Pseudorabies virus mutants lacking the essential glycoprotein gII can be complemented by glycoprotein gI of bovine herpesvirus 1.

The genome of pseudorabies virus (PrV) encodes at least seven glycoproteins. The glycoprotein complex gII consists of three related polypeptides, two of them derived by proteolytic cleavage from a common precursor and linked via disulfide bonds. It is homologous to herpes simplex virus (HSV) gB and is therefore thought to be essential for PrV replication, as is gB for HSV replication. To isolate PrV mutants deficient in gII expression, we established cell lines that stably carry the PrV gII gene. Line N7, of Vero cell origin, contains the gII gene under its own promoter and expresses gII after transactivation by herpesviral functions after infection. MDBK-derived line MT3 contains the gII gene under control of the mouse metallothionein promoter. However, it has essentially lost inducibility and constitutively produces high amounts of correctly processed glycoprotein gII. We used a beta-galactosidase expression cassette inserted into a partially deleted cloned copy of the gII gene for cotransfection with PrV DNA. gII- PrV mutants were isolated from viral progeny by taking advantage of their blue-plaque phenotype when incubated under an agarose overlay containing a chromogenic substrate. Analysis of these mutants proved that gII is indeed essential for PrV replication, since the gII- mutants grew normally on gII-complementing cells but were unable to produce plaques on noncomplementing cells. Surprisingly the PrV gII- mutants were also able to grow on a cell line constitutively expressing the gB-homologous glycoprotein gI from bovine herpesvirus 1 (BHV-1) to the same extent as on cells expressing PrV gII. gII- PrV propagated on cells expressing BHV-1 gI became susceptible to neutralization by anti-BHV-1 gI monoclonal antibodies. We also found that BHV-1 gI is present in the envelope of purified gII- pseudorabies virions grown on cells expressing BHV-1 gI, as judged by radioimmunoprecipitation and immunoelectron microscopy. These results prove that BHV-1 gI is integrated into the PrV envelope and can functionally replace glycoprotein gII of PrV.     

Glycoprotein gB (gII) of pseudorabies virus can functionally substitute for glycoprotein gB in herpes simplex virus type 1.

Glycoproteins homologous to gB of herpes simplex virus (HSV) constitute the most highly conserved family of herpesvirus glycoproteins. All gB homologs analyzed so far have been shown to play essential roles in penetration and direct viral cell-to-cell spread. In studies aimed at assessing whether the high sequence homology is also indicative of functional homology, we analyzed the ability of the gB-homologous glycoprotein (former designation gII) of pseudorabies virus (PrV) to complement a gB- HSV type 1 (HSV-1) mutant and vice versa. The results show that a PrV gB-expressing cell line phenotypically complemented the lethal defect in gB- HSV-1 whereas reciprocal complementation of a gB- PrV mutant by HSV-1 gB was not observed.     

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.     

Stable rescue of a glycoprotein gII deletion mutant of pseudorabies virus by glycoprotein gI of bovine herpesvirus 1.

Glycoproteins homologous to glycoprotein B (gB) of herpes simplex virus constitute the most highly conserved group of herpesvirus glycoproteins. This strong conservation of amino acid sequences might be indicative of a common functional role. Indeed, gB homologs have been implicated in the processes of viral entry and virus-mediated cell-cell fusion. Recently, we showed that pseudorabies virus (PrV) lacking the essential gB-homologous glycoprotein gII could be propagated on a cell line expressing the gB homolog of bovine herpesvirus 1, gI(BHV-1), leading to a phenotypic complementation of the gII defect (I. Rauh, F. Weiland, F. Fehler, G. Keil, and T.C. Mettenleiter, J. Virol. 65:621-631, 1991). However, this pseudotypic virus could still replicate only on complementing cell lines, thereby limiting experimental approaches to analyze the effects of the gB exchange in detail. We describe here the construction and isolation of a PrV recombinant, 9112C2, that lacks gII(PrV) but instead stably carries and expresses the gene encoding gI(BHV-1). The recombinant is able to replicate on noncomplementing cells with growth kinetics and final titers similar to those of its gII-positive wild-type PrV parent. Neutralization tests and immunoprecipitation analyses demonstrated incorporation of gI(BHV-1) into 9112C2 virions with concomitant absence of gII(PrV). Analysis of in vitro host ranges of wild-type PrV, BHV-1, and recombinant 9112C2 showed that in cells of pig, rabbit, canine, monkey, or human origin, the plating efficiency of 9112C2 was similar to that of its PrV parent. Exchange of gII(PrV) for gI(BHV-1) in recombinant 9112C2 or by phenotypic complementation of gII- PrV propagated on gI(BHV-1)-expressing cell lines resulted in penetration kinetics intermediate between those of wild-type PrV and BHV-1. In conclusion, we report the first isolation of a viral recombinant in which a lethal glycoprotein mutation has been rescued by a homologous glycoprotein of a different herpesvirus. Our data show that in gII- PrV, gI(BHV-1) in vitro fully complements the lethal defect associated with lack of gII(PrV). These results conclusively demonstrate that gI(BHV-1) in a PrV background can execute all essential functions normally provided by gII(PrV). They also indicate that the origin of gB-homologous glycoproteins influences the penetration kinetics of herpesviruses.     

