Truncation of the carboxy-terminal 28 amino acids of glycoprotein B specified by herpes simplex virus type 1 mutant amb1511-7 causes extensive cell fusion.

Three amber mutations were introduced proximal to the syn3 locus of the herpes simplex virus type 1 glycoprotein B (gB) gene specifying gB derivatives lacking the carboxy-terminal 28, 49, or 64 amino acids. A complementation system that utilized gBs expressed in COS cells to complement gB-null virus K delta T was established. The 49- or 64-amino-acid-truncated gBs failed to complement gB-null virus K delta T, while the 28-amino-acid-truncated gB complemented K delta T efficiently. Mutant herpes simplex virus type 1 KOS (amb1511-7) specifying the 28-amino-acid-truncated gB fused Vero cells extensively.     

Amino-terminal sequence, synthesis, and membrane insertion of glycoprotein B of herpes simplex virus type 1.

Glycoprotein B (gB) was purified from cells infected with two strains (KOS and F) of herpes simplex virus type 1. Determination of amino acid sequence at the NH2 termini revealed, by comparison with amino acid sequence deduced from previously published nucleotide sequence, that gB is made with a cleavable signal sequence of 29 or 30 amino acids, depending on the virus strain. Analysis of gB translated in vitro in the presence and absence of membranes showed that gB is inserted into membranes and glycosylated cotranslationally; a large portion of the gB polypeptide made in vitro is protected from proteolysis by membranes; the large protected fragment carries N-linked carbohydrate and is probably the NH2 terminus based on locations of signals for the addition of N-linked carbohydrate; and the size of the protected fragment is 93 kilodaltons (kDa) for gB made in vitro and associated with dog pancreas membranes, whereas both 93- and 98-kDa protected fragments can be detected for gB made in vivo. These last results are consistent with a previous proposal that gB may traverse the membrane three times.     

Expression of cell-associated and secreted forms of herpes simplex virus type 1 glycoprotein gB in mammalian cells.

The gene for glycoprotein gB1 of herpes simplex virus type 1 strain Patton was expressed in stable Chinese hamster ovary cell lines. Expression vectors containing the dihydrofolate reductase (dhfr) cDNA plus the complete gB1 gene or a truncated gene lacking the 194 carboxyl-terminal amino acids of gB1 were transfected into CHO DHFR-deficient cells. Radioimmunoprecipitation demonstrated that the complete gB1 protein expressed in CHO cell lines was cell associated, whereas the truncated protein was secreted from the cells due to deletion of the transmembrane and C-terminal domains of gB1. Cells expressing the truncated gB1 protein were subjected to stepwise methotrexate selection, and a cell line was isolated in which the gB1 gene copy number had been amplified 10-fold and the level of expression of gB1 had increased over 60-fold. The truncated gB1 protein was purified from medium conditioned by the amplified cell line. N-terminal amino acid sequence analysis of this purified protein identified the signal peptide cleavage site and predicted the cleavage of a 30-amino-acid signal sequence from the primary protein. The immunogenicity of the truncated gB1 protein was also tested in mice, and high levels of antibody and protection from virus challenge were observed.     

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.     

Amino-terminal sequence of glycoprotein D of herpes simplex virus types 1 and 2.

Glycoprotein D (gD) of herpes simplex virus is a structural component of the virion envelope which stimulates production of high titers of herpes simplex virus type-common neutralizing antibody. We carried out automated N-terminal amino acid sequencing studies on radiolabeled preparations of gD-1 (gD of herpes simplex virus type 1) and gD-2 (gD of herpes simplex virus type 2). Although some differences were noted, particularly in the methionine and alanine profiles for gD-1 and gD-2, the amino acid sequence of a number of the first 30 residues of the amino terminus of gD-1 and gD-2 appears to be quite similar. For both proteins, the first residue is a lysine. When we compared our sequence data for gD-1 with those predicted by nucleic acid sequencing, the two sequences could be aligned (with one exception) starting at residue 26 (lysine) of the predicted sequence. Thus, the first 25 amino acids of the predicted sequence are absent from the polypeptides isolated from infected cells.     

gA and gB glycoproteins of herpes simplex virus type 1: two forms of a single polypeptide.

