The Epstein-Barr virus glycoprotein 110 carboxy-terminal tail domain is essential for lytic virus replication.

To investigate the importance of the Epstein-Barr virus (EBV) glycoprotein 110 (gp110) tail domain in the intracellular localization of gp110 and virus lytic replication, three carboxy-terminal truncation mutants of gp110 were constructed. Deletion of 16 amino acids from the carboxyl-terminal tail resulted in gp110 intracellular localization which was indistinguishable from that of wild-type gp110, whereas deletion of either 41 or 56 amino acids from the carboxyl-terminal tail of gp110 resulted in loss of retention of gp110 in the endoplasmic reticulum and nuclear membrane. None of the gp110 truncation mutants was able to complement EBV(gp110-)+ lymphoblastoid cell lines in transformation assays, indicating the importance of the gp110 tail domain in virus lytic replication. In electron microscopy analysis, no nucleocapsids or enveloped viruses were detected in EBV(gp110-)+ lymphoblastoid cell lines induced for lytic replication.     

Oligomerization of herpes simplex virus glycoprotein B.

Glycoprotein B (gB) specified by herpes simplex virus can be extracted from virions or infected cells in the form of detergent-stable, heat-dissociable oligomers. The composition of the oligomers and requirements for their formation were investigated. Evidence is presented that the faster-migrating forms of the oligomers are homodimers of gB. Dimerization was shown to occur within minutes of polypeptide synthesis and did not depend on glycosylation, the expression of other viral proteins, or virion morphogenesis. The multiple, electrophoretically distinct forms of gB dimers differ in extent or rate of N-linked oligosaccharide processing and also have other differences that influence electrophoretic mobility.     

Glycoprotein B is a specific determinant of herpes simplex virus type 1 neuroinvasiveness.

Herpes simplex virus type 1 strains ANG and KOS lack neuroinvasiveness when inoculated on the footpads of mice, and because the strains are able to complement each other, the genes associated with this phenotype differ. In this study, we used marker rescue techniques to show that at least two genes cloned from ANG are required to restore neuroinvasiveness to KOS. One of the two fragments required is the 6.3-kb BamHI-A/EcoRI-D fragment (0.15 to 0.19 map units). The second has been identified as the sequence encoding glycoprotein B (gB) (UL27). Analysis of ANG and KOS DNA sequences in the relevant region of the gB gene revealed two nucleotide differences which result in amino acid differences in the gB protein. One appears to be unique to the strain of KOS used in our laboratory. The second, at codon 523 of the mature gB protein, encodes a valine in KOS and an alanine in ANG. Recombinant KOS viruses which contained ANG sequences in this region were constructed, and two independently selected recombinants demonstrated increased neuroinvasiveness in mice. From these results, we conclude that gB significantly influences neuroinvasiveness. Mechanisms by which this might occur are discussed.     

Varicella-zoster virus (VZV) open reading frame 10 protein, the homolog of the essential herpes simplex virus protein VP16, is dispensable for VZV replication in vitro.

Varicella-zoster virus (VZV) open reading frame 10 (ORF10) protein in the homolog of the herpes simplex virus type 1 (HSV-1) protein VP16. VZV ORF10 transactivates the VZV IE62 gene and is a tegument protein present in the virion. HSV-1 VP16, a potent transactivator of HSV-1 immediate-early genes and tegument protein, is essential for HSV-1 replication in vitro. To determine whether VZV ORF10 is required for viral replication in vitro, we constructed two VZV mutants which were unable to express ORF10. One mutant had a stop codon after the 61st codon of the ORF10 gene, and the other mutant was deleted for all but the last five codons of the gene. Both VZV mutants grew in cell culture to titers similar to that of the parental virus. To determine whether HSV-1 VP16 alters the growth of VZV, we constructed a VZV mutant in which VP16 was inserted in place of ORF10. Using immune electron microscopy, we found that HSV-1 VP16 was present in the tegument of the recombinant VZV virions. The VZV VP16 substitution mutant produced smaller plaques and grew to a lower titer than parental virus. Thus, VZV ORF10 is not required for growth of the virus in vitro, and substitution of HSV-1 VP16 for VZV ORF10 impairs the growth of VZV.     

Expression of the Marek''s disease virus homolog of herpes simplex virus glycoprotein B in Escherichia coli and its identification as B antigen.

