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.     

Characterization and mapping of a nonessential pseudorabies virus glycoprotein.

Antigenic variants of pseudorabies virus (PRV) containing mutations in a viral glycoprotein with a molecular weight of 82,000 (gIII) were isolated by selecting for resistance to a complement-dependent neutralizing monoclonal antibody (MCA82-2) directed against gIII. These mutants were completely resistant to neutralization with MCA82-2 in the presence of complement. Two mutants selected for further studies either did not express gIII or expressed an improperly processed form of the glycoprotein. The mutations were also associated with an altered plaque morphology (syncytium formation). The gIII gene was mapped by marker rescue of a gIII- mutant with cloned restriction enzyme fragments to the long unique region of the PRV genome between 0.376 and 0.383 map units. This corresponds to the map location of a glycoprotein described by Robbins et al. (J. Mol. Appl. Gen. 2:485-496, 1984). Since gIII is nonessential for viral replication in cell culture and has several other characteristics in common with the herpes simplex virus glycoprotein gC, gIII may represent the PRV equivalent to herpes simplex virus gC.     

Analysis of pseudorabies virus glycoprotein gIII localization and modification by using novel infectious viral mutants carrying unique EcoRI sites.

We have constructed two pseudorabies virus (PRV) mutants, each with a unique EcoRI restriction site in the nonessential gIII envelope glycoprotein gene. Since no natural PRV isolate has been reported to contain EcoRI sites, the isolation and single-step growth curve analysis of these mutants established that PRV can carry such a site with little ill effect in tissue culture. Virus carrying these defined mutations produced novel gIII proteins that enabled us to begin functional assignment of protein localization information within the gIII gene. Specifically, one viral mutant contained an in-frame synthetic EcoRI linker sequence that was flanked on one side by the first one-third of the gIII gene and on the other side by the last one-third of the gene. The resulting protein lacked the middle one-third of the parental species, including five of eight putative N-linked glycosylation signals, but was still glycosylated and found in enveloped virions; it was not secreted into the medium. A second viral mutant contained an in-frame synthetic EcoRI linker sequence that additionally specified a nonsense codon at position 158, producing a gIII protein that was glycosylated and secreted into the medium; the fragment was not found in enveloped virions. By endoglycosidase and pulse-chase analyses, we established a precursor-product relationship between the various forms of gIII expressed in the parental and mutant strains, and perhaps determined certain features of the gIII protein that are required for its efficient export within the cell.     

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.     

Pseudorabies virus envelope glycoprotein gI influences both neurotropism and virulence during infection of the rat visual system.

We previously demonstrated that intraocular injections of virulent and attenuated strains of pseudorabies virus (PRV) produce transneuronal infection of functionally distinct central visual circuits in the rat. The virulent Becker strain of PRV induces two temporally separated waves of infection that ultimately target all known retinorecipient neurons; the attenuated Bartha strain only infects a functionally distinct subset of these neurons. In this study, we demonstrate that deletion of a single viral gene encoding glycoprotein gI is sufficient to reproduce both the novel pattern of infectivity and the reduced neurovirulence of the Bartha strain of PRV. Glycoprotein gIII, a major viral membrane protein required for efficient adsorption of virus in cell culture, has no obvious role in determining the pattern of neuronal infectivity, but appears to function with gI to influence neurovirulence. These data suggest that neuroinvasiveness and virulence are the products of an interaction of viral envelope glycoproteins with as yet unidentified cellular receptors.     

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.     

Expression and regulation of glycoprotein C gene of herpes simplex virus 1 resident in a clonal L-cell line.

Ltk- cells were transfected with a plasmid containing the entire domain of glycoprotein C (gC), a true gamma or gamma 2 gene of herpes simplex virus 1 (HSV-1) and the methotrexate-resistant mouse dihydrofolate reductase mutant gene. The resulting methotrexate-resistant cell line was cloned; of the 39 clonal lines tested only 1, L3153(28), expressed gC after infection with HSV-1(MP), a gC- mutant, and none expressed gC constitutively. The induction of gC was optimal at multiplicities ranging between 0.5 and 2 PFU per cell, and the quantities produced were equivalent to or higher than those made by methotrexate-resistant gC- L cells infected with wild-type (gC+) virus. The gC gene resident in the L3153(28) cells was regulated as a beta gene inasmuch as the amounts of gC made in infected L3153(28) cells exposed to concentrations of phosphonoacetate that inhibited viral DNA synthesis were higher than those made in the absence of the drug, gC was induced at both permissive and nonpermissive temperatures by the DNA- mutant tsHA1 carrying a lesion in the gene specifying the major DNA-binding protein and which does not express gamma 2 genes at the nonpermissive temperature, and gC was induced only at the permissive temperature in cells infected with ts502 containing a mutation in the alpha 4 gene. The gC induced in L3153(28) cells was made earlier and processed faster to the mature form than that induced in a gC- clone of methotrexate-resistant cells infected with wild-type virus. Unlike virus stocks made in gC- cells, HSV-1(MP) made in L3153(28) cells was susceptible to neutralization by anti-gC monoclonal antibody.     

