Soluble forms of herpes simplex virus glycoprotein D bind to a limited number of cell surface receptors and inhibit virus entry into cells.

Herpes simplex virus type 1 (HSV-1) and HSV-2 plaque production was inhibited by treating cells with soluble forms of HSV-1 glycoprotein D (gD-1t) and HSV-2 glycoprotein D (gD-2t). Both glycoproteins inhibited entry of HSV-1 and HSV-2 without affecting virus adsorption. In contrast, a soluble form of HSV-2 glycoprotein B had no effect on virus entry into cells. Specific binding of gD-1t and gD-2t to cells was saturable, and approximately 4 x 10(5) to 5 x 10(5) molecules bound per cell. Binding of gD-1t was markedly reduced by treating cells with certain proteases but was unaffected when cell surface heparan sulfate glycosaminoglycans were enzymatically removed or when the binding was carried out in the presence of heparin. Together, these results suggest that gD binds to a limited set of cell surface receptors which may be proteins and that these interactions are essential for subsequent virus entry into cells. However, binding of gD to its receptors is not required for the initial adsorption of virus to the cell surface, which involves more numerous sites (probably including heparan sulfate) than those which mediate gD binding.     

Antigenic cross-reactions among herpes simplex virus types 1 and 2, Epstein-Barr virus, and cytomegalovirus.

Polyvalent rabbit antisera against herpes simplex virus type 1 and 2 (HSV-1 and HSV-2), cytomegalovirus (CMV), and Epstein-Barr virus (EBV), monospecific antisera against affinity-purified HSV-2 glycoproteins gB and gG, and a panel of monoclonal antibodies against HSV and EBV proteins were used to analyze cross-reactive molecules in cells infected with the four herpesviruses. A combination of immunoprecipitation and Western blotting with these reagents was used to determine that all four viruses coded for a glycoprotein that cross-reacted with HSV-1 gB. CMV coded for proteins that cross-reacted with HSV-2 gC, gD, and gE. Both CMV and EBV coded for proteins that cross-reacted with HSV-2 gG. Antigenic counterparts to the p45 nucleocapsid protein of HSV-2 were present in HSV-1 and CMV, and counterparts of the major DNA-binding protein and the ribonucleotide reductase of HSV-1 were present in all the viruses. The EBV virion glycoprotein gp85 was immunoprecipitated by antisera to HSV-1, HSV-2, and CMV. Antisera to CMV and EBV neutralized the infectivity of both HSV-1 and HSV-2 at high concentrations. This suggests that cross-reactivity between these four human herpesviruses may have pathogenic as well as evolutionary significance.     

Sequential isolation of proteoglycan synthesis mutants by using herpes simplex virus as a selective agent: evidence for a proteoglycan-independent virus entry pathway.

A novel mouse L-cell mutant cell line defective in the biosynthesis of glycosaminoglycans was isolated by selection for cells resistant to herpes simplex virus (HSV) infection. These cells, termed sog9, were derived from mutant parental gro2C cells, which are themselves defective in heparan sulfate biosynthesis and 90% resistant to HSV type 1 (HSV-1) infection compared with control L cells (S. Gruenheid, L. Gatzke, H. Meadows, and F. Tufaro, J. Virol. 67:93-100, 1993). In this report, we show that sog9 cells exhibit a 3-order-of-magnitude reduction in susceptibility to HSV-1 compared with control L cells. In steady-state labeling experiments, sog9 cells accumulated almost no [35S]sulfate-labeled or [6-3H]glucosamine-labeled glycosaminoglycans, suggesting that the initiation of glycosaminoglycan assembly was specifically reduced in these cells. Despite these defects, sog9 cells were fully susceptible to vesicular stomatitis virus (VSV) and permissive for both VSV and HSV replication, assembly, and egress. HSV plaques formed in the sog9 monolayers in proportion to the amount of input virus, suggesting the block to infection was in the virus entry pathway. More importantly, HSV-1 infection of sog9 cells was not significantly reduced by soluble heparan sulfate, indicating that infection was glycosaminoglycan independent. Infection was inhibited by soluble gD-1, however, which suggests that glycoprotein gD plays a role in the infection of this cell line. The block to sog9 cell infection by HSV-1 could be eliminated by adding soluble dextran sulfate to the inoculum, which may act by stabilizing the virus at the sog9 cell surface. Thus, sog9 cells provide direct genetic evidence for a proteoglycan-independent entry pathway for HSV-1, and results with these cells suggest that HSV-1 is a useful reagent for the direct selection of novel animal cell mutants defective in the synthesis of cell surface proteoglycans.     

