Conformationally Complex Epitope on Glycoprotein H
Transcription
Conformationally Complex Epitope on Glycoprotein H
JOURNAL OF VIROLOGY, May 1991, p. 2393-2401 Vol. 65, No. 5 0022-538X/91/052393-09$02.00/0 Copyright © 1991, American Society for Microbiology Characterization and Sequence Analyses of Antibody-Selected Antigenic Variants of Herpes Simplex Virus Show a Conformationally Complex Epitope on Glycoprotein H U. A. GOMPELS,t* A. L. CARSS, C. SAXBY, D. C. HANCOCK,: A. FORRESTER, AND A. C. MINSON Division of Virology, Department o Pathology, Cambridge University, Cambridge CB2 IQP, United Kingdom Received 2 November 1990/Accepted 30 January 1991 penetrate cells (8), while a temperature-sensitive gH mutant, tsQ26, at the nonpermissive temperature produces extracellular virus which lacks gH and is rendered noninfectious (17). gB has been extensively studied by use of temperaturesensitive mutants, antibody-resistant mutants, and insertion or deletion mutants (8, 9, 16, 25, 26, 31, 45, 48). Results indicate a role in virus penetration involving fusion (8, 9), although possible interactions with other glycoproteins during the adsorption process have been suggested (32, 45). In contrast, less is known concerning the function of gH as this glycoprotein has been defined only relatively recently (7, 22, 42). Furthermore, unlike gB or gD, few gH-specific monoclonal antibodies (MAbs) have been generated for the study of gH function (20, 22). However, the pronounced biological properties of gH-specific antibodies on virus replication in vitro suggest that gH is at least as important as gB in the process of virus entry. Like some antibodies specific for the nonconserved HSV type 1 (HSV-1) gD, the MAbs to gH strongly neutralize virus infectivity in the absence of complement and can inhibit cell fusion by syncytial virus strains Herpes simplex virus (HSV) encodes at least seven virion glycoproteins, gB, gC, gD, gE, gG, gH, and gI, which are located on both the virion and the infected cell surface where they can act to mediate infectivity (1, 3, 22, 29, 35, 47, 53). Glycoproteins homologous to HSV gB and gH have been identified in every herpesvirus examined including other human herpesviruses representing the a, 1B, and y subgroups-varicella-zoster virus, human cytomegalovirus (HCMV), and Epstein-Barr virus, respectively (13-15, 20, 36). Both gB and gH appear to be essential for virus replication; only the nonconserved gD has similar properties (34). A gB-null virus has been shown to bind to but not * Corresponding author. t Present address: Department of Medicine, Addenbrooke's Hospital, Level 5, Cambridge University, Hills Road, Cambridge CB2 2QQ, United Kingdom. t Present address: Biochemistry of the Cell Nucleus Laboratory, Imperial Cancer Research Fund Laboratories, Dominion House, Bartholomew Close, London EClA 7BE, United Kingdom. 2393 Downloaded from http://jvi.asm.org/ on January 21, 2015 by guest Thirteen antigenic variants of herpes simplex virus which were resistant to neutralization by monoclonal antibody 52S or LP11 were isolated and characterized. The antibodies in the absence of complement potently neutralize infectivity of wild-type virus as well as inhibit the transfer of virus from infected to uninfected cells ("plaque inhibition") and decrease virus-induced cell fusion by syncytial strains. The first variant isolated arose in vivo. Of 66 type 1 isolates analyzed from typing studies of 100 clinical isolates, one was identified as resistant to neutralization by LP11 antibody. The glycoprotein H (gH) sequence was derived and compared with those of wild-type and syncytial laboratory strains SC 16, strain 17, and HFEM. The sequences were highly conserved in contrast to the diversity observed between gH sequences from herpesviruses of different subgroups. Only four coding changes were present in any of the comparisons, and only one unique coding change was observed between the laboratory strains and the clinical isolate (Asp-168 to Gly). These sequences were compared with those of antigenic variants selected by antibody in tissue culture. Twelve variants were independently selected with antibody LP11 or 52S from parent strain SC16 or HFEM. For each variant, the gH nucleotide sequence was derived and a point mutation was identified giving rise to a single amino acid substitution. The LP11-resistant viruses encoded gH sequences with amino acid substitutions at sites distributed over one-half of the gH external domain, Glu-86, Asp-168, or Arg-329, while the 52S-resistant mutant viruses had substitutions at adjacent positions Ser-536 and Ala-537. One LP11 mutant virus had a point mutation in the gH gene that was identical to that of the clinical isolate, giving rise to a substitution of Asp-168 with Gly. Both LP11 and 52S appeared to recognize distinct gH epitopes as mutant virus resistant to neutralization and immunoprecipitation with LP11 remained sensitive to 52S and the converse was shown for the 52S-resistant mutant virus. This is consistent with previous studies which showed that while the 52S epitope could be formed in the absence of other virus products, virus gene expression was required for stable presentation of the LP11 epitope, and for transport of gH to the cell surface (Gompels and Minson, J. Virol. 63:4744-4755, 1989). All mutant viruses produced numbers of infectious particles that were similar to those produced by the wild-type virus, with the exception of one variant which produced lower yields. The antibody-resistant phenotype of the LP11 mutant viruses could be partially complemented by growth or titration on wild-type gH-producing cell lines, but the mutants differed in decreased resistance to plaque inhibition or neutralization of infectivity. Taken together, these results show that gH has a conformationally complex epitope which is part of a domain which has an essential function in virus infectivity and spread. This epitope is not stable when gH is expressed in the absence of virus infection, and this epitope may be differentially presented at the infected cell surface or on the viral envelope. 