Herpes simplex virus infection and propagation in a mouse L cell mutant lacking heparan sulfate proteoglycans.

We have isolated a variant line of mouse L cells, termed gro2C, which is partially resistant to infection by herpes simplex virus type 1 (HSV-1). Characterization of the genetic defect in gro2C cells revealed that this cell line harbors a specific defect in the heparan sulfate synthesis pathway. Specifically, anion-exchange high-performance liquid chromatography of metabolically radiolabeled glycosaminoglycans indicated that chondroitin sulfate moieties were synthesized normally in the mutant cells, whereas heparin-like chains were absent. Because of these properties, we have used these cells to investigate the role of heparan sulfate proteoglycans in the HSV-1 life cycle. In this report, we demonstrate that the partial block to HSV-1 infection in gro2C cells occurs in the virus entry pathway. Virus adsorption assays using radiolabeled HSV-1 (KOS) revealed that the gro2C cell surface is a relatively poor target for HSV-1 in that virus attachment was 85% lower in the mutant cells than in the parental L cell controls. A portion of the 15% residual virus adsorption was functional, however, insofar as gro2C cells were susceptible to HSV-1 infection in plaque assays and in single-step growth experiments. Moreover, although the number of HSV-1 plaques that formed in gro2C monolayers was reduced by 85%, the plaque morphology was normal, and the virus released from the mutant cells was infectious. Taken together, these results provide strong genetic evidence that heparan sulfate proteoglycans enhance the efficiency of HSV attachment to the cell surface but are otherwise not essential at any stage of the lytic cycle in culture. Moreover, in the absence of heparan sulfate, other cell surface molecules appear to confer susceptibility to HSV, leading to a productive viral infection.     

Suppression of herpes simplex virus 1 in MDBK cells via the interferon pathway

Herpes simplex virus (HSV) normally undergoes productive infection in culture, causing cell destruction and plaque formation. Here we characterize an unusual pattern of HSV type 1 (HSV-1) infection in MDBK cells which surprisingly results in suppression of replication, cell recovery, and maintenance of virus. Compared to Vero cells, MDBK cells supported a normal productive infection at a high multiplicity with complete cell destruction. At low multiplicity, HSV also showed an identical initial specific infectivity in the two cell types. Thereafter, the progression of infection was radically different. In contrast to the rapid plaque expansion and eventual destruction in Vero monolayers, in MDBK cells, after initial plaque formation, plaque size actually decreased and, with time, monolayers recovered. Using a green fluorescent protein (GFP)-VP16-expressing virus, we monitored infection in live individual plaques. After early stages of intense GFP-VP16 expression, expression regressed to a thin boundary at the edge of the plaques and was completely suppressed by 10 days. Cells lacking expression then began to grow into the plaque boundaries. Furthermore, following media replacement, individual cells expressing GFP-VP16 could be observed reinitiating infection. The results indicated the production of a potent inhibitory component during infection in MDBK cells, and we show the continued and prolonged presence of interferon in the medium, at times when there was no longer evidence of ongoing productive infection. We exploited the ability of V protein of simian virus 5 to degrade Stat1 and prevent interferon signaling. We established MDBK cells constitutively expressing the V protein with the resultant loss of Stat1. In comparison to the parental cells, infection in these cells now progressed at a rapid rate with expanding plaque formation. We believe the conclusions have significant implications for the study of HSV-1 and interferon signaling both in culture and in animal models.     

Sequential isolation of proteoglycan synthesis mutants by using herpes simplex virus as a selective agent: evidence for a proteoglycan-independent virus entry pathway.

A novel mouse L-cell mutant cell line defective in the biosynthesis of glycosaminoglycans was isolated by selection for cells resistant to herpes simplex virus (HSV) infection. These cells, termed sog9, were derived from mutant parental gro2C cells, which are themselves defective in heparan sulfate biosynthesis and 90% resistant to HSV type 1 (HSV-1) infection compared with control L cells (S. Gruenheid, L. Gatzke, H. Meadows, and F. Tufaro, J. Virol. 67:93-100, 1993). In this report, we show that sog9 cells exhibit a 3-order-of-magnitude reduction in susceptibility to HSV-1 compared with control L cells. In steady-state labeling experiments, sog9 cells accumulated almost no [35S]sulfate-labeled or [6-3H]glucosamine-labeled glycosaminoglycans, suggesting that the initiation of glycosaminoglycan assembly was specifically reduced in these cells. Despite these defects, sog9 cells were fully susceptible to vesicular stomatitis virus (VSV) and permissive for both VSV and HSV replication, assembly, and egress. HSV plaques formed in the sog9 monolayers in proportion to the amount of input virus, suggesting the block to infection was in the virus entry pathway. More importantly, HSV-1 infection of sog9 cells was not significantly reduced by soluble heparan sulfate, indicating that infection was glycosaminoglycan independent. Infection was inhibited by soluble gD-1, however, which suggests that glycoprotein gD plays a role in the infection of this cell line. The block to sog9 cell infection by HSV-1 could be eliminated by adding soluble dextran sulfate to the inoculum, which may act by stabilizing the virus at the sog9 cell surface. Thus, sog9 cells provide direct genetic evidence for a proteoglycan-independent entry pathway for HSV-1, and results with these cells suggest that HSV-1 is a useful reagent for the direct selection of novel animal cell mutants defective in the synthesis of cell surface proteoglycans.     

