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.     

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.     

Site-directed and linker insertion mutagenesis of herpes simplex virus type 1 glycoprotein H.

The gH-gL complex of herpes simplex virus type 1 (HSV-1) is essential for virion infectivity and virus-induced cell fusion, but functional domains of the gH molecule remain to be defined. We have addressed this question by mutagenesis. A set of linker insertion mutants in HSV-1 gH was generated and tested in transient assays for their ability to complement a gH-negative virus. Insertions at three sites in the C-terminal third of the external domain affected the ability of gH to function in cell-cell fusion and virus entry, while insertions at six sites in the N-terminal half of the external domain induced conformational changes in gH such that it was not recognized by monoclonal antibody LP11, although expression at the cell surface was unchanged. A recombinant virus in which a potential integrin-binding motif, RGD, in gH was changed to the triplet RGE entered cells as efficiently as the wild type, indicating that HSV-1 entry is not mediated by means of the gH-RGD motif binding to cell surface integrins. Furthermore, mutagenesis of the glycosylation site which is positionally conserved in all herpesvirus gH sequences in close proximity to the transmembrane domain generated a recombinant virus that grew in vitro with wild-type single-step kinetics.     

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.     

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.     

Interactome analysis of herpes simplex virus 1 envelope glycoprotein H

Herpes simplex virus 1 (HSV‐1) envelope glycoprotein H (gH) is important for viral entry into cells and nuclear egress of nucleocapsids. To clarify additional novel roles of gH during HSV‐1 replication, host cell proteins that interact with gH were screened for by tandem affinity purification coupled with mass spectrometry‐based proteomics in 293T cells transiently expressing gH. This screen identified 123 host cell proteins as potential gH interactors. Of these proteins, general control nonderepressive‐1 (GCN1), a trans‐acting positive effector of GCN2 kinase that regulates phosphorylation of the α subunit of translation initiation factor 2 (eIF2α), was subsequently confirmed to interact with gH in HSV‐1‐infected cells. eIF2α phosphorylation is known to downregulate protein synthesis, and various viruses have evolved mechanisms to prevent the accumulation of phosphorylated eIF2α in infected cells. Here, it was shown that GCN1 knockdown reduces phosphorylation of eIF2α in HSV‐1‐infected cells and that the gH‐null mutation increases eIF2α in HSV‐1‐infected cells, whereas gH overexpression in the absence of other HSV‐1 proteins reduces eIF2α phosphorylation. These findings suggest that GCN1 can regulate eIF2α phosphorylation in HSV‐1‐infected cells and that the GCN1‐binding viral partner gH is necessary and sufficient to prevent the accumulation of phosphorylated eIF2α. Our database of 123 host cell proteins potentially interacting with gH will be useful for future studies aimed at unveiling further novel functions of gH and the roles of cellular proteins in HSV‐1‐infected cells.     

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.     

Analysis of a membrane interacting region of herpes simplex virus type 1 glycoprotein H

Glycoprotein H (gH) of herpes simplex virus type I (HSV-1) is involved in the complex mechanism of membrane fusion of the viral envelope with the host cell. Membrane interacting regions and potential fusion peptides have been identified in HSV-1 gH as well as glycoprotein B (gB). Because of the complex fusion mechanism of HSV-1, which requires four viral glycoproteins, and because there are only structural data for gB and glycoprotein D, many questions regarding the mechanism by which HSV-1 fuses its envelope with the host cell membrane remain unresolved. Previous studies have shown that peptides derived from certain regions of gH have the potential to interact with membranes, and based on these findings we have generated a set of peptides containing mutations in one of these domains, gH-(626-644), to investigate further the functional role of this region. Using a combination of biochemical, spectroscopic, and nuclear magnetic resonance techniques, we showed that the alpha-helical nature of this stretch of amino acids in gH is important for membrane interaction and that the aromatic residues, tryptophan and tyrosine, are critical for induction of fusion.     

Oligomer formation of the gB glycoprotein of herpes simplex virus type 1.

Oligomer formation of the gB glycoprotein of herpes simplex virus type 1 was studied by sedimentation analysis of radioactively labeled infected cell and virion lysates. Fractions from sucrose gradients were precipitated with a pool of gB-specific monoclonal antibodies and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Pulse-labeled gB from infected cell was synthesized as monomers and converted to oligomers posttranslationally. The oligomers from infected cells and from virions sedimented as dimers, and there was no evidence of higher-molecular-weight forms. To identify amino acid sequences of gB that contribute to oligomer formation, pairs of mutant plasmids were transfected into Vero cells and superinfected with a gB-null mutant virus to stimulate plasmid-specified gene expression. Radioactively labeled lysates were precipitated with antibodies and examined by SDS-PAGE. Polypeptides from cotransfections were precipitated with an antibody that recognized amino acid sequences present in only one of the two polypeptides. A coprecipitated polypeptide lacking the antibody target epitope was presumed to contain the sequences necessary for oligomer formation. Using this technique, two noncontiguous sites for oligomer formation were detected. An upstream site was localized between residues 93 and 282, and a downstream site was localized between residues 596 and 711. Oligomer formation resulted from molecular interactions between two upstream sites, between two downstream sites, and between an upstream and a downstream site. A schematic diagram of a gB oligomer is presented that is consistent with these data.     