2394 GOMPELS ET AL. MATERIALS AND METHODS Antibodies. Anti-HSV-1 gH MAbs 52S (49) and LP11 (7) used as hybridoma supernatants or ascites fluid as described previously (22, 23). Hybridoma supernatants were concentrated 15 times by using a Centriprep 30 concentrator (Amicon, Danvers, Mass.) and supplemented with 0.02% azide. Viruses and cells. BHK, Vero, and HEp-2 cells were grown in Glasgow modified Eagle medium supplemented with 10% tryptose phosphate broth and 10% newborn calf serum. Cell lines F6 and B1.3 are Vero cells which have stably integrated multiple copies of the HSV-1 gH coding region under the control of the early gD promoter. The gH gene is expressed only after superinfection with virus, and levels of gH produced exceed that achieved during highmultiplicity infections analyzed 16 to 20 h postinfection. The cell lines were derived by cotransfection of the gH gene with the neomycin resistance gene followed by selection with the drug G418 (Geneticin; GIBCO) and will be described further elsewhere. The virus strains used were HSV-1 HFEM (56), SC16 (27), and 103/65 (6). Virus stocks were prepared by infections at a multiplicity between 0.01 and 0.1 PFU per cell of HEp-2 cells, and titers were determined by assay on BHK and Vero cells. HSV-1 103/65 is a clinical isolate which is resistant to the effects of MAb LP11 (6). Virus neutralizations. Dilutions of virus in a 100-,u volume were mixed with 5 pul of antibody and incubated at room temperature for 1 h before infection. Virus was then adsorbed to monolayers of cells for 1 h at 37°C and then washed twice with medium and overlaid with fresh medium with or without antibody diluted 1:200 and incubated for 2 were days at 37°C. Selection of antibody-resistant mutant virus. Antibodyresistant mutant viruses VAI, VAII, NillA, and NillB were selected independently by prior incubation of virus with antibody followed by infection of HEp-2 cells. A total of 106 PFU of HSV-1 strain HFEM were incubated with antibody LP11 as ascites diluted 1:20 for 1 h at room temperature and then mixed with 1 ml of medium and allowed to adsorb to monolayers of 5 x 106 HEp-2 cells for 1 h at 37°C. The inoculum was removed, and the cells were washed twice with 5 ml of medium and then overlaid with 5 ml of fresh medium and incubated at 37°C for 2 days. The virus was harvested and titers were determined in the presence or absence of antibody. This procedure was repeated until the virus population gave a similar titer in the presence or absence of antibody (three to four rounds of selection) and then was repeated twice more. Antibody-treated virus was cloned twice by limiting dilution. Antibody-resistant mutant viruses AD1, NiPIIIA, NiPIIIB, LP11A, and LP11B were independently selected as above except that antibody LP11 diluted 1:200 was also included in the medium overlay after virus neutralization and adsorption. Mutants LP11A and LP11B were derived from strain SC16. The virus populations thus obtained were completely resistant to infectivity neutralization by antibody after one round of selection. The procedure was repeated before virus was cloned twice as described above. Similarly, antibody-resistant mutant viruses 52SR, 5RA, and SRB were independently selected and cloned in the presence of 52S antibody before and during infection. Mutants 52SR and SRB were selected from strain HFEM, and mutant 5RA was selected from SC16. For each selection, 106 PFU of virus were incubated with an equal volume of 52S hybridoma supernatant. Antibody was included in the medium at a 1:20 dilution. Two additional VAI mutant viruses were selected from HFEM and SC16 parent strains by cotransfection of plasmid DNA containing the VAI mutant gH gene together with infected cell DNA of wild-type HFEM or SC16, using the calcium coprecipitation method in BES buffer (25 mM BES, 140 mM NaCl, 0.75 mM Na2HPO4) as described previously (23). After virus was harvested, antibody-resistant virus was selected and cloned as detailed above. Immunoprecipitations. HEp-2 cells were infected with each virus at a multiplicity of infection (MOI) of 5 PFU per cell. After 4 h, cells were labeled with [35S]methionine (800 Ci/mmol; Amersham) at 25 ,uCi/ml in methionine-free medium containing 1% fetal calf serum by incubation for a further 12 h at 37°C. Cells were lysed in immunoprecipitation buffer and incubated with antibody and then protein A-Sepharose, and immunoprecipitation products were separated on a 10% polyacrylamide gel with sodium dodecyl sulphate (SDS). The gel was then dried down and exposed to film as described previously (23). Plasmid clones. Infected cell DNA was extracted from proteinase K-SDS lysates with phenol and chloroform as described previously (22). The Bgl m fragment from each infected cell DNA was purified and ligated with BglIIdigested and phosphatased plasmid vector PKC7 (22). Preparations of plasmid DNA were made by the alkaline-SDS method (5). Derivation of nucleotide sequence and analyses. The nucleotide sequence of the gH gene for each virus was determined by the Sanger dideoxy-chain termination method from sequences cloned into bacteriophage M13mpl8 (38) by using methods detailed by Bankier and Barrell (2) and 14 synthetic 17-mer primers from the HFEM BglII m sequence (22) spanning the gH sequence at the following positions from the BglII recognition sequence: 1435, 1626, 1728, 1904, 2044, 2240, 2435, 2647, 2841, 2978, 3228, 3456, 3676, and 3837. Nru Downloaded from http://jvi.asm.org/ on January 21, 2015 by guest (7, 22, 40, 42, 43). Efficient neutralization of infectivity of HCMV or Epstein-Barr virus has also been achieved with antibodies specific for the respective gH homologs (reviewed in reference 20). One of these MAbs specific for EpsteinBarr virus gH appears to prevent fusion rather than adsorption (39), and a similar result has been recorded for interactions of HSV antibodies with HSV gH (19). Unlike most other neutralizing antibodies, MAbs to HSV-1 gH also inhibit the transfer of virus from infected to uninfected cells during plaque formation by both syncytial and nonsyncytial strains. It has been proposed that the interaction of antibody with gH expressed at the cell surface prevents its function in mediating cell-to-cell spread through formation of intercellular bridges. Studies of gH in mammalian cell expression systems have shown that gH requires interactions with other virus products for cell surface localization and formation of an antigenic structure important for its function in mediating infectivity (23). To identify protein domains involved in functions of gH, we are studying interactions of HSV-1 mutants with the type 1-specific MAbs 52S and LP11, which neutralize both infectivity and cell-to-cell spread. MAb 52S recognizes gH expressed in the absence of other virus proteins, whereas MAb LP11 only recognizes gH in the presence of other virus proteins (23). In the present study, 13 antibody-resistant mutants independently selected in the presence of either antibody were characterized. We show that LP11 recognizes a conformationally complex epitope in gH which spans one-half of the external domain. J. VIROL. VOL. 65, 1991 ANTIBODY-RESISTANT MUTANTS OF HSV-1 gH CD In ,O %a % 40 u, _ V v. . I I I I HFEM CT C GA SC16 f c f I 4.n C .n '0 N in m0 _ . (,.