Proteolytic cleavage of bovine herpesvirus 1 (BHV-1) glycoprotein gB is not necessary for its function in BHV-1 or pseudorabies virus.

Glycoprotein B homologs represent the most highly conserved group of herpesvirus glycoproteins. They exist in oligomeric forms based on a dimeric structure. Despite the high degree of sequence and structural conservation, differences in posttranslational processing are observed. Whereas gB of herpes simplex virus is not proteolytically processed after oligomerization, most other gB homologs are cleaved by a cellular protease into subunits that remain linked via disulfide bonds. Proteolytic cleavage is common for activation of viral fusion proteins, and it has been shown that herpesvirus gB homologs are essential for membrane fusion events during infection, e.g., virus penetration and direct viral cell-to-cell spread. To analyze the importance of proteolytic cleavage for the function of gB homologs, we isolated a mutant bovine herpesvirus 1 (BHV-1) expressing a BHV-1 gB that is no longer proteolytically processed because of a deletion of the proteolytic cleavage site and analyzed its phenotype in cell culture. We showed previously that BHV-1 gB can functionally substitute for the homologous glycoprotein in pseudorabies virus (PrV), based on the isolation of a PrV gB-negative PrV recombinant that expresses BHV-1 gB (A. Kopp and T. C. Mettenleiter, J. Virol, 66:2754-2762, 1992). Therefore, we also isolated a mutant PrV lacking PrV gB but expressing a noncleavable BHV-1 gB. Our results show that cleavage of BHV-1 gB is not essential for its function in either a BHV-1 or a PrV background. Compared with the PrV recombinant expressing cleavable BHV-1 gB, deletion of the cleavage site in the recombinant PrV did not detectably alter the viral phenotype, as analyzed by plaque assays, one-step growth kinetics, and penetration kinetics. In the BHV-1 mutant, the uncleaved BHV-1 gB was functionally equivalent to the wild-type protein with regard to penetration and showed only slightly delayed one-step growth kinetics compared with parental wild-type BHV-1. However, the resulting plaques were significantly smaller, indicating a role for proteolytic cleavage of BHV-1 gB in cell-to-cell spread of BHV-1.     

Pseudorabies virus protein homologous to herpes simplex virus type 1 ICP18.5 is necessary for capsid maturation.

In pseudorabies virus (PrV), an open reading frame that partially overlaps the gene for the essential glycoprotein gII has been shown to encode a protein homologous to the ICP18.5 polypeptide of herpes simplex virus type 1 (N. Pederson and L. Enquist, Nucleic Acids Res. 17:3597, 1989). To study the function of this protein during the viral replicative cycle, a PrV mutant which carries a beta-galactosidase expression cassette interrupting the ICP18.5(PrV) gene was constructed. This mutant could be propagated only on cell lines that were able to provide ICP18.5(PrV) in trans after transformation with a corresponding genomic PrV DNA fragment. Detailed analysis showed that inactivation of the ICP18.5(PrV) gene did not impair infection of noncomplementing cells, nor did it impair early or late gene expression, as shown by immunoprecipitation of glycoproteins gII, gIII, and gp50. Surface localization of glycoproteins as demonstrated by fluorescence-activated cell sorting analyses was also not affected. Southern blot hybridizations, however, showed that cleavage of replicative concatemeric viral DNA did not occur in noncomplementing cells infected by the ICP18.5 mutant PrV. In addition, electron microscopic analysis revealed an accumulation of empty capsids in the nucleus of mutant-infected noncomplementing cells. We conclude that the ICP18.5(PrV) protein is necessary for viral replication and plays an essential role in the process of mature capsid formation.     

