Role of glycoprotein B of herpes simplex virus type 1 in viral entry and cell fusion.

Glycoprotein B (gB) of herpes simplex virus type 1 is an envelope protein that is essential for viral growth. We previously reported the isolation of two gB-null viruses, which form gB-free virions in nonpermissive cells. In the present study, these gB-free virions were shown to bind to the cell surface at the same rate as the wild-type virus. They failed, however, to form plaques and to synthesize virus-specific proteins upon infection. Their plating efficiency was significantly enhanced by treatment with polyethylene glycol, a membrane fusion agent. Therefore, gB is required in a stage after viral attachment but before the expression of the virus-specific proteins. A gB-null syncytial virus was isolated, which contained a gB defect and a syncytial mutation in another genetic locus. It caused complete fusion of gB-transformed cells but no fusion on untransformed cells, indicating the essential role of gB in virus-induced cell fusion. Mutations located at two independent sites in the cytoplasmic domain of gB were transferred to viral DNA and shown to confer a syncytial phenotype to the virus. A transient-expression assay was developed to determine the ability of a set of plasmids containing addition and nonsense mutations in the gB gene to complement the cell-fusion defect in the gB-null syncytial virus. Mutations in plasmids, including those located in the extracytoplasmic domain of gB, were identified that reduced the fusion activity of gB. Therefore, gB contains different functional regions responsible for fusion induction and its inhibition.     

Herpes simplex virus type 1 gK is required for gB-mediated virus-induced cell fusion, while neither gB and gK nor gB and UL20p function redundantly in virion de-envelopment

Multiple amino acid changes within herpes simplex virus type 1 (HSV-1) gB and gK cause extensive virus-induced cell fusion and the formation of multinucleated cells (syncytia). Early reports established that syncytial mutations in gK could not cause cell-to-cell fusion in the absence of gB. To investigate the interdependence of gB, gK, and UL20p in virus-induced cell fusion and virion de-envelopment from perinuclear spaces as well as to compare the ultrastructural phenotypes of the different mutant viruses in a syngeneic HSV-1 (F) genetic background, gB-null, gK-null, UL20-null, gB/gK double-null, and gB/UL20 double-null viruses were constructed with the HSV-1 (F) bacterial artificial chromosome pYEBac102. The gK/gB double-null virus YEbacDeltagBDeltagK was used to isolate the recombinant viruses gBsyn3DeltagK and gBamb1511DeltagK, which lack the gK gene and carry the gBsyn3 or gBamb1511 syncytial mutation, respectively. Both viruses formed small nonsyncytial plaques on noncomplementing Vero cells and large syncytial plaques on gK-complementing cells, indicating that gK expression was necessary for gBsyn3- and gBamb1511-induced cell fusion. Lack of virus-induced cell fusion was not due to defects in virion egress, since recombinant viruses specifying the gBsyn3 or gKsyn20 mutation in the UL19/UL20 double-null genetic background caused extensive cell fusion on UL20-complementing cells. As expected, the gB-null virus failed to produce infectious virus, but enveloped virion particles egressed efficiently out of infected cells. The gK-null and UL20-null viruses exhibited cytoplasmic defects in virion morphogenesis like those of the corresponding HSV-1 (KOS) mutant viruses. Similarly, the gB/gK double-null and gB/UL20 double-null viruses accumulated capsids in the cytoplasm, indicating that gB, gK, and UL20p do not function redundantly in membrane fusion during virion de-envelopment at the outer nuclear lamellae.     

Effects of deletions in the carboxy-terminal hydrophobic region of herpes simplex virus glycoprotein gB on intracellular transport and membrane anchoring.

The gB glycoprotein of herpes simplex virus type 1 is involved in viral entry and fusion and contains a predicted membrane-anchoring sequence of 69 hydrophobic amino acids, which can span the membrane three times, near the carboxy terminus. To define the membrane-anchoring sequence and the role of this hydrophobic stretch, we have constructed deletion mutants of gB-1, lacking one, two, or three predicted membrane-spanning segments within the 69 amino acids. Expression of the wild-type and mutant glycoproteins in COS-1 cells show that mutant glycoproteins lacking segment 3 (amino acids 774 to 795 of the gB-1 protein) were secreted from the cells. Protease digestion and alkaline extraction of microsomes containing labeled mutant proteins further showed that segment 3 was sufficient for stable membrane anchoring of the glycoproteins, indicating that this segment may specify the transmembrane domain of the gB glycoprotein. Also, the mutant glycoproteins containing segment 3 were localized in the nuclear envelop, which is the site of virus budding. Deletion of any of the hydrophobic segments, however, affected the intracellular transport and processing of the mutant glycoproteins. The mutant glycoproteins, although localized in the nuclear envelope, failed to complement the gB-null virus (K082). These results suggest that the carboxy-terminal hydrophobic region contains essential structural determinants of the functional gB glycoprotein.     