- 0Wp in r~-_ -0'~ a r- a 2395 4 O) I I I v i rus 17 i c 103/65 C R R T I G C iT G C I Q G C I Gi C G C G CC Q T T T T R C C G R R P RT D R A T SR 6 U T FIG. 1. Comparisons of the gH nucleotide sequence from different HSV strains. Nucleotide sequence was derived for the gH gene from each strain. Comparisons were made to the strain HFEM nucleotide sequence and encoded amino acid sequence. Coding differences are underlined. Nucleotide and amino acid sequences are numbered from the initiating codon. Strain 17 and HFEM sequences are from references 37 and 22, respectively. A, Alanine; V, valine; P, proline; T, threonine; D, aspartic acid; G, glycine. subfragments of 2.3 and 2.0 kb were purified from plasmid DNA and ligated with SmaI-digested and phosphatased bacteriophage M13mpl8 DNA. The sequence was then determined by using universal and synthetic primers with 35S-dATP (Amersham) as a radioactive label, and the products were fractionated by Tris-borate-EDTA buffer gradient electrophoresis (4). The reaction products obtained with each individual chain-terminating dideoxynucleotide were electrophoresed in parallel to allow rapid and accurate identification of any nucleotide substitution. RESULTS Strain variation of HSV-1 gH sequences. The gH gene is a member of the subset of conserved herpesvirus genes. As determined by comparisons of encoded amino acid sequences, it is apparent that the homologous gH sequences from different herpesvirus subgroups (a, or -y) form one of the most divergent of these related protein families (15, 20, 21). To further examine this divergence and as a starting point to evaluate functional domains and the relevance of mutations which arise during selection with neutralizing antibody, we investigated the variation in gH sequence between strains of HSV-1. The gH gene coding sequence was derived for two recent clinical isolates, SC16 (27) and 103/65 (6), and compared with the sequence previously described for passaged laboratory strains HFEM (22, 56) and 17 (37) of syncytial and nonsyncytial plaque phenotypes, respectively. Strain SC16 was chosen for comparison since it is a virulent strain used to study pathogenicity in a mouse model (27). Strain 103/65 was selected from 100 clinical isolates which were serotyped by using MAbs to HSV-1 or HSV-2. Of 66 type 1 isolates, one was resistant to neutralization with the HSV-1 virus gH-specific MAb LP11. Examination of the nucleotide sequence of the gH genes of these viruses showed that, in contrast to the divergence seen between herpesviruses of different subgroups, the gH seI, quence was highly conserved between strains of HSV-1. For each comparison with the HFEM gH sequence, only 10 nucleotide differences were found, of which only 4 were coding changes (Fig. 1). The coding changes were clustered at the amino-terminal half of the external domain, a region not conserved between homologous gH sequences from herpesviruses of different subgroups (20). In comparisons between the LP11 antibody-resistant clinical isolate 103/65 and strain HFEM, only one of the four coding changes in the gH sequence was not present in strain 17. This change, adenine 503 to guanine, results in the substitution of glycine for aspartate 168. This was a potential site which conferred resistance to the virus-neutralizing effects of antibody LP11. Anti-gH antibody-resistant mutant virus. To further identify amino acids which may interact with neutralizing antibody and to determine sites on the protein molecule which function in mediating infectivity, we selected antibodyresistant mutant viruses in tissue culture for subsequent sequence analysis of their gH genes. Twelve independently selected antibody-resistant mutant viruses were isolated by treatment with antibody followed by limiting dilution cloning from infected tissue culture cells. Antibody-resistant viruses were selected in three ways. In the first method, antibodies LP11 and 52S were used only to neutralize initial infectivity, and the infected monolayer was then washed to dilute remaining antibody. In the second method, the ability of these antibodies to prevent infected cell-to-cell spread, (i.e., "plaque inhibition" [7, 22]) was used as an additional selection pressure by including antibody in the medium overlay of the infected cells after infection with antibodytreated virus. A third method was used to create two additional mutants by marker transfer. Cells were cotransfected with a mixture of cloned mutant gH DNA and DNA from cells infected with wild-type virus. Antibody-resistant mutants were selected from the resulting progeny and were characterized to confirm that antibody resistance was conferred by the mutation in the gH coding sequence. Each Downloaded from http://jvi.asm.org/ on January 21, 2015 by guest amino acid changes 2396 J. VIROL. GOMPELS ET AL. 244 LP1 1 Resistant mutants aa 86 1 Ila 168 ntd (2514) (838) E-[K D-G 256 G-A 503 A--G 9 VAl tsQ26 (HFEM) . NiP3B (HFEM) 1- I .103/65 clinical isolate lib D-N 502 G-A . LP11A (SC16) E-K LP 1 1 B (SC 16) 86 Ilia IIIb 329 R-W R- Q 985 986 Ni2B (HFEM) (HFEM) (HFEM) . Ni2A (HFEM) C-T * AD1 G-A VA2 * T III D-G D*N 168 R-'W LP 1 1 R R-sQ 329 T 800 IV S-L A-V 536,537 52SR FIG. 3. Summary of sites of amino acid substitutions found on the gH sequence of HSV gH mutant viruses. LP11R and 52R are LP11 resistant and 52S resistant, respectively. The single amino acid code is used as follows: A, alanine; C, cysteine; D, aspartic acid; E, glutamic acid; G, glycine; K, lysine; L, leucine; N, asparagine; Q, glutamine; V, valine; W, tryptophan. 52S Resistant mutants IVa IVb 536 S-L 537 A-V 1607 C -T 161 0 C-'T * 52SR (HFEM) * 5RA (SC 16) * 5RB (HFEM) Temperature-sensitive mutant 244 W-C 731 G-T ts 026 (KOS) FIG. 2. HSV gH mutants. Coding changes in antibody-selected antigenic variants and the temperature-sensitive mutant tsQ26 are shown. Sites are designated I through V. The names of the mutant viruses are indicated at the right. Viruses marked with a black dot were used as representatives for each group in all subsequent V assays. mutant virus isolated by one of these methods was completely resistant to neutralization by the MAb LP11 or 52S used for the selection. Antibody-resistant mutant virus, selected by using both antibody-mediated neutralization of infectivity and plaque inhibition, arose at a frequency of 10-6. Progeny virus obtained from cotransfection of plasmid DNA containing the mutant gH gene with wild type-infected cell DNA gave rise to resistant virus at a frequency of 10'3. Coding sequence of gH gene from antibody-resistant mutant viruses. The nucleotide sequence was derived for each mutant gH gene and compared with the gH sequence of the wild-type strains