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.     

An amino-terminal deletion mutation of pseudorabies virus glycoprotein gIII affects protein localization and RNA accumulation.

We have constructed a pseudorabies virus mutant that contains virtually a complete deletion of the predicted signal sequence coding region for a nonessential envelope glycoprotein, gIII. No signal sequence mutants have been reported previously for a herpesvirus glycoprotein. Through endoglycosidase treatments and pulse-chase analysis, we have determined that the mutant gIII protein is not posttranslationally modified like the wild-type polypeptide, but rather is present as a single, stable species within the infected cell. The mutant polypeptide cannot be detected in the virus envelope, nor is it aberrantly localized to the tissue culture medium. Immunofluorescence studies have indicated that the mutant protein also is not localized to the surfaces of infected cells. In addition, Northern (RNA) and slot blot analyses, as well as in vitro translation experiments using infected-cell cytoplasmic RNA, have indicated that the mutant gIII allele is expressed at lower levels than the wild-type gene is. This is despite the fact that no alterations have been made upstream of the gIII coding sequence. From these results, it appears that the first 22 amino acids of the wild-type gIII protein define a necessary signal peptide that is responsible for at least the correct initiation of translocation and subsequent glycosylation of the gIII envelope glycoprotein within infected cells.     

Pseudorabies virus gIII and bovine herpesvirus 1 gIII share complementary functions.

The gIII glycoproteins of bovine herpesvirus 1 (BHV-1) and of pseudorabies virus (PRV) are structurally homologous. Both proteins also play preeminent roles in mediating virus attachment to permissive cells. To directly compare the functional relation between these glycoproteins, we constructed a recombinant BHV-1 in which the BHV-1 gIII coding sequence was replaced by the PRV gene homolog. The resultant recombinant virus efficiently expressed PRV gIII and then incorporated it into its envelope. The levels of PRV gIII expression and incorporation were equivalent to those achieved by the wild-type virus for BHV-1 gIII. The recombinant virus was fully susceptible to neutralization by anti-PRV gIII neutralizing antibody. In addition, the virus attachment and penetration functions, as well as the virus replication efficiency, which were lost by deleting the BHV-1 gIII gene, were restored by expressing the PRV gIII homolog in its place. These results demonstrated that PRV gIII and BHV-1 gIII share complementary functions.     

The amino-terminal one-third of pseudorabies virus glycoprotein gIII contains a functional attachment domain, but this domain is not required for the efficient penetration of Vero cells.

We have examined the attachment and penetration phenotypes of several glycoprotein gIII mutants of pseudorabies virus (PRV) and have identified the first one-third of gIII as a region that mediates efficient virus attachment to PK15 and Vero cells. This portion of gIII, amino acids 25 through 157 of the wild-type sequence, appeared to support attachment by binding to heparinlike molecules on cell surfaces. Virions containing the first one-third of gIII were sensitive to heparin competition and showed greatly reduced infectivity on cells treated with heparinase. PRV virions lacking the first one-third of the mature glycoprotein exhibited only residual binding to cells if challenged by vigorous washing with phosphate-buffered saline at 2 h postinfection at 4 degrees C. This residual binding was resistant to heparin competition, and strains lacking the first one-third of gIII were able to infect cells treated with heparinase as effectively as untreated cells. When we determined the penetration phenotypes for each strain, we found that gIII-mediated virus attachment was necessary for timely penetration of PK15 cells but remarkably was not required for efficient virus penetration of Vero cells. Moreover, wild-type PRV was actually prohibited from rapid penetration of Vero cells by a gIII-heparan sulfate interaction. Our results indicate that initial virus binding to heparan sulfate via glycoprotein gIII is not required for efficient PRV infection of all cell types and may in fact be detrimental in some instances.     

Construction of an infectious pseudorabies virus recombinant expressing a glycoprotein gIII-β-galactosidase fusion protein

An infectious herpesvirus mutant has been constructed in which a major structural envelope glycoprotein gene was replaced by a hybrid gene encoding a novel fusion protein consisting of the N-terminus of the viral glycoprotein joined to Escherichia coli β-galactosidase (ßGal). Specifically, we fused DNA encoding the first 157 amino acids of the structural glycoprotein gIII from pseudorabies virus strain Becker to the E. coli lacZ gene in a bacterial expression vector. The resulting hybrid gene was then used to replace the wild-type gIII gene in the virus by cotransfection of plasmid and viral DNA. The desired viral recombinants were identified by their inability to react with specific monoclonal antibodies that recognized only wild-type gIII protein. One such mutant virus, PRV-Z1, was chosen for further analysis. PRV-Z1 expressed a glycosylated gIII-ßGal fusion protein after infection of PK15 cells. The fusion protein has no demonstrable ßGal activity and, although glycosylated, remains sensitive to the enzyme endo-β-N-acetylglucosaminidase H, unlike the mature gIII gene product, indicating that the fusion protein was incompletely processed.     

