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.     

Expression and regulation of glycoprotein C gene of herpes simplex virus 1 resident in a clonal L-cell line.

Ltk- cells were transfected with a plasmid containing the entire domain of glycoprotein C (gC), a true gamma or gamma 2 gene of herpes simplex virus 1 (HSV-1) and the methotrexate-resistant mouse dihydrofolate reductase mutant gene. The resulting methotrexate-resistant cell line was cloned; of the 39 clonal lines tested only 1, L3153(28), expressed gC after infection with HSV-1(MP), a gC- mutant, and none expressed gC constitutively. The induction of gC was optimal at multiplicities ranging between 0.5 and 2 PFU per cell, and the quantities produced were equivalent to or higher than those made by methotrexate-resistant gC- L cells infected with wild-type (gC+) virus. The gC gene resident in the L3153(28) cells was regulated as a beta gene inasmuch as the amounts of gC made in infected L3153(28) cells exposed to concentrations of phosphonoacetate that inhibited viral DNA synthesis were higher than those made in the absence of the drug, gC was induced at both permissive and nonpermissive temperatures by the DNA- mutant tsHA1 carrying a lesion in the gene specifying the major DNA-binding protein and which does not express gamma 2 genes at the nonpermissive temperature, and gC was induced only at the permissive temperature in cells infected with ts502 containing a mutation in the alpha 4 gene. The gC induced in L3153(28) cells was made earlier and processed faster to the mature form than that induced in a gC- clone of methotrexate-resistant cells infected with wild-type virus. Unlike virus stocks made in gC- cells, HSV-1(MP) made in L3153(28) cells was susceptible to neutralization by anti-gC monoclonal antibody.     

Blocking immune evasion as a novel approach for prevention and treatment of herpes simplex virus infection

Many microorganisms encode immune evasion molecules to escape host defenses. Herpes simplex virus type 1 glycoprotein gC is an immunoevasin that inhibits complement activation by binding complement C3b. gC is expressed on the virus envelope and infected cell surface, which makes gC potentially accessible to blocking antibodies. Mice passively immunized with gC monoclonal antibodies prior to infection were protected against herpes simplex virus challenge only if the gC antibodies blocked C3b binding. Mice treated 1 or 2 days postinfection with gC monoclonal antibodies that block C3b binding had less severe disease than control mice treated with nonimmune immunoglobulin G (IgG). Mice immunized with gC protein produced antibodies that blocked C3b binding to gC. Immunized mice were significantly protected against challenge by wild-type virus, but not against a gC mutant virus lacking the C3b binding domain, suggesting that protection was mediated by antibodies that target the gC immune evasion domain. IgG and complement from subjects immunized with an experimental herpes simplex virus glycoprotein gD vaccine neutralized far more mutant virus defective in immune evasion than wild-type virus, supporting the importance of immune evasion molecules in reducing vaccine potency. These results suggest that it is possible to block immune evasion domains on herpes simplex virus and that this approach has therapeutic potential and may enhance vaccine efficacy.     

Herpes simplex virus type 1 glycoprotein C-negative mutants exhibit multiple phenotypes, including secretion of truncated glycoproteins

