Heparan Sulfate Proteoglycan Binding by Herpes Simplex Virus Type 1 Glycoproteins B and C, Which Differ in Their Contributions to Virus Attachment, Penetration, and Cell-to-Cell Spread

Herpes simplex virus type 1 (HSV-1) mutants defective for envelope glycoprotein C (gC) and gB are highly impaired in the ability to attach to cell surface heparan sulfate (HS) moieties of proteoglycans, the initial virus receptor. Here we report studies aimed at defining the HS binding element of HSV-1 (strain KOS) gB and determining whether this structure is functionally independent of gB’s role in extracellular virus penetration or intercellular virus spread. A mutant form of gB deleted for a putative HS binding lysine-rich (pK) sequence (residues 68 to 76) was transiently expressed in Vero cells and shown to be processed normally, leading to exposure on the cell surface. Solubilized gBpK⁻ also had substantially lower affinity for heparin-acrylic beads than did wild-type gB, confirming that the HS binding domain had been inactivated. The gBpK⁻ gene was used to rescue a KOS gB null mutant virus to produce the replication-competent mutant KgBpK⁻. Compared with wild-type virus, KgBpK⁻ showed reduced binding to mouse L cells (ca. 20%), while a gC null mutant virus in which the gC coding sequence was replaced by the lacZ gene (KCZ) was substantially more impaired (ca. 65%-reduced binding), indicating that the contribution of gC to HS binding was greater than that of gB. The effect of combining both mutations into a single virus (KgBpK⁻gC⁻) was additive (ca. 80%-reduced binding to HS) and displayed a binding activity similar to that observed for KOS virus attachment to sog9 cells, a glycosaminoglycan-deficient L-cell line. Cell-adsorbed individual and double HS mutant viruses exhibited a lower rate of virus entry following attachment, suggesting that HS binding plays a role in the process of virus penetration. Moreover, the KgBpK⁻ mutant virus produced small plaques on Vero cells in the presence of neutralizing antibody where plaque formation depended on cell-to-cell virus spread. These studies permitted the following conclusions: (i) the pK sequence is not essential for gB processing or function in virus infection, (ii) the lysine-rich sequence of gB is responsible for HS binding, and (iii) binding to HS is cooperatively linked to the process of efficient virus entry and lateral spread but is not absolutely required for virus infectivity.     

Interaction of herpes simplex virus glycoprotein gC with mammalian cell surface molecules.

The entry of herpes simplex virus (HSV) into mammalian cells is a multistep process beginning with an attachment step involving glycoproteins gC and gB. A second step requires the interaction of glycoprotein gD with a cell surface molecule. We explored the interaction between gC and the cell surface by using purified proteins in the absence of detergent. Truncated forms of gC and gD, gC1(457t), gC2(426t), and gD1(306t), lacking the transmembrane and carboxyl regions were expressed in the baculovirus system. We studied the ability of these proteins to bind to mammalian cells, to bind to immobilized heparin, to block HSV type 1 (HSV-1) attachment to cells, and to inhibit plaque formation by HSV-1. Each of these gC proteins bound to conformation-dependent monoclonal antibodies and to human complement component C3b, indicating that they maintained the same conformation of gC proteins expressed in mammalian cells. Biotinylated gC1(457t) and gC2(426t) each bind to several cell lines. Binding was inhibited by an excess of unlabeled gC but not by gD, indicating specificity. The attachment of gC to cells involves primarily heparan sulfate proteoglycans, since heparitinase treatment of cells reduced gC binding by 50% but had no effect on gD binding. Moreover, binding of gC to two heparan sulfate-deficient L-cell lines, gro2C and sog9, both of which are mostly resistant to HSV infection, was markedly reduced. Purified gD1 (306t), however, bound equally well to the two mutant cell lines. In contrast, saturating amounts of gC1(457t) interfered with HSV-1 attachment to cells but failed to block plaque formation, suggesting a role for gC in attachment but not penetration. A mutant form of gC lacking residues 33 to 123, gC1(delta 33-123t), expressed in the baculovirus system, bound significantly less well to cells than did gC1(457t) and competed poorly with biotinylated gC1(457t) for binding. These results suggest that residues 33 to 123 are important for gC attachment to cells. In contrast, both the mutant and wild-type forms of gC bound to immobilized heparin, indicating that binding of these proteins to the cell surface involves more than a simple interaction with heparin. To determine that the contribution of the N-terminal region of gC is important for HSV attachment, we compared several properties of a mutant HSV-1 which contains gC lacking amino acids 33 to 123 to those of its parental virus, which contains full-length gC. The mutant bound less well to cells than the parental virus but exhibited normal growth properties.(ABSTRACT TRUNCATED AT 400 WORDS)     

