile myelomonocytic leukemia cells carrying KRAS2 or PTPN11
mutations. Gene Therapy 14, 699-703.
Mhamdi, M., Funk, A., Hohenberg, H., Will, H., and Sirma, H. (2007). Assembly
and budding of a hepatitis B virus is mediated by a novel type of intracellular
vesicles. Hepatology. 46, 95-106.
Milovic-Holm, K., Krieghoff, E., Jensen, K., Will, H., and Hofmann, T.G. (2007).
FLASH links the CD95 signaling pathway to the cell nucleus and nuclear bodies.
EMBO J 26, 391-401.
Münch J, Rajan D, Schindler M, Specht A, Rücker E, Novembre FJ, Nerrienet E,
Müller-Trutwin MC, Peeters M, Hahn BH, Kirchhoff F. (*Equal contribution)
(2007). Nef-mediated Enhancement of Virion Infectivity and Stimulation
of Viral Replication are Fundamental Properties of Primate Lentiviruses.
J Virol 81, 13852-13864.
159
Veröffentlichungen
Münch J, Rücker E, Ständker L, Adermann K, Goffinet C, Schindler M, Wildum S,
Chinnadurai R, Rajan D, Specht A, Giménez-Gallego G, Sánchez PC, Fowler DM,
Koulov A, Kelly JW, Mothes W, Grivel JC, Margolis L, Keppler OT, Forssmann
WG, Kirchhoff F. (2007). Semen-Derived Amyloid Fibrils Drastically Enhance
HIV Infection. Cell. 131, 1059-1071.
Niendorf, S., Oksche, A., Kisser, A., Löhler, J., Prinz, M., Schorle, H., Feller, S.,
Lewitzky, M., Horak, I., Knobeloch, K.P. (2007). Essential role of ubiquitinspecific protease 8 for receptor tyrosine kinase stability and endocytic trafficking
in vivo. Mol. Cell. Biol. 27, 5029-5039.
160
Reifenberg, K., Lehr, H.A., Torzewski, M., Steige, G., Wiese, E., Küpper, I., Becker,
C., Ott, S., Nusser, P., Yamamura, K., Rechtsteiner, G., Warger, T., Pautz, A.,
Kleinert, H., Schmidt, A., Pieske, B., Wenzel, P., Münzel, T., Löhler, J. (2007)
Interferon-gamma induces chronic active myocarditis and cardiomyopathy in
transgenic mice. Am. J. Pathol. 171, 463-472.
Rodenburg, M. Fischer, M., Engelmann, A., Ogryzlo-Harbers, S., Ziegler, M.,
Löhler, J. and Stocking, C. (2007). Importance of receptor usage, Fli1 activation,
and mouse strain in the “stem-cell” specificity of 10A1-MuLV leukemogenicity.
J. Virol. 81, 732-742.
Sarkar, I., Hauber, I., Hauber, J., and Buchholz, F. (2007). HIV-1 proviral DNA
excision using an evolved recombinase. Science 316, 1912-1915.
Schindler M, Rajan D, Specht A, Ritter C, Pulkkinen K, Saksela K, Kirchhoff
F. (2007). Association of Nef with p21-Activated Kinase 2 Is Dispensable for
Efficient Human Immunodeficiency Virus Type 1 Replication and Cytopathicity
in Ex Vivo-Infected Human Lymphoid Tissue. J Virol 81, 13005-13014.
Sieber, T., and Dobner, T. (2007). Adenovirus type 5 early region 1B 156R protein
promotes cell transformation independently of repression of p53-stimulated
transcription. J Virol 81, 95-105.
Sirma, H., Funk, A., Gerlich, W., and Schildgen, O. (2007). Does pre-treatment
with lamivudine prime for adefovir resistance of HBV infection? J. Antimicrobial
Chemotherapy 60, 448-449.
Strom, A., Wang, G.S., Reimer, R., Finegood, D.T., Scott, F.W. (2007). Pronounced
cytosolic aggregation of cellular prion protein in pancreatic beta-cells in
response to hyperglycemia. Lab Invest. 87, 139-149.
Tromsdorf, U.I., Bigall, N.C., Kaul, M.G., Bruns, O.T., Nikolic, M.S., Mollwitz, B.,
Sperling, R.A., Reimer, R., Hohenberg, H., Parak, W.J., Förster, S., Beisiegel, U.,
Adam, G., Weller, H. (2007). Size and surface effects on the MRI relaxivity of
manganese ferrite nanoparticle contrast agents. Nano Lett. 7, 2422-2427.
van Niekerk, EA., Willis, D.E., Chang, J.H., Reumann, K., Heise, T., and Twiss, J.L.
(2007). Sumoylation in axons triggers retrograde transport of the RNA binding
protein La. Proc. Natl. Acad. Sci. USA 104, 12913-12918.
Veröffentlichungen
Non Reviewed Publications, Reviews and Book Chapters
Bruns, M., and Maenz, C. (2007). Requirement of activation for hepatitis B virus
infection. Nature Precedings: http://hdl.nature.com/10101/npre.2007.221.1.
Groitl, P., and Dobner, T. (2007). Construction of adenovirus type 5 early region
1 and 4 virus mutants. In: Adenovirus Methods and Protocols, W.S. Wold, and
A.E. Tollefson, eds. (Totowa, NJ, Humana Press Inc.), pp. 29-39.
Nevels, M., and Dobner, T. (2007). Determination of the transforming activities
of adenovirus oncogenes. In: Adenovirus Methods and Protocols, W.S. Wold,
and A.E. Tollefson, eds. (Totowa, NJ, Humana Press Inc.), pp. 187-195.
Prassolov, V., Hein, S., Ivanov, D., Lohler, J., Spirin, P., and Stocking, C. (2007). The
M813 retrovirus belongs to a unique interference group and is highly fusogenic,
In Stem Cells and their Application for Clinical Therapy, B. Fehse, N. Bilko, C.
Stocking, W. Ostertag, and A. Zander, eds. (Dordrecht: Springer), pp.223-244.
Reimer, R., Warmer, M., Franz, N., Holstermann B., Hohenberg H. (2007) An
ImageJ Plugin for Stereoscopic Visualization of Three Dimensional Structures
on Images Obtained by Environmental Scanning Electron Microscopy (ESEM).
Microscopy and Microanalysis 13 (Suppl. 02), pp. 1676-1677.
Reimer, R., Warmer, M., Holstermann, B., Hohenberg, H. (2007). Strategies for
the Fine-Morphological Analysis of Fully Hydrated and Life-Like Preserved
Biological and Clinical Material in the SEM. Microscopy and Microanalysis 13
(Suppl. 02), pp. 1458-145.
Stocking, C., Niebuhr, B., Fischer, M., Täger, M., and Cammenga, J. (2007). The
RUNX1 transcription factor: A gatekeeper of acute leukemia, In Stem Cells
and their Potential for Clinical Application, B. Fehse, N. Bilko, C. Stocking, W.
Ostertag, and A. Zander, eds. (Dordrecht: Springer), pp. 183-199.
Sullivan CS, Grundhoff A. (2007). Identification of viral microRNAs. Methods
Enzymol 427, 3-23.
van Lunzen, J. (2007). How will CCR5 antagonists influence the recommendations
for the antiretroviral tratment of HIV-1 infection. Eur. J. Med. Res. 12, 435-440.
van Lunzen, J., and Hoffmann,C. (2007). Virological rebound and ist consequences during treatment interruption. Cur. Opin. HIV and AIDS 2, 1-5.
van Lunzen, J. (2007). Treatment of heavily antiretroviral-experienced HIVinfected patients. AIDS Rev. 9, 246-253.
Wepf, R., Droste, M.S., Schertel, A. and Hohenberg H. (2007). Zoom-in beyond
light microscopy: A new approach for biological structure research – correlative light and electron microscopy on one and the same sample” 12/2007 G.I.T.
Imaging & Microscopy, Spec. Ed. “Advances in Microscopy”.
Wepf, R., Richter, T., Biel, S., Schlüter, H., Fischer, F., Wittern, K.P., Hohenberg,
H. (2007). Multimodal imaging of skin structures: Imagining imaging of the
Skin. In: Bioengineering of the skin: Skin Imaging and Analysis 2nd Ed. 2007,
Informa Healthcare USA, New York.
161
Veröffentlichungen
2008
Peer-Reviewed Original Publications
Asang, C., Hauber, I., and Schaal, H. (2008). Insights into the selective activation
of alternatively used splice acceptors by the human immunodeficiency virus
type-1 bidirectional splicing enhancer. Nucleic Acids Res. 36, 1450-1463.
Blackford, A.N., Bruton, R.K., Dirlik, O., Stewart, G.S., Taylor, A.M., Dobner,
T., Grand, R.J., and Turnell, A.S. (2008). A role for E1B-AP5 in ATR signaling
pathways during adenovirus infection. J Virol 82, 7640-7652.
162
Blanchette, P., Kindsmüller, K., Groitl, P., Dallaire, F., Speiseder, T., Branton, P.E.,
and Dobner, T. (2008). Control of mRNA export by adenovirus E4orf6 and
E1B55K proteins during productive infection requires the E4orf6 ubiquitin
ligase activity. J Virol 82, 2642-2651.
Chemnitz, J., Pieper, D., Grüttner, C., and Hauber, J. (2008). Phosphorylation
of the HuR ligand APRIL by casein kinase 2 regulates CD83 expression. Eur. J.
Immunol. 39, 267-79.
Deppert, W. (2008). SIRT1 Protein Levels in Cancer: Tuning SIRT1 to the Needs
of a Cancer Cell. Cell Cycle 7, 2947-2948.
Engelmann, A., Speidel, D., Bornkamm, G.W., Deppert, W., and Stocking, C.
(2008). Gadd45ß is a Pro-Survival Factor Associated with Stress-Resistant
Tumors. Oncogene 27, 1429-1438.
Everett, R.D., Parada, C., Gripon, P., Sirma, H., and Orr, A. (2008). Replication of
ICP0-null-mutant herpes simplex virus type 1 Is restricted by both PML and
Sp100. J. Virol. 82, 2661-2672.
Funk, A., Mhamdi, M., Hohenberg, H., Heeren, J., Reimer, R., Lambert, C.,
Prange, R., and Sirma, H. (2008) Duck hepatitis B virus requires cholesterol for
endosomal escape during virus entry. J Virol. 82, 10532-10542.