A virus-neutralizing monoclonal antibody specific for glycoprotein C (gC) of herpes simplex virus type 1 strain KOS was used to select a number of neutralization-resistant mutants. A total of 103 of these mutants also were resistant to neutralization by a pool of gC-specific antibodies and thus were operationally defined as gC-. Analysis of mutant-infected cell mRNA showed that a 2.7-kilobase mRNA, comparable in size to the wild-type gC mRNA, was produced by nearly all mutants. However, six mutants, gC-5, gC-13, gC-21, gC-39, gC-46, and gC-98, did not produce the normal-size gC mRNA but rather synthesized a novel 1.1-kilobase RNA species. These mutants had deletions of 1.6 kilobases in the coding sequence of the gC structural gene, which explains their gC- phenotype. Despite the production of an apparently normal mRNA by the remaining 97 mutants, only 7 mutants produced a detectable gC polypeptide. In contrast to wild-type gC, which is a membrane-bound glycoprotein with an apparent molecular weight of 130,000 (130K), five of these mutants quantitatively secreted proteins of lower molecular weight into the culture medium. These were synLD70 (101K), gC-8 (109K), gC-49 (112K), gC-53 (108K), and gC-85 (106K). The mutant gC-3 secreted a protein that was indistinguishable in molecular weight from wild-type KOS gC. Another mutant, gC-44, produced a gC protein which also was indistinguishable from wild-type gC by molecular weight and which remained cell associated. Pulse-labeling of infected cells in the presence and absence of the glycosylation inhibitor tunicamycin demonstrated that these proteins were glycosylated and provided estimates of the molecular weights of the nonglycosylated primary translation products. The smallest of these proteins was produced by synLD70 and was 48K, about two-thirds the size of the wild-type polypeptide precursor (73K). Physical mapping of the mutations in synLD70 and gC-8 by marker rescue placed these mutations in the middle third of the gC coding sequence. Mapping of the mutations in other gC- mutants, including two in which no protein product was detected, also placed these mutations within or very close to the gC gene. The biochemical and genetic data available on mutants secreting gC gene products suggest that secretion is due to the lack of a functional transmembrane anchor sequence on these mutant glycoproteins.     

Antibodies to a synthetic oligopeptide that react with herpes simplex virus type 1 and 2 glycoprotein C

Nucleotide sequence and mRNA localization studies have allowed the prediction of the amino acid sequence of herpes simplex virus type 1 (HSV-1) glycoprotein C (gC). We immunized a rabbit with a conjugate of bovine serum albumin and a synthetic peptide having the same sequence as that deduced for amino acids 128 through 139 of HSV-1 gC. A very similar amino acid sequence has been predicted to exist in the related product, herpes simplex virus type 2 (HSV-2) gC, which was formerly designated gF. Preparations of crude antiserum and immunoaffinity-purified antibodies were obtained and shown to react in enzyme-linked immunosorbent assays with purified HSV-1 gC and HSV-2 gC. Although these antibodies did not detectably immunoprecipitate proteins from radiolabeled infected cell extracts, they reacted with HSV-1 gC and HSV-2 gC that were electrophoretically transferred to nitrocellulose membranes from polyacrylamide gels. These results confirm that HSV-1 gC and HSV-2 gC are immunologically related and also define a specific portion of HSV-1 gC that is conserved.     

Use of monoclonal antibodies against two 75,000-molecular-weight glycoproteins specified by herpes simplex virus type 2 in glycoprotein identification and gene mapping

We produced two monoclonal antibodies that precipitate different glycoproteins of similar apparent molecular weight (70,000 to 80,000) from extracts of cells infected with herpes simplex virus type 2. Evidence is presented that one of these glycoproteins is the previously characterized glycoprotein gE, whereas the other maps to a region of the herpes simplex virus type 2 genome collinear with the region in herpes simplex virus type 1 DNA that encodes gC.     

Identification of herpes simplex virus type 1 (HSV-1) glycoprotein gC as the immunodominant antigen for HSV-1-specific memory cytotoxic T lymphocytes

The frequency and fine specificity of herpes simplex virus (HSV)-reactive cytotoxic T lymphocytes (CTL) of C57BL/6 mice was investigated in limiting dilution culture. The reactivity patterns of virus-specific CTL were assayed on target cells infected with HSV type 1, strain KOS, HSV type 2, strain Mueller, and mutants of HSV-1 (KOS) antigenically deficient or altered in glycoproteins gC or gB, two of the four major HSV-1-encoded cell surface glycoprotein antigens. Most CTL clones recognized type-specific determinants on target cells infected with the immunizing HSV serotype. In addition, the majority of HSV-1-specific CTL did not cross-react with cells infected with syn LD70, a mutant of HSV-1 (KOS) deficient for the presentation of cell surface glycoprotein gC. These data are the first demonstration of the clonal specificity of HSV-1-reactive CTL, and they identify gC as the immunodominant antigen. The fine specificity of gC-specific CTL clones was analyzed on target cells infected with mutant viruses altered in the antigenic structure of gC. These mutants were selected by resistance to neutralization with monoclonal antibodies, referred to as monoclonal antibody-resistant (mar) mutants. Most mar mutations in gC did not affect recognition by the majority of CTL clones. This indicated that most epitopes recognized by CTL are distinct from those defined by antibodies. The finding, however, that one mar mutation in gC affected both CTL and antibody recognition of this antigen may help to define antigenic sites important to both humoral and cell-mediated immunity to herpesvirus infection.     