Novel cell adhesive glycosaminoglycan-binding proteins of Japanese encephalitis virus

Glycosaminoglycans (GAGs) are present in the extracellular matrix and/or tissue cell surface and, by binding to specified GAG-binding proteins, control many important cellular functions. Some animal viruses had evolved to use GAGs as part of their strategy to invade host cells. In this study, two putative GAG-binding proteins were identified from the E protein sequence of the live-attenuated strain CH2195LA of Japanese encephalitis virus (JEV): (i) the first GAG-binding region at residues from E-279 to E-297 (279KLTSGHLKCRLKMDKLALK297) and (ii) the second GAG-binding region at residues from E-397 to E-416 (397KAGSTLGKAFFSTTLKGAQR416). Four recombinant proteins with or without these two GAG-binding regions were expressed in Escherichia coli and purified to examine their GAG-binding properties. The first GAG binding region was demonstrated to exhibit a higher affinity in heparin-Sepharase column. Dose-dependent increases of BHK-21 cell binding were also demonstrated by cell binding enzyme-linked immunosorbent assay (ELISA). Immobilized on glass coverslips, the GAG-binding recombinant protein of JEV promoted BHK-21 cell adhesion and proliferation. The present studies demonstrate the recombinant GAG-binding proteins of JEV stimulate cell adhesive and proliferation with a potential for applications in tissue engineering.     

Recombinant Herpes Simplex Virus Type 1 Engineered for Targeted Binding to Erythropoietin Receptor-Bearing Cells

The utility of recombinant herpes simplex virus type 1 (HSV-1) vectors may be expanded by manipulation of the virus envelope to achieve cell-specific gene delivery. To this end, an HSV-1 mutant virus deleted for glycoprotein C (gC) and the heparan sulfate binding domain of gB (KgBpK⁻gC⁻) was engineered to encode different chimeric proteins composed of N-terminally truncated forms of gC and the full-length erythropoietin hormone (EPO). Biochemical analyses demonstrated that one gC-EPO chimeric molecule (gCEPO₂) was posttranslationally processed, incorporated into recombinant HSV-1 virus (KgBpK⁻gCEPO₂), and neutralized with antibodies directed against gC or EPO in a complement-dependent manner. Moreover, KgBpK⁻gCEPO₂ recombinant virus was specifically retained on a soluble EPO receptor column, was neutralized by soluble EPO receptor, and stimulated proliferation of FD-EPO cells, an EPO growth-dependent cell line. FD-EPO cells were nevertheless refractory to productive infection by both wild-type HSV-1 and recombinant KgBpK⁻gCEPO₂ virus. Transmission electron microscopy of FD-EPO cells infected with KgBpK⁻gCEPO₂ showed virus endocytosis leading to aborted infection. Despite the lack of productive infection, these data provide the first evidence of targeted HSV-1 binding to a non-HSV-1 cell surface receptor.     

Binding of complement component C3b to glycoprotein C is modulated by sialic acid on herpes simplex virus type 1-infected cells.

Neuraminidase treatment of cells infected with herpes simplex virus type 1 (HSV-1) markedly enhanced the binding of complement component C3b to HSV 1 glycoprotein C (gC). When HSV-1 was grown in BHK RicR14 cells in which glycoproteins had reduced amounts of N-linked complex oligosaccharides, including sialic acid, the binding of C3b to gC was markedly enhanced. We used neuraminidase treatment to demonstrate that cloning the gC gene from the HSV-1 F strain into an HSV-1 mutant which fails to express gC converted the mutant virus from C3b receptor negative to receptor positive. These results further support a role for gC as a C3b receptor and indicate that sialic acid modifies receptor activity.     