Goebel, T., Ulmer, D., Projahn, H., Kloeckner, J., Heller, E., Glaser, M., PonteSucre, A., Specht, S., Sarite, S.R., Hoerauf, A., Kaiser, A., Hauber, I., Hauber,
J., and Holzgrabe, U. (2008). In search of novel agents for therapy of tropical
diseases and human immunodeficiency virus. J. Med. Chem. 51, 238-250.
Härtl, B., Zeller, T., Blanchette, P., Kremmer, E., and Dobner, T. (2008). Adenovirus
type 5 early region 1B 55-kDa oncoprotein can promote cell transformation by a
mechanism independent from blocking p53-activated transcription. Oncogene
27, 3673-3684.
Heinlein, C., Krepulat, F., Löhler, J., Speidel, D., Deppert, W., and Tolstonog, G.
(2008). Mutant p53R20H Gain of Function Phenotype in a Mouse Model for
Oncogene-Induced Mammary Carcinogenesis. Int. J. Cancer 122, 1701-1709.
Veröffentlichungen
Henning, K., Heering, J., Schwanbeck, R., Schroeder, T., Helmbold, H, Schäfer,
H., Deppert, W., Kim, E., and Just, U. (2008) Notch1 Activation Reduces
Proliferation in the Multipotent Hematopoietic Progenitor Cell Line FDCP-mix
through a p53-Dependent Pathway but Notch1 Effects on Myeloid and Erythroid
Differentiation are Independent of p53. Cell Death Differ. 15, 298-407.
Horn, S., Bergholz, U., Jücker, M., McCubrey, J.A., Trümper, L. Stocking, C., and
Bäsecke, J.: (2008). Oncogenic function of mutations in the catalytic subunit of
PI3K in hematopoietic cells. Oncogene 27, 4096-4106.
Hoxha,E., Fehse B., Ortmeyer,G. Stocking,C., Mayr,G.W. and Jücker,M. (2008).
Overexpression of the p85α regulatory subunit of phosphatidylinositol 3-kinase
inhibits GM-CSF-dependent colony formation of CD34+ hematopoietic progenitor cells. Leuk. Lymphoma 22, 1-3.
Hutten, S., Wälde, S., Spillner, C., Hauber, J., and Kehlenbach, R.H. (2008). The
nuclear pore component Nup358 promotes transportin-dependent nuclear
import. J. Cell Sci., in press.
Kawamata, N., Ogawa, S., Zimmermann, M., Niebuhr, B., Stocking, C., Sanada,
M., Hemminki, K., Yamatomo, Nannya, Y., Koehler, R., Flohr, T., Miller, C.W.,
Harbott, J., Ludwig, W.-D., Stanulla, M., Schrappe, M., Bartram, C.R., and
Koeffler, H.P. (2008). Cloning of genes involved in chromosomal translocations
by high resolution single nucleotide polymorphism genomic microarray. Proc.
Natl. Acad. Sci. USA 105, 11921-6.
Kirchhoff F, Schindler M, Specht A, Arhel N, Münch J. (2008). Role of Nef in
primate lentiviral immunopathogenesis. Cell Mol Life Sci. 65, 2621-2636.
Krack, M., Hohenberg, H., Kornowski, A., Lindner, P., Weller, H., Förster, S.
(2008) Nanoparticle-loaded magnetophoretic vesicles. J Am Chem Soc. 130,
7315-7320.
Lehmann, C., Harper, J.M., Taubert, D., Hartmann, P., Fätkenheuer, G., Jung,N.,
van Lunzen, J., Stellbrink, H.J., Gallo, R.C., and Romerio, F. (2008). Increased
interferon alpha expression in circulating plasmacytoid dendritic cells of HIV1-infected patients. J. Acquir. Immune Defic. Syndr. 48, 522-530.
Metzner, A., Precht, C., Fehse, B., Fiedler, W., Stocking, C., Günther, A., Mayr,
G.W. and Jücker, M. (2008) Reduced proliferation of CD34+ cells from patients
with acute myeloid leukemia after gene transfer of INPP5D. Gene Therapy, 16,
570-573
Maenz, C., Loscher, C., Iwanski, A., and Bruns, M. (2008). Inhibition of duck
hepatitis B virus infection of liver cells by combined treatment with viral e antigen and carbohydrates. J. Gen. Virol. 89, 313-326.
Miller, A.D., Bergholz, U., Ziegler, M, and Stocking, C. (2008). Identification of
the myelin protein plasmolipin as the cell entry receptor for Mus caroli endogenous retrovirus. J. Virol. 82, 6862-6868.
Muller, S., and Dobner, T. (2008). The adenovirus E1B-55K oncoprotein induces
SUMO modification of p53. Cell Cycle 7, 754-758.
163
Veröffentlichungen
Netter, H.-J., Chang, S.-F., and Bruns, M. (2008). Host-range and pathogenicity
of hepatitis B virus. Future Virol. 3, 83-94.
Niebuhr, B., Fischer, M. Täger, M., Cammenga, J., and Stocking, C. (2008).
Gatekeeper function of the RUNX1 transcription factor in acute leukemia.
Blood Cells Mol. Dis. 40, 211-218.
Niebuhr, N., Iwanski, G.B., Schwieger, M., Roscher, S., Stocking C., Cammenga,
J. (2008). Investigation of C/EBPα function in human (versus murine) myelopoiesis provides novel insight into the impact of CEBPA mutations in acute
myelogenous leukemia (AML). Leukemia 23, 978-983
164
Pfuhl, T., Mamiani, A., Durr, M., Welter, S., Stieber, J., Ankara, J., Liss, M., Dobner,
T., Schmitt, A., Falkai, P., et al. (2008). The LARK/RBM4a protein is highly
expressed in cerebellum as compared to cerebrum. Neurosci Lett 444, 11-15.
Raimondo, G., Allain, JP., Brunetto, MR., Buendia, MA., Chen, DS., Colombo,
M., Craxì, A., Donato, F., Ferrari, C., Gaeta, GB., Gerlich, WH., Levrero, M.,
Locarnini, S., Michalak, T., Mondelli, MU., Pawlotsky, JM., Pollicino, T., Prati,
D., Puoti, M., Samuel, D., Shouval, D., Smedile, A., Squadrito, G., Trépo, C., Villa,
E., Will, H., Zanetti, AR., and Zoulim, F. (2008). Statements from the Taormina
expert meeting on occult hepatitis B virus infection. J. Hepatol. 49, 652-657.
Ruetze, M., Gallinat, S., Lim, I.J., Chow, E., Phan, T.T., Staeb, F., Wenck, H.,
Deppert, W, and Knott, A. (2008). Common Features of Umbilical Cord
Epithelial Cells and Epidermal Keratinocytes. J. Dermatol. Sci. 50, 227-231.
Schindler M, Schmökel J, Specht A, Li H, Münch J, Khalod M, Sodora DL, Hahn
BH, Silvestri G, Kirchhoff F. (2008). Inefficient Nef-mediated downmodulation
of CD3 and MHC-I correlates with loss of CD4+ T cells in natural SIV infection.
PLoS Pathog 4(7): e1000107. doi: 10.1371/journal.ppat. 1000107.
Schüler, A., Schwieger, M., Engelmann, A., Weber, K., Horn, S., Müller, U., Arnold,
M., Olson, E. and Stocking, C. (2008). The MADS transcription factor Mef2c is
a pivotal modulator of myeloid cell fate. Blood 111, 4532-4541.
Schwalbe, M., Ohlenschläger, O., Marchanka, A., Ramachandran, R., Häfner, S.,
Heise, T. and Görlach, M. (2008). Solution structure of the stem-loop of the
hepatitis B virus post-transcriptional regulatory element. Nucl. Acids Res. 36,
1681-1689.
Specht A, Degottardi MQ, Schindler M, Hahn B, Evans DT, Kirchhoff F. (2008).
Selective downmodulation of HLA-A and -B by Nef alleles from different
groups of primate lentiviruses. Virology 373: 229-237.
Specht, S., Sarite, S.R., Hauber, I., Hauber, J., Görbig, U.F., Meier, C., Bevec, D.,
Hoerauf, A., and Kaiser, A. (2008). The guanylhydrazone CNI-1493: an inhibitor
with dual activity against malaria-inhibition of host cell pro-inflammatory cytokine release and parasitic deoxyhypusine synthase. Parasitol. Res. 102, 1177-1184.
Toussaint H, Gobert FX, Schindler M, Banning C, Kozik Patrycja, Jouve M,
Kirchhoff F, Benaroch P. (2008). HIV-1 Nef expression prevents AP-2-mediated
internalization of the MHC II-associated invariant chain. J Virol 82, 8373-8782.
Veröffentlichungen
Weber, K., Bartsch, U., Stocking, C., Fehse, B. (2008). A multicolor panel of novel
lentiviral "Gene Ontology" (LeGO) vectors for functional gene analysis. Mol
Ther. 16, 698-706.
Weise, J.M., and Günes, C. (2008)e. Differential regulation of human and mouse
Telomerase Reverse Transcriptase (TERT) promoter activity during testis development. Mol. Reprod. Dev. Aug 26. [Epub ahead of print]
Wiegers, K., Schwarck, D., Reimer, R., and Bohn, W. (2008). Activation of the glucocorticoid receptor releases unstimulated PBMCs from an early block in HIV-1
replication. Virology 375, 73-84.
Zafrakas, M., Tarlatzis, B.C., Streichert, T., Pournaropoulos, F., Wölfle, U., Smeets,
S.J., Wittek, B., Grimbizis, G., Brakenhoff, R.H., Pantel, K., Bontis, J., and Günes,
C. (2008). Genome-wide microarray gene expression, array-CGH analysis, and
telomerase activity in advanced ovarian endometriosis: a high degree of differentiation rather than malignant potential. Int. J. Mol. Med. 21, 335-344.
Non Reviewed Publications, Reviews and Book Chapters
Kim, E., Giese, A., and Deppert, W. (2008)e. Wild-type p53 in Cancer Cells: When
a Guardian Turns into a Blackguard. Biochem. Pharmacol. [Epub 2008 Sep 3].
Nevels, M., and Dobner, T. (2008). Erreger-induzierte Tumoren. In: Klinische
Infektiologie. R. Marre, M. Trautmann & W. Zimmerli, eds. (München • Jena,
Verlag Urban und Fischer), pp. 53-66.
Schindler M. (2008). Evolution des lentiviralen Nef Proteins. VDM-Verlag, ISBN
978-3-639-01822-6.
van Lunzen, J. (2008). Ritonavir-boosted tipranavir (TPV/r) in combination with
new drug classes. Hospital Pharmacy Europe 39, 14-16.
