Characterization of the antigenic structure of herpes simplex virus type 1 glycoprotein C through DNA sequence analysis of monoclonal antibody-resistant mutants.

Earlier studies of a group of monoclonal antibody-resistant (mar) mutants of herpes simplex virus type 1 glycoprotein C (gC) operationally defined two distinct antigenic sites on this molecule, each consisting of numerous overlapping epitopes. In this report, we further define epitopes of gC by sequence analysis of the mar mutant gC genes. In 18 mar mutants studied, the mar phenotype was associated with a single nucleotide substitution and a single predicted amino acid change. The mutations were localized to two regions within the coding sequence of the external domain of gC and correlated with the two previously defined antigenic sites. The predicted amino acid substitutions of site I mutants resided between residues Gln-307 and Pro-373, whereas those of site II mutants occurred between amino acids Arg-129 and Glu-247. Of the 12 site II mutations, 9 induced amino acid substitutions within an arginine-rich segment of 8 amino acids extending from residues 143 to 151. The clustering of the majority of substituted residues suggests that they contribute to the structure of the affected sites. Moreover, the patterns of substitutions which affected recognition by antibodies with similar epitope specificities provided evidence that epitope structures are physically linked and overlap within antigenic sites. Of the nine epitopes defined on the basis of mutations, three were located within site I and six were located within site II. Substituted residues affecting the site I epitopes did not overlap substituted residues of site II, supporting our earlier conclusion that sites I and II reside in spatially distinct antigenic domains. A computer analysis of the distribution of charged residues and the predicted secondary structural features of wild-type gC revealed that the two antigenic sites reside within the most hydrophilic regions of the molecule and that the antigenic residues are likely to be organized as beta sheets which loop out from the surface of the molecule. Together, these data and our previous studies support the conclusion that the mar mutations identified by sequence analysis very likely occur within or near the epitope structures themselves. Thus, two highly antigenic regions of gC have now been physically and genetically mapped to well-defined domains of the protein molecule.     

Extensive homology between the herpes simplex virus type 2 glycoprotein F gene and the herpes simplex virus type 1 glycoprotein C gene.

The region of the herpes simplex virus type 2 (HSV-2) genome which maps colinearly with the HSV-1 glycoprotein C (gC) gene has been cloned, and the DNA sequence of a 2.29-kilobase region has been determined. Contained within this sequence is a major open reading frame of 479 amino acids. The carboxyterminal three-fourths of the derived HSV-2 protein sequence showed a high degree of sequence homology to the HSV-1 gC amino acid sequence reported by Frink et al. (J. Virol. 45:634-647, 1983). The amino-terminal region of the HSV-2 sequence, however, showed very little sequence homology to HSV-1 gC. In addition, the HSV-1 gC sequence contained 27 amino acids in the amino-terminal region which were missing from the HSV-2 protein. Computer-assisted analysis of the hydrophilic and hydrophobic properties of the derived HSV-2 sequence demonstrated that the protein contained structures characteristic of membrane-bound glycoproteins, including an amino-terminal signal sequence and carboxy-terminal hydrophobic transmembrane domain and charged cytoplasmic anchor. The HSV-2 protein sequence also contained seven putative N-linked glycosylation sites. These data, in conjunction with mapping studies of Para et al. (J. Virol. 45:1223-1227, 1983) and Zezulak and Spear (J. Virol. 49:741-747, 1984), suggest that the protein sequence derived from the HSV-2 genome corresponds to gF, the HSV-2 homolog of HSV-1 gC.     

Antigenic analysis of a major neutralization site of herpes simplex virus glycoprotein D, using deletion mutants and monoclonal antibody-resistant mutants.

Herpes simplex virus glycoprotein D is a component of the virion envelope and appears to be involved in attachment, penetration, and cell fusion. Monoclonal antibodies against this protein can be arranged in groups on the basis of a number of biological and biochemical properties. Group I antibodies are type common, have high complement-independent neutralization titers, and recognize discontinuous (conformational) epitopes; they are currently being used in several laboratories to study the functions of glycoprotein D. We have used a panel of neutralization-resistant mutants to examine the relationships between these antibodies in detail. We found that they can be divided into two subgroups, Ia and Ib, such that mutations selected with Ia antibodies have little or no effect on binding and neutralization by Ib antibodies and vice versa. In addition, Ia antibodies are able to bind deletion and truncation mutants of glycoprotein D that Ib antibodies do not recognize, suggesting that their epitopes are physically distinct. However, with one exception, Ia and Ib antibodies block each other strongly in binding assays with purified glycoprotein D, whereas antibodies from other groups have no effect. We have therefore defined the sum of the Ia and Ib epitopes as antigenic site 1.     