Functional regions and structural features of the gB glycoprotein of herpes simplex virus type 1. An analysis of linker insertion mutants

Glycoprotein B (gB) of Herpes simplex virus type 1 (HSV-1) plays an essential role in viral entry. A set of more than 100 HpaI (GTTAAC) linker insertion mutations and their derivatives were isolated in plasmids specifying the gB coding and flanking sequences. Mutations including addition, deletion and nonsense mutations at 34 independent sites were identified by DNA sequence analysis of 48 plasmids. A map was constructed for the ability of addition mutants to complement a gB-null virus. The expression of gB activity for some plasmids was temperature-dependent. Many complementation-negative plasmids inhibited the complementation activity of a plasmid specifying wild-type gB, suggesting an interaction between active and inactive molecules to form oligomers. The interaction was localized to 328 of the total of 904 amino acids comprising gB. Partial Endo H digestion of nonsense polypeptides revealed that five of the six potential N-linked oligosaccharide sites are glycosylated; the most C-terminal site appears not to be glycosylated. A number of mutations, including some on the cytoplasmic side, were identified that blocked processing, transport and secretion. Addition mutations that blocked processing of membrane polypeptides also blocked processing and secretion when combined into a nonsense mutant that by itself was processed and secreted. The previously predicted membrane spanning domain and the membrane orientation of the N-terminal portion of gB were confirmed.     

Proteolytic Processing of Human Cytomegalovirus Glycoprotein B Is Dispensable for Viral Growth in Culture

Glycoprotein B (gB) of human cytomegalovirus (HCMV), which is considered essential for the viral life cycle, is proteolytically processed during maturation. Since gB homologues of several other herpesviruses remain uncleaved, the relevance of this property of HCMV gB for viral infectivity is unclear. Here we report on the construction of a viral mutant in which the recognition site of gB for the cellular endoprotease furin was destroyed. Because mutagenesis of essential proteins may result in a lethal phenotype, a replication-deficient HCMV gB-null genome encoding enhanced green fluorescent protein was constructed, and complementation by mutant gBs was initially evaluated in transient-cotransfection assays. Cotransfection of plasmids expressing authentic gB or gB with a mutated cleavage site (gB-DeltaFur) led to the formation of green fluorescent miniplaques which were considered to result from one cycle of phenotypic complementation of the gB-null genome. To verify these results, two recombinant HCMV genomes were constructed: HCMV-BAC-DeltaMhdI, with a deletion of hydrophobic domain 1 of gB that appeared to be essential for viral growth in the cotransfection experiments, and HCMV-BACDeltaFur, in which the gB cleavage site was mutated by amino acid substitution. Consistent with the results of the cotransfection assays, only the DeltaFur mutant replicated in human fibroblasts, showing growth kinetics comparable to that of wild-type virus. gB in mutant-infected cells was uncleaved, whereas glycosylation and transport to the cell surface were not impaired. Extracellular mutant virus contained exclusively uncleaved gB, indicating that proteolytic processing of gB is dispensable for viral replication in cell culture.     

A cell-free recombination system for site-specific integration of multigenic shuttle plasmids into the herpes simplex virus type 1 genome.