Glycosaminoglycans are not indispensable for the anti-herpes simplex virus type 2 activity of lactoferrin

Lactoferrin has been recognized as a potent inhibitor of human herpetic viruses, such as herpes simplex type 1 (HSV-1) and 2 (HSV-2). In particular, bovine lactoferrin (bLf) has been found to prevent viral infection by binding to heparan sulphate (HS) glycosaminoglycans (GAGs) that in turn can act as cell receptors for human herpetic viruses. In this study we further investigate the mechanism of inhibiting activity of both human lactoferrin (hLf) and bLf against HSV-2. The antiviral effect of these proteins towards HSV-2 strain 333 and its glycoprotein C (gC)-truncated derivative HSV-2 gC-neg1 has been tested in monkey kidney cells. Our results indicate that the antiviral activity of bLf does not involve gC-HS interaction as there was no difference in its effectiveness towards wild type and mutant virus. As regards hLf, the mutant virus HSV-2 gC-neg1 was more sensitive compared to the wild type, suggesting that the human protein might interact with some viral structures that in wild-type viruses are masked by gC. When the modulation of HSV-2 infection by bLf and hLf was investigated under different experimental conditions, the bovine protein proved more effective than the human protein. Moreover, we found that, differently from what observed with HSV-1, bLf inhibited HSV-2 plaque-forming activity also in cells devoid of GAG expression. These results suggest that bLf may block a virus receptor of non-GAG nature and add new information on the anti-herpes virus activity of this protein, confirming it as an outstanding candidate for the treatment of herpetic infections.     

Novel mechanism of antibody-independent complement neutralization of herpes simplex virus type 1

The envelope surface glycoprotein C (gC) of HSV-1 interferes with the complement cascade by binding C3 and activation products C3b, iC3b, and C3c, and by blocking the interaction of C5 and properdin with C3b. Wild-type HSV-1 is resistant to Ab-independent complement neutralization; however, HSV-1 mutant virus lacking gC is highly susceptible to complement resulting in > or =100-fold reduction in virus titer. We evaluated the mechanisms by which complement inhibits HSV-1 gC null virus to better understand how gC protects against complement-mediated neutralization. C8-depleted serum prepared from an HSV-1 and -2 Ab-negative donor neutralized gC null virus comparable to complement-intact serum, indicating that C8 and terminal lytic activity are not required. In contrast, C5-depleted serum from the same donor failed to neutralize gC null virus, supporting a requirement for C5. EDTA-treated serum did not neutralize gC null virus, indicating that complement activation is required. Factor D-depleted and C6-depleted sera neutralized virus, suggesting that the alternative complement pathway and complement components beyond C5 are not required. Complement did not aggregate virus or block attachment to cells. However, complement inhibited infection before early viral gene expression, indicating that complement affects one or more of the following steps in virus replication: virus entry, uncoating, DNA transport to the nucleus, or immediate early gene expression. Therefore, in the absence of gC, HSV-1 is readily inhibited by complement by a C5-dependent mechanism that does not require viral lysis, aggregation, or blocking virus attachment.     

Localization on the herpes simplex virus type 1 genome of a region encoding proteins involved in adsorption to the cellular receptor.

We have previously shown that aminoglycosides such as neomycin and the polyamino acids polylysine and polyarginine selectively inhibit the binding of herpes simplex virus type 1 (HSV-1) to the cellular receptor, whereas HSV-2 infection is unaffected. In the present study we took advantage of this difference between HSV-1 and HSV-2 by using HSV(-1)-HSV(-2) intertypic recombinants to locate a region on the HSV-1 genome encoding proteins affecting the binding of the virion to the cellular receptor. The results were consistent with those obtained by marker rescue experiments. The identified region, which mapped between coordinates 0.580 and 0.687, contains two partial and eight complete genes, including the glycoprotein C (gC) gene and two others with potential transmembrane sequences. Various gC monoclonal antibody-resistant mutants of HSV-1 as well as a mutant completely lacking gC were found to be fully sensitive to neomycin, suggesting that gC is not the site of drug sensitivity and is not essential for adsorption of virus to the cellular receptor. However, the rate of adsorption was reduced in the absence of gC, indicating a facilitating function of the glycoprotein. The universal nature of this HSV-1 receptor binding was revealed by the similarity in drug sensitivity of infectivity in four different cell lines from various tissues and species.     