SC16 and HFEM. For each virus, there was a single point mutation in the gH coding sequence which gave rise to an amino acid substitution (Fig. 2 and 3). Mutants resistant to MAb LP11 had amino acid substitutions in their gH sequence at one of three sites, 86, 168, and 329, designated sites I, II, and III, respectively. Mutant NiP3B selected from wild-type strain HFEM had a point mutation in the gH gene that was identical to the nonconservative coding change found in the LP11-resistant clinical isolate 103/65 (Fig. 2). Mutant virus selected in the presence of 52S antibody had single point mutations which gave rise to amino acid substitutions at one of two adjacent amino acids at 536 and 537 (Fig. 2), designated sites IVa and IVb, respectively. Multiple independently derived mutants defined sites II, III, and IV. Mutants at sites II and III additionally have either of two different amino acid substitutions, designated a or b. A total of eight different gH mutant viruses from strains HFEM and SC16 were isolated and renamed mutant viruses I, Ila, Ilb, Illa, ITIb, IVa, and IVb depending on the amino acid substitution or antibody used (Fig. 2). Only one antibodyresistant virus was isolated with a point mutation at site I, but the resistance conferred by this encoded amino acid substitution was confirmed by marker transfer. Plasmid DNA containing the mutant VAI gH gene (site I) was cotransfected with infected cell DNA of wild-type strain SC16 or HFEM. Virus was selected with antibody LP11 and cloned by limiting dilution. Both these recombinant site I niutant viruses contained the identical site I mutation as determined by nucleotide sequence analysis. Properties of gH antibody-resistant mutant virus. The properties of the gH mutant viruses were examined. Because the mutants resistant to MAb 52S or LP11 had amino acid substitutions at distinct sites, the interactions of the mutant viruses with these antibodies were studied by virus neutralization and immunoprecipitation. Mutant virus resistant to LP11 could be neutralized by 52S but not by LP11, and conversely 52S-resistant virus could be neutralized by LP11 but not by 52S (Table 1). The ability of mutation at each site to confer resistance to neutralization appears to be the consequence of reduced affinity of the selecting antibody for the mutant gH molecule. Thus, Fig. 4 shows that each mutant gH could not be immunoprecipitated by the selecting antibody but remained precipitable by the nonselecting antibody. The result obtained with the HFEM IVa mutant is ambiguous because in this experiment, immunoprecipitation by the nonselecting antibody was weak. However, the equivalent mutant in strain SC16 (final two lanes in Fig. 4) gave an unambiguous result. To find whether mutations to antibody resistance resulted in growth impairment, HEp-2 cells were infected with parental or mutant viruses at high (MOI = 10) or low (MOI = 0.1) multiplicity and incubated at various temperatures (340C, 370C, 400C). Cultures were harvested after 16 h (high MOI) or 48 h (low MOI), and the virus yields were assayed. In some instances, intracellular and extracellular yields were assayed separately. Table 2 gives the results of a high MOI at 370C. None of the mutants with the exception of mutant I exhibited noticeable impairment of growth by comparison with the parental virus. Mutant I gave the lowest intracellu- Downloaded from http://jvi.asm.org/ on January 21, 2015 by guest NiP3A (HFEM) I 11 400 2397 ANTIBODY-RESISTANT MUTANTS OF HSV-1 gH VOL. 65, 1991 TABLE 2. Yield of wild-type and gH antibody-resistant mutant virus TABLE 1. Neutralization of antibody-resistant mutant virusa PFU after antibody treatment Virus Total PFU' 52S LP11 Strain 10 5 8 5 11 130 123 1 90 85 90 93 1 0 HFEM I Ila Illa IlIb IVa IVb Wild type 2.4 4.1 8.6 5.9 6.3 8.2 1.3 SC16 Ilb IVa Wild type 1.3 x 109 9.3 x 108 1.7 x 109 2.0 x 107 2.5 x 107 8.8 x 106 65 (Ila) 4.4 x 108 3.6 x 107 Intracellular HFEM (syn) Wild type I IIa Illa IlIb IVa IVb SC16 Wild type lIb IVa 0 70 0 1 0 97 WT mab 11 52 I 11 Ila 52 11 52 lila 11 52 x x x x x x x 105 106 107 107 106 107 107 to account for the altered growth phenotype of mutant I, and this is under further investigation. Properties of gH antibody-resistant mutant virus grown on gH-producing ceH lines. The phenotype of gH antibodyresistant mutant virus was further examined in the presence of wild-type gH. These rescue experiments tested whether the mutant gH could be complemented by wild-type gH in either the role of mediating infectivity or that of cell-to-cell spread. The available evidence suggests that gH functions in the fusion of the virion envelope with the plasma membrane during virus penetration and is also involved in the fusion of cellular plasma membranes in syncytium formation and cell-to-cell spread of virus. Antibodies to gH, in particular antibody LP11, inhibit both processes, but we do not know whether the mechanism of inhibition by LP11 is the same for both processes, nor indeed whether gH behaves similarly at the virion surface and the cell surface. We therefore decided SC 6 VW lVa lVb WT Illb H FEM vi rus 109 3.2 4.0 3.4 1.0 3.0 1.7 3.8 a HEp-2 cells (2 x 106) were infected at an MOI of 5, and virus was harvested 16 h postinfection and titrated. lar yield and gave a dramatically reduced yield of extracellular virus (underlined in Table 2). Experiments at other temperatures and at low multiplicity confirmed this result. Mutant I always gave the poorest yield (5- to 20-fold lower than wild type) and greatly reduced yields of extracellular virus (100-fold decrease). To determine whether the impaired growth of mutant I could be compensated by supplying wild-type gH, a high MOI experiment was repeated with two cell lines, Vero-F6 and Vero-B1.3, each of which provides wild-type gH in trans following infection (see next section). Table 3 shows that the growth of mutant I is impaired by comparison with wild-type virus regardless of the cell type used for infection or for assay of the progeny. Marker transfer of the site I mutation into the strain SC16 background yielded a virus which was resistant to neutralization with antibody LP11 but which exhibited no reproducible growth impairment. Thus, second-site mutations seem st rain 108 108 108 108 108 108 Illb 1 1 52 _W41 a. 11 52 11 52 11 5;2 11 52 1\Va 11 52 _ FIG. 4. Immunoprecipitation of 35S-labeled gH from mutant and wild-type virus-infected cells. Cells were infected at an MOI of 5, and lysates were prepared 16 h after infection. The lysates were treated with antibody LP11 or 52S, antibody-antigen complexes were purified with protein A-Sepharose, and the products were separated by electrophoresis on a 10% polyacrylamide