Expression of the Marek''s disease virus (MDV) homolog of glycoprotein B of herpes simplex virus by a recombinant baculovirus and its identification as the B antigen (gp100, gp60, gp49) of MDV.

A gene encoding a homolog of glycoprotein B of herpes simplex virus (gB homolog) has been identified on the Marek's disease virus (MDV) genome (L. J. N. Ross, M. Sanderson, S. D. Scott, M. M. Binns, T. Doel, and B. Milne, J. Gen. Virol. 70:1789-1804, 1989); however, the molecular and immunological characteristics of the gene product(s) are still not clear. In the present study, the gB homolog of MDV was expressed in insect cells by a recombinant baculovirus, and it was characterized to determine its molecular and antigenic properties. The expressed recombinant protein had three molecular sizes (88 to 110, 58, and 49 kDa) and was recognized by antisera from chickens inoculated with each of the three serotypes of MDV. By immunofluorescence analysis, it was shown that the protein was expressed in the cytoplasm and on the surface of the recombinant baculovirus-infected cells. The gB homolog of MDV was processed similarly to pseudorabies virus and varicella-zoster virus with respect to cleavage and the intramolecular disulfide bond between the cleaved products. Interestingly, the expressed protein reacted with monoclonal antibody M51, specific to the B antigen (gp100, gp60, gp49) of MDV, although the locations of the gene encoding the B antigen and of the gene encoding the gB homolog were reported to be different. Moreover, competitive experiments revealed that anti-gB homolog serum and monoclonal antibody M51 recognized the same molecules. From these results, the gB homolog and the B antigen of MDV seem to be the same glycoprotein.     

Protection of mice and swine from pseudorabies virus conferred by vaccinia virus-based recombinants.

Glycoproteins gp50, gII, and gIII of pseudorabies virus (PRV) were expressed either individually or in combination by vaccinia virus recombinants. In vitro analysis by immunoprecipitation and immunofluorescence demonstrated the expression of a gII protein of approximately 120 kDa that was proteolytically processed to the gIIb (67- to 74-kDa) and gIIc (58-kDa) mature protein species similar to those observed in PRV-infected cells. Additionally, the proper expression of the 90-kDa gIII and 50-kDa gp50 was observed. All three of these PRV-derived glycoproteins were detectable on the surface of vaccinia virus-PRV recombinant-infected cells. In vivo, mice were protected against a virulent PRV challenge after immunization with the PRV glycoprotein-expressing vaccinia virus recombinants. The coexpression of gII and gIII by a single vaccinia virus recombinant resulted in a significantly reduced vaccination dose required to protect mice against PRV challenge. Inoculation of piglets with the various vaccinia virus-PRV glycoprotein recombinants also resulted in protection against virulent PRV challenge as measured by weight gain. The simultaneous expression of gII and gp50 in swine resulted in a significantly enhanced level of protection as evaluated by weight evolution following challenge with live PRV.     

Identification and nucleotide sequence of the glycoprotein gB gene of equine herpesvirus 4.

