Neutralizing monoclonal antibodies specific for herpes simplex virus glycoprotein D inhibit virus penetration.

Nine monoclonal antibodies specific for glycoprotein D (gD) of herpes simplex virus type 1 were selected for their ability to neutralize virus in the presence of complement. Four of these antibodies exhibited significant neutralization titers in the absence of complement, suggesting that their epitope specificities are localized to site(s) which contribute to the role of gD in virus infectivity. Each of these antibodies was shown to effectively neutralize virus after virion adsorption to cell surfaces, indicating that neutralization did not involve inhibition of virus attachment. Although some of the monoclonal antibodies partially inhibited adsorption of radiolabeled virions, this effect was only observed at concentrations much higher than that required to neutralize virus and did not correlate with complement-independent virus-neutralizing activity. All of the monoclonal antibodies slowed the rate at which virus entered cells, further suggesting that antibody binding of gD inhibits virus penetration. Experiments were carried out to determine the number of different epitopes recognized by the panel of monoclonal antibodies and to identify epitopes involved in complement-independent virus neutralization. Monoclonal antibody-resistant (mar) mutants were selected by escape from neutralization with individual gD-specific monoclonal antibodies. The reactivity patterns of the mutants and antibodies were then used to construct an operational antigenic map for gD. This analysis identified a minimum of six epitopes on gD that could be grouped into four antigenic sites. Antibodies recognizing four distinct epitopes contained in three antigenic sites were found to neutralize virus in a complement-independent fashion. Moreover, mar mutations in these sites did not affect the processing of gD, rate of virus penetration, or the ability of the virus to replicate at high temperature (39 degrees C). Taken together, these results (i) confirm that gD is a major target antigen for neutralizing antibody, (ii) indicate that the mechanism of neutralization can involve inhibition of virus penetration of the cell surface membrane, and (iii) strongly suggest that gD plays a direct role in the virus entry process.     

Localization of epitopes of herpes simplex virus type 1 glycoprotein D.

We previously defined eight groups of monoclonal antibodies which react with distinct epitopes of herpes simplex virus glycoprotein D (gD). One of these, group VII antibody, was shown to react with a type-common continuous epitope within residues 11 to 19 of the mature glycoprotein (residues 36 to 44 of the predicted sequence of gD). In the current investigation, we have localized the sites of binding of two additional antibody groups which recognize continuous epitopes of gD. The use of truncated forms of gD as well as computer predictions of secondary structure and hydrophilicity were instrumental in locating these epitopes and choosing synthetic peptides to mimic their reactivity. Group II antibodies, which are type common, react with an epitope within residues 268 to 287 of the mature glycoprotein (residues 293 to 312 of the predicted sequence). Group V antibodies, which are gD-1 specific, react with an epitope within residues 340 to 356 of the mature protein (residues 365 to 381 of the predicted sequence). Four additional groups of monoclonal antibodies appear to react with discontinuous epitopes of gD-1, since the reactivity of these antibodies was lost when the glycoprotein was denatured by reduction and alkylation. Truncated forms of gD were used to localize these four epitopes to the first 260 amino acids of the mature protein. Competition experiments were used to assess the relative positions of binding of various pairs of monoclonal antibodies. In several cases, when one antibody was bound, there was no interference with the binding of an antibody from another group, indicating that the epitopes were distinct. However, in other cases, there was competition, indicating that these epitopes might share some common amino acids.     

Antibodies against synthetic peptides of herpes simplex virus type 1 glycoprotein D and their capability to neutralize viral infectivity in vitro.

Peptides corresponding to residues 1-13, 9-21, 18-30, 82-93, 137-150, 181-197, 232-243, 235-243, 267-281, 271-281 and 302-315 of glycoprotein D of herpes simplex virus type 1 (HSV-1) were chemically synthesized. These peptides were coupled to carrier proteins, and the resulting conjugates were used to immunize rabbits. An enzyme-linked immunosorbent assay was used to determine antipeptide antibody titers in serum collected after immunization. All peptides appeared to be immunogenic in rabbits. Western immunoblot analysis with detergent extracts of HSV-1-infected Vero cells showed that antibodies against each of the peptides were able to react with the parent glycoprotein under denaturing conditions. Antisera against peptides 1-13, 9-21, and 18-30 neutralized HSV-1 infectivity in vitro, peptide 9-21 being the most successful in this respect. Immunization with a mixture of peptides 9-21 and 267-281 yielded antisera which reacted strongly with glycoprotein gD in Western blot analysis and showed a more solid virus-neutralizing activity in vitro.     