Utilizing a combination of preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sodium dodecyl sulfate-hydroxylapatite column chromatography, we have separated and purified the gA and gB glycoproteins of the major virus-specific glycoprotein region from herpes simplex virus type 1-infected cells. By using purified antigen preparations, antisera specific to each of these glycoproteins were produced. Immunoprecipitation from detergent extracts of infected cells and radioimmune precipitation of the purified antigens have shown that the anti-gA and anti-gB sera each recognize both the gA and the gB glycoproteins. The anti-gA serum was also shown to neutralize virus despite the presence of only minute quantities of the gA glycoprotein in virions. Pulse-chase studies have indicated that the gA and gB glycoproteins are synthesized from a common precursor polypeptide. Together, these data demonstrate that the gA and gB glycoproteins of herpes simplex virus type 1 are antigenically similar but not identical and probably represent two different forms of the same polypeptide which differ in their degree of glycosylation.     

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

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

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.     

Effect of tunicamycin on the synthesis of herpes simplex virus type 1 glycoproteins and their expression on the cell surface.

Herpes simplex virus specifies five glycoproteins which have been found on the surface of both the intact, infected cells and the virion envelope. In the presence of the drug tunicamycin, glycosylation of the herpes simplex virus type 1 glycoproteins is inhibited. We present in this report evidence that the immunologically specificity of the glycoproteins designated gA, gB, and gD resides mainly in the underglycosylated "core" proteins, as demonstrated by the immunoblotting technique. We showed also that tunicamycin prevented exposure of the viral glycoproteins on the cell surface, as the individual glycoproteins lost their ability to participate as targets for the specific antibodies applied in the antibody-dependent, cell-mediated cytotoxicity test. Immunocytolysis was reduced between 73 and 97%, depending on the specificity of the antibodies used. The intracellular processing of the herpes simplex virus type 1-specific glycoprotein designated gC differed from the processing of gA, gB, and GD, as evidenced by the identification of an underglycosylated but immunochemically modified form of gC on the surface of infected cells grown in the presence of tunicamycin.     

Membrane anchoring domain of herpes simplex virus glycoprotein gB is sufficient for nuclear envelope localization.

We have used the glycoprotein gB of herpes simplex virus type 1 (gB-1), which buds from the inner nuclear membrane, as a model protein to study localization of membrane proteins in the nuclear envelope. To determine whether specific domains of gB-1 glycoprotein are involved in localization in the nuclear envelope, we have used deletion mutants of gB-1 protein as well as chimeric proteins constructed by replacing the domains of the cell surface glycoprotein G of vesicular stomatitis virus with the corresponding domains of gB. Mutant and chimeric proteins expressed in COS cells were localized by immunoelectron microscopy. A chimeric protein (gB-G) containing the ectodomain of gB and the transmembrane and cytoplasmic domains of G did not localize in the nuclear envelope. When the ectodomain of G was fused to the transmembrane and cytoplasmic domains of gB, however, the resulting chimeric protein (G-gB) was localized in the nuclear envelope. Substitution of the transmembrane domain of G with the 69 hydrophobic amino acids containing the membrane anchoring domain of gB allowed the hybrid protein (G-tmgB) to be localized in the nuclear envelope, suggesting that residues 721 to 795 of gB can promote retention of proteins in the nuclear envelope. Deletion mutations in the hydrophobic region further showed that a transmembrane segment of 21 hydrophobic amino acids, residues 774 to 795 of gB, was sufficient for localization in the nuclear envelope. Since wild-type gB and the mutant and chimeric proteins that were localized in the nuclear envelope were also retained in the endoplasmic reticulum, the membrane spanning segment of gB could also influence retention in the endoplasmic reticulum.     

Glycoprotein 110, the Epstein-Barr virus homolog of herpes simplex virus glycoprotein B, is essential for Epstein-Barr virus replication in vivo.

The Epstein-Barr virus (EBV) glycoprotein gp110 has substantial amino acid homology to gB of herpes simplex virus but localizes differently within infected cells and is essentially undetectable in virions. To investigate whether gp110, like gB, is essential for EBV infection, a selectable marker was inserted within the gp110 reading frame, BALF4, and the resulting null mutant EBV stain, B95-110HYG, was recovered in lymphoblastoid cell lines (LCLs). While LCLs infected with the parental virus B95-8 expressed the gp110 protein product following productive cycle induction, neither full-length gp110 nor the predicted gp110 truncation product was detectable in B95-110HYG LCLs. Infectious virus could not be recovered from B95-110HYG LCLs unless gp110 was provided in trans. Rescued B95-110HYG virus latently infected and growth transformed primary B lymphocytes. Thus, gp110 is required for the production of transforming virus but not for the maintenance of transformation of primary B lymphocytes by EBV.     

Use of transdominant mutants of the origin-binding protein (UL9) of herpes simplex virus type 1 to define functional domains.