The nucleotide sequence of the glycoprotein gB gene of equine herpesvirus 4 (EHV-4) was determined. The gene was located within a BamHI genomic library by a combination of Southern and dot-blot hybridization with probes derived from the herpes simplex virus type 1 (HSV-1) gB DNA sequence. The predominant portion of the coding sequences was mapped to a 2.95-kilobase BamHI-EcoRI subfragment at the left-hand end of BamHI-C. Potential TATA box, CAT box, and mRNA start site sequences and the translational initiation codon were located in the BamHI M fragment of the virus, which is located immediately to the left of BamHI-C. A polyadenylation signal, AATAAA, occurs nine nucleotides past the chain termination codon. Translation of these sequences would give a 110-kilodalton protein possessing a 5' hydrophobic signal sequence, a hydrophilic surface domain containing 11 potential N-linked glycosylation sites, a hydrophobic transmembrane domain, and a 3' highly charged cytoplasmic domain. A potential internal proteolytic cleavage site, Arg-Arg/Ser, was identified at residues 459 to 461. Analysis of this protein revealed amino acid sequence homologies of 47% with HSV-1 gB, 54% with pseudorabies virus gpII, 51% with varicella-zoster virus gpII, 29% with human cytomegalovirus gB, and 30% with Epstein-Barr virus gB. Alignment of EHV-4 gB with HSV-1 (KOS) gB further revealed that four potential N-linked glycosylation sites and all 10 cysteine residues on the external surface of the molecules are perfectly conserved, suggesting that the proteins possess similar secondary and tertiary structures. Thus, we showed that EHV-4 gB is highly conserved with the gB and gpII glycoproteins of other herpesviruses, suggesting that this glycoprotein has a similar overall function in each virus.     

Mutational analysis of the proteolytic cleavage site of glycoprotein B (gB) of Marek''s disease virus

The Marek's disease virus (MDV) glycoprotein B (gB) precursor, gp100, is proteolytically cleaved into two disulfide-linked subunits, gp60 and gp49. In the gB homologs of most other herpesviruses, a tetrapeptide, Arg-Xaa-Arg-Arg, is immediately upstream from the predicted cleavage site. We have investigated the specificity of the proteolytic cleavage in gplOO by introducing mutations within its predicted cleavage site (Arg-Leu-Arg-Arg) and expressed these mutants in recombinant fowlpox virus (FPV). The results show that all three Arg residues at the predicted cleavage site play an important role in the specific proteolytic cleavage of gp100. Furthermore, we demonstrated that the cleavage of gplOO is not necessary for transport of gB to the cell surface.     

Internalization of pseudorabies virus glycoprotein B is mediated by an interaction between the YQRL motif in its cytoplasmic domain and the clathrin-associated AP-2 adaptor complex

The cytoplasmic domain of pseudorabies virus (PRV) glycoprotein B (gB) contains three putative internalization motifs. Previously, we demonstrated that the tyrosine-based YQRL motif at positions 902 to 905, but not the YMSI motif at positions 864 to 867 or the LL doublet at positions 887 and 888, is required for correct functioning of gB during antibody-mediated internalization of PRV cell surface-bound glycoproteins. In the present study, we demonstrate that the YQRL motif is also crucial to allow spontaneous internalization of PRV gB, and thus, that spontaneous and antibody-mediated internalizations of PRV gB occur through closely related mechanisms. Furthermore, we found that PRV gB colocalizes with the cellular clathrin-associated AP-2 adaptor complex and that this colocalization depends on the YQRL motif. In addition, by coimmunoprecipitation assays, we found that during both spontaneous and antibody-dependent internalization, PRV gB physically interacts with AP-2, and that efficient interaction between gB and AP-2 required an intact YQRL motif. Collectively, these findings demonstrate for the first time that during internalization of an alphaherpesvirus envelope protein, i.e., PRV gB, a specific amino acid sequence in the cytoplasmic tail of the protein interacts with AP-2 and may constitute a common AP-2-mediated mechanism of internalization of alphaherpesvirus envelope proteins.     

Role of Envelope Protein gE Endocytosis in the Pseudorabies Virus Life Cycle

Several groups have reported that certain herpesvirus envelope proteins do not remain on the surface of cells that express them but rather are internalized by endocytosis in a recycling process. The biological function of membrane protein endocytosis in the virus life cycle remains a matter of speculation and debate. In this report, we demonstrate that some, but not all, membrane proteins encoded by the alphaherpesvirus pseudorabies virus (PRV) are internalized after reaching the plasma membrane. Glycoproteins gE and gB are internalized from the plasma membrane of cells, while gI and gC are not internalized efficiently. We show for gE that the cytoplasmic domain of the protein is required for endocytosis. While the gI protein is incapable of endocytosis on its own, it can be internalized when complexed with gE. We demonstrate that endocytosis of the gE-gI complex and gB occurs early after infection of tissue culture cells but that this process stops completely after 6 h of infection, a time that correlates with significant shutoff of host protein synthesis. We also show that gE protein internalized at 4 h postinfection is not present in virions formed at a later time. We discuss the differences in PRV gE and gI endocytosis compared to that of the varicella-zoster virus homologs and the possible roles of glycoprotein endocytosis in the virus life cycle.     