Schindler, M., Hohenberg, H. (2008). Virologische Fährtensuche. Kombinierte
Mikroskopietechniken zur Abbildung von Virusinfekti0onen. Bioforum 6, 2-6.
Stocking, C. and Kozak, C.A. (2008). Murine endogenous retrovirus. Cell. Life
Mol. Sci. 65, 3383-3398.
165
Habilitationen, Dissertationen,
Diplomarbeiten, Masterarbeiten,
Bachelorarbeiten
2007
Diplomarbeiten
Bohlmann, M.: „Identifizierung und Charaktisierung neuer aviärer Hepatitis B
– Virusisolate“, Diplomarbeit, Fakultät für Biowissenschaften (Biologie),
Universität Heidelberg (2007).
Gießelmann, K.: „Funktionelle Analyse des zellulären Proteins OTT/RBM15“,
Diplomarbeit, Fakultät für Biowissenschaften (Biologie), Universität Heidelberg
(2007).
166
Hildebrand, J.: „Regulation der LINE-1 Expression in humanen Tumorzelllinien
durch Wildtyp und mutiertes p53.“ Fachbereich Biologie, Diplomarbeit,
Universität Hamburg (2007).
Jahn, M.: „Beeinflussung der Expression eine Reportergens durch promoterflankierende transposable bzw. repetitive Sequenzen in einem episomalen Vektorsystem.“ Fachbereich Biologie, Diplomarbeit, Universität Münster
(2007).
Knauer, T.: „Charakterisierung nichtkodierender Transkripte in humanen
Glioblastoma Zellen.“ Fachbereich Humanbiologie, Diplomarbeit, Universität
Erlangen (2007).
Peters, C.: „Untersuchungen zur Wechselwirkung der Ubiquitin-spezifischen
Protease USP7 mit dem E1B-55K Protein humaner Adenoviren.“ Fakultät
für Mathematik, Informatik und Naturwissenschaften, Department Biologie,
Pieper, D.: „Untersuchungen zur adaptiven zellulären Immunantwort am Beispiel
der HIV/HCV-Coinfektion.” Naturwissenschaften, Diplomarbeit, Universität
Hamburg (2007).
Saker, J.: „Regulation und Funktion der Telomerase und der Telomerfaktoren
während der Differenzierung und Proliferation.“ Fachbereich Biologie,
Diplomarbeit, Universität Hamburg (2007).
Schaub, T.: „Analyse der Funktion der PEST-Domänen und der GSK-3ß vermittelten Phosphorylierung für die Stabilität des SPOC1-Proteins“, Diplomarbeit,
Institut für Biowissenschaften (Mathematisch Naturwissenschaftlicher
Fachbereich), Universität Rostock (2007).
Simon, N.: „Funktionelle Interaktionen von p53 mit Telomer- und TelomeraseKomponenten und Regulation der Telomerase-Aktivität durch p53.“ Fachbereich
Humanbiologie, Diplomarbeit, Uni Marburg (2007).
Wimmer, P. „Analyse der transformierenden Eigenschaften des E1B-55KProteins von Adenovirus Typ 5“. Naturwissenschaftliche Fakultät III. Universität
Regensburg (2007).
Bachelorarbeiten
Dissertationen
Bruhn, S.: „NAADP, CD38 und Ca2+-Signalling: Komplexes Wechselspiel bei
der Aktivierung von T-Lymphozyten.“ Fachbereich Chemie, Dissertation,
Fries, B.: „Einfluss des HuR-Liganden APRIL auf die CD83 Expression.“
Dissertation, Universität Erlangen-Nürnberg (2007).
Friese, O.: „Ein Mausmodell für die fehlende Erkennung methylierter DNA durch
das Mdb1-Protein (Methyl-CpG-bindendes Protein 1).“ Fachbereich Chemie,
Dissertation, Universität Hamburg (2007).
Iwanski, G.: „Untersuchungen zur Rolle des Transkripstionsfaktors C/EBPα bei
der normalen und aberranten Hämatopoese mit Hilfe der RNA-Interferenz
Methode.“ Dissertation, Fachbereich Medizin, Universität Hamburg (2007).
Kinkley, S.: „SPOC1: Characterization of a Novel Nuclear Protein with Implications
for a Functional Role in Mitotic Chromosome Condensation.“ Fachbereich
Biologie, Universität Hamburg (2007).
Kriegs, M.: „Untersuchung des Einflusses des Nicht-Strukturproteins 5A des
Hepatitis C Virus auf Zellwachstum, Apoptose und Karzinogese in vitro und in
vivo.“ Institut für Biologie, Humboldt-Universität zu Berlin (2007).
Lupberger, J.: „Cultivation of Hepatitis B Virus Producing Cell Line HepG2.2.15
on Microcarrier and Functional Characterization of the Hepatitis B Virus
Polymerase.“ Institut für Biologie, Humboldt Universität zu Berlin (2007).
Mhamdi, M.: „Characterization of the envelope-mediated Steps in the life cycle of
hepatitis B viruses (Intrazellulärer Transport und Morphogenese der Hepatitis B
Viren).“ Fachbereich Biologie, Universität Hamburg (2007).
Prechtel, A.: „Untersuchungen zur posttranskriptionellen Prozessierung der
Messenger RNA von CD83, dem Reifungsmarker Dendritischer Zellen.“
Dissertation, Naturwissenschaftliche Fakultät II (Biologie), Universität ErlangenNürnberg (2007).
M. Ritter: „A landmark-based method for the geometrical 3D calibration of scanning microscopes.” Fachbereich Informatik, TU-Berlin (2007).
Riu-Garcia, A.: „Functional Analysis of Hepatitis B Virus with Mutations in the
Envelope Proteins.” Fachbereich Chemie, Dissertation, Uni Hamburg (2007).
Sander, M.-S.: „Quantifizierung der viralen DNA-Synthese im produktiven
Infektionszyklus humaner Adenoviren durch Echtzeit-PCR.“ Medizinische
Fakultät der Universität Regensburg (2007).
Habilitationen
Dr. Günes, C.: „Regulation und Funktion der Telomerase in normalen und
Tumorzellen: Erkenntnisse aus in vitro und in vivo Modellen.“ Fachbereich
Medizin, Habilitation, Universität Hamburg (2007).
167
Bachelorarbeiten
2008
Bachelorarbeiten
Boukari, H.: „Zelluläre Determinanten der HBV Replikation.“ Bachelorarbeit,
Fachbereich Biologie, Universität Hamburg (2008).
Fischer, T.: „Charakterisierung des Adenovirus Serotyp 5 H5pm4182.“ Fakultät
für Mathematik, Informatik und Naturwissenschaften, Department Biologie,
168
Godau, J.: „Analysen zum transformierenden Potenzial des E1B-55K-Proteins
von Adenovirus Serotyp 5.“ Fakultät für Mathematik, Informatik und
Naturwissenschaften, Department Biologie, Universität Hamburg (2008).
Hofmann, S.: „Analyse der Tre-Rekombinase im HIV-1 LTR.“ Bachelorarbeit,
Fachbereich Biologie/Chemie der Justus-Liebig-Universität Giessen (2008).
Homann, L.: „Untersuchungen zur Dynaminabhängigkeit des Viruseintritts
von DHBV im primären Entenhepatozyten-System.“ Fachbereich Biologie,
Universität Marburg (2008).
Sekutowicz, M.: „Untersuchungen der Auswirkungen zellulärer Proteine auf
den adenoviralen Infektionsverlauf in humanen Hepatozyten mittels siRNATechniken.“ Fakultät für Mathematik, Informatik und Naturwissenschaften,
Department Biologie, Universität Hamburg (2008).
Shaikh, Y.: „Methodische Untersuchungen zur Erhaltung der eGFP Fluoreszenz
in biologischen Proben, während der Kryoprozessierung für die korrelative
Mikroskopie.“ Life Science, HAW Hamburg (2008).
Masterarbeiten
Kluth, A.: „p53-abhängige Differenzierung embryonaler Stammzellen in einem
Maustumorzellmodell.“ Studiengang Molekulare Biologie, Masterarbeit,
Universität Gelsenkirchen (2008).
Diplomarbeiten
Ching, W.: „Funktionelle Analysen zur Phosphorylierung des E1B-55K-Proteins
von Adenovirus Serotyp 5 durch Casein Kinase II.“ Fakultät für Mathematik,
Informatik und Naturwissenschaften, Department Chemie, Universität
Hamburg (2008).
Deing, V.: „Einfluss des Tumorsuppressorgens p53 auf die Qualität der Reparatur
von UV-induzierten DNA Schäden.“ Fakultät für Mathematik, Informatik und
Naturwissenschaften, Universität Hamburg (2008).
R. Getzieh: „Microbial Biofilms in Natural Environments and Medicine.
Development and Establishment of a new Technique for Correlative Light and
Electron Microscopy.“ Umwelt und Technik, Universität Lüneburg (2008).
Bachelorarbeiten
Keyser, B.: „Untersuchungen zur subzellulären Lokalisierung und zum intrazellulären Transport von ICP0 und ICP4 des Herpes Simplex-Virus 2 (HSV-2).“
Diplomarbeit, Fachbereich Biologie, Universität Hamburg (2008).
Kömm, N.: „Analyse der Funktionen von pRb2/p130 und pRb/p105 in der
Induktion zellulärer Seneszenz mittels RNA-Interferenz.“ Diplomarbeit,
Fachbereich Biologie, Universität Hamburg (2008).
Kopp, F.: „Charakterisierung der Interaktion des viralen E1B-55K-Proteins
mit Securin und dessen Einfluss auf den produktiven Infektionszyklus von
Adenovirus Serotyp 5.“ Mathematisch Naturwissenschaftliche Fakultät.
Universität Rostock (2008).
Korf, K.: „Zelluläre Parameter der Simian Virus 40 (SV40) Amplifikation
in Abhängigkeit des p53-Status der Wirtszelle.“ Fakultät für Mathematik,
Informatik und Naturwissenschaften, Universität Hamburg (2008).
Leinenkugel, F.: „Funktionelle Analysen der E1B-48K- und der -49K-Isoform des
Adenovirus Typ 5 E1B-55K-Proteins.“ Fakultät für Biologie. Albert-LudwigsUniversität Freiburg (2008).
Malitzky, A.: „Herstellung und Überprüfung eines neuartigen adenoviralen
Vektorsystems.“ Fakultät für Mathematik, Informatik und Naturwissenschaften,
Department Chemie, Universität Hamburg (2008).