This report describes a novel method for complementation studies of defective herpes simplex virus (HSV) genes. Viral test gene and nonviral reporter gene cassettes were rapidly integrated into the HSV genome in a site-specific and reversible manner by using the P1 phage-based Cre-lox recombination system. Shuttle plasmids contained a functional loxP recombination site, an expressible form of the bacterial lacZ gene, and a copy of the wild-type glycoprotein B (gB) gene or double mutant gB allele containing both a temperature-sensitive (ts) mutation and a syncytium (syn)-forming mutation. A recipient viral genome, K delta T::lox1, was constructed from the HSV type 1 (syn) gB-deficient mutant virus, K delta T, by marker transfer of the loxP recombination site into the viral thymidine kinase locus. Shuttle plasmids of up to 12.9 kb in length were recombined with high efficiency (11 to 20%) into the K delta T::lox1 genome in cell-free, Cre-mediated recombination reactions. Expression of a functional wild-type or double mutant gB polypeptide complemented the nonfunctional polypeptide expressed from the deleted, normal gB locus and allowed production of either wild-type or Syn- plaques on Vero cells. The latter recombinant virus was also ts for growth. The ability to express viral genes from plasmids which can be shuttled into and out of the HSV genome in cell-free recombination reactions makes this a powerful method for performing genetic studies of the biologic properties of viral gene products.     

Analysis of Epstein-Barr virus glycoprotein B functional domains via linker insertion mutagenesis

Epstein-Barr Virus (EBV) glycoprotein B (gB) is essential for viral fusion events with epithelial and B cells. This glycoprotein has been studied extensively in other herpesvirus family members, but functional domains outside of the cytoplasmic tail have not been characterized in EBV gB. In this study, a total of 28 linker insertion mutations were generated throughout the length of gB. In general, the linker insertions did not disrupt intracellular expression and variably altered cell surface expression. Oligomerization was disrupted by insertions located between residues 561 and 620, indicating the location of a potential site of oligomer contacts between EBV gB monomers. In addition, a novel N-glycosylated form of wild-type gB was identified under nonreducing Western blot conditions that likely represents a mature form of the protein. Fusion activity was abolished in all but three variants containing mutations in the N-terminal region (gB30), within the ectodomain (gB421), and in the intracellular C-terminal domain (gB832) of the protein. Fusion activity with variants gB421 and gB832 was comparable to that of the wild type with epithelial and B cells, and only these two mutants, but not gB30, were able to complement gB-null virus and subsequently function in virus entry. The mutant gB30 exhibited a low level of fusion activity with B cells and was unable to complement gB-null virus. The mutations generated here indicate important structural domains, as well as regions important for function in fusion, within EBV gB.     

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.     

Immunogenicity of herpes simplex virus glycoproteins gC and gB and their role in protective immunity.

The relative antigenicity of the individual herpes simplex virus type 1 (KOS) glycoproteins gC and gB was analyzed in BALB/c mice by using KOS mutants altered in their ability to present these antigens on cell surface membranes during infection. The mutants employed were as follows: syn LD70 , a non-temperature-sensitive mutant defective in the synthesis of cell surface membrane gC; tsF13 , a temperature-sensitive mutant defective in the processing of the precursor form of gB to the mature cell surface form at 39 degrees C; and ts606 , an immediate early temperature-sensitive mutant defective in the production of all early and late proteins including the glycoproteins. By comparing the relative susceptibility to immunolysis of mouse 3T3 cells infected at 39 degrees C with wild-type virus, presenting the full complement of the glycoprotein antigens, gC, gB, and gD, with target cells infected with mutants presenting only subsets of these antigens, we determined that a major portion of cytolytic antibody contained in hyperimmune anti-herpes simplex virus type 1 (KOS) mouse antiserum was directed against glycoproteins gC and gB. The relative immunogenicity of wild-type and mutant virus-infected cells also was compared in BALB/c mice. Immunogen lacking the mature form of gB induced a cytolytic antibody titer comparable to that of the wild-type virus, whereas that lacking the mature form of gC showed a 70% reduction in titer. The absence of the mature cell surface forms of gB and gC in immunogen preparations resulted in a 4- to 15-fold reduction in in virus neutralizing titer. Animals immunized with ts606 -infected cells (39 degrees C) induced relatively little virus-specific cytolytic and neutralizing antibody. Analysis of the glycoprotein specificities of these antisera by radioimmunoprecipitation showed that the antigens immunoprecipitated reflected the viral plasma membrane glycoprotein profiles of the immunogens. The absence of the mature forms of gC or gB in the immunizing preparation did not appreciably affect the immunoprecipitating antibody response to other antigens. Mice immunized with wild-type and mutant virus-infected cells were tested for their resistance to intracranial and intraperitoneal challenge with the highly virulent WAL strain of herpes simplex virus type 1. Despite the observed alterations in serum virus-specific antibody induced with the individual immunogens, all animals survived an intraperitoneal challenge of 10 50% lethal doses. However, differences in the survival of animals were obtained upon intracranial challenge.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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.     