Role of essential glycoproteins gII and gp50 in transneuronal transfer of pseudorabies virus from the hypoglossal nerves of mice.

The propagation of pseudorabies virus (PrV) mutants deficient in essential glycoproteins gp50 and gII was studied after inoculation of transcomplemented gp50- and gII- PrV into the motor hypoglossal (XII) nerves of mice. In this model, viral spread from the infected XII motoneurons involves specific transneuronal transfer to connected cells and local, nonspecific transfer. For comparison, a PrV mutant lacking the nonessential nonstructural glycoprotein gX was included. Although the efficiencies of first-cycle replication were similar for the three viruses, only gX- and gp50- progeny mutants could spread from XII motoneurons via transneuronal and local transfer. The extents of transfer of gX- and gp50- PrV were comparable. The results show that the absence of gp50 does not alter the pattern of transneuronal or local spread of PrV, whereas gII is essential for both processes.     

Sequence requirements for proteolytic processing of glycoprotein B of human cytomegalovirus strain Towne

Truncated versions of the human cytomegalovirus (CMV) strain Towne glycoprotein B (gB) gene were stably expressed in CHO cell lines. The calcium-specific ionophore A23187 inhibited proteolytic cleavage of C-terminal-truncated gB expressed by cell line 67.77. These inhibition studies also showed that the 93-kilodalton cleavage product most likely represents the N-terminal cleavage fragment of gB. The ionophore carboxyl cyanide m-chlorophenyl-hydrazone was used to show that proteolytic cleavage of gB did not occur in the endoplasmic reticulum. Two-dimensional polyacrylamide gel electrophoresis demonstrated that the N- and C-terminal cleavage products of gB remained associated by disulfide linkages after cleavage. Expression studies using constructs in which 80% or all of the N terminus was deleted demonstrated that the N terminus was required for secretion of the gB molecule. The amino acid sequence at the site of cleavage was shown to be critical for cleavage by a cellular protease. Our results indicate that an arginine-to-threonine change at either amino acid 457 or 460, a lysine-to-glutamine change at amino acid 459, or all three substitutions together block gB cleavage. The effect on proteolysis of the arginine-to-threonine amino acid change at residue 457 (position -4 relative to the cleavage site) demonstrated that a basic pair of amino acids at the endoproteolytic processing site is not the only requirement in cis for gB cleavage.     

A Network of Protein Interactions around the Herpes Simplex Virus Tegument Protein VP22

Assembly of the herpesvirus tegument is poorly understood but is believed to involve interactions between outer tegument proteins and the cytoplasmic domains of envelope glycoproteins. Here, we present the detailed characterization of a multicomponent glycoprotein-tegument complex found in herpes simplex virus 1 (HSV-1)-infected cells. We demonstrate that the tegument protein VP22 bridges a complex between glycoprotein E (gE) and glycoprotein M (gM). Glycoprotein I (gI), the known binding partner of gE, is also recruited into this gE-VP22-gM complex but is not required for its formation. Exclusion of the glycoproteins gB and gD and VP22''s major binding partner VP16 demonstrates that recruitment of virion components into this complex is highly selective. The immediate-early protein ICP0, which requires VP22 for packaging into the virion, is also assembled into this gE-VP22-gM-gI complex in a VP22-dependent fashion. Although subcomplexes containing VP22 and ICP0 can be formed when either gE or gM are absent, optimal complex formation requires both glycoproteins. Furthermore, and in line with complex formation, neither of these glycoproteins is individually required for VP22 or ICP0 packaging into the virion, but deletion of gE and gM greatly reduces assembly of both VP22 and ICP0. Double deletion of gE and gM also results in small plaque size, reduced virus yield, and defective secondary envelopment, similar to the phenotype previously shown for pseudorabies virus. Hence, we suggest that optimal gE-VP22-gM-gI-ICP0 complex formation correlates with efficient virus morphogenesis and spread. These data give novel insights into the poorly understood process of tegument acquisition.     

Specific pseudorabies virus infection of the rat visual system requires both gI and gp63 glycoproteins.