Resistance of herpes simplex virus type 2 to neomycin maps to the N-terminal portion of glycoprotein C.

Entry of herpes simplex virus (HSV) into cells is believed to be mediated by specific binding of envelope proteins to a cellular receptor. Neomycin specifically blocks this initial step in infection by HSV-1 but not HSV-2. Resistance of HSV-2 to this compound maps to a region of the genome encoding glycoprotein C (gC-2). We have studied the function of gC-2 in the initial interaction of the virus with the host cell, using HSV-2 mutants deleted for gC-2 and gC-2-rescued recombinants. Resistance to neomycin was directly linked to the presence of gC-2 within the viral genome. In addition, deletion of the gC-2 gene caused a marked delay in adsorption to cells relative to the wild-type virus. HSV-1 recombinants containing chimeric gC genes composed of HSV-1 and HSV-2 sequences were used to localize neomycin resistance within the N-terminal 223 amino acids of gC-2. This region of the glycoprotein comprises an important domain responsible for binding of HSV-2 to cell receptors in the presence of neomycin. A gC-2-negative mutant is still infectious, indicating that HSV-2 also has an alternative pathway of adsorption.     

A cellular function is required for pseudorabies virus envelope glycoprotein processing and virus egress.

The mouse L-cell mutant gro29 is defective for egress of herpes simplex virus type 1 (HSV-1) virions and is significantly reduced in HSV-1 glycoprotein export (B. W. Banfield and F. Tufaro, J. Virol. 64:5716-5729, 1990). In this report, we demonstrate that pseudorabies virus (PRV), a distantly related alphaherpesvirus, shows a distinctive set of defects after infection of gro29 cells. Specifically, we identify defects in the rate and extent of viral glycoprotein export, infectious particle formation, plaque formation, and virus egress. The initial rate of viral glycoprotein synthesis was unaffected in gro29 cells, but the extent of export from the endoplasmic reticulum to the Golgi apparatus was impaired and export through the Golgi apparatus became essentially blocked late in infection. Moreover, by using a secreted variant of a viral membrane protein, we found that export from the Golgi apparatus out of the cell was also defective in gro29 cells. PRV does not form plaques on gro29 monolayers. A low level of infectious virus is formed and released early after infection, but further virus egress is blocked. Taken together, these observations suggest that the gro29 phenotype involves either multiple proteins or a single protein used at multiple steps in viral glycoprotein export and virus egress from cells. Moreover, this host cell protein is required by both HSV and PRV for efficient propagation in infected cells.     

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.     

A novel herpes simplex virus glycoprotein, gL, forms a complex with glycoprotein H (gH) and affects normal folding and surface expression of gH.

A glycoprotein encoded by the UL1 gene of herpes simplex virus type 1 (HSV-1) was detected in infected cells with antipeptide sera. The UL1 gene has previously been implicated in virus-induced cell fusion (S. Little and P. A. Schaffer, Virology 112:686-697, 1981). Two protein species, a 30-kDa precursor form and a 40-kDa mature form of the glycoprotein, both of which were modified with N-linked oligosaccharides, were observed. This novel glycoprotein is the 10th HSV-1 glycoprotein to be described and was named glycoprotein L (gL). A complex was formed between gL and gH, a glycoprotein known to be essential for entry of HSV-1 into cells and for virus-induced cell fusion. Previously, it had been reported that gH expressed in the absence of other viral proteins was antigenically abnormal, not processed, and not expressed at the cell surface (U.A. Gompels and A. C. Minson, J. Gen. Virol. 63:4744-4755, 1989; A. J. Forrester, V. Sullivan, A. Simmons, B. A. Blacklaws, G. L. Smith, A. A. Nash, and A. C. Minson, J. Gen. Virol. 72:369-375, 1991). However, gH coexpressed with gL by using vaccinia virus recombinants was antigenically normal, processed normally, and transported to the cell surface. Similarly, gL was dependent on gH for proper posttranslational processing and cell surface expression. These results suggest that it is a hetero-oligomer of gH and gL which is incorporated into virions and transported to the cell surface and which acts during entry of virus into cells.     

