Complement component C3b binds directly to purified glycoprotein C of herpes simplex virus types 1 and 2

Cells infected with herpes simplex virus type 1 (HSV-1), but not HSV-2, express on their surfaces a receptor for the complement component C3b. Receptor activity is markedly enhanced by treatment of the infected cells with neuraminidase. Employing a direct binding assay, consisting of purified HSV glycoproteins immobilized on nitrocellulose and iodinated C3b as a probe, we found that C3b binds directly to gC-1, as well as to gC-2, but not to gB or gD from either serotype. C3b binding was enhanced by treatment of gC-1 or gC-2 with neuraminidase. Endo F or endo H treatment of gC-1 had no effect on C3b binding. However, treatment of gC-2 with these endoglycosidases had a marked negative effect on C3b binding. These results suggest that N-linked oligosaccharides are involved in binding of C3b to gC-2, but not gC-1. Alternatively, removal of N-linked oligosaccharides from gC-2 might adversely affect polypeptide conformation. Glycoprotein C-2 also differs from gC-1 in its effects on the complement cascade. Whereas gC-1 accelerated the decay of the alternative pathway C3 convertase and impaired the efficiency of lysis by the components C5 through C9, gC-2 stabilized the active C3 convertase and had little effect on the late-acting components. The dissimilarity of gC-1 and gC-2 with regard to their effects on the complement cascade may have implications regarding the role of these glycoproteins in confronting the host immune response.     

Direct binding of herpes simplex virus type 1 virions to complement C3

Glycoprotein C (gC) of type 1 herpes simplex virus (HSV-1) binds the human complement C3 as purified proteins, or when expressed on the surface of infected cells. However, it is not clear whether the purified HSV virion binds directly to C3. In this study, direct binding of purified virions, HSV-1(KOS) or HSV-1(hrR3), to C3-coated plate was demonstrated by an enzyme-linked immunosorbent assay (ELISA). Captured virions on C3-coated plates were still infectious as determined by adding Vero cells to allow for infection to occur. The binding of virions to C3 was abolished if C3 was heat-inactivated, confirming a requirement for complement. In addition, the interaction was inhibited by preincubation of purified virions with heparin. In conclusion, a direct interaction of C3 with the HSV-1 virions was demonstrated.     

Factors influencing the interaction of herpes simplex virus glycoprotein C with the third component of complement

Summary The factors influencing the interaction of herpes simplex virus (HSV) glycoprotein C (gC) with the third component of complement (C3) were investigated in this study. The ability of gC of HSV type 1 (gC-1) to bind to the C3b fragment of C3 was found to be influenced by cell specific processing of gC-1 in a different manner, binding being remarkably enhanced in some cell lines following removal of sialic acid residues. Testing several intertypic recombinants of HSV we found that only strains expressing gC-1 exhibited binding to C3b, even though their genome consisted mainly of HSV-2 sequences in some recombinants. Expression of type-2 glycoproteins gB, gD, gE, gG, gH, and gI did not alter the ability of gC-1 to bind to C3b. Rosetting of HSV-1 infected Vero cells with C3b-coated red blood cells (EAC) was found to be temperature dependent and could be inhibited with purified C3b and anti-C3 antibodies. Polyanions like heparin or dextran sulfate were also inhibitory in a dose dependent manner, whereas C3d, neomycin and other aminoglycoside antibiotics failed to block. As the tested polyanions are also known to inhibit the infectivity of HSV, it could be speculated, that the complement binding function and the heparin-binding/attachment function of gC might be related.     

Human antibodies to herpes simplex virus type 1 glycoprotein C are neutralizing and target the heparan sulfate-binding domain

Human antibodies specific for glycoprotein C (gC1) of herpes simplex virus type 1 (HSV-1) neutralized the virus infectivity and efficiently inhibited attachment of HSV-1 to human HaCaT keratinocytes and to murine mutant L cells expressing either heparan sulfate or chondroitin sulfate at the cell surface. Similar activities were observed with anti-gC1 monoclonal antibody B1C1. In addition to HaCaT and L cells, B1C1 antibody neutralized HSV-1 infectivity in simian GMK AH1 cells mildly pre-treated with heparinase III. Human anti-gC1 antibodies efficiently competed with the binding of gC1 to B1C1 antibody whose epitope overlaps a part of the attachment domain of gC1. Human anti-gC1 and B1C1 antibodies extended survival time of mice experimentally infected with HSV-1. We conclude that in HaCaT cells and in cell systems showing restricted expression of glycosaminoglycans, human and some monoclonal anti-gC1 antibodies can target the cell-binding domain of this protein and neutralize viral infectivity.     

