Herpes simplex virus (HSV) glycoprotein h is partially processed in a cell line that expresses the glycoprotein and fully processed in cells infected with deletion or is mutants in the known hsv glycoproteins

Cell lines that constitutively express herpes simplex virus 1 (HSV-1) glycoprotein H (gH-1 ) failed to synthesize the mature form of gH and accumulated a precursor-like form of the glycoprotein, which was retained intracellularly, most likely in RER. Fine-structure analysis of the oligosaccharides present in recombinant gH revealed oligosaccharides processed by RER enzymes; sialylated complex-type and biantennary oligosaccharides, which are assembled in the trans-Golgi, were absent. A small fraction had the characteristics of oligosaccharides processed by the early mannosidases of the Golgi. These findings suggest that a defect in the transport out of RER to the Golgi may account for the intracellular retention of the immature form of gH in cells that express the glycoprotein constitutively. Upon superinfection of cells expressing gH-1 with HSV-2, recombinant gH-1 underwent maturation, indicating that a viral function is required to attain full processing of gH. The known HSV glycoproteins do not appear to carry out this function, since in cells infected with deletion mutants in gD, gG, gE, and gE-gl, with a spontaneous gC- mutant, or with a temperature-sensitive mutant in gB, maturation of gH occurred independently of the presence or of the maturation of the single glycoproteins tested. The present findings together with previous observations on HSV, human CMV, and the EBV homologue of gH suggest that inability of gH to undergo full processing in the absence of viral protein (s) is a property of gH.     

Conservation of a gene cluster including glycoprotein B in bovine herpesvirus type 2 (BHV-2) and herpes simplex virus type 1 (HSV-1)

A library of subgenomic fragments of bovine herpesvirus type 2 (BHV-2) DNA was constructed in the expression cloning vector lambda gt11 and screened with monoclonal antibodies to the glycoprotein gb BHV-2, which is homologous to glycoprotein gB (gB-1) of herpes simplex virus type 1 (HSV-1). Lambda gt11 clones containing gB BHV-2-specific sequences were used to identify lambda EMBL3 vectors with DNA inserts which contained the complete gB BHV-2 gene. Nucleotide sequencing revealed that the gB BHV-2 gene is highly conserved compared to gB-1. The amino acid sequences and the predicted secondary structures of both glycoproteins are very similar. Two further open reading frames (ORF) in close vicinity to the gene encoding gB BHV-2 showed considerable homology to HSV-1 genes. They code for the major DNA-binding protein (dbp) of BHV-2 and a putative 72-kDa polypeptide. The gene of the latter protein corresponding to ICP18.5 of HSV-1 is interspersed between the ORFs of gB BHV-2 and the dbp of BHV-2. All three genes map in the unique long region of the genome. Their homology and the colinear arrangement compared to HSV-1 indicate a close relationship between the two viruses.     

Herpes simplex virus (HSV) glycoprotein H is partially processed in a cell line that expresses the glycoprotein and fully processed in cells infected with deletion or ts mutants in the known HSV glycoproteins.

Cell lines that constitutively express herpes simplex virus 1 (HSV-1) glycoprotein H (gH-1) failed to synthesize the mature form of gH and accumulated a precursor-like form of the glycoprotein, which was retained intracellularly, most likely in RER. Fine-structure analysis of the oligosaccharides present in recombinant gH revealed oligosaccharides processed by RER enzymes; sialylated complex-type and biantennary oligosaccharides, which are assembled in the trans-Golgi, were absent. A small fraction had the characteristics of oligosaccharides processed by the early mannosidases of the Golgi. These findings suggest that a defect in the transport out of RER to the Golgi may account for the intracellular retention of the immature form of gH in cells that express the glycoprotein constitutively. Upon superinfection of cells expressing gH-1 with HSV-2, recombinant gH-1 underwent maturation, indicating that a viral function is required to attain full processing of gH. The known HSV glycoproteins do not appear to carry out this function, since in cells infected with deletion mutants in gD, gG, gE, and gE-gI, with a spontaneous gC- mutant, or with a temperature-sensitive mutant in gB, maturation of gH occurred independently of the presence or of the maturation of the single glycoproteins tested. The present findings together with previous observations on HSV, human CMV, and the EBV homologue of gH suggest that inability of gH to undergo full processing in the absence of viral protein(s) is a property of gH.     