Pathogenicity of Glycoprotein C-Deficient Herpes Simplex Virus 1 Strain TN-1 Which Encodes Truncated Glycoprotein C

A clinical isolate of herpes simplex virus 1 (TN-1) from a stromal keratitis patient was found to be defective in the glycoprotein C (gC) gene (UL44), thus resulting in the production of truncated gC upon infection. To study the pathogenetic role of truncated gC, we prepared a recombinant LTN-8 derived from TN-1 with deletions of the 1.5 kilobase pairs of the gC gene including the initiation codon. A penetration assay revealed LTN-8 to be less efficient in its penetration ability than TN-1, the laboratory strain KOS and RTN-1-20-3, a recombinant derived from TN-1 with the KOS gC gene. The penetration of LTN-8 was facilitated by the addition of TN-1-infected culture medium. TN-1 virus preparations had no hemagglutinating activity. However, the animals infected with TN-1 did develop hemagglutination inhibition (HI) antibodies. The LTN-8-infected animals did not develop HI antibodies. The pathogenicity in BALB/c mice following either corneal, intraperitoneal or intracerebral inoculation did not significantly differ among TN-1, RTN-1-20-3 or LTN-8. Our results indicate that truncated gC was sufficient for the induction of HI antibodies and was also able to facilitate penetration in vitro. Although truncated gC might be a virulence factor acting as a decoy, both truncated gC and intact gC had little effect on the outcome following intracerebral, intraperitoneal or corneal inoculation.     

Antigenic variants of herpes simplex virus selected with glycoprotein-specific monoclonal antibodies.

Monoclonal antibodies specific for herpes simplex virus type 1 (HSV-1) glycoproteins were used to demonstrate that HSV undergoes mutagen-induced and spontaneous antigenic variation. Hybridomas were produced by polyethylene glycol-mediated fusion of P3-X63-Ag8.653 myeloma cells with spleen cells from BALB/c mice infected with HSV-1 (strain KOS). Hybrid clones were screened for production of HSV-specific neutralizing antibody. The glycoprotein specificities of the antibodies were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates of radiolabeled infected-cell extracts. Seven hybridomas producing antibodies specific for gC, one for gB, and one for gD were characterized. All antibodies neutralized HSV-1 but not HSV-2. Two antibodies, one specific for gB and one specific for gC, were used to select viral variants resistant to neutralization by monoclonal antibody plus complement. Selections were made from untreated and bromodeoxyuridine- and nitrosoguanidine-mutagenized stocks of a plaque-purified isolate of strain KOS. After neutralization with monoclonal antibody plus complement, surviving virus was plaque purified by plating at limiting dilution and tested for resistance to neutralization with the selecting antibody. The frequency of neutralization-resistant antigenic variants selected with monoclonal antibody ranged from 4 X 10(-4) in nonmutagenized stocks to 1 X 10(-2) in mutagenized stocks. Four gC and four gB antigenic variants were isolated. Two variants resistant to neutralization by gC-specific antibodies failed to express gC, accounting for their resistant phenotype. The two other gC antigenic variants and the four gB variants expressed antigenically altered glycoproteins and were designated monoclonal-antibody-resistant, mar, mutants. The two mar C mutants were tested for resistance to neutralization with a panel of seven gC-specific monoclonal antibodies. The resulting patterns of resistance provided evidence for at least two antigenic sites on glycoprotein gC.     

An ICP6::lacZ insertional mutagen is used to demonstrate that the UL52 gene of herpes simplex virus type 1 is required for virus growth and DNA synthesis.