A role for heparan sulfate 3-O-sulfotransferase isoform 2 in herpes simplex virus type 1 entry and spread

Heparan sulfate (HS) 3-O-sulfotransferase isoform-2 (3-OST-2), which belongs to a family of enzymes capable of generating herpes simplex virus type-1 (HSV-1) entry and spread receptors, is predominantly expressed in human brain. Despite its unique expression pattern, the ability of 3-OST-2 to mediate HSV-1 entry and cell-to-cell fusion is not known. Our results demonstrate that expression of 3-OST-2 can render Chinese hamster ovary K1 (CHO-K1) cells susceptible to entry of wild-type and mutant strains of HSV-1. Evidence for generation of gD receptors by 3-OST-2 were suggested by gD-mediated interference assay and the ability of 3-OST-2-expressing CHO-K1 cells to preferentially bind HSV-1 gD, which could be reversed by prior treatment of cells with HS lyases (heparinases II/III). In addition, 3-OST-2-expressing CHO-K1 cells acquired the ability to fuse with cells-expressing HSV-1 glycoproteins, a phenomenon that mimics a way of viral spread in vivo. Demonstrating specificity, the cell fusion was inhibited by soluble 3-O-sulfated forms of HS, but not unmodified HS. Taken together, our results raise the possibility of a role of 3-OST-2 in the spread of HSV-1 infection in the brain.     

Typing of herpes simplex virus types 1 and 2 by immunoblotting analysis using polyclonal antisera to herpes simplex virus glycoproteins

Herpes simplex virus (HSV) isolates were differentiated by immunoblotting analysis using a mixture of polyclonal antisera directed against HSV type 1 (HSV-1) and HSV type 2 (HSV-2) glycoprotein fractions (gB/gC of HSV-1 and gC/gE/84-kDa protein of HSV-2), since the mixed antisera recognized viral polypeptides with different molecular weights in HSV-1- and HSV-2-infected cells. Results of typing by immunoblotting analysis were consistent with those obtained by restriction endonuclease analysis of DNAs extracted from 10 HSV isolates. These results suggest that the immunoblotting technique will be applicable to reliable typing of HSV isolates using polyclonal antisera showing the difference in reaction patterns between HSV-1- and HSV-2-infected cells.     

Production, binding characteristics, and immunogenicity of heterologous anti-idiotypic antibody to herpes simplex virus glycoprotein C

A monoclonal antibody specific for glycoprotein C (gC) of herpes simplex virus type 1 (HSV-1) was used to prepare a heterologous anti-idiotypic antibody in rabbits. After absorption with normal mouse immunoglobulin (NMS) the anti-idiotypic (anti-id) antibody retained binding activity for MoAb D4.1, the immunogen. The anti-id (anti-id C) also demonstrated a cross-reactive binding activity, as shown by ELISA, for MoAb D4.2 and MoAb D4.8 which was specific for glycoprotein D (gD) and glycoprotein B (gB) of HSV-1, respectively. Also, anti-id C bound to and eluted from MoAb D4.2 and MoAb D4.8 affinity columns retained the ability to bind all three monoclonal antibodies. This cross-reactive anti-id could inhibit the binding of each of the three monoclonal antibodies to their respective proteins, suggesting an antigen combining site specificity. Subsequently, the idiotope on MoAb D4.8 was shown to be outside the antigen combining site, since anti-id C recognized MoAb D4.8 complexed with gB. The anti-id, however, did not bind MoAb D4.1 or MoAb D4.2, if these monoclonals were bound to gC or gD, respectively, suggesting the cross-reactive determinant was paratopic on those two monoclonals. Immunization of mice with anti-id C could prime splenocytes in vivo to proliferate in response to HSV antigen stimulation in vitro. Thus, spleen cells involved in the HSV immune response in vitro recognized the anti-idiotypic antibody in vivo.     

放射免疫沉淀试验鉴定单纯疱疹病毒糖蛋白C

用放射免疫沉淀试验和单纯疱疹病毒1型、2型(HSV—1/HSV-2)型间重组株作物理谱图的方法.鉴定出抗HSV的单克隆抗体(McAb) 1A12、Mad-2、2D11、CM-D3、2A8和1C_4的靶抗原是一组分子量在105～130Ka之间的糖蛋白,其编码基因定位于长单一序列(UL)上,在0.536～0.682遗传单位之间,与gC的编码基因部分重叠。故认为这些McAbs的靶抗原为gC,其中,Mad-2和CM-D3分别为HSV-1和HSV-2型特异性,其靶抗原分别为gC-1和gC-2。ELISA阻断试验结果表明,4株型共同性McAbs 1A12、2D11、2A8和1C4.抗gC上4个独立的抗原位点。     