gel with SDS. Mutant virus are designated by site of coding change. The gH from LP11-resistant mutant virus-infected cells is immunoprecipitated with 52S antibody, and the gH from the 52S-resistant mutant virus was immunoprecipitated with LP11 antibody. Downloaded from http://jvi.asm.org/ on January 21, 2015 by guest a Vero cells were infected with 250 PFU of virus treated with or without antibody (1:20 dilution of ascites fluid) for 1 h at room temperature. The virus-antibody mixture was removed, cells were washed twice with 5 ml of medium, and the overlay was replaced with 5 ml of fresh medium. The number of plaques are normalized from 100% for mock neutralizations. Assays were done in triplicate. x x x x x x x Extracellular 2398 GOMPELS ET AL. J. VIROL. TABLE 3. Growth and titration of antibody-resistant mutant virus I on gH-producing cell lines F6 and B1.3 Virus-infected cells HFEM in: Vero PFU as titrated in cell Vero Vero-F6 TABLE 4. Neutralization and plaque inhibition of antibodyresistant mutant virus grown on wild-type gH-producing cell lines % PFU after antibody treatment" line': Vero-B1.3 Vero-F6 Vero-B1.3 3.2 x 108 3.3 x 107 2.2 x 107 3.3 x 108 3.6 x 107 2.4 x 107 2.6 x 108 3.8 x 107 3.8 x 107 Mutant I in: Vero Vero-F6 Vero-B1.3 3.8 x 107 2.4 x 106 4.7 x 106 3.5 x 107 5.5 x 106 3.7 x 106 4.5 x 107 4.3 x 106 3.5 x 106 to ask whether the antibody-resistant phenotype of gH mutants was dominant or recessive to the wild-type phenotype and whether the same relationship would pertain at the virion surface and the cell surface. The experiments required virions and infected cells that contained mixtures of mutant and wild-type gH molecules, and this was achieved using a cell line, Vero-F6, that supplies wild-type gH in trans. The derivation and characterization of this cell line will be described elsewhere. Briefly, Vero-F6 cells contain multiple copies of the gH gene under the control of an HSV-1 promoter, and when these cells are infected with HSV, gH is synthesized from these resident genes. The following evidence shows that functional gH is produced by Vero-F6 cells: (i) a temperature-sensitive mutant (tsQ26 [17]) and a null mutant in gH both grow to normal yields on these cells, and (ii) HSV-2 grown on Vero-F6 cells is neutralized (>50%) by LP11, a type 1 gH-specific antibody. The rationale of our experiments is as follows. Mutant virus prepared on Vero-F6 cells and titrated on Vero cells can be used to measure wild-type gH-induced sensitivity to antibody-mediated neutralization because wild-type gH will be incorporated into mutant virions. On the other hand, virus prepared on Vero cells and titrated on Vero-F6 cells in the presence of antibody can be used to measure wild-type gH-induced sensitivity to antibody-mediated plaque inhibition because wild-type gH will be present on the cell surface. Vero or Vero-F6 cells were therefore infected with mutant or wild-type virus at an MOI of 1, and after 16 h, the virus was titrated on Vero or Vero-F6 cells with or without antibody treatment. Mutant IVa or IVb virus prepared on Vero or Vero gH cells showed no change in resistance to antibody 52S, although virus titrated on Vero gH cells showed reduced plaque size in the presence of 52S antibody. However, mutant I, IIa, Ilb, IIIa, and Illb viruses (the LP11-resistant mutant viruses) showed differing sensitivities to antibody-mediated virus neutralization or plaque inhibition in the presence of wild-type gH. Table 4 shows that these mutants yielded different results in these experiments and, in particular, that the same phenotype did not predominate in both assays. Thus, to take the extreme cases, for mutant IlIb the wild-type (sensitive) phenotype predominates in neutralization, whereas the mutant (resistant) phenotype predominates in plaque inhibition. By contrast, the results for mutant I (on a syncytial background) suggest that the mutant phenotype predominates in neutralization, while there is a strong influence of the wildtype phenotype in plaque inhibition. For mutant IIb, the wild-type (sensitive) phenotype is dominant in both assays. Vero/ Vero/ Vero-gH Vero-gH/ Vero Vero-gH/ Vero HFEM (syn) Wild type I Ila lIla IlIb 0 101 93 104 121 0 55+ 56+ 93+ 106+ 0 69 58 19 15 0 29+ 37+ 29+ 28+ SC16 Wild type Ilb 0 115 0 22+ 0 8 0 3+ Vero-gH a Vero or Vero-gH (Vero-F6 or Vero-B1.3) cells were infected with 250 PFU of virus treated with or without antibody LP11 (1:20 ascites fluid) for 1 h at room temperature. Infected cells were overlaid with medium containing antibody (1:200). The % PFU are normalized from 100% without antibody treatment. Plaque inhibition is indicated as + when plaque size was greatly reduced. Vero/Vero, cells for growth/cells for titration. These results imply that the effect of antibody LP11 at the virion surface and the cell surface is not the same and suggest that the function or presentation of gH at the cell and virion surface is different. DISCUSSION The results presented here show that gH has a conformationally complex epitope which is part of a domain important for the function of gH in mediating infectivity and spread from infected to uninfected cells. The gH-specific MAbs LP11 and 52S, in the absence of complement, neutralize infectivity, cell-to-cell spread, and virus-induced cell fusion (7, 22, 49). The antibodies are specific for conformationdependent epitopes and do not recognize denatured gH. Multiple independent isolates were selected in the presence of antibody 52S or LP11 from syncytial and nonsyncytial wild-type strains HFEM and SC16, respectively, and were characterized. These antibodies appeared to recognize distinct epitopes, as variants were resistant to neutralization only by the selecting antibody and the mutant gH could not be immunoprecipitated by the selecting antibody but remained sensitive to the other antibody. The genes for the MAb-selected antigenic variants were sequenced. Each mutant virus selected with MAb LP11 had a gH gene with a point mutation encoding a single amino acid substitution at one of three sites (residues 86, 168, and 329, designated sites I, II, and III) which span the N-terminal half of the external domain of the gH molecule (Fig. 2 and 3). Site II and III mutant viruses were confirmed by multiple independent isolates, and site II mutants were selected from strains HFEM and SC16. The validity of the site I mutant virus was confirmed by recombination of the mutant gH gene from HFEM mutant I with a different parent strain, SC16. Mutant virus selected with MAb 52S from both strains HFEM and SC16 encoded gH sequences with single amino acid substitutions at adjacent residues (536 and 537), site IV. It is implicit in our interpretation