Effect of monoclonal antibodies on limited proteolysis of native glycoprotein gD of herpes simplex virus type 1.

We examined the properties of 17 monoclonal antibodies to glycoprotein gD of herpes simplex type 1 (HSV-1) (gD-1) and HSV-2 (gD-2). The antibodies recognized eight separate determinants of gD, based on differences in radioimmuno-precipitation and neutralization assays. The determinants were distributed as follows: three were gD-1 specific, one was gD-2 specific, and four were type common. Several type-specific and type-common determinants appeared to be involved in neutralization. We developed a procedure for examining the effect that binding of monoclonal antibody has on proteolysis of native gD-1 by Staphylococcus aureus protease V8. We showed that several different patterns of protease V8 cleavage were obtained, depending on the monoclonal antibody used. The proteolysis patterns were generally consistent with the immunological groupings. With four groups of antibodies, we found that fragments of gD-1 remained bound to antibody after V8 treatment. A 38,000-dalton fragment remained bound to antibodies in three different groups of monoclonal antibodies. This fragment appeared to contain one type-common and two type-specific determinants. A 12,000-dalton fragment remained bound to antibodies belonging to one type-common group of monoclonal antibodies. Tryptic peptide analysis revealed that the 12,000-dalton fragment represented a portion of the 38,000-dalton fragment and was enriched in a type-common arginine tryptic peptide.     

Induction of signaling pathways by herpes simplex virus type 1 through glycoprotein H peptides

Eukaryotic cells respond to extracellular stimuli, such as viruses, by recruiting signal transduction pathways, many of which are mediated through activation of distinct mitogen-activated protein kinase (MAPK) cascades and activation of transductional regulation factors. The best characterized of this pathway are the extracellular signal regulated kinase (ERK), the c-Jun N-terminal kinase/stress activated protein kinase (JNK/SAPK), and the p38 MAPK cascade. Herpes simplex virus type 1 (HSV-1) encodes at least 11 envelope glycoproteins, which alone or in concert play different roles in viral adsorption, entry, cell-to-cell spread, and immune evasion. Of these proteins, three are designated glycoprotein B (gB), glycoprotein D (gD), and the gH/gL heterodimer, are clearly involved in attachment and entry, and therefore possible candidates in inducing early cellular activation.Nevertheless, the precise role of each glycoprotein and the cellular factor involved remain elusive. The signal transduction pathways involved, and the outcome of cellular activation on viral entry or postentry events, are still to be elucidated. To better understand the role of signal transduction pathways and phosphorylation events in HSV-1 entry, synthetic peptides modeled on HSV-1 gH were synthesized and tested for MEK1-MEK2/MAPK cascade activation. Our results show a major involvement of the JNK pathway in the intracellular signal transmission after stimulation with gH HSV-1 peptides.     

Use of synthetic peptides to map the antigenic determinants of glycoprotein D of herpes simplex virus.