The pseudorabies virus gII gene is closely related to the gB glycoprotein gene of herpes simplex virus.

We have looked for conserved DNA sequences between four herpes simplex virus type 1 (HSV-1) glycoprotein genes encoding gB, gC, gD, and gE and pseudorabies virus (PRV) DNA, HSV-1 DNA fragments representing these four glycoprotein-coding sequences were hybridized to restriction enzyme fragments of PRV DNA by the Southern blot procedure. Specific hybridization was observed only when HSV-1 gB DNA was used as probe. This region of hybridization was localized to a 5.2-kilobase (kb) region mapping at approximately 0.15 map units on the PRV genome. Northern blot (RNA blot) analysis, with a 1.2-kb probe derived from this segment, revealed a predominant hybridizing RNA species of approximately 3 kb in PRV-infected PK15 cells. DNA sequence analysis of the region corresponding to this RNA revealed a single large open reading frame with significant nucleotide homology with the gB gene of HSV-1 KOS 321. In addition, the beginning of the sequenced PRV region also contained the end of an open reading frame with amino acid homology to HSV-1 ICP 18.5, a protein that may be involved in viral glycoprotein transport. This sequence partially overlaps the PRV gB homolog coding sequence. We have shown that the PRV gene with homology to HSV-1 gB encoded the gII glycoprotein gene by expressing a 765-base-pair segment of the PRV open reading frame in Escherichia coli as a protein fused to beta-galactosidase. Antiserum, raised in rabbits, against this fusion protein immunoprecipitated a specific family of PRV glycoproteins of apparent molecular mass 110, 68, and 55 kilodaltons that have been identified as the gII family of glycoproteins. Analysis of the predicted amino acid sequence indicated that the PRV gII protein shares 50% amino acid homology with the aligned HSV-1 gB protein. All 10 cysteine residues located outside of the signal sequence, as well as 4 of 6 potential N-linked glycosylation sites, were conserved between the two proteins. The primary protein sequence for HSV-1 gB regions known to be involved in the rate of virus entry into the cells and cell-cell fusion, as well as regions known to be associated with monoclonal antibody resistance, were highly homologous with the PRV protein sequence. Furthermore, monospecific antibody made against PRV gII immunoprecipitated HSV-1 gB from infected cells. Taken together, these findings suggest significant conservation of structure and function between the two proteins and may indicate a common evolutionary history.     

Quantitation of latent varicella-zoster virus and herpes simplex virus genomes in human trigeminal ganglia

Using real-time fluorescence PCR, we quantitated the numbers of copies of latent varicella-zoster virus (VZV) and herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) genomes in 15 human trigeminal ganglia. Eight (53%) and 1 (7%) of 15 ganglia were PCR positive for HSV-1 or -2 glycoprotein G genes, with means of 2,902 +/- 1,082 (standard error of the mean) or 109 genomes/10(5) cells, respectively. Eleven of 14 (79%) to 13 of 15 (87%) of the ganglia were PCR positive for VZV gene 29, 31, or 62. Pooling of the results for the three VZV genes yielded a mean of 258 +/- 38 genomes/10(5) ganglion cells. These levels of latent viral genome loads have implications for virus distribution in and reactivation from human sensory ganglia.     

Discovery of potential diagnostic and vaccine antigens in herpes simplex virus 1 and 2 by proteome-wide antibody profiling

Routine serodiagnosis of herpes simplex virus (HSV) infections is currently performed using recombinant glycoprotein G (gG) antigens from herpes simplex virus 1 (HSV-1) and HSV-2. This is a single-antigen test and has only one diagnostic application. Relatively little is known about HSV antigenicity at the proteome-wide level, and the full potential of mining the antibody repertoire to identify antigens with other useful diagnostic properties and candidate vaccine antigens is yet to be realized. To this end we produced HSV-1 and -2 proteome microarrays in Escherichia coli and probed them against a panel of sera from patients serotyped using commercial gG-1 and gG-2 (gGs for HSV-1 and -2, respectively) enzyme-linked immunosorbent assays. We identified many reactive antigens in both HSV-1 and -2, some of which were type specific (i.e., recognized by HSV-1- or HSV-2-positive donors only) and others of which were nonspecific or cross-reactive (i.e., recognized by both HSV-1- and HSV-2-positive donors). Both membrane and nonmembrane virion proteins were antigenic, although type-specific antigens were enriched for membrane proteins, despite being expressed in E. coli.     