Schmid, M.: „Analysen regulatorischer Bereiche im E1B-55K Protein von
Adenovirus Serotyp 5 und ihre Rolle im produktiven Replikationszyklus.“
Naturwissenschaftliche Fakultät III, Universität Regensburg (2008).
Dissertationen
Ehlers, C.: „Einfluss des RNA bindenden Proteins AUF1 auf die CD83 Expression.“
Dissertation, Fakultät für Mathematik, Informatik und Naturwissenschaften,
Department Chemie der Universität Hamburg (2008).
Fischer, M.: „Etablierung eines in vivo Modells in Mus musculus (Linneaus 1758)
zur Untersuchung der onkongene Wirkung des Fusionsproteins TEL1/AML1.“
Dissertation, Fachbereich Biologie, Universität Hamburg (2008).
Hermannstädter, A.: „Die duale Rolle von p53 in der zellulären Immortalisierung
und der SV40 LT-vermittelten Transformation.” Fachbereich Chemie,
Horn, F.: „Hemmung der HIV-1-Rev- und HTLV-I-Rex-Funktion mit Hilfe von
Inhibitoren der Desoxyhypusin-Synthase.“ Dissertation, Medizinische Fakultät,
Universität Erlangen-Nürnberg (2008).
Iwanski, K.: „Funktionelle Interaktionen von p53 mit Telomerasekomponenten
und Regulation der Telomeraseaktivität durch p53.“ Fachbereich Medizin,
169
Bachelorarbeiten
Majlessi, A.; „Sequenzanalyse von Hepatitis B Virus in Patienten mit fibrosierender cholestatischer Hepatitis.“ Fachbereich Medizin, Universität Hamburg
(2008).
März, A.: „Mutp53 Interaktom in einem murinen Tumorzellmodell.” Fachbereich
Chemie, Dissertation, Universität Hamburg (2008).
Sieber, T.: „Untersuchung des funktionellen Potenzials der Adenovirus Typ 5
E1BN-Proteine.“ Naturwissenschaftliche Fakultät III, Universität Regensburg
(2008).
170
Tögel, L.: „Epigenetische Funktion des mutierten Tumorsuppressors p53 in
Mammakarzinoma.“ Fachbereich Biologie, Dissertation, Universität Hamburg
(2008).
Olotu, C.: „Analyse von virusspezifischen Resistenzfaktoren und phylogenetischer
Entwicklung von Hepatitis B Viren unter Therapie mit Adefovir.“ Dissertation,
Medizinische Fakultät, Universität Hamburg (2008).
Habilitationen
Dandri, M.: „Chronic HBV infection and hepatocarcinogenesis – Lessons from in
vitro and in vivo models.” Fachbereich Biologie, Universität Hamburg (2008).
Lehrtätigkeit
Thomas
Dobner
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ahlpflichtmodul: Molekulare Virologie und Zellbiologie (9 CP)
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Sc Biologie
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allstudie/ABKII: Methoden der molekularen Virologie und Zellbiologie
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Sc Molekulare & Funktionelle Biologie
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ingvorlesung: MSc Molekulare & Funktionelle Biologie. Virologie
Adam
Grundhoff
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Joachim
Hauber
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S eminar: Research Highlights in Molecular Virology and Immunology
Seminar: Experimental Approaches in Virology
Heinrich
Hohenberg
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FB Medizin Hamburg: Imagingtechniken in der Medizin
Michael
Schindler
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orlesung: Entstehung und Evolution des AIDS-Erregers HIV-1
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AIDS-Pathogenese
Hüseyin
Sirma
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Carol
Stocking
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Hans Will
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Vorlesung: RNAi: Gene Regulation by small RNAs, Aufbaustudiengang
Molekularbiologie (ZMNH Hamburg)
S eminar: Research Highlights in Molecular Hepatology and Immunology
Ko-Organisator der Seminarreihe der Forschungsinitiative
„Leberentzündungen: Infektion, Immunregulation und Konsequenzen“
S eminar: Research Highlights in Leukemia
Seminar: Experimental Approaches in Hematology
S eminar: Advances in Molecular and Cellular Biology
(H. Will, I. Dornreiter)
Advances in Virus and Tumor Research
(H. Will, I Dornreiter, G. Rohaly)
raktika: Practical Introduction in Experimental Research
P
(H. Will, I. Dornreiter, G. Rohaly, H. Staege)
Courses in Experimental Virology
(H. Will, I. Dornreiter, G. Rohaly)
orlesung: Hepatitis B Viruses (H. Will)
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Papovaviruses (I. Dornreiter, G. Rohaly)
171
Gutachtertätigkeit
Gutachtertätigkeit
für nationale
Vereinigungen
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Behring-Röntgen-Stiftung (J. Hauber)
BioPark Regensburg (J. Hauber)
Bundesministerium für Bildung und Forschung (H. Hohenberg, H. Will)
Bundesministerium für Gesundheit, Berlin (H. Hohenberg)
Charité – Universitätsmedizin Berlin (H. Will)
Deutsche Forschungsgemeinschaft (T. Dobner, J. Hauber, H. Hohenberg,
C. Stocking)
Deutsche Gesellschaft für Virologie (H. Will)
Deutsches Krebsforschungszentrum Heidelberg (H. Will)
Deutsche Krebshilfe (W. Deppert, A. Grundhoff)
Eberhard Karls Universität Tübingen, Medizinische Fakultät (H. Will)
Fond der Chemischen Industrie (H. Will)
Frankfurter Stiftung Krebskranker Kinder (W. Deppert)
Hamburger Kommission für Fragen der Gentechnik (A. Grundhoff)
Helmholtz-Zentrum für Infektionsforschung (J. Hauber)
Johann Wolfgang Goethe-Universität Frankfurt am Main, Medizinische
Fakultät (H. Will)
Justus-Liebig-Universität Gießen, Medizinische Fakultät (H. Will)
Leibniz-Gemeinschaft (H. Will)
Ludwig-Maximilians Universität München, Naturwissenschaftliche Fakultät
(T. Dobner, H.Will)
Max-Planck-Gesellschaft (W. Deppert, H. Will)
Medizinische Hochschule Hannover (C. Stocking)
Nationales Kompetenznetz HepNet (H. Will)
Peter Hans Hofschneider Stiftungsprofessur (W. Deppert)
Robert Koch Institut, Berlin (J. Hauber, H. Will)
Robert-Koch-Stiftung (J. Hauber)
Ruhr-Universität Bochum (W. Deppert)
Ruprecht-Karls-Universität Heidelberg, Medizinische Fakultät (H. Will)
Studienstiftung des Deutschen Volkes (T. Dobner, H. Will)
Technische Universität Berlin, Fachbereich Vermessungstechnik (H. Hohenberg)
Tumorzentrum Heidelberg/Mannheim (W. Deppert)
Universität des Saarlandes, Medizinische Fakultät (A .Grundhoff)
Universität Düsseldorf, Medizinische Fakultät (W. Deppert, J. Hauber)
Universität Frankfurt, Medizinische Fakultät (J. Hauber)
Universität Göttingen, Zentrum für Molekular Biowissenschaften (H. Will)
Universität Hamburg, Medizinische Fakultät (W. Deppert)
Universität Hamburg, Fakultät für Mathematik, Informatik und
Naturwissenschaften (W. Deppert, J. Hauber, H. Will)
Universität Heidelberg (H. Will)
Universität Leipzig, Medizinische Fakultät (H. Will)
Universität Mainz, Fachbereich Medizin (H. Will)
Universität Regensburg, Medizinische Fakultät (T. Dobner)
Universität Rostock (H. Will)
Wilhelm Sander-Stiftung (W. Deppert, T. Dobner)
Gutachtertätigkeit
Gutachtertätigkeit
für internationale
Vereinigungen
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American Society of Hematology (C. Stocking)
American Society of Microbiology (C. Stocking)
Associazione Italiana per la Ricerca sul Cancro (AIRC) (T. Dobner)
Cancer Research UK (W. Deppert, C. Stocking)
Chinesisch-Deutsches Zentrum für Wissenschaftsförderung, Peking (H. Will)
Consiglio Nazionale delle Richerche, Rom (H. Will)
European Inst. of Oncology, Milan, Italy (H. Will)
Förderung der wissenschaftlichen Forschung, (FWF) Austria. (T. Dobner)
Fudan University, Shanghai, China (H. Will)
Genesis Oncology Trust, Neuseeland (W. Deppert)
German-Israeli Foundation (W. Deppert)
INSERM, Institut Pasteur, Paris, France (H. Will)
Israel Science Foundation (T. Dobner)
Italian Association for Cancer Research (AIRC), Italy (H. Will)
L’Agence Nationale de la Recherche, Paris (H. Will)
Leukemic Research Foundation, London (C. Stocking)
Medizinische Universität Wien (J. Hauber)
Monash-University, Australia (H. Will)
Nanyang Technological University, Singapore (H. Will)
National Health Research Institute, Hinschu, Taiwan (H. Will)
National Research Foundation, Pretoria, South Africa (H. Will)
Northern Cancer Research Foundation, Canada (H. Will)
Rhode Island University Hospital, USA (H. Will)
The Kay Kendall Leukemia Fund, London (C. Stocking)
University of Adelaide, Australia (H. Will)
University of Bratislava, Czech Republic (H. Will)
University of New Foundland, Canada (H.Will)
University of the Witwatersrand, Johannesburg, South Africa (H. Will)
University of York, United Kingdom (W. Deppert)
US-Israel Binational Science Foundation (T. Dobner)
Wuhan University, China (H. Will)
173
Organisation von Meetings
und Chairman-Tätigkeiten
Wolfgang
Deppert
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J apanese-German Cancer Workshop in Kyoto, Japan 2007 (Organisation)
Thomas
Dobner
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M
olecular Life Sciences. Herbsttagung der Gesellschaft für Biochemie und
Molekularbiologie, Hamburg 2007 (Koorganisation)
T
agung: Adenovirus: Basic research and application. Ulm 2007 (Koorganisation
und Chair)
D
NA Tumor Virus Meeting. Madison, WI, USA. 2008
(Koorganisation und Chair)
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Joachim
Hauber
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M
olecular Life Sciences. Herbsttagung der Gesellschaft für Biochemie und
Molekularbiologie, Hamburg 2007 (Koorganisation)
Hüseyin
Sirma
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SFB Symposium on viral Hepatitis, Gießen, 2008.