Herpes Simplex Virus Glycoproteins: Participation of Individual Herpes Simplex Virus Type 1 Glycoprotein Antigens in Immunocytolysis and Their Correlation with Previously Identified Glycopolypeptides

Tissue culture cells infected with herpes simplex type 1 virus express virus-specified glycoprotein antigens on the plasma membrane. Three of these have been previously identified and have been designated as Ag-11, Ag-8, and Ag-6. In the present study, immunoglobulins to each of the antigens were shown to be capable of mediating immunocytolysis in the presence of either complement (antibody-dependent complement-mediated cytotoxicity) or peripheral blood mononuclear cells (antibody-dependent cell-mediated cytotoxicity [ADCC]). Two herpes simplex virus type 1 strains, VR-3 and F, reacted similarly in the ADCC test in the presence of immunoglobulins to Ag-11, Ag-8, and Ag-6 in both infected Chang liver cells and HEp-2 cells. Anti-Ag-6, however, produced a lower ADCC reaction in HEp-2 cells than in Chang liver cells, suggesting differences in the Ag-6 surface expression in, or release from, these cells. Chang liver and HEp-2 cells infected with the MP mutant strain of herpes simplex virus type 1 showed reduced ADCC in the presence of anti-Ag-11 and anti-Ag-8, but no reactivity at all with anti-Ag-6. Crossed immunoelectrophoretic analysis showed that MP-infected cell extracts contain Ag-11 and Ag-8, but lack Ag-6. Polypeptide analysis of herpes simplex virus type 1 strains F, VR-3, and MP showed that Ag-11 consists of the glycoproteins gA and gB, that Ag-8 consists of gD, and that Ag-6 consists of gC. In conclusion, the present study demonstrates that either one of the glycoproteins (gC, gD, and a mixture of gA and gB) can function as a target for immunocytolysis and that the antibody preparation to gC (Ag-6) does not cross-react with any of the other glycoproteins.     

Characterization of murine gammaherpesvirus 68 glycoprotein B (gB) homolog: similarity to Epstein-Barr virus gB (gp110).

Murine gammaherpesvirus 68 (MHV-68) is a natural pathogen of murid rodents and displays similar pathobiological characteristics to those of the human gammaherpesvirus Epstein-Barr virus (EBV). However, in contrast to EBV, MHV-68 will replicate in epithelial cells in vitro. It has therefore been proposed that MHV-68 may be of use as a model for the study of gammaherpesviruses, EBV in particular, both in vitro and in vivo. The EBV homolog of herpes simplex virus glycoprotein B (gB), termed gp110, is somewhat unusual compared with those of many other herpesviruses. We therefore decided to characterize the homolog of gB encoded by MHV-68 (termed MHV gB) to observe the properties of a gammaherpesvirus gB produced in epithelial cells and also to test the relatedness of MHV-68 and EBV. The MHV gB-coding sequence was determined from cloned DNA. The predicted amino acid sequence shared closest homology with gammaherpesvirus gB homologs. Biochemical analysis showed that MHV gB was a glycoprotein with a molecular weight of 105,000. However, the glycans were of the N-linked, high-mannose type, indicating retention in the endoplasmic reticulum. In line with this, MHV gB was localized to the cytoplasm and nuclear margins of infected cells but was not detected on the cell surface or in virions. Additionally, anti-MHV gB antisera were nonneutralizing. Thus, the MHV gB was unlike many other herpesvirus gBs but was extremely similar to the EBV gB. This highlights the close relationship between MHV-68 and EBV and underlines the potential of MHV-68 as a model for EBV in epithelial cells both in vitro and in vivo.     

Monoclonal antibodies suppress replication of herpes simplex virus type 1 in trigeminal ganglia.