Herpes simplex virus types 1 and 2 differ in their interaction with heparan sulfate

Cell surface heparan sulfate (HS) serves as an initial receptor for many different viruses, including herpes simplex virus types 1 and 2 (HSV-1 and 2, respectively). Glycoproteins C and B (gC and gB) are the major components of the viral envelope that mediate binding to HS. In this study, purified gB and gC homologous proteins as well as purified HSV-1 and HSV-2 virions were compared for the ability to bind isolated HS receptor molecules. HSV-1 gC and HSV-2 gC bound comparable amounts of HS. Similarly, HSV-1 gB and its HSV-2 counterpart showed no difference in the HS-binding capabilities. Despite the similar HS-binding potentials of gB and gC homologs, HSV-1 virions bound more HS than HSV-2 particles. Purified gC and gB proteins differed with respect to sensitivity of their interaction with HS to increased concentrations of sodium chloride in the order gB-2 > gB-1 > gC-1 > gC-2. The corresponding pattern for binding of whole HSV virions to cells in the presence of increased ionic strength of the medium was HSV-2 gC-neg1 > HSV-1 gC(-)39 > HSV-1 KOS 321 > HSV-2 333. These results relate the HS-binding activities of individual glycoproteins with the cell-binding abilities of whole virus particles. In addition, these data suggest a greater contribution of electrostatic forces for binding of gB proteins and gC-negative mutants compared with binding of gC homologs and wild-type HSV strains. Binding of wild-type HSV-2 virions was the least sensitive to increased ionic strength of the medium, suggesting that the less extensive binding of HS molecules by HSV-2 than by HSV-1 can be compensated for by a relatively weak contribution of electrostatic forces to the binding. Furthermore, gB and gC homologs exhibited different patterns of sensitivity of binding to cells to inhibition with selectively N-, 2-O-, and 6-O-desulfated heparin compounds. The O-sulfate groups of heparin were found to be more important for interaction with gB-1 than gB-2. These results indicate that HSV-1 and HSV-2 differ in their interaction with HS.     

The Amino Terminus of Herpes Simplex Virus 1 Glycoprotein K Is Required for Virion Entry via the Paired Immunoglobulin-Like Type-2 Receptor Alpha

The herpes simplex virus 1 (HSV-1) glycoprotein K (gK)/UL20 protein complex is incorporated into virion envelopes and cellular membranes and functions during virus entry and cell-to-cell spread. To investigate the role of gK/UL20 in the context of a highly neurovirulent virus strain, the HSV-1(McKrae) genome was cloned into a bacterial artificial chromosome plasmid (McKbac) and utilized to construct the mutant virus McK(gKΔ31-68), carrying a 37-amino-acid deletion within the gK amino terminus. The McKbac virus entered efficiently into Chinese hamster ovary (CHO) cells constitutively expressing HSV-1 human receptors, nectin-1, herpesvirus entry mediator (HVEM), or paired immunoglobulin-like type-2 receptor alpha (PILRα). In contrast, the McK(gKΔ31-68) virus failed to enter into CHO-PILRα cells, while it entered CHO cells expressing HVEM and nectin-1 more efficiently than the McKbac virus. Both McKbac and McK(gKΔ31-68) viruses entered all CHO cells expressing HSV-1 receptors via a pH-independent pathway. The HSV-1(F) gBΔ28syn mutant virus, encoding a carboxyl-terminal truncated gB, causes extensive cell fusion. Previously, we showed that the gKΔ31-68 amino acid deletion abrogated gBΔ28syn virus-induced cell fusion, indicating that the amino terminus of gK is required for gB-mediated virus-induced cell fusion (V. N. Chouljenko, A. V. Iyer, S. Chowdhury, D. V. Chouljenko, and K. G. J. Kousoulas, Virology 83:12301–12313, 2009). Surprisingly, the gKΔ31-68/gBΔ28syn virus caused extensive fusion of CHO-nectin-1 cells but limited cell fusion of CHO-PILRα cells. Coimmunoprecipitation experiments revealed that both gK and PILRα bound gB in infected cells. Collectively, these results indicate that the amino terminus of gK is functionally and physically associated with the gB-PILRα protein complex and regulates membrane fusion of the viral envelope with cellular membranes during virus entry as well as virus-induced cell-to-cell fusion.     