Cysteine mutants of herpes simplex virus type 1 glycoprotein D exhibit temperature-sensitive properties in structure and function.

We previously constructed seven mutations in the gene for glycoprotein D (gD) of herpes simplex virus type 1 in which the codon for one of the cysteine residues was replaced by a serine codon. Each of the mutant genes was cloned into a eucaryotic expression vector, and the proteins were transiently expressed in mammalian cells. We found that alteration of any of the first six cysteine residues had profound effects on protein conformation and oligosaccharide processing. In this report, we show that five of the mutant proteins exhibit temperature-sensitive differences in such properties as aggregation, antigenic conformation, oligosaccharide processing, and transport to the cell surface. Using a complementation assay, we have now assessed the ability of the mutant proteins to function in virus infection. This assay tests the ability of the mutant proteins expressed from transfected plasmids to rescue production of infectious virions of a gD-minus virus, F-gD beta, in Vero cells. Two mutant proteins, Cys-2 (Cys-106 to Ser) and Cys-4 (Cys-127 to Ser), were able to complement F-gD beta at 31.5 degrees C but not at 37 degrees C. The rescued viruses, designated F-gD beta(Cys-2) and F-gD beta(Cys-4), were neutralized as efficiently as wild-type virus by anti-gD monoclonal antibodies, indicating that gD was present in the virion envelope in a functional form. Both F-gD beta(Cys-2) and F-gD beta(Cys-4) functioned normally in a penetration assay. However, the infectivity of these viruses was markedly reduced compared with that of the wild type when they were preincubated at temperatures above 37 degrees C. The results suggest that mutations involving Cys-106 or Cys-127 in gD-1 confer a temperature-sensitive phenotype on herpes simplex virus. These and other properties of the cysteine-to-serine mutants allowed us to predict a disulfide bonding pattern for gD.     

Passive immune protection by herpes simplex virus-specific monoclonal antibodies and monoclonal antibody-resistant mutants altered in pathogenicity.

Virus-neutralizing monoclonal antibodies specific for 13 different genetically defined epitopes of glycoproteins gC, gB, and gD of herpes simplex virus type 1, strain KOS-321, were compared for their ability to provide passive immunity to DBA-2 mice challenged intracranially. Protection was highly specific, since individual monoclonal antibodies failed to protect against infection with monoclonal antibody-resistant (mar) mutants altered in the single epitope recognized by the injected antibody. The dose-response kinetics of passive immunity paralleled the in vitro neutralization titers for each antibody. No correlation was observed between immune protection and antibody isotype or complement-dependent in vitro neutralization titers. This suggests that virus neutralization was not the protective mechanism. In general, antibodies reactive with epitopes of gC were protective at the lowest antibody doses, antibodies specific for gB were less efficient in providing immunity, and antibodies against gD were the least effective. mar mutants with single epitope changes in gC and multiple epitope changes in gB showed highly reduced pathogenicity, requiring up to 5 X 10(6) PFU to kill 50% of infected animals. These findings indicated that antigenic variation affects virus growth and spread in the central nervous system. Thus, mutations which affect antigenic structure also can alter virus pathogenicity. The alteration of these epitopes does not, however, appreciably reduce the development of resistance to infection. Infection of mice with these mutants or inoculation of mice with UV-inactivated, mutant-infected cells before challenge rendered the animals resistant to infection with wild-type herpes simplex virus type 1.     

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 herpes simplex virus type 1 glycoprotein D deletion mutants in mammalian cells.