The effect of monoclonal antibodies on the growth of herpes simplex virus type 1 in trigeminal ganglia was investigated. Four-week-old mice were infected on an abrased cornea with herpes simplex virus type 1. Forty-eight hours after infection, trigeminal ganglia ipsilateral with infected eyes were removed and placed in culture. Incubation of infected ganglia in the presence of a pool of nonneutralizing monoclonal antibodies specific for glycoproteins of gB and gE suppressed virus growth by greater than 90%. This was comparable to the amount of suppression observed when infected ganglia were incubated in hyperimmune serum. Individual monoclonal antibodies were less efficient, being able to inhibit virus growth by only two- to threefold. The mechanism of suppression was examined. Reduction in virus growth was observed under conditions in which all susceptible ganglion cells were infected in vitro before nonneutralizing monoclonal antibody was added. Similar results were obtained in tests with virus-infected neuroblastoma cells. Furthermore, suppression of infectious progeny was seen in the absence of complement and immunologically reactive cells. Thus, neither virus neutralization nor immunocytolysis could account for the effects of antibody on virus growth. Rather, the data suggest that antibody can bind to herpes simplex virus type 1-infected neuronal cells and suppress intracellular virus replication.     

Hydrophobic residues that form putative fusion loops of Epstein-Barr virus glycoprotein B are critical for fusion activity

To test the importance of the hydrophobic residues within the putative Epstein-Barr virus (EBV) glycoprotein B (gB) fusion loops in membrane fusion, WY(112-113) and WLIW(193-196) were mutated into alanine, glutamic acid, or the analogous residues from herpes simplex virus type 1 (HSV-1) gB (HR and RVEA). All gB variants exhibited cell surface expression, demonstrating that the substitutions did not perturb gB trafficking. None of six gB variants was, however, capable of mediating fusion with either epithelial or B cells. These data demonstrate that the bulky and hydrophobic EBV loop residues, which differ from the more hydrophilic HSV-1 residues and appear more compatible with membrane insertion, are essential for EBV gB-dependent fusion.     

Characterization of two TCR transgenic mouse lines specific for herpes simplex virus

To better understand the T cell-mediated processes involved in the immune response to herpes simplex virus type 1 (HSV-1)infection, two HSV-specific T cell receptor (TCR) transgenic mouse lines were produced. These mice (gBT-I.1 and gBT-I.3) are MHC class I-restricted and specific for the immunodominant peptide from HSV glycoprotein B (gB), gB498-505. Although derived from the same clone, the mice differ in the chromosomal location of the TCR transgenes and show marked differences in TCR alpha/beta expression on both CD4+ and CD8+ cells in the thymus. Despite this, peripheral CD8+ Tcells from both mice express equally high levels of the transgenic TCR and bind the KbgB498-505 tetramer to the same degree. In concordance with this, both were shown to respond equally well in vitro upon stimulation with the gB498-505 peptide or HSV-infected cells. These data show that selection of broadly equivalent peripheral T-cell subsets can occur in the presence of distinctly different thymic T-cell subsets.     

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.     

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.     

Effects of mutations in the cytoplasmic domain of herpes simplex virus type 1 glycoprotein B on intracellular transport and infectivity

Herpes simplex virus type 1 (HSV-1) is a human pathogen of the alphaherpesvirus family which infects and spreads in the nervous system. Glycoproteins play a key role in the process of assembly and maturation of herpesviruses, which is essential for neuroinvasion and transneuronal spread. Glycoprotein B (gB) is a main component of the HSV-1 envelope and is necessary for the production of infectious particles. The cytoplasmic domain of gB, the longest one among HSV-1 glycoproteins, contains several highly conserved peptide sequences homologous to motifs involved in intracellular sorting. To determine the specific roles of these motifs in processing, subcellular localization, and the capacity of HSV-1 gB to complement a gB-null virus, we generated truncated or point mutated forms of a green fluorescent protein (GFP)-tagged gB. GFP-gB with a deletion in the acidic cluster DGDADEDDL (amino acids [aa] 896 to 904) behaved the same as the parental form. Deletion or disruption of the YTQV motif (aa 889 to 892) abolished internalization and reduced complementation by 60%. Disruption of the LL motif (aa 871 to 872) impaired the return of the protein to the trans-Golgi network (TGN) while enhancing its recycling to the plasma membrane. Truncations from residue E 857 abolished transport and processing of the truncated proteins, which had null complementation activity, through the Golgi complex. Altogether, our results favor a model in which HSV-1 gets its final envelope in the TGN, and they suggest that endocytosis, albeit not necessary, might play a role in infectivity.