Herpes Simplex Virus 2 ICP0? Mutant Viruses Are Avirulent and Immunogenic: Implications for a Genital Herpes Vaccine

Herpes simplex virus 1 (HSV-1) ICP0 ⁻ mutants are interferon-sensitive, avirulent, and elicit protective immunity against HSV-1 (Virol J, 2006, 3:44). If an ICP0 ⁻ mutant of herpes simplex virus 2 (HSV-2) exhibited similar properties, such a virus might be used to vaccinate against genital herpes. The current study was initiated to explore this possibility. Several HSV-2 ICP0 ⁻ mutant viruses were constructed and evaluated in terms of three parameters: i. interferon-sensitivity; ii. virulence in mice; and iii. capacity to elicit protective immunity against HSV-2. One ICP0 ⁻ mutant virus in particular, HSV-2 0ΔNLS, achieved an optimal balance between avirulence and immunogenicity. HSV-2 0ΔNLS was interferon-sensitive in cultured cells. HSV-2 0ΔNLS replicated to low levels in the eyes of inoculated mice, but was rapidly repressed by an innate, Stat 1-dependent host immune response. HSV-2 0ΔNLS failed to spread from sites of inoculation, and hence produced only inapparent infections. Mice inoculated with HSV-2 0ΔNLS consistently mounted an HSV-specific IgG antibody response, and were consistently protected against lethal challenge with wild-type HSV-2. Based on their avirulence and immunogenicity, we propose that HSV-2 ICP0 ⁻ mutant viruses merit consideration for their potential to prevent the spread of HSV-2 and genital herpes.     

Resistance of herpes simplex virus type 2 to neomycin maps to the N-terminal portion of glycoprotein C.

Entry of herpes simplex virus (HSV) into cells is believed to be mediated by specific binding of envelope proteins to a cellular receptor. Neomycin specifically blocks this initial step in infection by HSV-1 but not HSV-2. Resistance of HSV-2 to this compound maps to a region of the genome encoding glycoprotein C (gC-2). We have studied the function of gC-2 in the initial interaction of the virus with the host cell, using HSV-2 mutants deleted for gC-2 and gC-2-rescued recombinants. Resistance to neomycin was directly linked to the presence of gC-2 within the viral genome. In addition, deletion of the gC-2 gene caused a marked delay in adsorption to cells relative to the wild-type virus. HSV-1 recombinants containing chimeric gC genes composed of HSV-1 and HSV-2 sequences were used to localize neomycin resistance within the N-terminal 223 amino acids of gC-2. This region of the glycoprotein comprises an important domain responsible for binding of HSV-2 to cell receptors in the presence of neomycin. A gC-2-negative mutant is still infectious, indicating that HSV-2 also has an alternative pathway of adsorption.     

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.     

Binding of complement component C3b to glycoprotein gC of herpes simplex virus type 1: mapping of gC-binding sites and demonstration of conserved C3b binding in low-passage clinical isolates.

The sites on glycoprotein gC of herpes simplex virus type 1 (HSV-1) which bind complement component C3b were evaluated by using anti-gC monoclonal antibodies and mutants which have alterations at defined regions of the glycoprotein. Monoclonal antibodies were incubated with HSV-1-infected cells in a competitive assay to block C3b binding. Each of 12 different monoclonals, which recognize the four major antigenic sites of gC, completely inhibited C3b binding. With this approach, no one antigenic group on gC could be assigned as the C3b-binding region. Next, 21 gC mutants were evaluated for C3b binding, including 1 which failed to synthesize gC, 4 which synthesized truncated forms of the glycoprotein such that gC did not insert into the cell's membrane, and 16 which expressed gC on the cell's surface but which had mutations in various antigenic groups. Eleven strains did not bind C3b. This included the 1 strain which did not synthesize gC, the 4 strains which secreted gC without inserting the glycoprotein into the cell membrane, and 6 of 16 strains which expressed gC on the cell surface. In these six strains, the mutations were at three different antigenic sites. One hypothesis to explain these findings is that C3b binding is modified by changes in the conformation of gC which develop either after antibodies bind to gC or as a result of mutations in the gC gene. Attachment of C3b to gC was also evaluated in 31 low-passage clinical isolates of HSV-1. Binding was detected with each HSV-1 isolate, but not with nine HSV-2 isolates. Therefore, although mutants that lack C3b binding are readily selected in vitro, the C3b-binding function of gC is maintained in vivo. These results indicate that the sites on gC that bind C3b are different from those that bind monoclonal antibodies, that antibodies directed against all sites on gC block C3b binding, and that C3b binding is a conserved function of gC in vivo.     