Glycoprotein D (gD) is a viron envelope component of herpes simplex virus types 1 and 2. We have previously defined seven monoclonal antibody (MAb) groups which recognize distinct epitopes on the mature gD-1 protein of 369 amino acids. MAb groups VII, II, and V recognize continuous epitopes at residues 11-19, 272-279, and 340-356, respectively. MAb groups I, III, IV, and VI recognize discontinuous epitopes. Recent studies have focused on epitopes I, III, and VI. Using truncated forms of gD generated by recombinant DNA methods and proteolysis, epitopes III, IV, and VI were located within amino acids 1-233. A portion of discontinuous epitope I was located in a region within residues 233-275. For this study, we used recombinant DNA methods to create mutations in the gD-1 gene and studied the effects of those mutations on gD as expressed in mammalian cells. Plasmid pRE4, containing the coding sequence of gD-1 and the Rous sarcoma virus long terminal repeat promoter, was transfected into mammalian cells. The expressed protein, gD-1-(pRE4), was identical in size and antigenic properties to gD-1 from infected cells. Six in-frame deletion mutations were subsequently constructed by using restriction enzymes to excise portions of the gD-1 gene. Plasmids carrying these mutated forms were transfected into cells, and the corresponding proteins were examined at 48 h posttransfection for antigenicity and glycosylation patterns. Three deletions of varying size were located downstream of residue 233. Analysis of these mutants showed that amino acids within the region 234-244 were critical for binding of DL11 (group I), but not for other MAb groups. Three other deletion mutants lost all ability to bind MAbs which recognize discontinuous epitopes. In addition, much of the gD expressed by these mutants was observed to migrate as high-molecular-weight aggregated forms in nondenaturing gels. Each of these mutations involved the loss of a cysteine residue, suggesting that disulfide linkages play an essential role in the formation of discontinuous epitopes. The extent of glycosylation of the mutant gD molecules accumulated at 48 h posttransfection suggested altered carbohydrate processing. In one case, there was evidence for increased O-linked glycosylation. Those proteins which had lost a cysteine residue as part of the deletion did not accumulate molecules processed beyond the high-mannose stage. The results suggest that carbohydrate processing during synthesis of gD is very sensitive to alterations in structure, particularly changes involving cysteine residues.     

Molecular basis of the glycoprotein C-negative phenotypes of herpes simplex virus type 1 mutants selected with a virus-neutralizing monoclonal antibody.

Previously (Holland et al., J. Virol. 52:566-574, 1984; Kikuchi et al., J. Virol. 52:806-815, 1984) we described the isolation and partial characterization of over 100 herpes simplex virus type 1 mutants which were resistant to neutralization by a pool of glycoprotein C- (gC) specific monoclonal antibodies. The genetic basis for the inability of several of these gC- mutants to express an immunoreactive envelope form of gC is reported here. Comparative nucleotide sequence analysis of the gC gene of the six mutants gC-3, gC-8, gC-49, gC-53, gC-85, and synLD70, which secrete truncated gC polypeptides, with that of the wild-type KOS 321 gC gene revealed that these mutant phenotypes were caused by frameshift or nonsense mutations, resulting in premature termination of gC translation. Secretion of the gC polypeptide from cells infected with these mutants was due to the lack of a functional transmembrane anchor sequence. The six secretor mutants were tested for suppression of amber mutations in mixed infection with a simian virus 40 amber suppressor vector. Mutant gC-85 was suppressed and produced a wild-type-sized membrane-bound gC. Nucleotide sequence analysis of the six gC deletion mutants gC-5, gC-13, gC-21, gC-39, gC-46, and gC-98 revealed that they carried identical deletions which removed 1,702 base pairs of the gC gene. The deletion, which was internal to the gC gene, removed the entire gC coding sequence and accounted for the novel 1.1-kilobase mRNA previously seen in infections with these mutants. The mutant gC-44 was previously shown to produce a membrane-bound gC protein indistinguishable in molecular weight from wild-type gC. This mutant differed from wild-type virus in that it had reduced reactivity with virus-neutralizing monoclonal antibodies. Nucleotide sequence analysis of the gC gene of mutant gC-44 demonstrated a point mutation which changed amino acid 329 of gC from a serine to a phenylalanine.     

Varicella-zoster virus complements herpes simplex virus type 1 temperature-sensitive mutants.

Varicella-zoster virus (VZV) can complement temperature-sensitive mutants of herpes simplex virus. Of seven mutants tested, two, carrying mutations in the immediate-early ICP4 and ICP27 proteins, were complemented. This complementation was not seen in coinfections with adenovirus type 5 or cytomegalovirus. Following transfection into CV-1 cells, a DNA fragment containing the VZV short repeat sequence complemented the ICP4 mutant. These data demonstrate a functional relationship between VZV and herpes simplex virus and have allowed localization of a putative VZV immediate-early gene.     

Characterization of a herpes simplex virus type 2 75,000-molecular-weight glycoprotein antigenically related to herpes simplex virus type 1 glycoprotein C.