of the data that those mutations that confer resistance to LP11 identify sequences that contribute to the LP11 epitope. This view is supported by the results of immunoprecipitation experiments which show that mutations at any one of these noncontiguous sites (site I, II, or III) greatly reduce LP11 binding. Of course, it is conceiv- Downloaded from http://jvi.asm.org/ on January 21, 2015 by guest a Cell lines Vero-F6 and Vero-B1.3 are inducible for gH expression. Cells (2 x 106) were infected at an MOI of 5, and virus was harvested 16 h postinfection and titrated on monolayers of the indicated cell lines. Virus VOL. 65, 1991 ANTIBODY-RESISTANT MUTANTS OF HSV-1 gH able that a mutation distant from an epitope will result in a conformational change that acts at long distance to modify the epitope, but there is no precedent for this scenario from studies of other viral transmembrane glycoproteins and it seems likely that such long-range effects would modify glycoprotein function. None of the observed mutations significantly modified the growth properties of the virus, and we consider that the simple interpretation of the data is that sites I, II, and III all contribute to the LP11 epitope. Conformationally complex sites, in which different parts of a polypeptide chain contribute to an antigenic site, have been identified in other glycoproteins, notably in the hemagglutination molecule of influenza virus (reviewed in reference 51). Nevertheless, the LP11 epitope of HSV gH seems unusually complex in that a single antibody can select mutations in three distant regions of the polypeptide over a span of 243 amino acid residues. In contrast, the 52S epitope, which is also conformation dependent, appears more simple in that resistant mutants contained mutations at a single site. It may be that examination of a larger number of mutants would reveal additional, distant, sites. However, we have previously shown that whereas the 52S epitope can form when gH is expressed in the absence of other HSV-1 proteins, the epitope recognized by LP11 is formed only when other HSV-1 proteins are expressed (23). It is possible that interaction of gH with a second viral protein is required to stabilize the complex epitope seen by LP11, but direct evidence for such an interaction has yet to be demonstrated. We also know that expression of other viral products is required for transport of gH to the cell surface; thus, interactions that are necessary for gH conformation may be related to cell surface transport (23). For pseudorabies virus gB and CMV gB, oligomerization is a requisite for export from the endoplasmic reticulum to the Golgi apparatus, where subsequent modifications are followed by transport to the cell surface (52, 55). Some antigenic sites are dependent on this oligomerization (10). For HSV-1 gH, it follows that interactions with another virus product or virus-induced cellular product may allow export to the Golgi apparatus and cell surface with concomitant conformational change leading to formation of the LP11 epitope. Although sequences which confer retention in the endoplasmic reticulum have been described (44, 54), none of these are applicable to HSV-1 gH. Furthermore, although gH appears to be distributed throughout the cytoplasm as expressed in the temperaturesensitive COS cell and vaccinia virus systems (18a, 23), the prominent localization around the nuclear membrane is visually more akin to the nuclear membrane lamin proteins than to the resident endoplasmic reticulum protein markers, and this may imply a subcompartmentalization of the endoplasmic reticulum (28). gH, like other HSV glycoproteins, is acquired by the virion envelope and by the infected cell plasma membrane by different routes. HSV capsids appear to be enveloped at the inner nuclear membrane, while viral glycoproteins are expressed at the cell surface by the more familiar pathway from the endoplasmic reticulum via the Golgi apparatus. It is therefore conceivable that the presentation of gH at the virion membrane and the plasma membrane is different. Some support for this view comes from studies of the gH mutants for growth, neutralization, and titration on cell lines producing wild-type gH. LP11-resistant mutant viruses prepared on gH cell lines and titrated on normal Vero cells varied in resistance to antibody neutralization. HFEM site I and II mutant viruses remained more resistant to neutralization of infectivity than site III mutant viruses. In contrast, LP11-resistant mutant virus prepared on Vero cells and titrated on gH cell lines varied in resistance to antibodymediated plaque inhibition. In this system, wild-type gH is provided in the cell postinfection and thus effects on plaque inhibition can be separated from those on infectivity as described above. Here, site III mutant viruses were more resistant to plaque inhibition in the presence of wild-type gH than site I or II mutant viruses (Table 4). Thus, antigenic variants may differ depending on where the selecting antibody interacts with gH, either on the cell surface or on the viral envelope. Changes at any of the three sites cause changes in the LP11 epitope giving rise to resistance to neutralization, but in the presence of wild-type gH these effects may vary. Further studies of mutant gH in expression systems will be required to investigate the mechanism giving rise to these different phenotypes. The envelope glycoproteins of many viruses are targets for antibody-mediated neutralization, and in some cases these proteins can vary to evade the immune response. This has been shown for influenza viruses (51) and is strongly suggested for gpl20 of human immunodeficiency virus (11, 12, 18, 33, 41, 46). Neutralizing antibodies to HSV protect animals from experimental infection and may contribute to the control of recurrences (e.g., see reference 50), but there is little evidence for antigenic variation of HSV glycoproteins in response to immune selection. Antibodies to gH have been shown to be strongly protective in experimental infection (50), but gH appears to be a poor immunogen (at least in mice), and we know nothing about the response to gH in natural infection of humans. Our data suggest that gH exhibits little variation since only four conservative changes were identified in comparisons between four isolates, two of which have a long passage history. This is in contrast to the extreme variation in gH sequences from herpesviruses of different subgroups (20), in which gH is one of the most divergent of subgroup-common proteins and similarities are found only around cysteine residues in the C-terminal half of the external domain. Nevertheless, it is interesting that 1 of 66 clinical