Carol
Stocking
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1 4th International Runx Meeting 2007; Singapore, 19.-22. August 2007
(Chairperson)
3 6th Annual Scientific meeting of the ISEH Society for Hematology and Stem
Cells; Hamburg, 28.-30. September 2007 (Koorganisation)
M
olecular Life Sciences: Herbsttagung der GBM; Hamburg, 16.-19. September
2007 (Chairperson)
I ACRLRD Meeting – From Molecular Pathogenesis to Targeted Therapy in
Leukemia and Sold Tumors; Freiburg, 7.-11. September 2007 (Chairperson)
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VII. Wilsede Meeting “Modern Trends in Human Leukemia” 14.-18. Juni
2008 (Koorganisation)
S ummer School “Mechansims of early differentiation; embryonegenesis,
myogenesis, and lymphohematopoiesis”; Barsinghausen, 1.-5. September 2008
(Chairperson)
1 5th International Runx Meeting 2008; Provincetown, MA, USA. 14.-17.
September 2008 (Chairperson)
A
merican Society of Hematology (ASH) Meeting, San Francisco, CA, USA.
5.-9. Dezember 2008 (Chairperson)
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Organisation von Meetings
und Chairman-Tätigkeiten
Hans Will
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4th Workshop „Hot Topics in Hepatology“, Modena, Italien, 25.-26.01.2007
(Chairman)
3nd Workshop “Function of SPOC1 in Cells and Cancer”, Universität
München, 24.-26.3.2007 (Co-Organisator and Chairman)
2nd Sino-German Symposium on Hepatology “Viral Hepatitis from Basic
Research to Clinical Application”, Wuhan, China, 18.-19.05.2007 (Chairman)
Meeting on “Molecular Life Science 2007” of the German Society
of Biochemistry and Molecular Biology”, Hamburg, 16.-19.09.2007
(Co-Organisator)
New insights in HBV Diversity, Pathogenesis, Diagnosis and Treatment,
Ghent, Belgien, 12.-14.12.2007 (Chairman)
3rd Workshop “Function of SPOC1 in Cells and Cancer”, Dortmund,
08.02.2008 (Co-Organisator and Chairman)
International Symposium on “Occult Hepatitis B virus Infection: Biology and
Clinical Impact”, Taormina, Italien, 07.-08.03.2008 (Chairman)
4th Virgil Symposium, Lyon Frankreich, 26.-27.05.2008 (Co-Organisator)
International Symposium on Hepatitis B Virus Diversity, University
Witwatersrand, Johannesburg, Südafrika, 27.07-03.08.2008 (Co-Organisator
and Chairman)
International Symposium on Viral Hepatitis and Liver Diseases, 20.-24.3.2009
Washington, USA
175
Mitherausgabe
wissenschaftlicher Zeitschriften
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iochemistry and Cell Biology (W. Deppert)
B
Journal of Hepatitis (Editorial Board, H. Will)
Oncology (W. Deppert)
Virus Genes (Editorial Board, H. Will)
Drittmittelprojekte
2007-2008
Internationale Förderung
German-Israeli Foundation
for Scientific Research
and Development (GIF)
„Structural, biophysical and functional
studies on mutant p53 and its interaction
with DNA“
Wolfgang Deppert
Tokyo Metropolitan
Organization for
Medical Research
„Risk assessment of hematopoietic stem
cell (HSC) genes for leukemia induction,
referred to „the Study“.
Carol Stocking
Europäische Förderung
Fondazione Poieo
Onkologische Adenoviren (Virobori)
177
Thomas Dobner
EU
„Mutant p53 as target for improved
cancer treatment“
Wolfgang Deppert
EU
„Manipulating tumor suppression:
a key to improve cancer treatment“
Wolfgang Deppert
EU
„Molecular signatures as diagnostic
and therapeutic targets for disseminated
epithelial malignancies“
Wolfgang Deppert
Bundesministerium
für Bildung
und Forschung
„Entwicklung und Umsetzung eines Verwertungs-
konzeptes zur Verbesserung der schutzrechtlichen
Sicherung und Professionalisierung der Verwertung
von Forschungsergebnissen am Heinrich-Pette-Institut
für Experimentelle Virologie und Immunologie (HPI)“
Bundesministerium
für Bildung
und Forschung
„Analyse der cccDNA von Hepatitis B-Viren:
Bildung, Stabilität, pathogebetische Bedeutung
und Modulation durch antivirale Medikamente“
Verbundprojekt: Kompetenznetz Infektionskrankheiten (Hep-Net)
Bundesministerium
für Bildung
und Forschung
„GO-Bio 2: Entwicklung und Kommerzialisierung
eines biotechnologischen Verfahrens zur Eradikation
proviraler HIV-1 DNA aus Patientenzellen“
Joachim Hauber
Bundesministerium
für Bildung
und Forschung
„Behandlung der chronischen myeloischen Leukämie
und HIV-Infektion: Analyse der Wirkungsweise von
elF-5A (TP1)“
Joachim Hauber
Heinrich Hohenberg
Hans Will
Drittmittelprojekte
Bundesministerium
für Bildung
und Forschung
Behörde für
Wissenschaft
und Forschung
Verbund TOMCAT – Entwicklung und Verbesserung
von spezifischen magnetischen Nanopartikel zur
Detektion maligner Tumoren, Teilvorhaben HPI
Heinrich Hohenberg
Zuwendung für die Einrichtung einer Kooperationstelle
im Leibnitz- Zentrum für Infektionsforschung (LZIF),
für die Zeit vom 01.11.2007 bis 31.03.2010
Heinrich Hohenberg
Deutsche Krebshilfe e.V. Bonn
178
„Studien zur Funktion von zellulären und viralen Proteinen
in ND-10 Kerndomänen bei akuter promyelozytischer
Leukämie und anderen maligen Erkrankungen“
Hans Will
„Funktionsanalyse von SPOC1: Ein neuer mit der
Entstehung und Progression von Ovarialkarzinom
assoziierter Transkriptionsregulator und mögliches
Therapieziel von Tumorerkrankungen“
Hans Will
„Co-Regulation invasionsassoziierter Gene humaner
Gliome durch das zelluläre Proto-Onkogen Ets-1 und
Tumorsuppressor p53“
Wolfgang Deppert
„Beteiligung von Tumorstamm-/progenitorzellen
am Aufbau ihrer zellulären Umgebung und der
metastatischen Nische“
Wolfgang Deppert
„Dissecting the mechanism of apoptosis-resistance in
hematopoietic malignancies expressing wild-type p53“
Wolfgang Deppert
„Induktion zellulärer Seneszenz in Glioblastomzellen
des Menschen“
Wolfgang Bohn
„Regulation und Funktion der Telomerase in
Mammakarzinomen im WAP-T Mausmodell“
Cagatay Günes
„Funktionelle Analyse der Rex Transaktivierung
des Humanen T Zell Leukämievierus Typ I“
„Mouse models to study the collaboration between FLT3
activation and RUNX1 mutations in AML induction“
Joachim Hauber
Carol Stocking
Drittmittelprojekte
„Mausmodelle für Untersuchungen zu FLT3-abhängigen
Signaltransferwegen und kooperierenden Onkogenen,
die für die Aufrechterhaltung einer akuten Leukämie
erforderlich sind“
Carol Stocking
„Dissecting the mechanism of apoptosis-resistance in
hematopoietic malignancies expressing wild-type p53“
Carol Stocking
Einfluss des Tumorsuppressorgens p53 auf die Regulation
der Reparatur von DNA-Doppelstrangbrüchen und die
Kopplung der Reparatur an den Zellzyklus (Folgeantrag)
Irene DornreiterNicholas
179
Deutsche Forschungsgemeinschaft
„Hepatitis B-Viren als molekulare Sonden für die
systembiologische Analyse der hepatozellulären
Differenzierung und Polarität“
Hüseyin Sirma
„Molekulare Mechanismen der infektiösen
Aufnahme und des zellulären Transports von
Hepatitis B-Viren und deren Bedeutung
für den viralen Wirts- und Lebertropismus“
Hüseyin Sirma
„Regulation und Funktion des schadeninduzierten
Polymerase alpha-Primase-wtp53 Komplexes im
ATM/ATR-vermittelten intra-S-Phasen Kontrollpunkt“
Irene DornreiterNicholas
„Analysis of the metastatic cascade using mouse
models for ductal mammary carcinoma“
Wolfgang Deppert
„Modulation of Tumor Angiogenesis by CEACAM1
in a Mouse Model for Mammary Carcinogenesis
(WAP-T Mice)“
Wolfgang Deppert
„Analysis off tumor progression and metastasis in
transgenic mouse models for SV40 induced mammary
carcinogenesis“
Wolfgang Deppert
„Funktionelle Analyse der HIV – Rev Transaktivierung“
Joachim Hauber
Drittmittelprojekte
„Inhibition des eukaryotischen Initiationsfaktors
elF-A aus Plasmodien und humanen Zellen –
Mechanismen der Apoptose-Induktion und
Zielstruktur für einen gemeinsamen Therapieansatz
zur Behandlung der Malaria und der HIV-Infektion“
„Aktivitätskontrolle und zelluläre Funktionen
von Transglutaminasen“
„Role of viral microRNAs in Kaposi`s Sarcomaassociated Herpesvirus (KSHV) infection and
KSHV-associated Disease“
180
„Molekulare Analyse der CD83 Expression in
dendritischen Zellen“
Joachim Hauber
Heinz Lother
Adam Grundhoff
Jan Chemnitz
Förderung durch Vereine und Stiftungen
„Disseminated Tumor Cells as Targets for Inhibiting
Metastasis Epithelial Tumours“
Verein zur Förderung
der Krebsforschung e.V.
Horst Müggenburg
Stiftung
Stiftung zur Bekämpfung
neuroviraler Erkrankungen
„Studien zur Tumorgenese und Metastasenbildung
beim Eierstockkrebs: Beitrag des SPOC1-Proteins“
„Virus factories: Zentrale Plattformen für
Assemblierung, Knospung und Sekretion von Viren“
„Epigenetische Inaktivierung von p53 in
Glioblastomen des Menschen“
Wolfgang Deppert
Hans Will
Hüseyin Sirma
Wolfgang Deppert
Seminarmittel und wissenschaftliche Retreats
Heinrich Hohenberg
Mittel für Heinrich Pette Lectures 2007
Heinrich Hohenberg
„Hemmung der Herpes Virus Reaktivierung durch
PML-Kerndomänen-vermittelten epigenetischen
Repression und ihre feinmorphologische Analyse.“
Heinrich Hohenberg
Hüseyin Sirma
„Funktionelle Analyse der Interaktion des sehr frühen
Herpes Simplex-Virus Regulatorproteins ICP0 mit der
zellulären E3-Ligase SIAH-1“
Joachim Hauber
„Hit & Run Transformation“
Thomas Dobner
Drittmittelprojekte
„Bedeutung der Interaktion des HIV-1 Vpr mit dem
Glukokortikoidrezeptor für die virale Replikation und
die neuronale AIDS Pathogenese“
„Induktion genomischer Instabilität als Grundlage
Virus-vermittelter Onkogenese“
Michael Schindler
Thomas Dobner
W. Sander-Stiftung
Erich und Gertrud
Roggenbuck-Stiftung
„Analyse von mutiertem p53 als Progressionsfaktor
in einem WAP-mutp53 transgenen, induzierbaren
Mausmodell für das Mammakarzinom“
„Analyse der Telomeraseregulation während der Entstehung und Progression von Mammakarzinomen“
„Adenovirus LDL-Rezeptor“
Hamburger Stiftung
zur Förderung der
Krebsbekämpfung
Deutsche José Carreras
Leukämie-Stiftung e.V.