The predictive algorithm Surfaceplot (J.M.R. Parker, D. Guo, and R.S. Hodges, Biochemistry 25:5425-5432, 1986) was used to examine glycoprotein D of herpes simplex virus type 1 (HSV-1) for amino acid residues with a high probability of being exposed on the molecular surface. Based on these data, 11 different peptides corresponding to 10-residue segments in the primary sequence of glycoprotein D and one 20-residue segment were synthesized, conjugated to carrier proteins, and used to generate specific antisera in rabbits. Two synthetic peptides predicted not to be on the surface of glycoprotein D were included as negative controls. The polyclonal antisera against individual synthetic peptide conjugates were in turn evaluated for their ability to recognize both isolated glycoprotein D and intact HSV-1 virions in an enzyme-linked immunosorbent assay. Based on Surfaceplot predictions, eight linear antigenic sites on glycoprotein D were thereby defined from the 12 antipeptide antisera prepared. Four of these sites contained epitopes to which complement-independent neutralizing antibodies could be generated. The latter sites corresponded to sequences 12 to 21, 267 to 276, 288 to 297, and 314 to 323 of the mature protein. An additional peptide sequence, 2 to 21, was found to generate antisera which had potent virus-neutralizing capacity in the presence of complement. Identification of a neutralizing epitope in the sequence 314 to 323 makes it likely that the membrane-spanning region of glycoprotein D is within the subsequent sequence, 323 to 339. Antipeptide antisera prepared in this study from 12 synthetic peptides contained 13 surface sites predicted by Surfaceplot, of which 7 were not predicted by the parameters of Hopp and Woods (Proc. Natl. Acad. Sci. USA 78:3824-3828, 1981). Of these seven sites not predicted by the Hopp and Woods plot, all generated antipeptide antibodies that bound to HSV-1 virions and three of these seven sites generated neutralizing antibodies. In total, 8 of 12 synthetic peptides containing surface regions produced antipeptide antibodies that bound to HSV-1 virions and 5 of these generated neutralizing antibodies. These results suggest the advantages of Surfaceplot in mapping antigenic determinants in proteins.     

Synthesis and comparison of antibody recognition of conjugates containing herpes simplex virus type 1 glycoprotein D epitope VII

Synthetic oligopeptides comprising linear or continuous topographic B-cell epitope sequences of proteins might be considered as specific and small size antigens. It has been demonstrated that the strength and specificity of antibody binding could be altered by conjugation to macromolecules or by modification in the flanking regions. However, no systematic studies have been reported to describe the effect of different carrier macromolecules in epitope conjugates. To this end, the influence of carrier structure and topology on antibody recognition of attached epitope has been studied by comparing the antibody binding properties of a new set of conjugates with tetratuftsin analogue (H-[Thr-Lys-Pro-Lys-Gly](4)-NH(2), T20) sequential oligopeptide carrier (SOC(n)), branched chain polypeptide, poly[Lys(Ser(i)-DL-Ala(m))] (SAK), multiple antigenic peptide (MAP), and keyhole limpet hemocyanine (KLH). In these novel constructs, peptide (9)LKNleADPNRFRGKDL(22) ([Nle(11)]-9-22) representing an immunodominant B cell epitope of herpes simplex virus type 1 glycoprotein D (HSV-1 gD) was conjugated to polypeptides through a thioether or amide bond. Here we report on the preparation of sequential and polymeric polypeptides possessing chloroacetyl groups in multiple copies at the alpha- and/or epsilon-amino group of the polypeptides and its use for the conjugation of epitope peptides possessing Cys at C-terminal position. We have performed binding studies (direct and competitive ELISA) with monoclonal antibody (Mab) A16, recognizing the HSV gD-related epitope, [Nle(11)]-9-22, and conjugates containing identical and uniformly oriented epitope peptide in multiple copies attached to five different macromolecules as carrier. Data suggest that the chemical nature of the carrier and the degree of substitution have marked influence on the strength of antibody binding.     

Glycoproteins of herpes simplex virus type 2 as defined by monoclonal antibodies.

We used monoclonal antibodies reacting with glycoproteins specified by herpes simplex virus type 2 (HSV-2) to characterize the individual antigens in terms of structure, processing, and kinetics of synthesis in BHK or Vero infected cells. Our results provided a direct demonstration of the structural identity of the gA and gB proteins of HSV-2 as well as confirmation of the existence of type-specific and type-common domains within the gD molecule. They also show that, with the exception of gC, processing of the viral glycoproteins differs to some extent in Vero and BHK infected cells, possibly as a result of different efficiency of glycosylation or different processing of underglycosylated and unglycosylated products in the two cell types. Finally, we showed that individual HSV-2 glycoproteins are synthesized at greatly different times during the infectious cycle, possibly in response to their different roles in virus replication and assembly.     

Cysteine mutants of herpes simplex virus type 1 glycoprotein D exhibit temperature-sensitive properties in structure and function.