isolates contained a mutation identical to that obtained by in vitro selection with antibody LP11, and it would be worth screening a large number of isolates with this antibody. As the gH homologs of varicella-zoster virus, HCMV, and Epstein-Barr virus are also targets for strong neutralization of infectivity by respective gH-specific antibodies, a common function in virus entry is implicated; however, gH in these systems may interact with different cellular or viral proteins to function in mediating virus infectivity and spread. We showed here that conformational epitopes in the nonconserved N-terminal half of the external domain of HSV-1 gH are recognized by neutralizing antibodies and previously showed a requirement for interactions with virus or virus-induced products to form one of these sites and to allow transport of gH to the cell surface (23). In this regard, it is of interest that some evidence for interactions with cellular or viral proteins with CMV gH (gp86K) have been presented (24, 30). Whether this is related to observations for HSV gH or any other herpesvirus gH remains to be determined. 2399 United Kingdom. Downloaded from http://jvi.asm.org/ on January 21, 2015 by guest ACKNOWLEDGMENTS We are grateful to Christine Lelliott for expert cell culture assistance, Susanne Bell for purifying and testing oligonucleotide primers, and both Anita Hancock and Mary Wright for professional typing of the manuscript. This work was supported by the Medical Research Council of the 2400 GOMPELS ET AL. 19. Fuller, A. O., R. E. Santos, and P. G. Spear. 1989. Neutralizing antibodies specific for glycoprotein H or herpes simplex virus permit viral attachment to cells but prevent penetration. J. Virol. 63:3435-3443. 20. Gompels, U. A., M. A. Craxton, and R. W. Honess. 1988. Conservation of glycoprotein H (gH) in herpesviruses: nucleotide sequence of the gH gene from herpesvirus saimiri. J. Gen. Virol. 69:2819-2829. 21. Gompels, U. A., M. A. Craxton, and R. W. Honess. 1988. Conservation of gene organization in the lymphotropic herpesviruses herpesvirus saimiri and Epstein-Barr virus. J. Virol. 62:757-767. 22. Gompels, U., and A. Minson. 1986. The properties and sequence of glycoprotein H of herpes simplex virus type 1. Virology 153:230-247. 23. Gompels, U. A., and A. C. Minson. 1989. Antigenic properties and cellular localization of herpes simplex virus glycoprotein H synthesized in a mammalian cell expression system. J. Virol. 63:4744-4755. 24. Gretch, D. R., B. Kari, L. Rasmussen, R. Gehrz, and M. F. Stinski. 1988. Identification and characterization of three distinct families of glycoprotein complexes in the envelope of human cytomegalovirus. J. Virol. 62:875-881. 25. Highlander, S. L., W. Cai, S. Person, M. Levine, and J. C. Glorioso. 1988. Monoclonal antibodies define a domain as herpes simplex virus glycoprotein B involved in virus penetration. J. Virol. 62:1881-1888. 26. Highlander, S. L., D. J. Dorney, P. J. Gage, T. C. Holland, W. Cai, S. Person, M. Levine, and J. C. Glorioso. 1989. Identification of mar mutations in herpes simplex virus type 1 glycoprotein B which alter antigenic structure and function in virus penetration. J. Virol. 63:730-738. 27. Hill, T. J., H. J. Field, and W. A. Blyth. 1975. Acute and recurrent infection with herpes simplex virus in the mouse: a model for studying latency and recurrent disease. J. Gen. Virol. 28:341-353. 28. Holtz, D., R. A. Tanaka, J. Hartwig, and F. McKeon. 1989. The CaaX motif of lamina A functions in conjunction with the nuclear localisation signal to target assembly to the nuclear envelope. Cell 59:969-977. 29. Johnson, D. C., M. C. Frame, M. W. Ligas, A. M. Cross, and N. Stow. 1988. Herpes simplex virus immunoglobulin G Fc receptor activity depends on a complex of two viral glycoproteins, gE and gI. J. Virol. 62:1347-1354. 30. Keay, S., T. C. Merigan, and L. Rasmussen. 1989. Identification of cell surface receptors for the 86-kilodalton glycoprotein of human cytomegalovirus. Proc. Natl. Acad. Sci. USA 86:1010010103. 31. Kousoulas, K. G., H. Bin, and L. Pereira. 1988. Antibody resistant mutations in cross-reactive and type-specific epitopes of herpes simplex virus type 1 glycoprotein B map in separate domains. Virology 166:423-431. 32. Kuhn, J. E., M. D. Kramer, W. Willenbeacher, H. Wieland, E. U. Lorentzen, and R. W. Braun. 1990. Identification of herpes simplex virus type 1 glycoproteins interacting with the cell surface. J. Virol. 64:2491-2497. 33. Lasky, L., G. Nakamura, D. Smith, C. Fennie, C. Shimasaki, E. Patzer, P. Berman, T. Gregory, and D. Capon. 1987. Neutralisation of the AIDS retrovirus by antibodies to a recombinant envelope glycoprotein. Cell 50:975-985. 34. Ligas, M. W., and D. C. Johnson. 1988. A herpes simplex virus mutant in which glycoprotein D sequences are replaced by ,-galactosidase sequences binds to but is unable to penetrate into cells. J. Virol. 62:1486-1494. 35. Longnecker, R., S. Chatterjee, R. J. Whitley, and B. Roizman. 1987. Identification of a herpes simplex virus 1 glycoprotein gene within a gene cluster dispensable for growth in cell culture. Proc. Natl. Acad. Sci. USA 84:4303-4307. 36. McGeoch, D. J., M. A. Dalrymple, A. J. Davison, A. Dolan, M. C. Frame, D. M. McNab, L. Perry, J. E. Scott, and P. Taylor. 1988. The complete DNA sequence of the long unique region in the genome of herpes simplex virus type 1. J. Gen. Virol. 69:1531-1574. Downloaded from http://jvi.asm.org/ on January 21, 2015 by guest REFERENCES 1. Ackerman, M., R. Longnecker, B. Roizman, and L. Pereira. 1986. Identification, properties and gene location of a novel glycoprotein specified by herpes simplex virus 1. Virology 150:207-220. 2. Bankier, A. T., and B. G. Barrell. 1983. Shotgun DNA sequencing, p. 1-33. In R. A. Flavell (ed.), Techniques in the life sciences, vol. B5. Elsevier/North-Holland Publishing Co., Amsterdam. 3. Bell, S., M. Cranage, L. Borysiewicz, and A. Minson. 1990. Induction of immunoglobulin G Fc receptors by recombinant vaccinia virus expressing glycoproteins E and I of herpes simplex virus type 1. J. Virol. 64:2181-2186. 4. Biggin, M. D., T. J. Gibson, and C. F. Hong. 1983. Buffer gradient gels and 35S-label as an aid to rapid DNA sequence determination. Proc. Natl. Acad. Sci. USA 80:3963-3965. 5. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513-1523. 6. Buckmaster, E. A., M. P. Cranage, C. S. McLean, R. R. A. Coombs, and A. C. Minson. 1984. The use of monoclonal antibodies to differentiate isolates of herpes simplex types 1 and 2 by neutralisation and reverse passive haemagglutination tests. J. Med. Virol. 13:193-202. 7. Buckmaster, E. A., U. Gompels, and A. C. Minson. 