Herpes simplex virus glycoproteins E and I facilitate cell-to-cell spread in vivo and across junctions of cultured cells.

Herpes simplex virus (HSV) glycoproteins E and I (gE and gI) can act as a receptor for the Fc domain of immunoglobulin G (IgG). To examine the role of HSV IgG Fc receptor in viral pathogenesis, rabbits and mice were infected by the corneal route with HSV gE- or gI- mutants. Wild-type HSV-1 produced large dendritic lesions in the corneal epithelium and subsequent stromal disease leading to viral encephalitis, whereas gE- and gI- mutant viruses produced microscopic punctate or small dendritic lesions in the epithelium and no corneal disease or encephalitis. These differences were not related to the ability of the gE-gI oligomer to bind IgG because the differences were observed before the appearance of anti-HSV IgG and in mice, in which IgG binds to the Fc receptor poorly or not at all. Mutant viruses produced small plaques on monolayers of normal human fibroblasts and epithelial cells. Replication of gE- and gI- mutant viruses in human fibroblasts were normal, and the rates of entry of mutant and wild-type viruses into fibroblasts were similar; however, spread of gE- and gI- mutant viruses from cell to cell was significantly slower than that of wild-type HSV-1. In experiments in which fibroblast monolayers were infected with low multiplicities of virus and multiple rounds of infection occurred, the presence of neutralizing antibodies in the culture medium caused the yields of mutant viruses to drop dramatically, whereas there was a lesser effect on the production of wild-type HSV. It appears that cell-to-cell transmission of wild-type HSV-1 occurs by at least two mechanisms: (i) release of virus from cells and entry of extracellular virus into a neighboring cell and (ii) transfer of virus across cell junctions in a manner resistant to neutralizing antibodies. Our results suggest that gE- and gI- mutants are defective in the latter mechanism of spread, suggesting the possibility that the gE-gI complex facilitates virus transfer across cell junctions, a mode of spread which may predominate in some tissues. It is ironic that the gE-gI complex, usually considered an IgG Fc receptor, may, through its ability to mediate cell-to-cell spread, actually protect HSV from IgG in a manner different than previously thought.     

Flow cytometric evaluation of anti-herpes drugs

A rapid means of screening drugs for toxicity and anti-herpes simplex virus activity was developed based on the flow cytometric detection of HSV induced changes in cellular DNA content. Subconfluent monolayers of human diploid fibroblasts (HEL 299) were assayed for DNA content with propidium iodide 24 h after infection with HSV-1 (multiplicity of infection 1-10) and treatment with the drug to be tested. Infection was detected by a broadening of the normal diploid and tetraploid peaks and presence of greater than 4-n DNA staining. Inhibition of viral DNA synthesis and maintenance of the normal growth pattern of control cells was indication of antiviral activity. Toxicity of the compound was indicated by the loss of S phase and tetraploid cell populations. Using this assay, we evaluated the activities of one experimental and two established antiviral agents.     

细胞周期蛋白依赖性蛋白激酶2在单纯疱疹病毒复制中的作用

细胞周期蛋白依赖性蛋白激酶(CDK)与单纯疱疹病毒(HSV)等多种重要人类疾病病毒的复制密切相关.但具体哪种CDK是病毒复制所必需的还不清楚.本文用不同剂量的HSV-1-KOS株(以下简称HSV)感染CDK2功能缺陷型宿主细胞,结果发现HSV在CDK2功能缺陷型宿主细胞中的复制具有感染剂量依赖性;一步生长曲线分析结果表明其在CDK2功能缺陷型宿主细胞中的复制较在正常细胞延迟3h;感染6h时CDK2活性被诱导,9h时活性最大;CDK2活性增加后HSV-1即进入快速的裂解性复制.提示CDK2可能在HSV复制的启动中起着某种重要作用.     