Analysis of the intracellular maturation of the herpes simplex virus type 1 glycoprotein gH in infected and transfected cells.

We have expressed the HSV-1 glycoprotein, gH, in transiently transfected COS-1 cells. The expressed protein was retained intracellularly, contained unprocessed carbohydrate, and was unrecognized by the monoclonal antibody, LP11. In addition, the protein was aggregated. These properties suggest that unlike other HSV glycoproteins, gH is misfolded in transfected cells. Pulse-chase studies of HSV-1-infected cells indicate that the kinetics of processing of gH are comparable to those of gB, gC, and gD. Rescue studies suggest that gH may interact with another protein during maturation in infected cells. However, we were unable to detect any stable interaction, although analysis of gH on neutral sucrose gradients shortly after synthesis indicated a possible transient association with a high molecular weight molecule or complex. The processing and cell surface expression of gH were also analyzed in HSV-1 virus mutants lacking gB, gC, or gD. Our results indicate that the maturation and cell surface transport of gH did not require the presence of these HSV-1 glycoproteins. In addition, three truncation mutants were constructed by linker insertion mutagenesis. Each of the three truncated proteins was synthesized, but the proteins were aggregated, contained only endo H-sensitive carbohydrate, and none were secreted.     

Computer prediction of antigenic and topogenic domains in HSV-1 and HSV-2 glycoprotein B (gB)

The envelope glycoprotein B (gB) coded for by the herpes simplex virus type 1 (HSV-1) U_L27 gene is similar to the amino acid (aa) sequence of the gB coded by a homologous gene in HSV-2 DNA. The putative antigenic domains in HSV-1 and HSV-2 gB glycoproteins were analyzed on a comparative basis by suitable computer programs, which allowed the prediction of putative antigenic and topogenic domains. The computer-derived domains were compared to experimentally reported antigenic domains in HSV-1 gB glycoprotein. The computer-predicted antigenic domains in the HSV-1 gB glycoprotein matched well with the reported experimentally derived antigenic domains. The aa sequence of antigenic domain 1 was noted to resemble the amino acid sequence in ApoE that is involved in the attachment of this protein to LDL receptors. The clusters of hydrophobic aa domains are conserved in the two viral glycoproteins and are signals for transfer of the viral proteins through the cellular membrane.     

Common epitopes of glycoprotein B map within the major DNA-binding proteins of bovine herpesvirus type 2 (BHV-2) and herpes simplex virus type 1 (HSV-1)

Bovine herpesvirus 2 (BHV-2) specifies a glycoprotein of 130 kDa (gB BHV-2) which shows extensive homology to glycoprotein B (gB-1) of herpes simplex virus 1 (HSV-1). The BHV-2-specific 130-kDa glycoprotein is able to induce cross-reacting antibodies, some of which even cross-neutralize HSV-1. In order to determine the genome localization of gB BHV-2 and in order to identify conserved antigenic domains in both glycoproteins, we established libraries of subgenic fragments of BHV-2 and HSV-1 DNA in the prokaryotic expression vector lambda gt11 and screened them with cross-reacting monoclonal antibodies which allowed us to identify recombinant lambda gt11 clones expressing gB fusion protein. Nucleotide sequencing of inserted DNA fragments within these recombinant lambda gt11 clones revealed that they originated from the carboxy-terminal part of the major DNA-binding proteins (dbp) of BHV-2 (dbp BHV-2) and its counterpart ICP8 in HSV-1. Antisera raised against the beta-galactosidase fusion protein of recombinant phage lambda-113/2 coding for an 84 amino acid (aa) polypeptide originating from dbp BHV-2 neutralized infectivity of BHV-2 and HSV-1 in the presence of complement and precipitated [3H] glucosamine-labeled gB BHV-2 and gB-1. This antiserum also reacts with ICP8 and presumably with dbp BHV-2. Two hypotheses are discussed to explain this unexpected result: (i) epitopes in the carboxy-terminal part of gB BHV-2 and gB-1 are similar to antigenic determinants in the amino-terminal region of the gBs, thus providing cross-reacting antibody-binding sites; (iii) during gene expression a carboxy-terminal part of dbp BHV-2 and ICP8 genes might be spliced to the amino-terminal region of the glycoproteins gB BHV-2 and gB-1.     