A single amino acid substitution in the cytoplasmic tail of the glycoprotein B of herpes simplex virus 1 affects both syncytium formation and binding to intracellular heparan sulfate

Herpes simplex virus 1 (HSV-1) (S) is a spontaneous syncytial mutant derived from the prototype HSV-1(F) after extensive plaque purification, and produces large syncytial plaques on Vero cells. Marker transfer experiments and DNA sequence analysis mapped the syncytial phenotype to a T-C base substitution at codon 787 of the cytoplasmic domain of mature gB, that results in Leu to Pro substitution and consequently belongs to the syn 3 locus. Both the cytoplasmic and the extracellular domains of gB are active in the fusion event since the addition of anti-gB monoclonal antibodies that recognize the extracellular domain of gB prevent HSV-1(S) induced cell fusion. Similarly, gD also participates in cell fusion since addition of anti-gD monoclonal antibodies also prevent HSV-1(S) induced cell fusion. Furthermore the glycoproteins B and D formed complexes in cells infected with mutant or wild type viruses. The amount of gB bound to total heparan sulfate is lower in the mutant than in the wild type strain. This difference becomes particularly profound when gB is associated with a portion of heparan sulfate intercalated to the membranes. The discrepancy in the binding of the mutant and wild type gB to heparan sulfate may be related to the mechanism of cell fusion induced by HSV-1(S).     

Characterization of glycoprotein C of HSZP strain of herpes simplex virus 1

Sequences of UL44 genes of strains HSZP, KOS and 17 of herpes simplex virus 1 (HSV-1) were determined and the amino acid sequences of corresponding glycoproteins (gC) were deduced. In comparison with the 17 strain, the HSZP strain showed specific changes in 3 nucleotides and in 2 amino acids (aa 139 and 147, both from Arg to Trp) in the antigenic locus LII. The change at aa 147 was situated within the GAG-binding epitope. In a similar comparison, KOS strain had changes in 3 nucleotides and 3 amino acids (aa 3, 14, and 300). The UL44 genes of HSZP and KOS strains were expressed in insect Sf-21 cells by means of the baculovirus (Bac-to-Bac) expression system. As shown by immunoblot analysis, both the recombinant baculoviruses (B1-HSZP and B6-KOS) expressed a glycosylated gC, the M(r) of which (116 K) was lower than that of gC synthesized in Vero cells (129 K) infected with strains HSZP or KOS. In addition, smaller gC-specific proteins (of apparent M(r) of 50-58 K and 98 K) corresponding to a non-glycosylated precursor polypeptide and/or incomplete forms of the partially glycosylated gC were found. When Balb/c mice were immunized with Sf-21 cells expressing gC, the recombinant gC-HSZP represented a more efficient immunogen possibly due to its stronger expression in these cells. The corresponding gC-HSZP antiserum reacted in enzyme-linked immunosorbent assay (ELISA) equally well with HSZP and KOS virion antigens and neutralized HSZP strain at a low titer. Both gC-HSZP and gC-KOS antisera detected the homologous as well as the heterologous gC antigens in Vero cells regardless whether infected with strains HSZP, KOS or 17, revealing the presence of gC from 6 to 16 hrs post infection (p.i.) in the cytoplasm, on the nuclear membrane and at the cell surface.     

Serological analysis of herpes simplex virus types 1 and 2 with monoclonal antibodies.

A panel of monoclonal antibodies to herpes simplex virus glycoproteins was used for serological analysis of 130 strains. Based on specific immunological determinants, strains of each serotype clustered into subgroups. Monoclonal antibodies were suitable reagents for serotyping and have potential application to epidemiology of herpes simplex virus infections.     

Detection of type-specific antibody to herpes simplex virus type 1 by radioimmunoassay with herpes simplex virus type 1 glycoprotein C purified with monoclonal antibody.

Herpes simplex virus type 1 (HSV-1) and HSV-2 specify at least four glycoproteins designated gA/gB, gC, gD, and gE. Previous studies have shown that gC produced by HSV-1 is antigenically distinct from the corresponding HSV-2 glycoprotein. With the exception of gC, the glycoproteins of both serotypes share antigenic sites. Standard serological assays fail to differentiate the antibody to the shared antigenic determinants from the type-specific antibody. In this paper, we describe a procedure for purifying gC from HSV-1-infected cell extracts with an immunoadsorbent prepared with an HCL monoclonal antibody. When used in a solid-phase radioimmunoassay, gC proved to be a type-specific antigen for quantitation of antibody to HSV-1. Among individuals who had no antibody to HSV at the onset of infection, all of those with primary HSV-1 infection developed antibody to gC. Subjects with primary HSV-2 infection failed to develop antibody reactive with gC of HSV-1 (P less than 0.01). Both immunoglobulin G and M antibodies against gC were detected in sera from subjects with either primary or recurrent HSV-1 infection. Higher antibody titers to gC were found in sera from individuals with recurrent infection than in sera from those with primary HSV-1 infection.     