Growth of Rio Bravo Virus in Cell Cultures

The growth of Rio Bravo virus (RBV) in eight cell culture systems was studied. Highest yields of virus were produced in BHK-21 (C13), L, and Vero cell lines, but L cells were resistant to low doses of virus. LLC-MK(2), HeLa, and human embryo skin cells produced moderate amounts of virus, but FL amnion and primary chick embryo fibroblasts supported little virus growth. Virus was rapidly inactivated by exposure to pH values below 7.0. Single-cycle growth in BHK-21, L, and LLC-MK(2) cell monolayers was characterized by a latent period of about 12 hr followed by rapid virus production that peaked at 36 to 48 hr. Vero cell cultures can remain chronically infected with RBV for more than 100 days. Such cultures show evidence of cell destruction, and their supernatant fluids contain virus at 10(4) to 10(5) log(10) per ml.     

Soluble V domain of Nectin-1/HveC enables entry of herpes simplex virus type 1 (HSV-1) into HSV-resistant cells by binding to viral glycoprotein D

Interaction of herpes simplex virus (HSV) glycoprotein D (gD) with specific cellular receptors is essential for HSV infection of susceptible cells. Virus mutants that lack gD can bind to the cell surface (attachment) but do not enter, implying that interaction of gD with its receptor(s) initiates the postattachment (entry) phase of HSV infection. In this report, we have studied HSV entry in the presence of the gD-binding variable (V) domain of the common gD receptor nectin-1/HveC to determine whether cell association of the gD receptor is required for HSV infection. In the presence of increasing amounts of the soluble nectin-1 V domain (sNec1(123)), increasing viral entry into HSV-resistant CHO-K1 cells was observed. At a multiplicity of 3 in the presence of optimal amounts of sNec1(123), approximately 90% of the cells were infected. The soluble V domain of nectin-2, a strain-specific HSV entry receptor, promoted entry of the HSV type 1 (HSV-1) Rid-1 mutant strain, but not of wild-type HSV-1. Preincubation and immunofluorescence studies indicated that free or gD-bound sNec1(123) did not associate with the cell surface. sNec1(123)-mediated entry was highly impaired by interference with the cell-binding activities of viral glycoproteins B and C. While gD has at least two functions, virus attachment to the cell and initiation of the virus entry process, our results demonstrate that the attachment function of gD is dispensable for entry provided that other means of attachment are available, such as gB and gC binding to cell surface glycosaminoglycans.     

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.     

Multiplicity reactivation of alkylating agent damaged herpes simplex virus (type I) in human cells

Herpes simplex virus type I (Strain KOS) is inactivated by treatment with MMS, MNNG and HN2 as determined by plaque assay on Vero cell monolayers, or by an infectious center assay with FS2 cells, human foreskin fibroblast line. At a given dose of MMS and MNNG, survival of the virus was significantly higher at a multiplicity of infection of 1.0 PFU/cell compared to 0.01 PFU/cell. These results indicate that HSV-1 infected human cells are capable of repairing chemically induced lesions by way of multiplicity reactivation. No evidence for multiplicity reactivation with HN2-treated virus could be obtained, however.     

Herpes simplex viruses lacking glycoprotein D are unable to inhibit virus penetration: quantitative evidence for virus-specific cell surface receptors.

Herpes simplex virus (HSV) glycoprotein D (gD) plays an essential role in the entry of virus into cells. HSV mutants unable to express gD were constructed. The mutants can be propagated on VD60 cells, which supply the viruses with gD; however, virus particles lacking gD were produced in mutant-infected Vero cells. Virus particles with or without gD adsorbed to a large number (greater than 4 x 10(4] of sites on the cell surface; however, virions lacking gD did not enter cells. Cells pretreated with UV-inactivated virions containing gD (approximately 5 x 10(3) particles per cell) were resistant to infection with HSV type 1 (HSV-1) and HSV-2. In contrast, cells pretreated with UV-inactivated virions lacking gD could be infected with HSV-1 and HSV-2. If infectious HSV-1 was added prior to UV-inactivated virus particles containing gD, the infectious virus entered cells and replicated. Therefore, virus particles containing gD appear to block specific cell surface receptors which are very limited in number. Particles lacking gD are presumably unable to interact with these receptors, suggesting that gD is an essential receptor-binding polypeptide.     

A herpes simplex virus mutant in which glycoprotein D sequences are replaced by beta-galactosidase sequences binds to but is unable to penetrate into cells.