Marek's disease (MD) is an oncogenic disease of chickens caused by MD virus (MDV). Among the major glycoproteins found in MDV-infected cells are gp100, gp60, and gp49, detected by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis with antisera previously shown to be reactive with B antigen in immunodiffusion analysis. Following treatment with tunicamycin (TM), an inhibitor of N-linked glycosylation, the same sera were reported to detect two molecules called pr88 and pr44. However, the gene encoding B antigen was not unequivocally identified. Recently, an MDV homolog of the gene encoding herpes simplex virus glycoprotein B (gB) was identified and sequenced (L. J. N. Ross, M. Sanderson, S. D. Scott, M. M. Binns, T. Doel, and B. Milne, J. Gen. Virol. 70:1789-1804, 1989). To determine whether the MDV gB homolog gene might encode the B antigen, antisera against trpE fusion proteins of the MDV gB homolog (trpE-MDV-gB) were prepared. These antisera immunoprecipitated gp100, gp60, gp49, and a 92-kDa precursor polypeptide (pr88, now designated 92-kDa pr88, in the presence of TM) from MDV-infected cell lysates. On the basis of size comparison, trpE-MDV-gB competition and blocking assays, and the fact that gp100, gp60, gp49, and 92-kDa pr88 could be detected in MDV-infected cells with antisera specific to both MDV B antigen and the gB homolog, it was concluded that (i) the MDV gB homolog gene encodes MDV B antigen and (ii) 92-kDa pr88 is the primary precursor polypeptide. The antisera against trpE-MDV-gB also contained antibody reactive with the herpesvirus of turkey gB homolog, consistent with the known antigenic relatedness between the MDV and herpesvirus of turkey B antigens. TM inhibition data and results from pulse-chase analysis with MDV-infected cells show that MDV gB homolog processing involves cotranslational glycosylation of 92-kDa pr88 to form gp100, which is then cleaved to form gp60 and gp49, the N- and C-terminal halves, respectively, of gp100. This processing pathway is consistent with those of other gB homologs, further supporting the gene identification described above. The conclusions of this study will facilitate future research on the immunobiology of MD, especially studies on the mechanism of immunoprotection.     

Glycoprotein D of herpes simplex virus encodes a domain which precludes penetration of cells expressing the glycoprotein by superinfecting herpes simplex virus.

Earlier studies have shown that herpes simplex viruses adsorb to but do not penetrate permissive baby hamster kidney clonal cell lines designated the BJ series and constitutively expressing the herpes simplex virus 1 (HSV-1) glycoprotein D (gD). To investigate the mechanism of the restriction, the following steps were done. First, wild-type HSV-1 strain F [HSV-1(F)] virus was passaged blindly serially on clonal line BJ-1 and mutant viruses [HSV-1(F)U] capable of penetration were selected. The DNA fragment capable of transferring the capacity to infect BJ cells by marker transfer contains the gD gene. The mutant gD, designated gDU, differed from wild-type gD only in the substitution of Leu-25 by proline. gDU reacted with monoclonal antibodies which neutralize virus and whose epitopes encompass known functional domains involved in virus entry into cells. It did not react with the monoclonal antibody AP7 previously shown to react with an epitope which includes Leu-25. Second, cell lines expressing gDU constitutively were constructed and cloned. Unlike the clonal cell lines constitutively expressing gD (e.g., the BJ cell line), those expressing gDU were infectable by both HSV-1(F) and HSV-1(F)U. Lastly, exposure of BJ cells to monoclonal antibody AP7 rendered the cells capable of being infected with HSV-1(F). The results indicate that (i) gD expresses a specific function, determined by sequences at or around Leu-25, which blocks entry of virus into cells synthesizing gD, (ii) the gD which blocks penetration by superinfecting virus is located in the plasma membrane, (iii) the target of the restriction to penetration is the identical domain of the gD molecule contained in the envelope of the superinfecting virus, and (iv) the molecular basis of the restriction does not involve competition for a host protein involved in entry, as was previously thought.     

Deletions in herpes simplex virus glycoprotein D define nonessential and essential domains.

Herpes simplex virus glycoprotein D (gD) is a major component of the virion envelope and infected cell membranes and is essential for virus entry into cells. We have recently shown that gD interacts with a limited number of cell surface receptors which are required for virus penetration into cells. To define domains of gD which are required for aspects of virus replication including receptor binding, deletion mutations of 5 to 14 amino acids were constructed by using oligonucleotide-directed mutagenesis. Plasmids containing mutant genes for gD were assayed for the ability to rescue a recombinant virus, F-gD beta, in which beta-galactosidase sequences replace gD-coding sequences. Effects on global folding and posttranslational processing of the molecules were assessed by using a panel of monoclonal antibodies which recognize both continuous and discontinuous epitopes. A region near the amino terminus (residues 7 to 21) of gD which is recognized by monoclonal antibodies able to neutralize herpes simplex virus in the absence of complement was not essential for function. In addition, virtually all of the cytoplasmic domain of gD and an extracellular domain close to the membrane were dispensable. In contrast, deletion mutations in the central region of the molecule, save for one exception, led to alterations in global folding of the molecule and maturation of the protein was inhibited.     