Specificity of human natural killer cells in limiting dilution culture for determinants of herpes simplex virus type 1 glycoproteins.

The frequency and specificity of human cells with natural killer (NK) cytotoxic activity for herpes simplex virus type 1 (HSV-1)-infected targets was measured by limiting dilution culture. The frequency of NK cell precursors (NK-p) reactive with HSV-1-infected cells was 2- to 11-fold higher than that of NK-p reactive with mock-infected cells. The frequency of NK-p reactive with infected target cells lacking viral glycoprotein C or presenting an antigenically altered glycoprotein B was approximately twofold lower than that with wild-type virus-infected cells. Specificity analysis demonstrated that NK cells with a high statistical probability of being monoclonal were reactive with either glycoprotein B or C. These results provide the first evidence that cells with human NK activity possess clonal specificity for HSV-1-infected target cells.     

Use of lambda gt11 to isolate genes for two pseudorabies virus glycoproteins with homology to herpes simplex virus and varicella-zoster virus glycoproteins.

A library of pseudorabies virus (PRV) DNA fragments was constructed in the expression cloning vector lambda gt11. The library was screened with antisera which reacted with mixtures of PRV proteins to isolate recombinant bacteriophages expressing PRV proteins. By the nature of the lambda gt11 vector, the cloned proteins were expressed in Escherichia coli as beta-galactosidase fusion proteins. The fusion proteins from 35 of these phages were purified and injected into mice to raise antisera. The antisera were screened by several different assays, including immunoprecipitation of [14C]glucosamine-labeled PRV proteins. This method identified phages expressing three different PRV glycoproteins: the secreted glycoprotein, gX; gI; and a glycoprotein that had not been previously identified, which we designate gp63. The gp63 and gI genes map adjacent to each other in the small unique region of the PRV genome. The DNA sequence was determined for the region of the genome encoding gp63 and gI. It was found that gp63 has a region of homology with a herpes simplex virus type 1 (HSV-1) protein, encoded by US7, and also with varicella-zoster virus (VZV) gpIV. The gI protein sequence has a region of homology with HSV-1 gE and VZV gpI. It is concluded that PRV, HSV, and VZV all have a cluster of homologous glycoprotein genes in the small unique components of their genomes and that the organization of these genes is conserved.     

Specificity and affinity of binding of herpes simplex virus type 2 glycoprotein B to glycosaminoglycans.

Herpes simplex virus type 2 (HSV-2) interacts with cell surface glycosaminoglycans during virus attachment. Glycoprotein B of HSV-2 can potentially mediate the interaction between the virion and cell surface glycosaminoglycans. To determine the specificity, kinetics, and affinity of these interactions, we used plasmon resonance-based biosensor technology to measure HSV-2 glycoprotein binding to glycosaminoglycans in real time. The recombinant soluble ectodomain of HSV-2 gB (gB2) but not the soluble ectodomain of HSV-2 gD bound readily to biosensor surfaces coated with heparin. The affinity constants (Kds) were determined for gB2 (Kd = 7.7 x 10(-7) M) and for gB2 deltaTM (Kd = 9.9 x 10(-7) M), a recombinant soluble form of HSV-2 gB in which only its transmembrane domain has been deleted. gB2 binding to the heparin surface was competitively inhibited by low concentrations of heparin (50% effective dose [ED50] = 0.08 microg/ml). Heparan sulfate and dermatan sulfate glycosaminoglycans have each been suggested as cell surface receptors for HSV. Our biosensor analyses showed that both heparan sulfate and dermatan sulfate inhibited gB2 binding (ED50 = 1 to 5 microg/ml), indicating that gB2 interacts with both heparin-like and dermatan sulfate glycosaminoglycans. Chondroitin sulfate A, in contrast, inhibited gB2 binding to heparin only at high levels (ED50 = 65 microg/ml). The affinity and specificity of gB2 binding to glycosaminoglycans demonstrated in these studies support its role in the initial binding of HSV-2 to cells bearing heparan sulfate or dermatan sulfate glycosaminoglycans.     