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.     

Replacement of the pseudorabies virus glycoprotein gIII gene with its postulated homolog, the glycoprotein gC gene of herpes simplex virus type 1.

gIII, the major envelope glycoprotein of pseudorabies virus (PRV), shares approximately 20% amino acid similarity with glycoprotein gC of herpes simplex virus type 1 (HSV-1) and HSV-2. We describe here our first experiments on the potential conservation of function between these two genes and gene products. We constructed PRV recombinants in which the gIII gene and regulatory sequences have been replaced with the entire HSV-1 gC gene and its regulatory sequences. The gC promoter functions in the PRV genome, and authentic HSV-1 gC protein is produced, albeit at a low level, in infected cells. The gC protein is present at the cell surface but cannot be detected in the PRV envelope.     

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.     

Structure and expression of the herpes simplex virus type 2 glycoprotein gB gene.

The gene for glycoprotein gB2 of herpes simplex virus type 2 strain 333 was cloned, sequenced, and expressed in mammalian cells. The gB2 protein had an overall nucleotide and amino acid sequence homology of 86% with the cognate gB1 protein. However, of the 125 amino acid substitutions or deletions, only 12.5% were conservative replacements. These differences were clustered within an NH2-terminal region, a central region, and a COOH-terminal region, resulting in domains of near identity broken by small regions of marked divergence. Regions of greatest homology included a 90-amino-acid stretch starting at residue 484 and 39 amino acids spanning residues 835 to 873, which cover a rate-of-entry locus mapped to Ala-552 and a syn locus mapped to Arg-857, respectively, in gB1 by Bzik et al. (D. J. Bzik, B. A. Fox, N. A. DeLuca, and S. Person, Virology 133:301-314, 1984). Pellett et al. (P. E. Pellett, K. G. Kousoulas, L. Pereira, and B. Roizman, J. Virol. 53:243-253, 1985) mapped the mutations in three monoclonal antibody-resistant gB1 mutants between amino acids 273 and 443. These epitopes are included in a region of 98 residues identical between gB1 and gB2. The identity of this protein was verified by placing a truncated gene lacking the 303 carboxyl-terminal amino acids of gB2 into mammalian COS and CHO cells. Expression was demonstrated by immunofluorescence and radioimmunoprecipitation. This protein will be purified from the stable CHO cell lines and compared with gB1 for immunogenicity and protective efficacy in animal challenge models.     

Linker-insertion nonsense and restriction-site deletion mutations of the gB glycoprotein gene of herpes simplex virus type 1.

To study the effects of missense, nonsense, and deletion mutations of the gB glycoprotein gene of herpes simplex virus type 1, a gB-transformed cell line was isolated that, after virus infection, would express sufficient quantities of gB from the cellular chromosome to complement temperature-sensitive gB mutants. The transformed cell line was then used as a permissive cell to transfer two gB mutations from plasmid to viral DNA. One of the mutants, K082, harbored an HpaI linker insertion that introduced one new amino acid and a chain terminator codon within amino acid residue 43. The other mutant contained a 969-base-pair deletion in a part of the gene that includes the membrane-spanning region; a correspondingly shorter gB polypeptide was detected by sodium dodecyl sulfate-gel electrophoresis after immunoprecipitation of infected-cell extracts with four pooled monoclonal antibodies. No polypeptide was observed from K082-infected cells. The shortened gB polypeptide was efficiently processed and secreted into the growth medium. Each of the four monoclonal antibodies precipitated full-length gB, and three of the four precipitated the shortened polypeptide. Enveloped virus particles could be purified after infection of nonpermissive cells with either mutant virus. Virus particles appeared to possess normal polypeptide and glycopeptide profiles except for the absence of gB. Therefore, the presence of gB is not essential for viral assembly, including envelopment. Recombinants in virus stocks grown on the gB-transformed cells occurred at frequencies on the order of 10(-7) to 10(-5), compared with a frequency of approximately 10(-2) in mixed infections with the two mutants.     

Mapping of the structural gene for the herpes simplex virus type 2 counterpart of herpes simplex virus type 1 glycoprotein C and identification of a type 2 mutant which does not express this glycoprotein

The gene encoding glycoprotein F (gF) of herpes simplex virus type 2 (HSV-2) was mapped to the region of the viral genome from 0.62 to 0.64 map units. This region is colinear with, and partially homologous to, the region of the HSV-1 genome previously shown to encode gC. Mapping of the gF gene was done by insertion of HSV-2 DNA fragments into the thymidine kinase gene of an HSV-1 virus and screening of the resultant recombinant viruses for the expression of gF. In this way, DNA sequences necessary for the expression of gF in infected cells were also delimited. Because several plaque morphology mutants (syncytial mutants) of HSV-1 have previously been shown to be gC-, a syncytial mutant of HSV-2 (GP) was tested for the expression of gF. It was found to be gF-, indicating that gF is not essential for replication of HSV-2 in cell culture, just as gC is not essential for replication of HSV-1. This result also suggests that the gF- and gC- phenotypes are related in the same, as yet undefined, way to the expression of a syncytial marker. A proposal to change the name of HSV-2 gF to gC (gC-2) is discussed.     