Transneuronal transport of pseudorabies virus (PRV) from the retina to visual centers that mediate visual discrimination and reflexes requires specific genes in the unique short region of the PRV genome. In contrast, these same viral genes are not required to infect retinorecipient areas of the brain involved in circadian rhythm regulation. In this report, we demonstrate that viral mutants carrying defined deletions of the genes encoding glycoprotein gI or gp63, or both, result in the same dramatic transport defect. Efficient export of either gI or gp63 from the endoplasmic reticulum to the Golgi apparatus in a fibroblast cell line requires the presence of both proteins. We also show that gI and gp63 physically interact, as demonstrated by pulse-chase and sucrose gradient sedimentation experiments. Complex formation is rapid compared with homodimerization of PRV glycoprotein gII. We suggest that gI and gp63 function in concert to affect neurotropism in the rat visual circuitry and that a heterodimer is likely to be the unit of function.     

Processing of pseudorabies virus glycoprotein gII.

The glycoprotein complex gII of pseudorabies virus was isolated by immunoprecipitation with the monoclonal antibody M5, which was covalently linked to protein A-Sepharose. After sodium dodecyl sulfate-polyarylamide gel electrophoresis under reducing conditions and blotting onto poly(vinylidene difluoride) membrane, its subunits, gIIa, gIIb, and gIIc, were subjected to N-terminal sequencing. gIIa and gIIb start at position 59 and gIIc starts at position 503 according to the amino acid sequence deduced from the gene, indicating that there is one major protein (gIIa) which is cleaved into the two protein fragments gIIb and gIIc. Protein labeling with 14C-amino acids gave no indication that the three proteins (gIIa, gIIb, and gIIc) of the complex are present in equimolar ratios. It seems that gIIa is only a minor component of the complex, whereas gIIb and gIIc are contained in equimolar amounts.     

Demonstration of three major species of pseudorabies virus glycoproteins and identification of a disulfide-linked glycoprotein complex.

The glycoproteins of pseudorabies virus (PRV) Phylaxia were characterized with monoclonal antibodies as specific reagents. Three major structural glycoproteins with molecular weights of 155,000 (155K) (gC), 122K (gA), and 90K (gB) could be identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. We investigated the processing of glycoproteins gA, gB, and gC by in vitro translation, pulse-chase experiments, and in the presence of the ionophore monensin which inhibits glycosylation. gA and gB were found to compose a single polypeptide, whereas gC was found to be a disulfide-linked glycoprotein complex. Immunoprecipitates formed with the aid of anti-gC monoclonal antibodies gave rise to three glycoprotein bands (gC0 [120K], gC1 [67K], and gC2 [58K]) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Limited proteolysis of gC0, gC1, and gC2 resulted in peptide maps of gC0 related to those of both gC1 and gC2. No common peptide bands between gC1 and gC2, however, were seen. We suggest that (i) gC1 and gC2 arise by proteolytic cleavage from the same precursor molecule and stay joined via disulfide bridges and (ii) gC0 is an uncleaved precursor.     

Pseudorabies virus glycoproteins gII and gp50 are essential for virus penetration.

Pseudorabies virus (PrV) glycoproteins gII and gp50 are major constituents of the viral envelope and targets of neutralizing monoclonal antibodies. Both are homologs of essential glycoproteins found in herpes simplex virus, gB (gII) and gD (gp50). We recently isolated a gII-negative PrV deletion mutant on complementing cell lines and established the essential character of gII for PrV replication (I. Rauh, F. Weiland, F. Fehler, G. Keil, and T.C. Mettenleiter, J. Virol. 65: 621-631, 1991). In this report, we describe the isolation of a gp50-negative PrV mutant after constructing cell lines that constitutively express gp50 and phenotypically complement the gp50 defect. Analysis of the gp50- mutant proved that gp50 is essential for PrV replication. Further studies showed that both gII and gp50 are required for viral penetration into target cells. The penetration defect in the gII and gp50 deletion mutants could be overcome by experimental polyethylene glycol-induced membrane fusion. Surprisingly, whereas gII proved to be essential for both penetration and cell-cell spread of the virus, gp50 was required only for penetration and appeared dispensable for direct cell-cell spread.