1984. Characterisation and physical mapping of an HSV-1 glycoprotein of approximately 115 x 103 molecular weight. Virology 139:408-413. 8. Cai, W., G. Baohua, and S. Person. 1988. Role of glycoprotein B of herpes simplex virus type 1 in viral entry and cell fusion. J. Virol. 62:2596-2604. 9. Cai, W. Z., S. Person, C. Debroy, and B. H. Gu. 1988. Functional regions and structural features of the gB glycoprotein of herpes simplex virus type 1. An analysis of linker insertion mutants. J. Mol. Biol. 201:575-588. 10. Chapsal, J. M., and L. Pereira. 1988. Characterisation of epitopes on native and denatured forms of herpes simplex virus glycoprotein B. Virology 164:427-434. 11. Cheng-Mayer, C., M. Quiroga, J. W. Tung, D. Dina, and J. A. Levy. 1990. Viral determinants of human immunodeficiency virus type 1 T-cell or macrophage tropism, cytopathogenicity, and CD4 antigen modulation. J. Virol. 64:4390-4398. 12. Cordonnier, A., L. Montagnier, and M. Emerman. 1989. Single amino acid changes in HIV envelope affect viral tropisms and receptor binding. Nature (London) 340:571-574. 13. Cranage, M. P., T. Kouzarides, A. T. Bankier, S. Satchwell, K. Weston, P. Tomlinson, B. Barrell, H. Hart, S. E. Bell, A. C. Minson, and G. L. Smith. 1986. Identification of the human cytomegalovirus glycoprotein B gene and induction of neutralising antibodies via its expression in recombinant vaccinia virus. EMBO J. 5:3057-3063. 14. Cranage, M. P., G. L. Smith, S. E. Bell, H. Hart, C. Brown, A. T. Bankier, T. Tomlinson, B. G. Barrell, and A. C. Minson. 1988. Identification and expression of a human cytomegalovirus glycoprotein with homology to the Epstein-Barr virus BXLF2 product, varicella-zoster virus gpIII, and herpes simplex virus type 1 glycoprotein H. J. Virol. 62:1416-1422. 15. Davison, A. J., and P. Taylor. 1987. Genetic relations between varicella-zoster virus and Epstein-Barr virus. J. Gen. Virol. 68:1067-1079. 16. DeLuca, N., S. Person, D. J. Bzik, and W. Snipes. 1984. Genome locations of temperature-sensitive mutants in glycoprotein gB of herpes simplex virus type 1. Virology 137:382-389. 17. Desai, P. J., P. A. Schaffer, and A. C. Minson. 1988. Excretion of non-infectious virus particles lacking glycoprotein H by a temperature sensitive mutant of herpes simplex virus type 1: evidence that gH is essential for virion infectivity. J. Gen. Virol. 69:1147-1156. 18. Dowbenko, D. G., G. Nakamura, C. Fennie, C. Shimasaki, L. Riddle, R. Harris, T. Gregory, and L. Lasky. 1988. Epitope mapping of the human immunodeficiency virus type 1 gpl20 with monoclonal antibodies. J. Virol. 62:4703-4711. 18a.Forrester, A., and A. C. Minson. J. Gen. Virol., in press. J. VIROL. VOL. 65, 1991 ANTIBODY-RESISTANT MUTANTS OF HSV-1 gH 37. McGeoch, D. J., and A. J. Davison. 1986. DNA sequence of the herpes simplex virus type 1 gene encoding glycoprotein H and identification of homologues in the genomes of varicella-zoster virus and Epstein-Barr virus. Nucleic Acids Res. 14:4281-4292. 38. Messing, J. 1983. New M13 vectors for cloning. Methods Enzymol. 10:20-78. 39. Miller, N., and L. M. Hutt-Fletcher. 1988. A monoclonal antibody to glycoprotein gp85 inhibits fusion but not attachment of Epstein-Barr virus. J. Virol. 62:2366-2372. 40. Minson, A. C., T. C. Hodgman, P. Digard, D. C. Hancock, S. E. Bell, and E. A. Buckmaster. 1986. An analysis of the biological properties of monoclonal antibodies against glycoprotein D of herpes simplex virus and identification of amino acid substitutions that confer resistance to neutralisation. J. Gen. Virol. 67:1001-1013. 41. Myers, G., S. F. Josephs, J. A. Berzofsky, A. B. Rabson, T. F. Smith, and F. Wong-Staal (ed.). 1989. Human retroviruses and AIDS 1989-compilation and analysis of nucleic acid and amino acid sequences. Los Alamos National Laboratory, Los Alamos, N.M. 42. Noble, A. G., G. T.-Y. Lee, R. Sprague, M. L. Parish, and P. G. Spear. 1983. Anti-gD monoclonal antibodies inhibit cell fusion induced by herpes simplex virus type 1. Virology 129:218-244. 43. Para, M. F., M. L. Parish, A. G. Noble, and P. G. Spear. 1985. Potent neutralizing activity associated with anti-glycoprotein D specificity among monoclonal antibodies selected for binding to herpes simplex virions. J. Virol. 55:483-488. 44. Pelham, H. R. B. 1989. Control of protein exit from the endoplasmic reticulum. Annu. Rev. Cell Biol. 5:1-25. 45. Pereira, L., M. Ali, K. Kousoulas, B. Huo, and T. Banks. 1989. Domain structure of herpes simplex virus 1 glycoprotein B: neutralising epitopes map in regions of continuous and discontinuous residues. Virology 172:11-24. 46. Reitz, M. S., Jr., C. Wilson, C. Naugle, R. C. Gallo, and M. Robert-Guroff. 1988. Generation of a neutralisation-resistant variant of HIV-1 is due to selection for a point mutation in the envelope gene. Cell 54:57-63. 47. Richman, D. D., A. Buckmaster, S. Bell, C. Hodgeman, and A. C. Minson. 1986. Identification of a new glycoprotein of herpes simplex virus type 1 and genetic mapping of the gene that codes for it. J. Virol. 57:647-655. 48. Sarmiento, M., and P. G. Spear. 1979. Membrane proteins specified by herpes simplex viruses. IV. Conformation of the virion glycoprotein designated VP7 (B2). J. Virol. 34:1159-1167. 49. Showalter, S. D., M. Zweig, and B. Hampar. 1981. Monoclonal antibodies to herpes simplex virus type 1 proteins, including the immediate-early protein ICP4. Infect. Immun. 34:684-692. 50. Simmons, A., and A. A. Nash. 1985. Role of antibody in primary and recurrent herpes simplex virus infection. J. Virol. 53:944948. 51. Skehel, J. J. 1986. Antigenic variation in Hong Kong influenza virus haemagglutinins, p. 19-24. In T. H. Birkbeck and C. W. Penn (ed.), Antigenic variation in infectious diseases. IRL Press, Oxford. 52. Spaete, R. R., A. Saxena, P. I. Scott, G. I. Song, W. S. Probert, W. J. Britt, W. Gibson, L. Rasmussen, and C. Pachl. 1990. Sequence requirement for proteolytic processing of glycoprotein B of human cytomegalovirus strain Towne. J. Virol. 64: 2922-2931. 53. Spear, P. G. 1984. Glycoproteins specified by herpes simplex virus, p. 315-356. In B. Roizman (ed.), The herpesviruses, vol. 3. Plenum Publishing Corp., New York. 54. Stirzaker, S. C., and G. W. Both. 1989. The signal peptide of the rotavirus glycoprotein VP7 is essential for its retention in the ER as an integral membrane protein. Cell 56:741-747. 55. Whealey, M. E., A. K. Robbins, and L. W. Enquist. 1990. The export pathway of the pseudorabies virus gB homolog glI involves oligomer formation in the endoplasmic reticulum and protease processing in the Golgi apparatus. J. Virol. 64:19461955. 56. Wildy, P., W. C. Russell, and R. W. Horne. 1960. The morphology of herpesviruses. Virology 12:204-222. 2401 Downloaded from http://jvi.asm.org/ on January 21, 2015 by guest