We previously constructed seven mutations in the gene for glycoprotein D (gD) of herpes simplex virus type 1 in which the codon for one of the cysteine residues was replaced by a serine codon. Each of the mutant genes was cloned into a eucaryotic expression vector, and the proteins were transiently expressed in mammalian cells. We found that alteration of any of the first six cysteine residues had profound effects on protein conformation and oligosaccharide processing. In this report, we show that five of the mutant proteins exhibit temperature-sensitive differences in such properties as aggregation, antigenic conformation, oligosaccharide processing, and transport to the cell surface. Using a complementation assay, we have now assessed the ability of the mutant proteins to function in virus infection. This assay tests the ability of the mutant proteins expressed from transfected plasmids to rescue production of infectious virions of a gD-minus virus, F-gD beta, in Vero cells. Two mutant proteins, Cys-2 (Cys-106 to Ser) and Cys-4 (Cys-127 to Ser), were able to complement F-gD beta at 31.5 degrees C but not at 37 degrees C. The rescued viruses, designated F-gD beta(Cys-2) and F-gD beta(Cys-4), were neutralized as efficiently as wild-type virus by anti-gD monoclonal antibodies, indicating that gD was present in the virion envelope in a functional form. Both F-gD beta(Cys-2) and F-gD beta(Cys-4) functioned normally in a penetration assay. However, the infectivity of these viruses was markedly reduced compared with that of the wild type when they were preincubated at temperatures above 37 degrees C. The results suggest that mutations involving Cys-106 or Cys-127 in gD-1 confer a temperature-sensitive phenotype on herpes simplex virus. These and other properties of the cysteine-to-serine mutants allowed us to predict a disulfide bonding pattern for gD.     

Soluble glycoprotein D blocks herpes simplex virus type 1 infection of rat eyes.

Herpes simplex virus type 1 (HSV-1) ocular infection in rats was blocked by treating the eyes with UV-inactivated virions containing glycoprotein D (gD) prior to ocular challenge. In contrast, rats treated with UV-inactivated virions lacking gD were not protected. A soluble, truncated form of HSV-2 gD (gD-2t) also protected against ocular infection. Treatment with gD-2t not only reduced mortality but also restricted progression of pathology and reduced the amount of viral antigen in the cornea. Host antibody or alpha/beta interferon responses to the gD-2t treatment were not detected. These results are similar to those observed in cell culture (D. C. Johnson, R. L. Burke, and T. Gregory, J. Virol. 64:2569-2576, 1990). The in vivo effect of exogenous gD is consistent with blocking of a cell surface gD receptor or with an inhibitory interaction of gD with virions.     

Characterization of a herpes simplex virus type 2 75,000-molecular-weight glycoprotein antigenically related to herpes simplex virus type 1 glycoprotein C.

Evidence is presented that the herpes simplex virus type 2 glycoprotein previously designated gF is antigenically related to herpes simplex virus type 1 gC (gC-1). An antiserum prepared against type 1 virion envelope proteins immunoprecipitated gF of type 2 (gF-2), and competition experiments revealed that the anti-gC-1 component of the antiserum was responsible for the anti-gF-2 cross-reactivity. An antiserum prepared against fully denatured purified gF-2, however, and three anti-gF-2 monoclonal antibodies failed to precipitate any type 1 antigen, indicating that the extent of cross-reactivity between gC-1 and gF-2 may be limited. Several aspects of gF-2 synthesis and processing were investigated. Use of the enzymes endo-beta-N-acetylglucosaminidase H and alpha-D-N-acetylgalactosaminyl oligosaccharidase revealed that the fully processed form of gF-2 (about 75,000 [75K] apparent molecular weight) had both complex-type N-linked and O-linked oligosaccharides, whereas newly synthesized forms (67K and 69K) had only high-mannose N-linked oligosaccharides. These last two forms were both reduced in size to 54K by treatment with endo-beta-N-acetylglucosaminidase H and therefore appear to differ only in the number of N-linked chains. Neutralization tests and radioiodination experiments revealed that gF-2 is exposed on the surfaces of virions and that the 75K form of gF-2 is exposed on cell surfaces. The similarities and differences of gF-2 and gC-1 are discussed in light of recent mapping results which suggest collinearity of their respective genes.     