Expression of herpes simplex virus type 1 glycoprotein B in insect cells. Initial analysis of its biochemical and immunological properties.

A recombinant baculovirus (vAc-gB1) was constructed which expresses the glycoprotein B (gB) gene of herpes simplex virus type 1 (HSV-1). When Sf9 cells were infected with these recombinant viruses, a protein that was close in size to authentic HSV-1 gB was detected by gB polyclonal antibody. The recombinant gB was found on the membrane of Sf9 cells and was susceptible to tunicamycin, glycosidase F (PNGase F) and partially susceptible to Endo-H. Antibodies raised in mice to this recombinant recognized viral gB and neutralized the infectivity of HSV-1 in vitro. Mice inoculated with the recombinant gB were protected from lethal challenge with HSV-1.     

Conserved antigenic and functional domains on glycoprotein B of herpes simplex virus 1 and bovine herpesvirus 2

Summary A panel of monoclonal antibodies to glycoprotein B (gB) of herpes simplex virus 1 (HSV-1) reacted in immune precipitation and in immunofluorescence tests with a glycoprotein 130,000 in apparent molecular weight specified by bovine herpesvirus 2 (BHV-2). Two cross reactive antibodies neutralized the heterologous virus. These results suggest that the BHV-2 glycoprotein is the homologue of HSV-1 gB and shares structural and functional domains expressed on the gB gene product.     

The nucleotide sequence of the gB glycoprotein gene of HSV-2 and comparison with the corresponding gene of HSV-1

The nucleotide sequence of the gB glycoprotein gene of HSV-2 has been determined and compared with the homologous gene of HSV-1. The two genes are specified by the same total number of codons (904); eight additional codons of the HSV-1 gene are found within the signal sequence, and eight additional codons of the HSV-2 gene are found at three different sites in the gene. The signal cleavage, membrane-spanning, and eight potential N-linked oligosaccharide sites, as well as 5'- and 3'-regulatory signals are largely conserved. The overall amino acid homology is 85%; least conserved are the N- and C-terminal regions of the protein. Secondary structure plots were determined for the two proteins, and the structures were compared with each other and with alterations in structure due to several mutations in the HSV-1 gB gene for which sequence analysis is available. The high homology in primary and secondary structure suggests a conserved, essential function for the gene.     

The glycoprotein B gene and its syn3 locus of herpes simplex virus type 1 are involved in the synthesis of virus-associated growth factor (HSGF-1).

A putative growth factor (HSGF-1) associated with herpes simplex virus type 1 (HSV-1), which is similar to PRGF associated with pseudorabies virus, and/or HSGF-2 associated with HSV-2, was described. Experiments with four syncytial (syn) and four nonsyncytial (syn+) HSV-1 strains showed that the ability of this virus to produce HSGF-1 in infected cells is associated with the syn+ phenotype. Double infection of cells with syn+ and syn strain resulted either in enhancement or complete inhibition of HSGF-1 production, depending on the chosen pair of syn+ and syn strains. The studies with the recombinants between the syn+ strain KOS and syn strain ANGpath in the gene for glycoprotein B (gB) and syn3 locus revealed that the gB gene and its syn3 locus play a role in the HSGF-1 synthesis.     

Immunogenicity of purified glycoprotein gB of herpes simplex virus

Summary The efficacy of a herpes simplex virus (HSV) component vaccine consisting of viral glycoprotein gB was examined in a mouse system. Immunization of mice with HSV type 1 (HSV-1) gB emulsified in Freund's complete adjuvant or with HSV-1 gB adsorbed to aluminum gel was fully protective against subsequent challenge with HSV-1 or HSV type 2. Latent infection in the trigeminal ganglion was also prevented by immunization with gB.With 6 Figures     

Synthesis of the gB and gC glycoproteins of herpes simplex virus type 1 in the absence of viral DNA synthesis

Using an inhibitor of DNA synthesis as well as a temperature-sensitive (ts), DNA negative mutant of herpes simplex virus type 1 (HSV-1), we have examined the role of viral DNA synthesis on the expression of the virus-specific glycoproteins. Analysis by immunoblotting employing monospecific antisera revealed that only gC glycoprotein synthesis was completely inhibited in the absence of viral DNA synthesis. In contrast, the gB glycoprotein was detectable in significant, albeit somewhat reduced, amounts. These data suggest that gC is a ‘true-late’ protein of HSV-1 while gB is not. In addition, temperature shift-up experiments with the ts mutant (tsA1) suggest that some viral gene product(s) is needed continuously to achieve wild-type levels of gC synthesis.     