Antibodies Are Required for Complete Vaccine-Induced Protection against Herpes Simplex Virus 2

Herpes simplex virus 2 (HSV-2) 0ΔNLS is a live HSV-2 ICP0 ^- mutant vaccine strain that is profoundly attenuated in vivo due to its interferon-hypersensitivity. Recipients of the HSV-2 0ΔNLS vaccine are resistant to high-dose HSV-2 challenge as evidenced by profound reductions in challenge virus spread, shedding, disease and mortality. In the current study, we investigated the requirements for HSV-2 0ΔNLS vaccine-induced protection. Studies using (UV)-inactivated HSV-2 0ΔNLS revealed that self-limited replication of the attenuated virus was required for effective protection from vaginal or ocular HSV-2 challenge. Diminished antibody responses in recipients of the UV-killed HSV-2 vaccine suggested that antibodies might be playing a critical role in early protection. This hypothesis was investigated in B-cell-deficient μMT mice. Vaccination with live HSV-2 0ΔNLS induced equivalent CD8⁺ T cell responses in wild-type and μMT mice. Vaccinated μMT mice shed ~40-fold more infectious HSV-2 at 24 hours post-challenge relative to vaccinated wild-type (B-cell⁺) mice, and most vaccinated μMT mice eventually succumbed to a slowly progressing HSV-2 challenge. Importantly, passive transfer of HSV-2 antiserum restored full protection to HSV-2 0ΔNLS-vaccinated μMT mice. The results demonstrate that B cells are required for complete vaccine-induced protection against HSV-2, and indicate that virus-specific antibodies are the dominant mediators of early vaccine-induced protection against HSV-2.     

The PK Domain of the Large Subunit of Herpes Simplex Virus Type 2 Ribonucleotide Reductase (ICP10) Is Required for Immediate-Early Gene Expression and Virus Growth

The large subunit of herpes simplex virus (HSV) ribonucleotide reductase (RR), RR1, contains a unique amino-terminal domain which has serine/threonine protein kinase (PK) activity. To examine the role of the PK activity in virus replication, we studied an HSV type 2 (HSV-2) mutant with a deletion in the RR1 PK domain (ICP10ΔPK). ICP10ΔPK expressed a 95-kDa RR1 protein (p95) which was PK negative but retained the ability to complex with the small RR subunit, RR2. Its RR activity was similar to that of HSV-2. In dividing cells, onset of virus growth was delayed, with replication initiating at 10 to 15 h postinfection, depending on the multiplicity of infection. In addition to the delayed growth onset, virus replication was significantly impaired (1,000-fold lower titers) in nondividing cells, and plaque-forming ability was severely compromised. The RR1 protein expressed by a revertant virus [HSV-2(R)] was structurally and functionally similar to the wild-type protein, and the virus had wild-type growth and plaque-forming properties. The growth of the ICP10ΔPK virus and its plaque-forming potential were restored to wild-type levels in cells that constitutively express ICP10. Immediate-early (IE) genes for ICP4, ICP27, and ICP22 were not expressed in Vero cells infected with ICP10ΔPK early in infection or in the presence of cycloheximide, and the levels of ICP0 and p95 were significantly (three- to sevenfold) lower than those in HSV-2- or HSV-2(R)-infected cells. IE gene expression was similar to that of the wild-type virus in cells that constitutively express ICP10. The data indicate that ICP10 PK is required for early expression of the viral regulatory IE genes and, consequently, for timely initiation of the protein cascade and HSV-2 growth in cultured cells.     