UL9, the origin-binding protein of herpes simplex virus type 1, contains six sequence motifs conserved in a large superfamily of RNA and DNA helicases. Single-amino-acid substitution mutations in these motifs inactivate UL9 function in vivo (R. Martinez, L. Shao, and S. K. Weller, J. Virol. 66:6735-6746, 1992). Overexpression of wild-type UL9 is inhibitory to plaque formation in a transfection assay which measures viral plaque formation by infectious herpes simplex virus type 1 DNA. Constructs containing mutations in motif I, II, or VI exhibit even stronger inhibitory effects in the same assay and thus can be considered strong transdominant inhibitors of plaque formation by the wild-type virus. The transdominant phenotype can be relieved by introducing a second mutation in the DNA-binding domain or by deleting the N-terminal 35 amino acids of the protein. The inhibitory effects of wild-type UL9 can also be partially relieved by deletion of amino acids 292 to 404. We propose that the N-terminal 35 amino acids of UL9 and residues 292 to 404 may define new functional domains of the UL9 protein.     

The nucleotide sequence of herpes simplex virus type 2 (333) glycoprotein gB2 and analysis of predicted antigenic sites

The gene coding for glycoprotein B2 (gB2) of herpes simplex virus type 2 (HSV-2) strain 333 was mapped and its nucleotide sequence determined. Open reading frame analysis deduced a polypeptide consisting of 902 amino acids and having close homology to gB1 of HSV type 1. Several predicted features of gB2 are consistent with a membrane-bound glycoprotein, i.e., a signal peptide sequence, a hydrophilic extracellular domain containing possible N-linked glycosylation sites, a hydrophobic membrane spanning sequence, and a cytoplasmic domain. Computer analysis on hydrophilicity, accessibility, and flexibility of the gB2 amino acid sequence, produced a composite surface value plot. At least nine major antigenic regions were predicted on the extracellular domain. The amino acids between residues 59-74, 127-139, 199-205, 460-476, and 580-594 exhibited the highest surface values. Comparison of the primary sequence with gB1 revealed localized regions showing amino acid diversity. Several of these locations correspond to major antigenic regions. Chou and Fasman analyses indicated that the amino acid substitutions, between positions 57-66, 461-472, and 473-481, induced changes in the secondary structure of gB. These sites could represent site-specific epitopes in the gB polypeptide.     

Contribution of endocytic motifs in the cytoplasmic tail of herpes simplex virus type 1 glycoprotein B to virus replication and cell-cell fusion

The use of endocytic pathways by viral glycoproteins is thought to play various functions during viral infection. We previously showed in transfection assays that herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) is transported from the cell surface back to the trans-Golgi network (TGN) and that two motifs of gB cytoplasmic tail, YTQV and LL, function distinctly in this process. To investigate the role of each of these gB trafficking signals in HSV-1 infection, we constructed recombinant viruses in which each motif was rendered nonfunctional by alanine mutagenesis. In infected cells, wild-type gB was internalized from the cell surface and concentrated in the TGN. Disruption of YTQV abolished internalization of gB during infection, whereas disruption of LL induced accumulation of internalized gB in early recycling endosomes and impaired its return to the TGN. The growth of both recombinants was moderately diminished. Moreover, the fusion phenotype of cells infected with the gB recombinants differed from that of cells infected with the wild-type virus. Cells infected with the YTQV-mutated virus displayed reduced cell-cell fusion, whereas giant syncytia were observed in cells infected with the LL-mutated virus. Furthermore, blocking gB internalization or impairing gB recycling to the cell surface, using drugs or a transdominant negative form of Rab11, significantly reduced cell-cell fusion. These results favor a role for endocytosis in virus replication and suggest that gB intracellular trafficking is involved in the regulation of cell-cell fusion.     

Amino acids 143 to 150 of the herpes simplex virus type 1 scaffold protein are required for the formation of portal-containing capsids

The herpes simplex virus type 1 (HSV-1) portal is composed of a dodecamer of UL6 protein molecules whose incorporation into the capsid is mediated by interaction with the HSV-1 UL26.5 scaffold protein. Previous results with an in vitro capsid assembly assay demonstrated that nine amino acids (amino acids 143 to 151) of the UL26.5 protein are required for its interaction with UL6 and for incorporation of the portal complex into capsids. In the present study an HSV-1 mutant, bvFH411, was isolated and contained a deletion that removed the codons for UL26.5 amino acids 143 to 150. The mutant virus failed to produce infectious virus in noncomplementing cells, and only B capsids that contained only minor amounts of portal protein were made. These data corroborate our previous in vitro studies and demonstrate that amino acids 143 to 150 of UL26.5 are required for the formation of portal-containing HSV-1 capsids.     