Protection by herpes simplex virus glycoprotein D against Fas-mediated apoptosis: role of nuclear factor kappaB

Signals involved in protection against apoptosis by herpes simplex virus 1 (HSV-1) were investigated. Using U937 monocytoid cells as an experimental model, we have demonstrated that HSV-1 rendered these cells resistant to Fas-induced apoptosis promptly after infection. UV-inactivated virus as well as the envelope glycoprotein D (gD) of HSV-1, by itself, exerted a protective effect on Fas-induced apoptosis. NF-kappaB was activated by gD, and protection against Fas-mediated apoptosis by gD was abolished in cells stably transfected with a dominant negative mutant I-kappaBalpha, indicating that NF-kappaB activation plays a role in the antiapoptotic activity of gD in our experimental model. Moreover, NF-kappaB-dependent protection against Fas-mediated apoptosis was associated with decreased levels of caspase-8 activity and with the up-regulation of intracellular antiapoptotic proteins.     

Glycoprotein gE of herpes simplex virus type 1: effects of anti-gE on virion infectivity and on virus-induced fc-binding receptors.

An Fc-binding glycoprotein, designated gE, was detected previously in cells infected with herpes simplex virus type 1 (HSV-1) and in virion preparations isolated from infected cells. For the studies reported here, we purified gE from HSV-1 strain HFEM(syn) by affinity chromatography and preparative electrophoresis and then immunized a rabbit to produce an antiserum to glycoprotein gE. We found that this antiserum selectively precipitated gE and its precursors from detergent-solubilized extracts of HSV-1 strain HFEM(syn)-infected HEp-2 cells, from extracts of other cell lines infected with the same virus, and from extracts of HEp-2 cells infected with several other HSV-1 strains. The antiserum did not precipitate any proteins from uninfected cells. The several forms of gE detected by immunoprecipitation accumulated in variable quantities in different cells infected with the different virus strains and also varied slightly with respect to electrophoretic mobility, suggesting some differences in the gE's from different HSV-1 strains and some effects of the host cell on the nature and extent of post-translational processing. One of the electrophoretic forms of gE previously detected in purified preparations of virions could be precipitated by anti-gE from extracts of purified HSV-1 strain HFEM(syn) virions. Moreover, anti-gE neutralized HSV-1 infectivity, but only in the presence of complement. Finally, F(ab')2 fragments of the anti-gE immunoglobulin partially inhibited the binding of 125I-labeled immunoglobulin G to the Fc receptors on HSV-1-infected cells.     

Locations of herpes simplex virus type 2 glycoprotein B epitopes recognized by human serum immunoglobulin G antibodies.

Herpes simplex virus type 2 (HSV-2) glycoprotein B (gB-2) gene segments were expressed as recombinant proteins in Escherichia coli. gB-2 recombinant proteins were reacted with human serum immunoglobulin G (IgG) antibodies in Western immunoblot assays. Initially, samples were tested for the presence of HSV-1-specific antibodies and HSV-2-specific antibodies by using HSV-infected cell lysates as antigen targets in Western blot assays. Serum samples that contained HSV-2-specific IgG (n = 58), HSV-1-specific IgG (n = 33), or no detectable HSV antibodies (n = 31) were tested for reactivities with the gB-2 recombinant proteins. In 58 of 58 samples that contained HSV-2-specific IgG, antibodies were present that reacted strongly with a gB-2 amino-proximal segment between amino acids (aa) 18 and 75. Three of 33 serum samples that contained HSV-1- and not HSV-2-specific IgG (as defined by the HSV lysate Western blot assay) reacted with this segment. Both HSV-2 antibodies and HSV-1 antibodies reacted strongly with a carboxy-terminal gB-2 segment between aa 819 and 904; a second minor cross-reactive region was mapped to a gB-2 segment between aa 564 and 626. The gB-2 segment from aa 18 to 75 may constitute a useful reagent for the virus type-specific serodiagnosis of HSV-2 infections. Further studies will be required to determine the relative sensitivities and specificities of the assay for gB-2 aa 18 to 75, HSV gG assays, and HSV lysate Western blot assays for detecting virus type-specific antibody responses in acute and chronic HSV-2 infections.     