Identification of a site on herpes simplex virus type 1 glycoprotein D that is essential for infectivity.

Herpes simplex virus glycoprotein D (gD) plays an essential role during penetration of the virus into cells. There is evidence that it recognizes a specific receptor after initial attachment of virions to cell surface heparan sulfate and also that gD-1, gD-2, and gp50 (the pseudorabies virus gD homolog) bind to the same receptor. Although the antigenic structure of gD has been studied intensively, little is known about functional regions of the protein. Antigenic site I is a major target for neutralizing antibodies and has been partially mapped by using deletion mutants and neutralization-resistant viruses. Working on the assumption that such a site may overlap with a functional region of gD, we showed previously that combining two or more amino acid substitutions within site I prevents gD-1 from functioning and is therefore lethal. We have now used a complementation assay to measure the functional activity of a panel of deletion mutants and compared the results with an antigenic analysis. Several mutations cause gross changes in protein folding and destroy functional activity, whereas deletions at the N and C termini have little or no effect on either. In contrast, deletion of residues 234 to 244 has only localized effects on antigenicity but completely abolishes functional activity. This region, which is part of antigenic site Ib, is therefore essential for gD-1 function. The complementation assay was also used to show that a gD-negative type 1 virus can be rescued by gD-2 and by two gD-1-gD-2 hybrids but not by gp50, providing some support for the existence of a common receptor for herpes simplex virus types 1 and 2 but not pseudorabies virus. Alternatively, gp50 may lack a signal for incorporation into herpes simplex virions.     

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.     

Cross-reactivity between herpes simplex virus glycoprotein B and a 63,000-dalton varicella-zoster virus envelope glycoprotein.

Cross-reactive monoclonal antibodies recognizing both herpes simplex virus (HSV) glycoprotein B and a major 63,000-dalton varicella-zoster virus (VZV) envelope glycoprotein were isolated and found to neutralize VZV infection in vitro. None of the other VZV glycoproteins was recognized by any polyclonal anti-HSV serum tested. These results demonstrate that HSV glycoprotein B and the 63,000-dalton VZV glycoprotein share antigenic epitopes and raise the possibility that these two proteins have a similar function in infection.     

Topoisomerase II cleavage of herpes simplex virus type 1 DNA in vivo is replication dependent

The genome of herpes simplex virus type 1 contains a large number of recognition sites for eucaryotic DNA type II topoisomerase. Topoisomerase II sites were identified by means of the consensus sequence described previously (J.R. Spitzner and M.T. Muller, Nucleic Acids Res. 16:5553-5556, 1988) and then confirmed by sequencing DNA cleavages introduced by purified topoisomerase II. In vivo, host topoisomerase II also introduced double-stranded DNA breaks in the viral genome at sites predicted by the consensus sequence. Host topoisomerase II acted on all immediate-early genes as well as on genes from other temporal classes; however, cleavages were not detected until 4 to 5 h postinfection and were most intense at 10 h postinfection. Topoisomerase II cleavages were not detected when viral DNA replication was prevented with phosphonoacetic acid. These data indicate that, although progeny viral genomes are acted upon by host topoisomerase II, this enzyme either does not act on parental viral genomes before DNA replication or acts on them with such low efficiency that cleavages are beyond our limit of detection. The findings suggest that host topoisomerase II is involved in aspects of viral replication at late times in the infectious cycle.     

The Epstein-Barr virus BMRF1 gene is essential for lytic virus replication

The Epstein-Barr virus (EBV) BMRF1 protein is a DNA polymerase processivity factor. We have deleted the BMRF1 open reading frame from the EBV genome and assessed the DeltaBMRF1 EBV phenotype. DeltaBMRF1 viruses were replication deficient, but the wild-type phenotype could be restored by BMRF1 trans-complementation. The replication-deficient phenotype included impaired lytic DNA replication and late protein expression. DeltaBMRF1 and wild-type viruses were undistinguishable in terms of their ability to transform primary B cells. Our results provide genetic evidence that BMRF1 is essential for lytic replication of the EBV genome.     

Identification of functional domains in herpes simplex virus 2 glycoprotein B

Glycoprotein B (gB) is one of four membrane proteins that are essential for the entry of herpes simplex viruses (HSV) into cells, and coexpression of the same combination of proteins in transfected cells results in cell fusion. The latter effect is reminiscent of the ability of virus infection to cause cell fusion, particularly since the degree of fusion is greatly increased by syncytial mutations in gB. Despite intensive efforts with the gB homologs of HSV and some other herpesviruses, information about functionally important regions in the 700-amino-acid ectodomain of this protein is very limited at present. This is largely due to the misfolding of the majority of the mutants examined. It was shown previously that the percentage of correctly folded mutants could be increased by targeting only predicted loop regions (i.e., not alpha-helix or beta-strand), and by using this approach new functional domains in HSV-2 gB have now been identified.     