Unusual lectin-binding properties of a herpes simplex virus type 1-specific glycoprotein

Lysates from herpes simplex virus type 1-infected cells were subjected to affinity chromatography on soybean and Helix pomatia lectins. One of the virus-specified glycoproteins, probably the herpes simplex virus type 1-specific gC glycoprotein, bound to the lectins and was eluted with N-acetylgalactosamine. The affinity chromatography permitted a high degree of purification of the type-specific glycoprotein with respect to both host cell components and other viral glycoproteins. The lectin affinity pattern of this glycoprotein indicates the presence of a terminal alpha-N-acetylgalactosamine in an oligosaccharide, a finding not reported previously for glycoproteins of enveloped viruses.     

Extensive homology between the herpes simplex virus type 2 glycoprotein F gene and the herpes simplex virus type 1 glycoprotein C gene.

The region of the herpes simplex virus type 2 (HSV-2) genome which maps colinearly with the HSV-1 glycoprotein C (gC) gene has been cloned, and the DNA sequence of a 2.29-kilobase region has been determined. Contained within this sequence is a major open reading frame of 479 amino acids. The carboxyterminal three-fourths of the derived HSV-2 protein sequence showed a high degree of sequence homology to the HSV-1 gC amino acid sequence reported by Frink et al. (J. Virol. 45:634-647, 1983). The amino-terminal region of the HSV-2 sequence, however, showed very little sequence homology to HSV-1 gC. In addition, the HSV-1 gC sequence contained 27 amino acids in the amino-terminal region which were missing from the HSV-2 protein. Computer-assisted analysis of the hydrophilic and hydrophobic properties of the derived HSV-2 sequence demonstrated that the protein contained structures characteristic of membrane-bound glycoproteins, including an amino-terminal signal sequence and carboxy-terminal hydrophobic transmembrane domain and charged cytoplasmic anchor. The HSV-2 protein sequence also contained seven putative N-linked glycosylation sites. These data, in conjunction with mapping studies of Para et al. (J. Virol. 45:1223-1227, 1983) and Zezulak and Spear (J. Virol. 49:741-747, 1984), suggest that the protein sequence derived from the HSV-2 genome corresponds to gF, the HSV-2 homolog of HSV-1 gC.     

Localization of a binding site for herpes simplex virus glycoprotein D on herpesvirus entry mediator C by using antireceptor monoclonal antibodies

The human herpesvirus entry mediator C (HveC), also known as the poliovirus receptor-related protein 1 (PRR1) and as nectin-1, allows the entry of herpes simplex virus type 1 (HSV-1) and HSV-2 into mammalian cells. The interaction of virus envelope glycoprotein D (gD) with such a receptor is an essential step in the process leading to membrane fusion. HveC is a member of the immunoglobulin (Ig) superfamily and contains three Ig-like domains in its extracellular portion. The gD binding site is located within the first Ig-like domain (V domain) of HveC. We generated a panel of monoclonal antibodies (MAbs) against the ectodomain of HveC. Eleven of these, which detect linear or conformational epitopes within the V domain, were used to map a gD binding site. They allowed the detection of HveC by enzyme-linked immunosorbent assay, Western blotting, and biosensor analysis or directly on the surface of HeLa cells and human neuroblastoma cell lines, as well as simian Vero cells. The anti-HveC V-domain MAbs CK6, CK8, and CK41, as well as the previously described MAb R1.302, blocked HSV entry. Their binding to soluble HveC was blocked by the association of gD with the receptor, indicating that their epitopes overlap a gD binding site. Competition assays on an optical biosensor showed that CK6 and CK8 (linear epitopes) inhibited the binding of CK41 and R1.302 (conformational epitopes) to HveC and vice versa. Epitope mapping showed that CK6 and CK8 bound between residues 80 and 104 of HveC, suggesting that part of the gD binding site colocalizes in the same region.     

Characterization of baculovirus-expressed herpes simplex virus type 1 glycoprotein K.