Wolfgang Deppert
Cagatay Günes
Thomas Dobner
„Untersuchung der Funktionen des pathogenen
Potentials der microRNAs des Epstein-Barr-Virus“
Adam Grundhoff
„Synergitische Wirkung der Transkriptionsfaktoren
MEF2C und ICSBP bei der Entstehung der akuten
myeloischen Leukämie (AML)“
Carol Stocking
„Die Bedeutung der MEF2C und MEF2D Transkriptionsfaktoren bei der Entstehung der akuten Leukämie“
Carol Stocking
„Mausmodelle für die Entstehung der durch die (12;21)
Translokation verursachten akuten Iymphoblastischen
Leukämie (ALL)“
Carol Stocking
„Mausmodelle für die Entstehung der durch die (12;21)
Translokation verursachten akuten Iymphoblastischen
Leukämie (ALL)“ (Folgeprojekt)“
Carol Stocking
181
Drittmittelprojekte
Industriekooperationen
Dade Behring
Marburg GmbH
Biomedizinische
Forschungsgesellschaft mbH Wien
182
Axxima
Pharmaceuticals
ASTRAZENECA UK
Limited
FEI Company
Bal-Tec AG
Media Cybernetics,
Inc.
Point Electronics
GmbH
Nikon GmbH
Es sollen HBs-Expressionsvektoren hergestellt werden,
mit denen die mutierten HBs-Antigene der gelieferten
Hepatitis B Virus DNAs in eukaryontischen Zellen
exprimiert werden sollen.
Hans Will
„Untersuchung der subzellulären Lokalisation sehr
früher Genprodukte des humanen Herpesvirus Typ 1
und Typ 2 (Herpes-Simplex-Virus 1 und 2)“
Joachim Hauber
Zusammenarbeit auf dem Gebiet potentieller HSV
Impfantigene
Joachim Hauber
„Charakterisierung von durch Axxima ausgewählten
chemischen Wirkstoffen zur Erforschung von Möglichkeiten zur Hemmung von HIV-Infektionen und Aufklärung ihres Einflusses auf das zelluläre System; Analyse
der Apoptose ausgelöst von durch Axxima ausgewählten
chemischen Wirkstoffen in HIV-infizierten Zellen“
Joachim Hauber
„WHERAS, AstraZeneca wishes to engage in scientific
research related to the response of mutant c-Kit receptors
to the tyrosine kinase inhibitor AZD2171“
Carol Stocking
„Ausarbeitung gemeinsamer Konzepte zur „Systemischen
Mikroskopie“ inklusive der Entwicklung und Modifikation
von elektronenoptischen Abbildungssystemen“
Heinrich
Hohenberg
„Konzeption, Konstruktion und Realisation einer neuen
kompakten Hochdruckgefrierenanlage“
Heinrich
Hohenberg
Adaption des “Blind Deconvolution” – Algorithmus für
konventionelle und Environmental-REM-Daten
Heinrich
Hohenberg
„Konzeption und Realisierung neuer Chip-Detektoren
für die hochauflösende 3D Raster-Elektronenmikroskopie“.
Heinrich
Hohenberg
„Optimierung der Nikon Biostation für ein breites
Spektrum neuer biologischer Applikationen.“
Heinrich
Hohenberg
Erfindungsmeldungen
und Patente
2007
Joachim Hauber,
Ilona Hauber
„Piperidone derivatives and uses thereof“ (Europäische Patentanmeldung),
gemeinsam mit Universität Würzburg (U. Holzgrabe, J. Klöckner)
Joachim Hauber,
Ilona Hauber
„Use of a tailored recombinase for the treatment of retroviral infections“
(Europäische Patentanmeldung), gemeinsam mit MPI und Technische
Universität Dresden (F. Buchholz, I. Sarkar, F.A. Stewart)
2008
Ilona Hauber,
Heinrich Hohenberg,
Joachim Hauber
183
„Topical formulation for preventing sexual transmission of viral infection”
(Europäische Patentanmeldung)
Finanzielle Förderung
Endstand 2007
Institutionelle Förderung
Summe Drittmittel
Gesamtes HPI
184
davon:
Bereich
Grundfinanzierung
Bereich
Gebäude und Technik
Bereich
Wissenschaft
Summe Inst. Förderung
Drittmitteleinnahmen
MiGeb DFG
MiGeb Diverse
MiGeb EU
MiGeb International
MiGeb Krebshilfe
MiGeb Öffentliche
MiGeb Stiftungen
MiGeb Unternehmen
Summe Drittmittel
Einnahmen
Personalkosten
Sachkosten Investitionen
Gesamtwert
8.182.886
4.420.941
2.544.645
1.850.227
8.815.813
2.268.589 1.407.989
644.484
0
2.052.473
10.451.476 5.828.929 3.189.129 1.850.227 10.868.285
7.722.886 1.100.569
462.601
0
1.563.170
460.000 247.301
929.220
1.197.854
2.374.375
0
3.073.071
1.152.824
652.373
4.878.267
8.182.886 4.420.941
2.544.645
1.850.227
8.815.813
567.833
142.114
148.101
6.577
644.868
430.182
328.915
0
430.406
10.198
221.372
0
484.161
47.584
214.267
0
132.865
106.934
87.452
6.555
181.306
8.156
105.123
16.093
0
0
0
0
0
0
0
0
563.271
117.132
308.825
6.555
665.468
55.740
319.389
16.093
2.268.589
1.407.989
644.484
0
2.052.473
Finanzielle Förderung
Vorläufiger Endstand 2008
Institutionelle Förderung
Summe Drittmittel
Gesamtes HPI
davon:
Bereich
Grundfinanzierung
Bereich
Gebäude und Technik
Bereich
Wissenschaft
Summe Inst. Förderung
Einnahmen
Personalkosten
10.201.746
4.653.040
2.567.549
2.265.760
9.486.350
2.486.191
1.606.583
791.309
39.865
2.437.757
12.687.936
6.259.623
3.358.859
2.305.625
11.924.106
7.820.475
1.085.682
503.522
0
1.589.204
1.889.250
262.474
936.777
1.601.510
2.800.761
492.021
3.304.884
1.127.250
664.250
5.096.384
10.201.746
4.653.040
2.567.549
2.265.760
9.486.350
17.174
43.188
Drittmitteleinnahmen
Internationale
Förderungen
51.820
Europäische
Förderungen
308.056
Bundesförderungen
605.039
Deutsche Forschungsgemeinschaft SPP
82.700
Deutsche Forschungsgemeinschaft
493.794
Deutsche Krebshilfe
495.350
Josè Carreras Stiftung
Wilhelm SanderStiftung
65.200
Förderung durch
weitere Stiftungen
210.850
Industriekooperationen
480
diverse Mittelgeber
172.902
Summe Drittmittel
2.486.191
26.014
174.293
254.094
Sachkosten Investitionen
39.865
237.768
557.296
63.152
29.598
92.750
375.798
457.953
87.063
125.574
142.388
22.385
501.372
600.342
109.448
22.055
10.692
32.747
110.424
19.451
16.284
59.901
8.126
48.659
170.326
27.577
64.943
1.606.583
63.475
263.337
Gesamtwert
791.309
39.865
2.437.757
185
Seminare
11.01.07 Michael Schwarz, Institut für Pharmakologie und Toxikologie der
Universität Tübingen, „Genotype-phenotype relationships in normal
liver and liver cancer“
18.01.07 Marc Sitbon, Institut de Genetique Moleculaire de Montpellier CNRS
UMR 5535, Montpellier, Frankreich, „HTLV-1 tropism and envelope
receptor“
25.01.07 Martin Eilers, Institute for Molecular Biology and Tumor Research
(IMT), Universität Marburg, „Control of cell proliferation and growth
by myc proteins“
186
01.02.07 Felix Wieland, Biochemie-Zentrum der Universität Heidelberg (BZH),
„Formation of a transport vesicle: players and rules“
08.02.07 Thomas von Zglinicki, Cellular Gerontology, University of Newcastle,
School of Clinical Medical Sciences, Henry Wellcome Laboratory for
Biogerontology Research, England, „Telomeres and mitochondria:
Strange bedfellows in senescence“
02.03.07 Markus Manz, Institute for Research in Biomedicine (IRB), Bellinzona,
Schweiz, „Human immune system Rag2-/-γc-/- mice: New options to
study human lymphotropic viruses in vivo“
18.04.07 Thomas Sternsdorf, The Salk Institute for Biological Studies, La Jolla,
California, „Artificial oncogenes as tools to dissect leukomogenesis“
19.04.07 Umberto Galderisi, Dept. Experimental Medicine, Second University of
Naples, Napoli, Italy, „Cell cycle regulation in the biology of mesenchymal stem cells“
10.05.07 Nicole Fischer, Institut für Med. Mikrobiologie, Virologie und Hygiene,
UKE Hamburg, „Virus detection and discovery using DNA microarrays“
30.05.07 Michael Schindler, Institut für Virologie, Ulm, „The role of Nef in
primate lentiviral pathogenesis“
Xavier Dervillez, Laboratoire de Biologie et Thérapeutique des Pathologies Immunitaires, Hopital Pitié, Paris, „New vaccine strategies
against HIV/AIDS: Development of (i) peptide or (ii) VLP-based multiepitope vaccine candidates, expressing a hybrid FeLV-A p15E/HIV-1
gp41 MPER retroviral envelope“
14.06.07 Thomas Hofmann, Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, „HIPK2: an emerging regulator of DNA damage signaling“
21.06.07 Michael Nevels, Institut für Medizinische Mikrobiologie und Hygiene,
Universität Regensburg, „Epigenetic control of human cytomegalovirus
infection“
28.06.07 Herbert Tschochner, Lehrstuhl Biochemie II, Universität Regensburg,
„Aspects of ribosome biosynthesis in eukaryotes“
Seminare
05.07.07 Stefan Müller, Max-Planck-Institute of Biochemistry, Dept. Molecular
Cell Biology, München, „The SUMO system in the control of cell cycle
progression and cell division“
12.07.07 Christoph Englert, Leibniz-Institut für Altersforschung – FritzLipmann-Institut, Jena, „The Wilms tumor suppressor Wt1 in urogenital development: Insights from mice and zebrafish“
19.07.07 Rolf Renne, University of Florida, Dept. of Molecular Genetics and