Herpes simplex virus (HSV) glycoprotein gD is a major component of the virion envelope and is thought to play an important role in the initial stages of viral infection and stimulates the production of high titers of neutralizing antibodies. We assumed that gD plays an essential role in virus replication, and so to complement viruses with mutations in the gD gene we constructed a cell line, denoted VD60, which is capable of expressing high levels of gD after infection with HSV. A recombinant virus, designated F-gD beta, in which sequences encoding gD and a nonessential glycoprotein, gI, were replaced by Escherichia coli beta-galactosidase sequences, was selected on the basis that it produced blue plaques on VD60 cell monolayers under agarose overlays containing 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal). F-gD beta was able to replicate normally on complementing VD60 cells. However, F-gD beta was unable to form plaques on noncomplementing Vero cells. Virions lacking gD were produced in normal amounts by Vero cells infected with F-gD beta, and the virus particles were distributed throughout the cytoplasm and on the cell surface, suggesting that gD is not essential for HSV envelopment and egress. Virions lacking gD were able to bind to cells, but were unable to initiate synthesis of viral early polypeptides. Plaque production of F-gD beta particles lacking gD was enhanced by polyethylene glycol treatment, suggesting that gD is essential for penetration of HSV into cells. Other HSV glycoproteins have been implicated in the entry of virus into cells, and thus this process appears to involve multiple interactions at the cell surface.     

Herpes simplex virus glycoprotein K promotes egress of virus particles.

Herpes simplex virus (HSV) glycoprotein K (gK) is thought to be intimately involved in the process by which infected cells fuse because HSV syncytial mutations frequently alter the gK (UL53) gene. Previously, we characterized gK produced in cells infected with wild-type HSV or syncytial HSV mutants and found that the glycoprotein was localized to nuclear and endoplasmic reticulum membranes and did not reach the cell surface (L. Hutchinson, C. Roop, and D. C. Johnson, J. Virol. 69:4556-4563, 1995). In this study, we have characterized a mutant HSV type 1, denoted F-gK beta, in which a lacZ gene cassette was inserted into the gK coding sequences. Since gK was found to be essential for virus replication, F-gK beta was propagated on complementing cells which can express gK. F-gK beta produced normal plaques bounded by nonfused cells when plated on complementing cells, although syncytia were observed when the cells produced smaller amounts of gK. In contrast, F-gK beta produced only microscopic plaques on Vero cells and normal human fibroblasts (which do not express gK) and these plaques were reduced by 10(2) to 10(6) in number. Further, large numbers of nonenveloped capsids accumulated in the cytoplasm of F-gK beta-infected Vero cells, virus particles did not reach the cell surface, and the few enveloped particles that were produced exhibited a reduced capacity to enter cells and initiate an infection of complementing cells. Overexpression of gK in HSV-infected cells also caused defects in virus egress, although particles accumulated in the perinuclear space and large multilamellar membranous structures juxtaposed with the nuclear envelope were observed. Together, these results demonstrate that gK regulates or facilitates egress of HSV from cells. How this property is connected to cell fusion is not clear. In this regard, gK may alter cell surface transport of viral particles or other viral components directly involved in the fusion process.     

Human cytotoxic T cell clones directed against herpes simplex virus-infected cells. II. Bifunctional clones with cytotoxic and virus-induced proliferative activities exhibit herpes simplex virus type 1 and 2 specific or type common reactivities

Human cytotoxic T lymphocyte (CTL) clones directed against herpes simplex virus (HSV)-infected cells were generated after stimulation of peripheral blood lymphocytes (PBL) with HSV type 1 (HSV-1) and HSV type 2 (HSV-2). These CTL clones were studied with regard to HSV type specificity and with regard to whether they also express helper cell activity. Some clones, generated after stimulation with HSV-1, were cytotoxic for autologous cells infected with either HSV-1 or HSV-2 ("HSV type common clones"), whereas other clones lysed HSV-1-infected cells only ("type-specific clones"). Similarly, after HSV-2 stimulation, both HSV-2 specific and HSV type common clones were obtained, indicating the heterogeneity of human cytotoxic T cells to HSV. All CTL clones tested were found to be bifunctional in that they also proliferated in response to stimulation with HSV. The HSV type specificity of the proliferative response was identical to that of the cytotoxic activity of the clones. An HSV type common clone, when stimulated with either HSV-1 or HSV-2, and an HSV-1 specific clone, when stimulated with HSV-1 but not with HSV-2, produced a factor, presumably interleukin 2 (IL 2), which induced proliferation of CTLL, an IL 2-dependent T cell line, providing evidence that our HSV-directed CTL clones also express helper cell activity. CTL clones that we previously reported were restricted in cytotoxic activity by HLA class II DR-1 or MB-1 antigens were found, in this study, to be restricted in proliferative response to HSV by these same HLA antigens. These results suggest that our bifunctional T cell clones directed against HSV may recognize the same viral antigenic determinants and the same HLA antigens for both cytotoxic and virus-induced proliferative activities. This is the first demonstration of human HSV type specific and HSV type common T cell clones and HSV specific T cell clones with both cytotoxic and helper cell activities.     