Sequence of a bovine herpesvirus type-1 glycoprotein gene that is homologous to the herpes simplex gene for the glycoprotein gB

The 130-kDa bovine herpesvirus-1 (BHV-1) glycoprotein GVP 6 was found to cross-react immunologically with the herpes simplex glycoprotein gB. Antibodies in polyclonal serum against gB immunoprecipitated GVP 6 and its cleavage products from a lysate of BHV-1-infected cells. Conversely, polyclonal serum against GVP 6 precipitated gB from HSV-1-infected cell lysates. Sera against the other glycoproteins did not demonstrate cross-reactivity. A 3.6-kb Kpnl-Hpal fragment of BHV-1 DNA that hybridized to the gene for gB was cloned and the nucleotide sequence of both strands was determined. The longest codon reading frame in the fragment coded for a protein that showed extensive homology with gB1 and related sequences from pseudorabies virus, varicella-zoster virus, cytomegalovirus, and Epstein-Barr virus. The strongest homologies were observed in two segments of the ectodomain, the transmembrane domain, and sequences adjacent to the transmembrane domain.     

Proliferative T-cell response to glycoprotein B of the human herpes viruses: the influence of MHC and sequence of infection on the pattern of cross-reactivity.

Oligopeptides of the highly conserved herpes virus glycoprotein B (gB) were expressed from DNA fragments of the EBV gB (BALF4) and HSV-2 gB open reading frames as fusion proteins with the lambda CII protein and beta-galactosidase (GZ), respectively, in Escherichia coli. After immunopurification using anti-gB or anti-GZ affinity columns, the fusion proteins were used in vitro to stimulate human peripheral blood lymphocytes (PBL) or murine lymph node cells that have been primed with EBV, HSV-1, HSV-2, VZV or HCMV (all human herpes viruses) to proliferate. Results obtained in BALB/c mice indicate that different herpes viruses induce different levels of T-cell response to each other and to gB, over a range of type-specific and cross-reactive T-cell epitopes. There is a lack of correlation of immunogenicity and antigenicity in the generation of T-cell responses between some of the viruses. Major T-cell epitopes are located at the C terminal half of the gB molecule. The T-cell response to gB in healthy individuals seropositive for various combinations of the five herpes viruses differed markedly from individual to individual, even when they are seropositive to the same set of herpes viruses. However, two individuals with high proliferative T-cell response to VZV and sharing HLA A2, B7, DR2 and DQw1 are also good responders for cross-reactive gB/fragments and for virus antigen of all the five herpes viruses. Therefore the data obtained demonstrated that the MHC and the immune interaction arising from cross-reactive T-cell response evoked by other herpes viruses may determine the pathogenesis of a herpes virus infection.     

Estimation of the B lymphocyte precursor frequencies to herpes simplex type 1 glycoproteins by a limiting dilution assay

The precursor frequency of B lymphocytes from Balb/c mice producing HSV-1 glycoprotein B (gB), glycoprotein C (gC), and glycoprotein D (gD) antibody was determined by limiting dilution analysis under conditions to detect antibody from the clonal progeny of a single B cell precursor. In spleens of naive mice the average gC frequency was 1/48,917 +/- 5,550, while gD was 1/73,330 +/- 15,898, and gB frequency was in excess of 1/100,000. Immunization with live HSV-1 (KOS) increased the B cell frequencies of all three glycoproteins to approximately 1:3,000; however, the serum gB antibody ELISA titer was fivefold higher than gC or gD.     