Microbiology, Gainesville, FL, U.S.A., „Kaposi´s sarcoma-associated
herpesvirus-encoded microRNAs and their potential role in pathogenesis“
14.09.07 Urs Greber, Universität Zürich, Zoologisches Institut, „Computational
systems analyses of adenovirus trafficking“
20.09.07 Larry Gerace, Depts. of Cell and Molecular Biology, The Scripps
Research Institute, La Jolla, U.S.A., „The nuclear lamina and signaling
during myogenic differentiation“
27.09.07 Stefan Heinrichs, Dept. of Pediatric Oncology, Dana-Farber Cancer
Institute, Boston, MA, U.S.A., „Identification of Structural Aberrations
in Cancer by SNP Array Analysis“
01.10.07 Helga Hofmann, Dept. of Pediatric Oncology, Dana-Farber Cancer
Institute, Boston, MA, U.S.A., „Identification of Structural Aberrations
in Cancer by SNP Array Analysis“
11.10.07 Wulf Schneider, Institut für Medizinische Mikrobiologie und Hygiene,
Universität Regensburg, „Adenoviral inhibition of TNF-r1-induced
apoptosis signaling“
29.11.07 Stefan Müller, Max-Planck-Institute of Biochemistry, Dept. Molecular
Cell Biology, München, „Ten years of SUMO-ubiquitin´s cousin“
17.12.07 Marta Szaszak, Leibniz-Institut für Molekulare Pharmakologie (FMP),
Campus Berlin-Buch, „Intracellular trafficking of the angiotensin AT
1-receptor“
24.01.08 Walter Birchmeier, Max-Delbrueck-Center for Molecular Medicine
(MDC), Berlin, „Wnt/beta-catenin signals in development and cancer“
31.01.08 Jo Milner, YCR P53 Research Group, Department of Biology, York,
England, „Reciprocal regulation between p53 and SIRT1“
07.02.08 Ivo P. Touw, Experimental Hematology, Erasmus University Medical
Center, Rotterdam, Niederlande, „Granulocyte colony-stimulating
factor and its receptor in normal myeloid cell development, leukemia
and related blood cell disorders“
14.02.08 Andreas Meyerhans, Institut für Virologie, Universität des Saarlandes,
Homburg/Saar, „A microscopic and macroscopic view on viral infections“
187
Seminare
15.02.08 Juana Diez, Virology Group, Department of Experimental and Health
Sciences, Universitat Pompeu Fabra, Barcelona, Spain, „Effect of P-body
components in positive strand DNA virus replication“
15.02.08 Kevin V. Morris, Dept. Molecular Medicine, The Scripps Research
Institute, La Jolla, CA, USA, „Small RNA mediated transcriptional regulation of gene expression in human cells“
21.02.08 Mukesh Kumar, Dept. Plant Molecular Biology, MFPL, Wien, Österreich,
„Arabidopsis Heat Shock Factors: Understanding their Function“
06.03.08 Alex Matter, Novartis Institute for Tropical Diseases, Singapore, „New
drugs for neglected diseases“
188
03.04.08 Jochen Dahm-Daphi, Institut für Strahlenbiologie und Experimentelle
Radioonkologie, Universitätsklinikum Hamburg-Eppendorf, „Regulation of DNA Double-Strand Break Repair“
10.04.08 Andreas Möller, Peter MacCallum Cancer Centre, Melbourne, Australien,
„Siah regulates hypoxia signalling in cancer and inflammation“
17.04.08 Tristam Parslow, Emory University, Dept. of Pathology and Laboratory
Medicine, Atlanta, GA, U.S.A., „Packaging the genome of influenza virus“
15.05.08 Bernard P. Mahon, Institute of Immunology, National University of
Ireland Maynooth, „Mesenchymal stem cells, overcoming immunological barriers to therapeutic application“
22.05.08 Roger D. Everett, MRC Virology Unit, Glasgow, England, „STAT-1 and
IRF-3 dependent pathways are not essential for repression of ICP0-null
mutant herpes simplex virus type 1 in human fibroblasts“
12.06.08 Alexander Strom, Chronic Disease Program, Ottawa Health Research
Institute (OHRI) Ottawa, Ontario, Canada, „Diabetes meets mad cow
disease: Pancreatic ß-cells and cellular prion protein“
26.06.08 Barbara Schmidt, Institut für Klinische und Molekulare Virologie, Universität Erlangen-Nürnberg, „Plasmacytoid dendritic cell (PDC) innate
immune defense against HIV“
01.07.08 Dean Jackson, Faculty of Life Science, Manchester, UK, „Using advanced imaging techniques to explore nuclear structure and genome stability in mammalian cells“
03.07.08 Albert Heim, Medizinische Hochschule Hannover, Abt. für Virologie,
„Disseminated adenovirus infections of immunosuppressed patients:
Molecular virology and pathophysiology“
17.07.08 Gunhild Sommer, Medical University of South Carolina, Biochemistry
& Molecular Biology, Charleston, South Carolina, USA, “The fusion
protein OTTMAL induces R-loop formation and genomic instability“
Seminare
24.07.08 Tilman Heise, Medical University of South Carolina, Department of
Biochemistry & Molecular Biology, Charleston, South Carolina, USA,
„Translation elongation of cyclin D1 requires an RNA chaperone“
25.07.08 Stuart Le Grice, RT Biochemistry Section, HIV Drug Resistance Program,
National Cancer Institute – Frederick, U.S.A., „Reverse transcriptaseassociated ribonuclease H – from single molecules to crystallization“
04.08.08 Rajesh K. Gaur, Division of Molecular Biology, Graduate School of
Biological Sciences, Beckman Research Institute of the City of Hope,
Duarte, CA, U.S.A., „Modulation of alternative splicing: a potential
therapy for cancer and other diseases“
18.09.08 Harald Wodrich, Institut de Genetique de Montpellier, MontpellierCedex, Frankreich, „Protein VI hires an ESCRT service for adenovirus
during entry“
08.10.08 Wolfgang Kemmner, Max-Delbrück-Center for Molecular Medicine,
Charité, Campus Buch, „Microarray examination of a transgenic mouse
model for the ductal carcinoma in situ (DCIS) of the mammary gland“
13.11.08 Jan Münch, Institut für Virologie, Universitätsklinikum Ulm, „Natural
inhibitors and enhancers of HIV infection“
25.11.08 Michael Nevels, Universität Regensburg, Institut für Medizinische
Mikrobiologie und Hygiene, Regensburg, „Innate, Intrinsic and
Epigenetic Control of Human Cytomegalovirus Infection“
Renate König, Infectious and Inflammatory Disease Centre, Burnham
Institute for Medical Research, La Jolla, CA, USA, „HIV-host interactions – discovering host factors by high-throughput systems-based
analysis“
Gülsah Gabriel, University of Oxford, Sir William Dunn School of
Pathology, Oxford – United Kingdom, „Adaptation of a highly pathogenic avian influenza virus to the mammalian host: virus-host interactions“
Luka Cicin-Sain, Vaccine and Gene Therapy Institute, Oregon Health
and Science University, Beaveron, OR, USA, „Environmental contributors to immune aging“
03.12.08 Christine Stahl-Hennig, Deutsches Primatenzentrum Göttingen – DPZ,
„Animal Models for Lentivirus Infection“
04.12.08 Ludger Hengst, Division of Medical Biochemistry Biocenter, Innsbruck
Medical University, „CDK-inhibitors – connecting signal transduction
and cell cycle control“
11.12.08 Peter Hemmerich, Leibniz-Institut für Altersforschung-Fritz-LipmannInstitut e.V., Jena, „Superresolution microscopy of PML nuclear body
assembly“
189
Mitglieder des
Wissenschaftlichen Beirats
2007-2008
Name
Institution
Amtszeit
Prof. Dr. Hans Wolf
(Vorsitzender)
Institut für med. Mikrobiologie
und
Hygiene, Universität
Regensburg, Deutschland
Seit 2003
Prof. Dr. Thomas F.
Schulz
(stellv. Vorsitzender)
Institut für Virologie,
Medizinische Hochschule
Hannover, Deutschland
Seit 2008
Prof. Dr. Ueli Aebi
Biozentrum M.E. MüllerInstitut Universität Basel/
Schweiz
Seit 2003
Prof. Dr. J. Valerie
Bosch
ATV FO200, Deutsches
Krebsforschungszentrum
Heidelberg/Deutschland
Seit 2003
Dr. Roger Everett
MRC Virology Unit,
University of Glasgow, Scotland
UK
Seit 2008
Prof. Dr. Wolfram
Gerlich
Institut für Medizinische
Virologie, FB 20,
Universitätsklinikum
Giessen, Deutschland
20.03.2003 - 19.03.2007
Dr. Dean A. Jackson
University of Manchester Dept.
Of Biomoleculare Sciences/UK
20.03.2003 - 19.03.2007
190
Neubestellung
20.03.2007 - 19.03.2011
MD, PhD Tristram
G. Parslow
Microbial Pathogenesis
HIV Pathogenesis,
Pathology and Laboratory
Medicine, University
Hospital Atlanta, USA
23.01.2008 - 22.01.2012
Prof. Dr. med. Axel
Rethwilm
Universität Würzburg,
Institut für Virologie und
Immunologie, Deutschland
20.03.2003 - 19.03.2007
(fettgedruckte Namen markieren aktuelle Mitglieder des Wissenschaftlichen Beirats)
Mitglieder des Kuratoriums
2007-2008
Name
Institution
Amtszeit
Wilfried Mohr
(Vorsitzender)
Medac GmbH, Hamburg
Seit 2003
Senatsdirektor Gerhard
Schneider (Stellv.