Antigenic cross-reactions among herpes simplex virus types 1 and 2, Epstein-Barr virus, and cytomegalovirus.

Polyvalent rabbit antisera against herpes simplex virus type 1 and 2 (HSV-1 and HSV-2), cytomegalovirus (CMV), and Epstein-Barr virus (EBV), monospecific antisera against affinity-purified HSV-2 glycoproteins gB and gG, and a panel of monoclonal antibodies against HSV and EBV proteins were used to analyze cross-reactive molecules in cells infected with the four herpesviruses. A combination of immunoprecipitation and Western blotting with these reagents was used to determine that all four viruses coded for a glycoprotein that cross-reacted with HSV-1 gB. CMV coded for proteins that cross-reacted with HSV-2 gC, gD, and gE. Both CMV and EBV coded for proteins that cross-reacted with HSV-2 gG. Antigenic counterparts to the p45 nucleocapsid protein of HSV-2 were present in HSV-1 and CMV, and counterparts of the major DNA-binding protein and the ribonucleotide reductase of HSV-1 were present in all the viruses. The EBV virion glycoprotein gp85 was immunoprecipitated by antisera to HSV-1, HSV-2, and CMV. Antisera to CMV and EBV neutralized the infectivity of both HSV-1 and HSV-2 at high concentrations. This suggests that cross-reactivity between these four human herpesviruses may have pathogenic as well as evolutionary significance.     

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.     

Pseudorabies virus glycoprotein M inhibits membrane fusion

A transient transfection-fusion assay was established to investigate membrane fusion mediated by pseudorabies virus (PrV) glycoproteins. Plasmids expressing PrV glycoproteins under control of the immediate-early 1 promoter-enhancer of human cytomegalovirus were transfected into rabbit kidney cells, and the extent of cell fusion was quantitated 27 to 42 h after transfection. Cotransfection of plasmids encoding PrV glycoproteins B (gB), gD, gH, and gL resulted in formation of polykaryocytes, as has been shown for homologous proteins of herpes simplex virus type 1 (HSV-1) (A. Turner, B. Bruun, T. Minson, and H. Browne, J. Virol. 72:873-875, 1998). However, in contrast to HSV-1, fusion was also observed when the gD-encoding plasmid was omitted, which indicates that PrV gB, gH, and gL are sufficient to mediate fusion. Fusogenic activity was enhanced when a carboxy-terminally truncated version of gB (gB-008) lacking the C-terminal 29 amino acids was used instead of wild-type gB. With gB-008, only gH was required in addition for fusion. A very rapid and extended fusion was observed after cotransfection of plasmids encoding gB-008 and gDH, a hybrid protein consisting of the N-terminal 271 amino acids of gD fused to the 590 C-terminal amino acids of gH. This protein has been shown to substitute for gH, gD, and gL function in the respective viral mutants (B. G. Klupp and T. C. Mettenleiter, J. Virol. 73:3014-3022, 1999). Cotransfection of plasmids encoding PrV gC, gE, gI, gK, and UL20 with gB-008 and gDH had no effect on fusion. However, inclusion of a gM-expressing plasmid strongly reduced the extent of fusion. An inhibitory effect was also observed after inclusion of plasmids encoding gM homologs of equine herpesvirus 1 or infectious laryngotracheitis virus but only in conjunction with expression of the gM complex partner, the gN homolog. Inhibition by PrV gM was not limited to PrV glycoprotein-mediated fusion but also affected fusion induced by the F protein of bovine respiratory syncytial virus, indicating a general mechanism of fusion inhibition by gM.     

Fine structure analysis of type-specific and type-common antigenic sites of herpes simplex virus glycoprotein D

The fine structure of the antigenic determinants of herpes simplex virus type 1 and 2 glycoprotein D (gD) was analyzed to determine whether structural differences underlie the differential immunogenicity of these glycoproteins. A region common to herpes simplex virus type 1 and 2 gD (amino acid residues 11 to 19) and two sites specific for herpes simplex virus type 2 gD (one determined by proline at position 7, the other determined by asparagine at position 21) were localized within the N-terminal 23 amino acids of gD by synthesis of peptides and comparison of their cross-reactivity with antisera raised to herpes simplex virus type 1 and 2 gD. The secondary structure of these peptides, as predicted by computer analysis, is discussed in relation to their immunogenicity.