Evidence is presented that the herpes simplex virus type 2 glycoprotein previously designated gF is antigenically related to herpes simplex virus type 1 gC (gC-1). An antiserum prepared against type 1 virion envelope proteins immunoprecipitated gF of type 2 (gF-2), and competition experiments revealed that the anti-gC-1 component of the antiserum was responsible for the anti-gF-2 cross-reactivity. An antiserum prepared against fully denatured purified gF-2, however, and three anti-gF-2 monoclonal antibodies failed to precipitate any type 1 antigen, indicating that the extent of cross-reactivity between gC-1 and gF-2 may be limited. Several aspects of gF-2 synthesis and processing were investigated. Use of the enzymes endo-beta-N-acetylglucosaminidase H and alpha-D-N-acetylgalactosaminyl oligosaccharidase revealed that the fully processed form of gF-2 (about 75,000 [75K] apparent molecular weight) had both complex-type N-linked and O-linked oligosaccharides, whereas newly synthesized forms (67K and 69K) had only high-mannose N-linked oligosaccharides. These last two forms were both reduced in size to 54K by treatment with endo-beta-N-acetylglucosaminidase H and therefore appear to differ only in the number of N-linked chains. Neutralization tests and radioiodination experiments revealed that gF-2 is exposed on the surfaces of virions and that the 75K form of gF-2 is exposed on cell surfaces. The similarities and differences of gF-2 and gC-1 are discussed in light of recent mapping results which suggest collinearity of their respective genes.     

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.     

Genome sequence of herpes simplex virus 1 strain KOS

Herpes simplex virus type 1 (HSV-1) strain KOS has been extensively used in many studies to examine HSV-1 replication, gene expression, and pathogenesis. Notably, strain KOS is known to be less pathogenic than the first sequenced genome of HSV-1, strain 17. To understand the genotypic differences between KOS and other phenotypically distinct strains of HSV-1, we sequenced the viral genome of strain KOS. When comparing strain KOS to strain 17, there are at least 1,024 small nucleotide polymorphisms (SNPs) and 172 insertions/deletions (indels). The polymorphisms observed in the KOS genome will likely provide insights into the genes, their protein products, and the cis elements that regulate the biology of this HSV-1 strain.     

Characterization of baculovirus-expressed herpes simplex virus type 1 glycoprotein K.

The DNA region encoding the complete herpes simplex virus type 1 (HSV-1) glycoprotein K (gK) was inserted into a baculovirus transfer vector, and recombinant viruses expressing gK were isolated. Four gK-related recombinant baculovirus-expressed peptides of 29, 35, 38, and 40 kDa were detected with polyclonal antibody to gK. The 35-, 38-, and 40-kDa species were susceptible to tunicamycin treatment, suggesting that they were glycosylated. The 38- and 40-kDa species corresponded to partially glycosylated precursor gK (pgK) and mature gK, respectively. The 29-kDa peptide probably represented a cleaved, unglycosylated peptide. The 35-kDa peptide probably represented a cleaved, glycosylated peptide that may be a precursor to pgK. Indirect immunofluorescence with polyclonal antibody to gK peptides indicated that the recombinant baculovirus-expressed gK was abundant on the surface of the insect cells in which it was expressed. Mice vaccinated with the baculovirus-expressed gK produced very low levels (< 1:10) of HSV-1 neutralizing antibody. Nonetheless, these mice were partially protected from lethal challenge with HSV-1 (75% survival). This protection was significant (P = 0.02). Despite some protection against death, gK-vaccinated mice showed no protection against the establishment of latency. Surprisingly, gK-vaccinated mice that were challenged ocularly with a stromal disease-producing strain of HSV-1 had significantly higher levels of ocular disease (herpes stromal keratitis) than did mock-vaccinated mice. In summary, this is the first report to show that vaccination with HSV-1 gK can provide protection against lethal HSV-1 challenge and that vaccination with an HSV-1 glycoprotein can significantly increase the severity of HSV-1-induced ocular disease.     

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.     

Genetic analysis of temperature-sensitive mutants which define the gene for the major herpes simplex virus type 1 DNA-binding protein.

We have assigned eight temperature-sensitive mutants of herpes simplex virus type 1 to complementation group 1-1. Members of this group fail to complement mutants in herpes simplex virus type 2 complementation group 2-2. The mutation of one member of group 1-1, tsHA1 of strain mP, has been shown to map in or near the sequence which encodes the major herpes simplex virus type 1 DNA-binding protein (Conley et al., J. Virol. 37:191-206, 1981). The mutations of five other members of group 1-1 map in or near the sequence in which the tsHA1 mutation maps, a sequence which lies near the center of UL between the genes for the viral DNA polymerase and viral glycoprotein gAgB. These mutants can be divided into two groups; the mutations of one group map between coordinates 0.385 and 0.398, and the mutations of the other group map between coordinates 0.398 and 0.413. At the nonpermissive temperature mutants in group 1-1 are viral DNA negative, and mutant-infected cells fail to react with monoclonal antibody to the 130,000-dalton DNA-binding protein. Taken together, these data indicate that mutants in complementation groups 1-1 and 2-2 define the gene for the major herpes simplex virus DNA-binding protein, an early gene product required for viral DNA synthesis.