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.     

Viral replication is required for induction of ocular immunopathology by herpes simplex virus.

Corneal infection of BALB/c mice with herpes simplex virus type 1 results in a chronic inflammatory response in the stroma termed herpetic stromal keratitis (HSK). This disease is considered to be immunopathological and mediated primarily by CD4+ T cells of the type 1 cytokine profile. However, the nature of the antigens, virus or host derived, which drive the inflammatory response remains in doubt. In this study, the relevance of infection with replicating virus for the subsequent development of HSK was evaluated with immunocompetent mice as well as with SCID mice reconstituted with herpes simplex virus-immune CD4+ T cells. In the corneas of immunocompetent mice, infectious virus, viral antigen, and mRNA expression were detectable for only a brief period of time (< or = 7 days postinfection), and all were undetectable by the time clinical lesions were evident (10 to 15 days). Viral replication, however, was necessary for the development of HSK in both models, since infection with UV-inactivated virus or with mutant viruses which were incapable of multiple rounds of replication in vivo failed to induce HSK. The inactivated and mutant viral preparations did, however, stimulate T-cell immune responses in immunocompetent mice. The results are discussed in terms of possible involvement of host antigens exposed in response to transient progeny virion replication in the immune-privileged cornea.     

The carboxy-terminal 41 amino acids of herpes simplex virus type 1 glycoprotein B are not essential for production of infectious virus particles.

Glycoprotein B (gB) is a virally encoded protein that is found in the envelope of herpes simplex virus type 1 and membranes of cells infected with herpes simplex virus type 1. It is essential for the production of infectious virus particles. An amber mutation was introduced into the gB gene by oligonucleotide-directed mutagenesis at the codon for amino acid 863 of the protein. Virus carrying this mutation should synthesize gB molecules lacking the last 41 amino acids of the cytoplasmic domain. Immunoprecipitation of infected cell extracts demonstrated the synthesis of appropriately truncated gB molecules. Characterization of the mutant virus indicated that the loss of the carboxy-terminal 41 amino acids has little effect on gB function.     

The extreme carboxyl terminus of the equine herpesvirus 1 homolog of herpes simplex virus VP16 is essential for immediate-early gene activation.

Gene 12 of equine herpesvirus 1 (EHV-1), the homolog of herpes simplex virus (HSV) VP16 (alpha TIF, Vmw65), was cloned into a eukaryotic expression vector by PCR and used in transactivation studies of both the EHV-1 and HSV-1 IE1 promoters. Results demonstrated that the product of gene 12 is a potent transactivator of immediate-early gene expression of both viruses, which requires sequences in the upstream HSV-1 promoter for activity. Mutational analysis of the gene 12 open reading frame indicated that removal of the C-terminal 7 amino acids, which contain a short region of homology with the extreme C terminus of VP16, inactivated the protein. Within this region, only a single methionine residue appeared to be essential for activity, implying that gene 12 may have a modular array of organization similar to that of VP16. However, fusion of the gene 12 C terminus to a truncated form of VP16, which contained the complex formation domain, did not restore activity to the HSV-1 protein. These data demonstrate that the EHV-1 immediate-early transactivator may not be functionally colinear with VP16, with transactivation requiring both the C terminus and another region(s) present within the N-terminal portion.     

Glycoprotein H of herpes simplex virus type 1 requires glycoprotein L for transport to the surfaces of insect cells.

In mammalian cells, formation of heterooligomers consisting of the glycoproteins H and L (gH and gL) of herpes simplex virus type 1 is essential for the cell-to-cell spread of virions and for the penetration of virions into cells. We examined whether formation of gH1/gL1 heterooligomers and cell surface expression of the complex occurs in insect cells. Three recombinant baculoviruses, expressing gL1, gH1, and truncated gH1 (gH1t), which lacks the transmembrane region, were constructed. It was shown that recombinant gH1/gL1 and gH1t/gL1 heterooligomers were produced in insect cells. As in mammalian cells, gH1 and gH1t were not detected on the surfaces of insect cells in the absence of gL1. When coexpressed with gL1, recombinant gH1 was displayed on the surfaces of insect cells. Coexpression of gH1t and gL1 resulted in secretion of the gH1t/gL1 complex into the cell culture medium, indicating that gH1t is also transported to the surfaces of insect cells. Our results indicate that the process of folding and intracellular transport of gH1 and gL1 is comparable in insect cells and mammalian cells and that the baculovirus expression system can be used to examine the complex formation and the intracellular transport of gH1 and gL1. The availability of secreted gH1t/gL1 complex offers the opportunity to further investigate the immunological properties of this complex.     

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

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