The DNA region encoding the complete herpes simplex virus type 1 (HSV-1) glycoprotein K (gK) was inserted into a baculovirus transfer vector, and recombinant viruses expressing gK were isolated. Four gK-related recombinant baculovirus-expressed peptides of 29, 35, 38, and 40 kDa were detected with polyclonal antibody to gK. The 35-, 38-, and 40-kDa species were susceptible to tunicamycin treatment, suggesting that they were glycosylated. The 38- and 40-kDa species corresponded to partially glycosylated precursor gK (pgK) and mature gK, respectively. The 29-kDa peptide probably represented a cleaved, unglycosylated peptide. The 35-kDa peptide probably represented a cleaved, glycosylated peptide that may be a precursor to pgK. Indirect immunofluorescence with polyclonal antibody to gK peptides indicated that the recombinant baculovirus-expressed gK was abundant on the surface of the insect cells in which it was expressed. Mice vaccinated with the baculovirus-expressed gK produced very low levels (< 1:10) of HSV-1 neutralizing antibody. Nonetheless, these mice were partially protected from lethal challenge with HSV-1 (75% survival). This protection was significant (P = 0.02). Despite some protection against death, gK-vaccinated mice showed no protection against the establishment of latency. Surprisingly, gK-vaccinated mice that were challenged ocularly with a stromal disease-producing strain of HSV-1 had significantly higher levels of ocular disease (herpes stromal keratitis) than did mock-vaccinated mice. In summary, this is the first report to show that vaccination with HSV-1 gK can provide protection against lethal HSV-1 challenge and that vaccination with an HSV-1 glycoprotein can significantly increase the severity of HSV-1-induced ocular disease.     

Expression of herpes simplex virus type 1 glycoprotein D deletion mutants in mammalian cells.

Glycoprotein D (gD) is a viron envelope component of herpes simplex virus types 1 and 2. We have previously defined seven monoclonal antibody (MAb) groups which recognize distinct epitopes on the mature gD-1 protein of 369 amino acids. MAb groups VII, II, and V recognize continuous epitopes at residues 11-19, 272-279, and 340-356, respectively. MAb groups I, III, IV, and VI recognize discontinuous epitopes. Recent studies have focused on epitopes I, III, and VI. Using truncated forms of gD generated by recombinant DNA methods and proteolysis, epitopes III, IV, and VI were located within amino acids 1-233. A portion of discontinuous epitope I was located in a region within residues 233-275. For this study, we used recombinant DNA methods to create mutations in the gD-1 gene and studied the effects of those mutations on gD as expressed in mammalian cells. Plasmid pRE4, containing the coding sequence of gD-1 and the Rous sarcoma virus long terminal repeat promoter, was transfected into mammalian cells. The expressed protein, gD-1-(pRE4), was identical in size and antigenic properties to gD-1 from infected cells. Six in-frame deletion mutations were subsequently constructed by using restriction enzymes to excise portions of the gD-1 gene. Plasmids carrying these mutated forms were transfected into cells, and the corresponding proteins were examined at 48 h posttransfection for antigenicity and glycosylation patterns. Three deletions of varying size were located downstream of residue 233. Analysis of these mutants showed that amino acids within the region 234-244 were critical for binding of DL11 (group I), but not for other MAb groups. Three other deletion mutants lost all ability to bind MAbs which recognize discontinuous epitopes. In addition, much of the gD expressed by these mutants was observed to migrate as high-molecular-weight aggregated forms in nondenaturing gels. Each of these mutations involved the loss of a cysteine residue, suggesting that disulfide linkages play an essential role in the formation of discontinuous epitopes. The extent of glycosylation of the mutant gD molecules accumulated at 48 h posttransfection suggested altered carbohydrate processing. In one case, there was evidence for increased O-linked glycosylation. Those proteins which had lost a cysteine residue as part of the deletion did not accumulate molecules processed beyond the high-mannose stage. The results suggest that carbohydrate processing during synthesis of gD is very sensitive to alterations in structure, particularly changes involving cysteine residues.     

Cross-linking of glycoprotein oligomers during herpes simplex virus type 1 entry.