Recombinant Herpes Simplex Virus Type 1 Engineered for Targeted Binding to Erythropoietin Receptor-Bearing Cells

The utility of recombinant herpes simplex virus type 1 (HSV-1) vectors may be expanded by manipulation of the virus envelope to achieve cell-specific gene delivery. To this end, an HSV-1 mutant virus deleted for glycoprotein C (gC) and the heparan sulfate binding domain of gB (KgBpK⁻gC⁻) was engineered to encode different chimeric proteins composed of N-terminally truncated forms of gC and the full-length erythropoietin hormone (EPO). Biochemical analyses demonstrated that one gC-EPO chimeric molecule (gCEPO₂) was posttranslationally processed, incorporated into recombinant HSV-1 virus (KgBpK⁻gCEPO₂), and neutralized with antibodies directed against gC or EPO in a complement-dependent manner. Moreover, KgBpK⁻gCEPO₂ recombinant virus was specifically retained on a soluble EPO receptor column, was neutralized by soluble EPO receptor, and stimulated proliferation of FD-EPO cells, an EPO growth-dependent cell line. FD-EPO cells were nevertheless refractory to productive infection by both wild-type HSV-1 and recombinant KgBpK⁻gCEPO₂ virus. Transmission electron microscopy of FD-EPO cells infected with KgBpK⁻gCEPO₂ showed virus endocytosis leading to aborted infection. Despite the lack of productive infection, these data provide the first evidence of targeted HSV-1 binding to a non-HSV-1 cell surface receptor.     

Antigenic structure of soluble herpes simplex virus (HSV) glycoprotein D correlates with inhibition of HSV infection.

Soluble forms of herpes simplex virus (HSV) glycoprotein D (gD) block viral penetration. Likewise, most HSV strains are sensitive to gD-mediated interference by cells expressing gD. The mechanism of both forms of gD-mediated inhibition is thought to be at the receptor level. We analyzed the ability of different forms of soluble, truncated gD (gDt) to inhibit infection by different strains of HSV-1 and HSV-2. Strains that were resistant to gD-mediated interference were also resistant to inhibition by gDt, thereby suggesting a link between these two phenomena. Virion gD was the major viral determinant for resistance to inhibition by gDt. An insertion-deletion mutant, gD-1(delta 290-299t), had an enhanced inhibitory activity against most strains tested. The structure and function of gDt proteins derived from the inhibition-resistant viruses rid1 and ANG were analyzed. gD-1(ridlt) and gD-1(ANGt) had a potent inhibitory effect on plaque formation by wild-type strains of HSV but, surprisingly, little or no effect on their parental strains. As measured by quantitative enzyme-linked immunosorbent assay with a diverse panel of monoclonal antibodies, the antigenic structures of gD-1(rid1t) and gD-1(ANGt) were divergent from that of the wild type yet were similar to each other and to that of gD-1 (delta 290-299t). Thus, three different forms of gD have common antigenic changes that correlate with enhanced inhibitory activity against HSV. We conclude that inhibition of HSV infectivity by soluble gD is influenced by the antigenic conformation of the blocking gDt as well as the form of gD in the target virus.     

Comparative structural analysis of glycoprotein gD of herpes simplex virus types 1 and 2.

We studied the synthesis and processing of the type-common glycoprotein gD in herpes simplex virus type 2 (HSV-2) and compared it structurally to glycoprotein gD of herpes simplex virus type 1 (HSV-1). We demonstrated that in HSV-2, gD undergoes posttranslational processing from a lower-molecular-weight precursor (pgD51) to a higher-molecular-weight product (gD56). Tryptic peptide analysis by cation-exchange chromatography indicated that this processing step altered neither the methionine nor the arginine tryptic peptide profile of gD of HSV-2. Comparative tryptic peptide analysis of gD of HSV-1 and HSV-2 showed that the methionine and arginine tryptic peptide profiles of these two proteins were very similar, but not identical. Some of the resolved peptides coeluted from the cation-exchange column, suggesting that some amino acid sequences of the two proteins might be very similar. However, each protein also appeared to possess several type-specific tryptic peptides. The structural similarity of these two glycoproteins correlates well with their antigenic cross-reactivity since monoprecipitin antibody to gD of HSV-1 also immunoprecipitates gD of HSV-2 and neutralizes the infectivity of both viruses to approximately the same extent.