Mapping of the structural gene for the herpes simplex virus type 2 counterpart of herpes simplex virus type 1 glycoprotein C and identification of a type 2 mutant which does not express this glycoprotein

The gene encoding glycoprotein F (gF) of herpes simplex virus type 2 (HSV-2) was mapped to the region of the viral genome from 0.62 to 0.64 map units. This region is colinear with, and partially homologous to, the region of the HSV-1 genome previously shown to encode gC. Mapping of the gF gene was done by insertion of HSV-2 DNA fragments into the thymidine kinase gene of an HSV-1 virus and screening of the resultant recombinant viruses for the expression of gF. In this way, DNA sequences necessary for the expression of gF in infected cells were also delimited. Because several plaque morphology mutants (syncytial mutants) of HSV-1 have previously been shown to be gC-, a syncytial mutant of HSV-2 (GP) was tested for the expression of gF. It was found to be gF-, indicating that gF is not essential for replication of HSV-2 in cell culture, just as gC is not essential for replication of HSV-1. This result also suggests that the gF- and gC- phenotypes are related in the same, as yet undefined, way to the expression of a syncytial marker. A proposal to change the name of HSV-2 gF to gC (gC-2) is discussed.     

Herpes simplex virus type 1 evades the effects of antibody and complement in vivo

Herpes simplex virus type 1 (HSV-1) encodes a complement-interacting glycoprotein, gC, and an immunoglobulin G (IgG) Fc binding glycoprotein, gE, that mediate immune evasion by affecting multiple aspects of innate and acquired immunity, including interfering with complement components C1q, C3, C5, and properdin and blocking antibody-dependent cellular cytotoxicity. Previous studies evaluated the individual contributions of gC and gE to immune evasion. Experiments in a murine model that examines the combined effects of gC and gE immune evasion on pathogenesis are now reported. Virulence of wild-type HSV-1 is compared with mutant viruses defective in gC-mediated C3 binding, gE-mediated IgG Fc binding, or both immune evasion activities. Eliminating both activities greatly increased susceptibility of HSV-1 to antibody and complement neutralization in vitro and markedly reduced virulence in vivo as measured by disease scores, virus titers, and mortality. Studies with C3 knockout mice indicated that other activities attributed to these glycoproteins, such as gC-mediated virus attachment to heparan sulfate or gE-mediated cell-to-cell spread, do not account for the reduced virulence of mutant viruses. The results support the importance of gC and gE immune evasion in vivo and suggest potential new targets for prevention and treatment of HSV disease.     

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.     

The UL13 and US3 Protein Kinases of Herpes Simplex Virus 1 Cooperate to Promote the Assembly and Release of Mature,Infectious Virions

Herpes simplex virus type 1 (HSV-1) encodes two bona fide serine/threonine protein kinases, the US3 and UL13 gene products. HSV-1 ΔUS3 mutants replicate with wild-type efficiency in cultured cells, and HSV-1 ΔUL13 mutants exhibit <10-fold reduction in infectious viral titers. Given these modest phenotypes, it remains unclear how the US3 and UL13 protein kinases contribute to HSV-1 replication. In the current study, we designed a panel of HSV-1 mutants, in which portions of UL13 and US3 genes were replaced by expression cassettes encoding mCherry protein or green fluorescent protein (GFP), respectively, and analyzed DNA replication, protein expression, and spread of these mutants in several cell types. Loss of US3 function alone had largely negligible effect on viral DNA accumulation, gene expression, virion release, and spread. Loss of UL13 function alone also had no appreciable effects on viral DNA levels. However, loss of UL13 function did result in a measurable decrease in the steady-state levels of two viral glycoproteins (gC and gD), release of total and infectious virions, and viral spread. Disruption of both genes did not affect the accumulation of viral DNA, but resulted in further reduction in gC and gD steady-state levels, and attenuation of viral spread and infectious virion release. These data show that the UL13 kinase plays an important role in the late phase of HSV-1 infection, likely by affecting virion assembly and/or release. Moreover, the data suggest that the combined activities of the US3 and UL13 protein kinases are critical to the efficient assembly and release of infectious virions from HSV-1-infected cells.