Herpes simplex virus (HSV) has 10 glycoproteins in its envelope. Glycoprotein B (gB), gC, gD, gH, and gL have been implicated in virus entry. We previously used chemical cross-linking to show that these five glycoproteins were close enough to each other to be cross-linked into homodimeric and hetero-oligomeric forms; hetero-oligomers of gB-gC, gC-gD, gD-gB, gH-gL, gC-gL and gD-gL were found in purified virions. To better understand the roles of these glycoproteins in viral entry, we have modified a standard HSV penetration assay to include cross-linkers. This allowed us to examine changes in associations of viral glycoproteins during the entry process. HSV-1(KOS) was adsorbed at 4 degrees C to human neuroblastoma cells (SY5Y). The temperature was raised to 37 degrees C and cells were treated with cross-linker at various times after the temperature shift. Cytoplasmic extracts were examined by Western blotting (immunoblotting) for viral glycoproteins. We found that (i) as in virus alone, the length and concentration of the cross-linking agent affected the number of specific complexes isolated; (ii) the same glycoprotein patterns found in purified virions were also present after attachment of virions to cells; and (iii) the ability to cross-link HSV glycoproteins changed as virus penetration proceeded, e.g., gB and gD complexes which were present during attachment disappeared with increasing time, and their disappearance paralleled the kinetics of penetration. However, this phenomenon appeared to be selective since it was not observed with gC oligomers. In addition, we examined the cross-linking patterns of gB and gD in null viruses K082 and KOSgD beta. Neither of these mutants, which attach but cannot penetrate, showed changes in glycoprotein cross-linking over time. We speculate that these changes are due to conformational changes which preclude cross-linking or spatial alterations which dissociate the glycoprotein interactions during the penetration events.     

Fusogenic domains in herpes simplex virus type 1 glycoprotein H

Infection of eukaryotic cells by enveloped viruses requires fusion between the viral envelope and the cellular plasma or endosomal membrane. The actual merging of the two membranes is mediated by viral envelope glycoproteins, which generally contain a highly hydrophobic region termed the fusion peptide. The entry of herpesviruses is mediated by three conserved proteins: glycoproteins B, H (gH), and L. However, how fusion is executed remains unknown. Herpes simplex virus type 1 gH exhibits features typical of viral fusion glycoproteins, and its ectodomain seems to contain a putative internal fusion peptide. Here, we have identified additional internal segments able to interact with membranes and to induce membrane fusion of large unilamellar vesicles. We have applied the hydrophobicity-at-interface scale proposed by Wimley and White (Wimley, W. C., and White, S. H. (1996) Nat. Struct. Biol. 3, 842-848) to identify six hydrophobic stretches within gH with a tendency to partition into the membrane interface, and four of them were able to induce membrane fusion. Experiments in which equimolar mixtures of gH peptides were used indicated that different fusogenic regions may act in a synergistic way. The functional and structural characterization of these segments suggests that herpes simplex virus type 1 gH possesses several fusogenic internal peptides that could participate in the actual fusion event.     

Herpes simplex virus glycoprotein D mediates interference with herpes simplex virus infection.

We showed that the expression of a single protein, glycoprotein D (gD-1), specified by herpes simplex virus type 1 (HSV-1) renders cells resistant to infection by HSV but not to infection by other viruses. Mouse (LMtk-) and human (HEp-2) cell lines containing the gene for gD-1 under control of the human metallothionein promoter II expressed various levels of gD-1 constitutively and could be induced to express higher levels with heavy metal ions. Radiolabeled viruses bound equally well to gD-1-expressing and control cell lines. Adsorbed viruses were unable to penetrate cells expressing sufficient levels of gD-1, based on lack of any cytopathic effects of the challenge virus and on failure to detect either the induction of viral protein synthesis or the shutoff of host protein synthesis normally mediated by a virion-associated factor. The resistance to HSV infection conferred by gD-1 expression was not absolute and depended on several variables, including the amount of gD-1 expressed, the dosage of the challenge virus, the serotype of the challenge virus, and the properties of the cells themselves. The interference activity of gD-1 is discussed in relation to the role of gD-1 in virion infectivity and its possible role in permitting escape of progeny HSV from infected cells.