An insertional mutagen was developed which consists of the lacZ gene of Escherichia coli under the control of the regulatory elements of the large subunit of ribonucleotide reductase (ICP6). This ICP6::lacZ cassette was used to create a mutation in a gene designated UL52 (D. J. McGeoch, M. A. Dalrymple, A. Dolan, D. McNab, L. J. Perry, P. Taylor, and M. D. Challberg, J. Virol. 62:444-453, 1988), which is predicted to encode a 114,000-molecular-weight protein. To isolate and propagate this mutant, we generated a cell line, BL-1, by cotransfection of Vero cells with pSV2neo and a plasmid containing the herpes simplex virus type 1 KOS strain BamHI L fragment (coordinates 0.708 to 0.745). An ICP6::lacZ insertion mutant, hr114, was capable of growing in BL-1 cells but not in normal Vero cells. In addition, hr114 was defective in the synthesis of viral DNA and late proteins; however, this mutant appeared to exhibit normal early gene expression. Thus, the results presented in this report show that the UL52 gene product is required for viral DNA synthesis. Furthermore, our studies indicate that the ICP6::lacZ cassette will provide a useful tool for obtaining mutants of other herpes simplex virus genes.     

Functional expression of a cloned herpes simplex virus type 1 DNA polymerase gene.

A vector which expresses the herpes simplex virus type 1 (HSV-1) (strain 17) DNA polymerase gene was constructed by ligating two separately cloned HSV DNA restriction fragments into an intermediate plasmid and then mobilizing the intact polymerase gene-encoding sequence into a pSV2 derivative. The expression vector (pD7) contains a functional simian virus 40 replication origin and early enhancer-promoter upstream from the HSV DNA polymerase-encoding sequence. COS-1 cells transfected with pD7 contained an RNA species, shown by Northern blot analysis to hybridize specifically with an HSV DNA pol probe and to be the same size (4.3 kilobases) as the pol mRNA found in HSV-1-infected COS-1 cells. A genetic complementation test was used to establish that pD7 expresses a functional pol gene product. COS-1 cells transfected with pD7 were able to partially complement the growth defect of an HSV-1 (KOS) temperature-sensitive mutant, tsC7, in the DNA polymerase gene at the nonpermissive temperature.     

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)     

Biochemical characterization of peptides from herpes simplex virus glycoprotein gC: loss of CNBr fragments from the carboxy terminus of truncated, secreted gC molecules.

A biochemical characterization of peptides from herpes simplex virus type 1 glycoprotein gC was carried out. We utilized simple micromethods, based on immunological isolation of biosynthetically radiolabeled gC, to obtain gC in pure form for biochemical study. CNBr fragments of gC were prepared, isolated, and characterized. These CNBr fragments were resolved into six peaks by chromatography on Sephacryl S-200 in 6 M guanidine hydrochloride. Only three of the CNBr fragments contained carbohydrate side chains, as judged from the incorporation of [14C]glucosamine. Radiochemical microsequence analyses were carried out on the gC molecule and on each of the CNBr fragments of gC. A comparison of this amino acid sequence data with the amino acid sequence predicted from the DNA sequence of the gC gene showed that the first 25 residues of the predicted sequence are not present in the gC molecule isolated from infected cells and allowed alignment of the CNBr fragments in the gC molecule. Glycoprotein gC was also examined from three gC mutants, synLD70, gC-8, and gC-49. These mutants lack an immunoreactive envelope form of gC but produce a secreted, truncated gC gene product. Glycoprotein gC from cells infected with any of these gC- mutants was shown to have lost more than one CNBr fragment present in the wild-type gC molecule. The missing fragments included the one containing the putative transmembrane anchor sequence. Glycoprotein gC from the gC-8 mutant was also shown, by tryptic peptide map analysis, to have lost more than five major arginine-labeled tryptic peptides arginine-labeled tryptic peptides present in the wild-type gC molecule and to have gained a lysine-labeled tryptic peptide not present in wild-type gC.