Vorsitzender)
Behörde für Wissenschaft und
Forschung (BWF), Hamburg
2003 - 2007
Leitender Regierungs
direktor Dr. Rolf Greve
(Stell. Vorsitzender)
Behörde für Wissenschaft und
Forschung (BWF), Hamburg
2008 - 2011
Prof. Dr. Hans Wolf
Institut für Medizinische
Mikrobiologie und Hygiene,
Regensburg
Seit 2003
Prof. Dr. Michael
Kramer
Bundesministerium für
Gesundheit (BMG), Bonn
2003 - 2007
PD Dr. Lars Schaade
Bundesministerium für
Gesundheit (BMG), Berlin
Seit 2007
Regierungsdirektor
Lutz Klinghammer
BMG, Berlin
2006 - 2007
Ministerialrätin
Maria Becker
BMG, Berlin
Seit 2007
Dr. Ingeborg
Kirchhoff
Behörde für Soziales,
Familie, Gesundheit und
Verbraucherschutz (BSG),
Hamburg
Seit 2006
Regierungsdirektor
Frank Laubert
Universität Hamburg
2003 - 2008
Regierungsdirektor
Dr. Harald Schlüter
Universität Hamburg
Seit 2008
Prof. Dr. Nikolaus
MüllerLantsch
Universität des
Saarlandes, Homburg
2005 - 2008*
Prof. Dr. Hans Peter
Mühlbach
Institut für Botanik, Universität
Hamburg
2003 - 2008*
(fettgedruckte Namen markieren aktuelle Mitglieder des Kuratoriums)
191
Kaufmännische Abteilung
Leiter: Dr. Volker Uhl
Die Kaufmännische Abteilung hat in den vergangenen Jahren einen umfassenden
Wandel der personellen und organisatorischen Struktur vollzogen.
Der eingeschlagene Weg der Ausgliederung administrativer Prozesse im Personal
bereich ist in 2008 weiter entwickelt worden. Danach wird Anfang 2009 das
Vertragsmanagement komplett durch einen externen Dienstleister übernommen,
wodurch eine weitere Stelle in dieser Arbeitsgruppe eingespart werden kann.
192
Nachdem die umfangreichen Neu- und Umbauprojekte zwischen 2006 und 2008
von den Mitarbeitern der Technik realisiert werden konnten, wird in den nächs
ten Jahren das Bestandsmanagement in den Vordergrund treten. Die natürliche
Fluktuation aufgrund von Ruhestandsregelungen, wird für eine Neuorganisation
dieses Bereichs genutzt werden können. Konkret wird hier demnach von den zwei
frei werdenden Stellen nur eine neu besetzt. Zur Verstärkung des Technikerteams
ist geplant, eine Fachkraft aus dem Bereich Heizungs- und Installationstechnik
einzustellen.
Die Dimensionierung der Arbeitsgruppe Finanz- und Rechnungswesen mit vier
qualifizierten Fachkräften entspricht den institutsspezifischen Arbeitsanforde
rungen. Dabei wurde das Team in 2007 und 2008 durch einen externen Wirt
schaftsprüfer und Steuerberater unterstützt, der für Beratungs- und Fachaufgabe
flexibel eingesetzt werden kann. Sowohl die Prüfungen des Finanz- und Rechnungswesens durch behördliche Einrichtungen als auch durch externe Wirtschaftsprüfer haben zu keinen Beanstandungen geführt.
Das in den letzten beiden Jahren gebildete Einkaufsteam hat sich in seiner personellen Zusammensetzung bewährt und eine hohe Akzeptanz in den Forschungsabteilungen erreicht. Eine Mitarbeiterin des Einkaufsteams wird zukünftig im
Vertretungsfall die Personalbetreuung übernehmen.
Die neu geschaffene Stelle des System-Administrators wird in 2009 auch verstärkt
für die Unterstützung der wissenschaftlichen EDV im HPI eingesetzt werden.
Dadurch kann eine weitere Personalaufstockung in der wissenschaftlichen EDV
durch hausinterne Ressourcen abgedeckt werden. Die Stelle eines Auszubildenden soll zum Herbst 2009 besetzt werden. Insbesondere der Aufbau eines EDVgestützten Personalcontrollingsystems zur Steuerung des Stellenplans und des
Budgets haben die Planungssicherheit im kaufmännischen Bereich erheblich verbessert.
Die Mitarbeiterin des Eingangsbereichs und der Warenannahme wird auch nach
ihrer Verrentung weiter für das HPI tätig sein, so dass keine weitere Stelle für
diese Funktion zur Verfügung gestellt werden muss. Dadurch kann ab Mitte 2009
zusätzlich eine Tierpflegerin eingestellt werden.
Der Umbau der Kaufmännischen Abteilung in Richtung einer serviceorientierten
Dienstleistungseinrichtung ist weitgehend abgeschlossen.
Personal der Kaufmännischen
Abteilung und Allgemeine Dienste
Kaufmännische Abteilung
193
Leitung
Sekretariat
Dr. Volker Uhl
Kira Feldhinkel
Dorothea Stille*
Finanz- und
Rechnungswesen
Meike Alisch
Sylvia Bräu
Helena Kuhn
Lilia Spanagel
Personalabteilung
Kristin Gessler
Jörg Schinkel
Einkauf
Ingo Jackstien
Katja Krauß
Nina Lenk
Petra Plarre
EDV
Heiko Juretzka
Wissenschaftliche Stabsstellen
194
EDV
Großgeräte
Presse- und
Öffentlichkeitsarbeit
Verstandsreferenz
und LCI-Koordination
Norbert Zangenberg, Dr. rer. nat.
Arne Düsedau, Dipl.-Ing.
Angela Homfeld, Dr. rer. nat.
Nicole Nolting, Dr. rer. nat.
Technische Dienste
Leitung
Mitarbeiter
Carl-Heinz Lohm
Ingo Gregorzewski
Michael Habenicht
Wolfgang Möller
Olaf Nehls
Tierhaltung
195
Hasso Mündt
Anke Dorendorf
Beate Miksche
Matthias Timmermann
Spülküchen
Radica Karalic
Svetlana Dragicevic-Slankamenac
Irmgard Haye
Margarete Houillon
Radmilla Kiel
Maria-Cristina Moreno
Monika Müller
Semsa Reus
Beauftragte 2007-2008
• Bestrahlungsgerät LISA
• Bibliothek
• Biolog. Sicherheit
• Datenschutz
• Entsorgung
• Gleichstellung
196
• Großgeräte
• Hygiene Beauftr. S-3
• Isotopenkoordinator
• Laserschutz
• Ombudspersonen
• S 3-Labor
• Seminar
• Sicherheit
• Spülküche
• Tierhaltung
• Tierschutz
Günes, Cagatay
Bohn, Wolfgang
Stellv.: Hintz-Malchow, Martina
Grundhoff, Adam
Stellv.: Hauber, Ilona
Düsedau, Arne
Dornreiter, Irene
Iwanski, Alicja
Stellv.: Neumann, Ute
Düsedau, Arne
Hauber, Joachim
Tessmer, Uwe
Stellv.: Wiegers, Klaus-Jochen
Düsedau, Arne
Stocking, Carol
Stellv.: Hohenberg, Heinrich
Hauber, Ilona
Bohn, Wolfgang
Tryba, Martin
Stellv.: Nehls, Olaf
Bergholz, Ursula
Stocking, Carol
Stellv.: Müller, Ursula
Bruns, Michael
Technische Dienste
C.-H. Lohm
Einkauf
K. Krauß
I. Jackstien
Finanz- und
Rechnungswesen
S. Bräu
Personal
J. Schinkel
Leiter
Dr. V. Uhl
Kaufmännische
Abteilung
Öffentlichkeitsarbeit
Dr. A. Homfeld
Vorstandsreferentin
Dr. N. Nolting
Stabsstellen
Kuratorium
Vorstand
Leiter
Dr. C. Stocking-Harbers
Molekulare
Pathologie
Forschungsgruppe
Leiter
Prof. Dr. J. Hauber
Zellbiologie
und Virologie
Abteilung
Kollegium
Vorsitzender
O. Nehls
Betriebsrat
Leiter
Prof. Dr. W. Deppert
Tumorvirologie
Abteilung
Stv. Vorsitzender
Dr. M. Schindler
Vorsitzender
Prof. Dr. T. Dobner
A. Iwanski
Gleichstellungsbeauftragte
Organigramm, Stand: Dezember 2008
Leiter
Dr. M. Schindler
Leiter
PD Dr. H. Lother
Leiter
Dr. H. Hohenberg
Leiter
Dr. A. Grundhoff
Somatische
Stammzellgenetik
ElektronenMikroskopie und
Mikrotechnologie
VirusPathogenese
Forschungsgruppe
Forschungsgruppe
Zelluläre
Virusabwehr
Leiter
Prof. Dr. H. Will
Leiter
Prof. Dr. T. Dobner
Nachwuchsgruppe
Allgemeine
Virologie
Molekulare
Virologie
Nachwuchsgruppe
Abteilung
Abteilung
Abteilungen, Forschungs- und Nachwuchsgruppen
Stv. Kaufmännischer Leiter
J. Schinkel
Stv. Wissenschaftl. Direktor
Prof. Dr. T. Dobner
Vorsitzender
Prof. Dr. H. Wolf
Kaufmännischer Leiter
Dr. V. Uhl
Wiss. Beirat
Wissenschaftlicher Direktor
Dr. H. Hohenberg
Vorsitzender
W. Mohr
197
Laborküche
W. Möller
R. Karalic
Zentrale/Empfang
P. Plarre
Großgeräte
A. Düsedau
Tierhaltung
H. Münd
EDV
Dr. N. Zangenberg
H. Juretzka
Biologische
Sicherheit
Dr. A. Grundhoff
Allgemeine
Dienste
Gemälde von Edith Pette, das neben dem Abbild ihres Mannes hängt.
Beide Gemälde schmücken nun das Foyer des HPI im neuen Ersatzund Erweiterungsbau II.
Impressum
Verantwortlich
für den Inhalt
Dr. Heinrich Hohenberg
Redaktion
Dr. Angela Homfeld
Dr. Nicole Nolting
Grafik & Layout
AlsterWerk MedienService GmbH
Hamburg
Druck
Hartung Druck + Medien GmbH
Hamburg
Titelbild
Neu gestaltete Fassade
des Seuchenlaborgebäudes
Tätigkeitsbericht
Tätigkeitsbericht 200 7/ 2008
2007/2008
Stiftung bürgerlichen Rechts
Tel.: +49 (0) 40 480 51-0 · Fax: +49 (0) 40 480 51-103
hpi@hpi.uni-hamburg.de · www.hpi-hamburg.de

Activity Report 2007/08 - Heinrich-Pette

Transcription

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