The UL45 gene product is required for herpes simplex virus type 1 glycoprotein B-induced fusion.

Herpes simplex virus type 1 (HSV-1) syncytial (syn) mutants cause formation of giant polykaryocytes and have been utilized to identify genes promoting or suppressing cell fusion. We previously described an HSV-1 recombinant, F1 (J.L. Goodman, M. L. Cook, F. Sederati, K. Izumi, and J. G. Stevens, J. Virol. 63:1153-1161, 1989), which has unique virulence properties and a syn mutation in the carboxy terminus of glycoprotein B (gB). We attempted to replace this single-base-pair syn mutation through cotransfection with a 379-bp PCR-generated fragment of wild-type gB. The nonsyncytial viruses isolated were shown by DNA sequencing not to have acquired the expected wild-type gB sequence. Instead, they had lost their cell-cell fusion properties because of alterations mapping to the UL45 gene. The mutant UL45 gene is one nonsyncytial derivative of F1, A4B, was found to have a deletion of a C at UL45 nucleotide 230, resulting in a predicted frame shift and termination at 92 rather than 172 amino acids. Northern (RNA) analysis showed that the mutant UL45 gene was normally transcribed. However, Western immunoblotting showed no detectable UL45 gene product from A4B or from another similarly isolated nonsyncytial F1 derivative, A61B, while another such virus, 1ACSS, expressed reduced amounts of UL45. When A4B was cotransfected with the wild-type UL45 gene, restoration of UL45 expression correlated with restoration of syncytium formation. Conversely, cloned DNA fragments containing the mutant A4B UL45 gene transferred the loss of cell-cell fusion to other gB syn mutants, rendering them UL45 negative and nonsyncytial. We conclude that normal UL45 expression is required to allow cell fusion induced by gB syn mutants and that the nonessential UL45 protein may play an important role as a mediator of fusion events during HSV-1 infection.     

Disulfide bonds of herpes simplex virus type 2 glycoprotein gB.

Glycoprotein B (gB) is the most highly conserved envelope glycoprotein of herpesviruses. The gB protein is required for virus infectivity and cell penetration. Recombinant forms of gB being used for the development of subunit vaccines are able to induce virus-neutralizing antibodies and protective efficacy in animal models. To gain structural information about the protein, we have determined the location of the disulfide bonds of a 696-amino-acid residue truncated, recombinant form of herpes simplex virus type 2 glycoprotein gB (HSV gB2t) produced by expression in Chinese hamster ovary cells. The purified protein, which contains virtually the entire extracellular domain of herpes simplex virus type 2 gB, was digested with trypsin under nonreducing conditions, and peptides were isolated by reversed-phase high-performance liquid chromatography (HPLC). The peptides were characterized by using mass spectrometry and amino acid sequence analysis. The conditions of cleavage (4 M urea, pH 7) induced partial carbamylation of the N termini of the peptides, and each disulfide peptide was found with two or three different HPLC retention times (peptides with and without carbamylation of either one or both N termini). The 10 cysteines of the molecule were found to be involved in disulfide bridges. These bonds were located between Cys-89 (C1) and Cys-548 (C8), Cys-106 (C2) and Cys-504 (C7), Cys-180 (C3) and Cys-244 (C4), Cys-337 (C5) and Cys-385 (C6), and Cys-571 (C9) and Cys-608 (C10). These disulfide bonds are anticipated to be similar in the corresponding gBs from other herpesviruses because the 10 cysteines listed above are always conserved in the corresponding protein sequences.     

Sequence characteristics of a gene in equine herpesvirus 1 homologous to glycoprotein H of herpes simplex virus.

A gene in equine herpesvirus 1 (EHV-1, equine abortion virus) homologous to the glycoprotein H gene of herpes simplex virus (HSV) was identified and characterised by its nucleotide and derived amino acid sequence. The EHV-1 gH gene is located at 0.47-0.49 map units and contains an open reading frame capable of specifying a polypeptide of 848 amino acids, including N- and C-terminal hydrophobic domains consistent with signal and membrane anchor regions respectively, and 11 potential sites for N-glycosylation. Alignment of the amino acid sequence with those published for HSV gH, varicella zoster virus gpIII, Epstein Barr virus gp85 and human cytomegalovirus p86 shows similarity of the EHV gene with the 2 other alpha-herpesviruses over most of the polypeptide, but only the C-terminal half could be aligned for all 5 viruses. The identical positioning of 6 cysteine residues and a number of highly conserved amino acid motifs supports a common evolutionary origin of this gene and is consistent with its role as an essential glycoprotein of the herpesvirus family. An origin of replication is predicted to occur at approximately 300 nucleotides downstream of the EHV-1 gH coding region, on the basis of similarity to other herpesvirus origins.