The receptor-binding domain of pseudorabies virus glycoprotein gC is composed of multiple discrete units that are functionally redundant.

Many herpesviruses attach to cells in a two-step process, using the glycoprotein gC family of homologs to bind the primary receptor, heparan sulfate (HS) proteoglycan, and glycoprotein gD homologs to bind an unknown secondary receptor. We have previously shown by deletion analysis that the amino-terminal one-third of gC from pseudorabies virus (PRV), a swine herpesvirus, includes at least the principal HS receptor-binding domain. This portion of PRV gC contains three discrete clusters of basic residues that exactly or nearly match proposed consensus sequences for heparin-binding domains (HBDs); four additional potential HBDs lie in the distal two-thirds of the glycoprotein. We now specifically implicate each of the three amino-terminal HBDs in virus attachment. Mutational analysis demonstrated that any one of the three HBDs could mediate efficient virus infectivity; HS-dependent PRV attachment to cells was eliminated only after all three amino-terminal HBDs were altered. Furthermore, the binding dysfunction was due to a disruption of the specific HBDs and not to total charge loss. Thus, unlike previously described viral receptor-binding domains, the PRV gC receptor-binding domain is composed of multiple, discrete units that can function independently of one another. These units may function redundantly either to increase binding affinity or perhaps to effectively increase the virus's host range.     

DNA sequence of the Herpes simplex virus type 2 glycoprotein D gene

We describe a 1635-bp Herpes simplex virus type 2 (HSV-2) DNA sequence containing the entire coding region of glycoprotein D (gD-2). The amino acid sequence of gD-2, deduced from the nucleotide sequence, was compared to that of the analogous Herpes simplex virus type 1 (HSV-1) glycoprotein (gD-1). The two glycoproteins are 85% homologous and contain highly conserved regions of as much as 49 amino acids in length. Comparison of DNA sequences upstream from gD-1 and gD-2 coding regions identified possible conserved regulatory sequences.     

Novel mechanism of antibody-independent complement neutralization of herpes simplex virus type 1

The envelope surface glycoprotein C (gC) of HSV-1 interferes with the complement cascade by binding C3 and activation products C3b, iC3b, and C3c, and by blocking the interaction of C5 and properdin with C3b. Wild-type HSV-1 is resistant to Ab-independent complement neutralization; however, HSV-1 mutant virus lacking gC is highly susceptible to complement resulting in > or =100-fold reduction in virus titer. We evaluated the mechanisms by which complement inhibits HSV-1 gC null virus to better understand how gC protects against complement-mediated neutralization. C8-depleted serum prepared from an HSV-1 and -2 Ab-negative donor neutralized gC null virus comparable to complement-intact serum, indicating that C8 and terminal lytic activity are not required. In contrast, C5-depleted serum from the same donor failed to neutralize gC null virus, supporting a requirement for C5. EDTA-treated serum did not neutralize gC null virus, indicating that complement activation is required. Factor D-depleted and C6-depleted sera neutralized virus, suggesting that the alternative complement pathway and complement components beyond C5 are not required. Complement did not aggregate virus or block attachment to cells. However, complement inhibited infection before early viral gene expression, indicating that complement affects one or more of the following steps in virus replication: virus entry, uncoating, DNA transport to the nucleus, or immediate early gene expression. Therefore, in the absence of gC, HSV-1 is readily inhibited by complement by a C5-dependent mechanism that does not require viral lysis, aggregation, or blocking virus attachment.     

Binding of complement component C3b to glycoprotein C is modulated by sialic acid on herpes simplex virus type 1-infected cells.

Neuraminidase treatment of cells infected with herpes simplex virus type 1 (HSV-1) markedly enhanced the binding of complement component C3b to HSV 1 glycoprotein C (gC). When HSV-1 was grown in BHK RicR14 cells in which glycoproteins had reduced amounts of N-linked complex oligosaccharides, including sialic acid, the binding of C3b to gC was markedly enhanced. We used neuraminidase treatment to demonstrate that cloning the gC gene from the HSV-1 F strain into an HSV-1 mutant which fails to express gC converted the mutant virus from C3b receptor negative to receptor positive. These results further support a role for gC as a C3b receptor and indicate that sialic acid modifies receptor activity.