Domain structure of herpes simplex virus 1 glycoprotein B: neutralizing epitopes map in regions of continuous and discontinuous residues

Herpes simplex virus 1 (HSV-1) glycoprotein B (gB) is a multifunctional glycoprotein required for infectivity; it is thought to promote fusion of the viral envelope with the cell membrane and entry of virions into cells. To map the antigenic and functional domains on gB, we constructed amino terminal derivatives lacking the entire carboxyl terminus and internal deletion mutants lacking defined regions of the extracellular and transmembrane domains. Transient expression of the mutants in COS-1 cells revealed that the amino terminal derivatives were released into the medium whereas those with deletions in the extracellular domain were mostly retained within the transfected cells. Analysis of intact gB and the amino terminal derivatives showed that the intact molecule formed dimers whereas the mutant derivatives did not. Reactions of the derivatives with a panel of well-characterized monoclonal antibodies to gB showed that the neutralizing epitopes cluster in two domains. The first maps in the amino terminal 190 residues and contains seven continuous epitopes, five of which are HSV-1-specific. Reactions of antibodies with a set of oligopeptides fine-mapped the epitopes between residues 1 and 47. The second domain is composed of discontinuous epitopes and was expressed by amino terminal derivatives that were at least 457 residues in length or longer. Eleven epitopes map in this region, including those of four potent neutralizing antibodies whose cognitive sites mapped between residues 273 and 298 in mapping studies using antibody-resistant mutants. Results of the present study indicate that the cognitive sites of these antibodies are assembled into the discontinuous domain by juxtaposing residues from the amino-terminal half of gB monomers.     

Glycoprotein B from strain 17 of herpes simplex virus type I contains an invariant chain homologous sequence that binds to MHC class II molecules

Major histocompatibility complex class I (MHCI) molecules are major targets of virus evasion strategies because they introduce antigens from the biosynthesis pathway into the antigen-processing and presentation pathways for immune recognition by CD8+ T cells. Little is known about viral strategies that interfere with the MHC class II (MHCII) antigen presentation pathway. We identified a six amino acid sequence from type I herpes simplex virus (HSV-1) glycoprotein B (gB) that is identical to a sequence of human leucocyte antigen D (HLA-D) -associated invariant chain (Ii). In addition, this gB sequence is adjacent to a highly conserved HLA-DR1 binding motif. Both viral sequences together resemble the class II binding site of human Ii, consisting of a MHCII groove binding segment and a promiscuous binding site. We cloned gB from HSV-1 strain 17 and demonstrate association of the virus envelope protein to three HLA-DR allotypes. With chimeric Ii/gB fusion proteins we identified gB sequences that mediate promiscuous or allotype-specific binding to the HLA-DR peptide-binding domain. Mutation of two Lys residues in the viral segment of Ii/gB abolished promiscuous binding to HLA-DR heterodimers. The result indicates promiscuous binding of the virus sequence to HLA-DR molecules and suggests a potential for HSV-1 to manipulate antigen processing and presentation.     

A mutation in the ectodomain of herpes simplex virus 1 glycoprotein B causes defective processing and retention in the endoplasmic reticulum.

Herpes simplex virus 1 (HSV-1) glycoprotein B (gB) is one of several envelope glycoproteins required for virion infectivity and is the only one known to oligomerize into homodimers. To study the conformational constraints for translocation of HSV-1 gB to the surface of eukaryotic cells, we analyzed the transport through the exocytic pathway of the wild-type glycoprotein and of mutant forms with insertions in the ectodomain and intracellular carboxy terminus. Transient expression of the glycoproteins in COS-1 cells showed that an insertion at position 479 in the amino-terminal ectodomain of gB, shown previously by reactions with monoclonal antibodies to have altered the conformation of the molecule, also had a drastic effect on transport, precluding exit of the mutant from the endoplasmic reticulum (ER) and transport to the Golgi and the plasma membrane. The fact that the mutant, gB-(Lk479), formed dimers suggests that local changes in assembled regions caused the transport defect. Mutants containing insertions at residues 600 of the ectodomain and 810 in the intracellular domain were slightly retarded in their rate of transport from the ER to the Golgi. The glucose-regulated proteins GRP78 and GRP94, which are resident proteins of the ER, associated with partially glycosylated, faster-migrating forms of gB but not with the fully processed, more slowly migrating product. GRP78 and GRP94 formed complexes with the mutant gB-(Lk479), which was degraded in the ER. Our results indicate that GRP78, and perhaps also GRP94, acts as a chaperone in the assembly of native gB oligomers and also binds to aberrant forms of the molecule, arresting their transport from the ER and possibly serving as markers for protein degradation in this compartment of the exocytic pathway.