Defective glycoprotein C of the syncytial strain of herpes simplex virus type 1

Glycoproteins of the herpes simplex virus type 1 (HSV-1) non-syncytial strain Kupka showed two times higher affinity for the Helix pomatia (HP) lectin than glycoproteins of the syncytial strain HSZP. As revealed by the results of immunoprecipitation test and by the comparison of the synthesis of virus glycoproteins, glycoprotein C (gC) was synthesized in sufficient amounts in the HSZP strain-infected cells. The difference in the binding to HP lectin was found to be caused by decreased affinity of gC of the strain HSZP for HP lectin. The decreased affinity for HP lectin was brought about by lower proportion of O-linked oligosaccharides in the gC molecule. The glycoproteins of either HSV-1 strain under study synthesized in BHK cells displayed higher affinity for HP lectin than those synthesized in VERO cells.     

Type-common and type-specific monoclonal antibodies to herpes simplex virus types 1 and 2.

Seventeen monoclonal antibodies (Mabs) reacting specifically with the cells infected with herpes simplex viruses type 1 (HSV-1) and type 2 (HSV-2) were characterized by a variety of immunological tests such as radioimmunoprecipitation, immunoblotting and virus-neutralization. The majority of Mabs was directed against glycoprotein B (anti-gB), six reacted with glycoprotein C (anti-gC) and one with glycoprotein G (anti-gG). Six anti-gB Mabs reacted with both types of HSV (anti-gB-1,2), two anti-gB and all the six anti-gC Mabs have been specific for HSV-1 (anti-gB-1 and anti-gC-1). The remaining two anti-gB Mabs and the anti-gG have been specific to HSV-2 (anti-gB-2 and anti-gG). Only three out of the seventeen examined Mabs neutralized the virus.     

Comparison of foldback sequences of herpes simplex virus types 1 and 2 DNA

The DNAs of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) were separately denatured and allowed to renature briefly. The intrastand foldback structures that resulted from base pairing of inverted repeated sequences on otherwise single-stranded (ss) DNA were visualized in the electron microscope. The two genomes were found to contain similar size classes of small duplex stem DNA sequences. However, HSV-2 DNA appeared to possess an additional, larger size class of foldback structures not found on HSV-1 DNA. Both HSV DNAs were found to contain stem-plus-loop structures; the larger stem-plus-loop structures of the two genomes had similar stem lengths but dissimilar loop lengths. Thus, a comparison of the genomes of HSV-1 and HSV-2 showed that they possessed similar size classes of foldback sequences.     

Discrimination of antibody to herpes B virus from antibody to herpes simplex virus types 1 and 2 in human and macaque sera

The antigenic cross-reactive characteristics of herpes B virus and herpes simplex virus (HSV) type 1 (HSV-1) and HSV-2 are responsible for false-positive diagnoses by serological assays in humans and macaques. In the present study, we developed a fluorometric indirect enzyme-linked immunosorbent assay (ELISA) with recombinant herpes B virus glycoprotein D (gD) and HSV-1 and HSV-2 gG (gG-1 and gG-2, respectively) to discriminate between the three primate herpesvirus infections. The secreted form of gD, gDdTM, was used to detect antibody to herpes B virus gD. Sera positive for herpes B virus, HSV-1, and HSV-2 showed specific reactions to gD, gG-1, and gG-2, respectively. Sera collected from humans and rhesus macaques were investigated for the presence of antibodies to the recombinant proteins of the three herpesviruses. The results suggested that the approach is able to discriminate between herpes B virus and HSV infections. The ELISA was also found to be able to detect infections with multiple primate herpesviruses and may have the potential to identify a subsequent infection in individuals that have already been infected with another herpesvirus. In addition, we found evidence of a greater cross-reactivity of herpes B virus with HSV-1 than with HSV-2. It is suggested that the ELISA with the recombinant antigens is useful not only for the serodiagnosis of primate herpesvirus infections but also for elucidation of the seroprevalence of herpesviruses in humans and primates.     

A related epitope is consistently present on glycoprotein C of herpes simplex virus type 1 and 2.

A monoclonal antibody (VE8) directed to glycoprotein C of herpes simplex virus type 1 (HSV-1) cross-reacted with all HSV type 2 (HSV-2) strains tested. Positive reaction was also observed with all investigated HSV-1 strains, indicating that the related epitope is consistently present in HSV-1 and HSV-2.