Persistence of herpes simplex virus type 1 in rat neurotumor cells.

Herpes simplex virus type 1 (HSV-1) infection of a rat central nervous system tumor cell line led to almost complete destruction of the cells. Cells that survived the infection could be isolated and shown to produce infectious HSV particles for variable lengths of time in culture ranging from 20 to 57 passages. Even though infectious virus production eventually ceased, the cell lines continued to produce herpes-specified proteins as measured by immunological techniques. These cells also showed herpesvirus-like structures in the electron microscope. The persistently infected cells that produced HSV antigens and bore HSV sequences were resistant to superinfection by HSV-1. The resistance was not due to failure of adsorption of the virus or to the production of interferon by the cells. The nature of the block in HSV replication in these neurotumor cells, which contain and partially express the HSV genome, is unknown, but may offer an interesting parallel to the known latency of HSV in neural tissues.     

Herpes simplex virus type 2 glycoprotein G is targeted by the sulfated oligo- and polysaccharide inhibitors of virus attachment to cells

Variants of herpes simplex virus type 2 (HSV-2) generated by virus passage in GMK-AH1 cells in the presence of the sulfated oligosaccharide PI-88 were analyzed. Many of these variants were substantially resistant to PI-88 in their initial infection of cells and/or their cell-to-cell spread. The major alteration detected in all variants resistant to PI-88 in the initial infection of cells was a frameshift mutation(s) in the glycoprotein G (gG) gene that resulted in the lack of protein expression. Molecular transfer of the altered gG gene into the wild-type background confirmed that the gG-deficient recombinants were resistant to PI-88. In addition to PI-88, all gG-deficient variants of HSV-2 were resistant to the sulfated polysaccharide heparin. The gG-deficient virions were capable of attaching to cells, and this activity was relatively resistant to PI-88. In addition to having a drug-resistant phenotype, the gG-deficient variants were inefficiently released from infected cells. Purified gG bound to heparin and showed the cell-binding activity which was inhibited by PI-88. Many PI-88 variants produced syncytia in cultured cells and contained alterations in gB, including the syncytium-inducing L792P amino acid substitution. Although this phenotype can enhance the lateral spread of HSV in cells, it conferred no virus resistance to PI-88. Some PI-88 variants also contained occasional alterations in gC, gD, gE, gK, and UL24. In conclusion, we found that glycoprotein gG, a mucin-like component of the HSV-2 envelope, was targeted by sulfated oligo- and polysaccharides. This is a novel finding that suggests the involvement of HSV-2 gG in interactions with sulfated polysaccharides, including cell surface glycosaminoglycans.     

Characterization of a BHK(TK-) cell clone resistant to postattachment entry by herpes simplex virus types 1 and 2.

BHK(TK-) cells selected for resistance to polyethylene glycol-mediated fusion give rise to clones that are resistant to herpes simplex virus (HSV) infection. We have characterized one such clone, designated 95-19, and found that it is resistant to entry of HSV type 1 (HSV-1), HSV-2, and the related alphaherpesvirus pseudorabies virus (PRV). Single-step growth experiments show no detectable replication of multiple strains of HSV-1 and HSV-2 on 95-19 cells. Three lines of evidence suggest that these cells are resistant to postattachment entry. (i) Measurements of binding of radiolabeled virus show that heparin-sensitive binding of HSV-1 and HSV-2 to 95-19 cells is identical to binding to BHK(TK-) cells, suggesting that the block to replication occurs after attachment to heparan sulfate proteoglycan. (ii) 95-19 cells exposed to HSV-1 or HSV-2 at high multiplicity show no detectable immediate-early (IE) mRNA expression. (iii) Exposure of attached virus and cells to polyethylene glycol results in partial recovery of both IE gene expression and virus yield in single-step growth. The degrees of recovery of single-step yield and IE gene expression are similar, suggesting that the only block to single-step replication is at the point of virus entry and that these cells are deficient in some cellular factor required for efficient postattachment entry of free virus. 95-19 cells are also highly resistant to entry by cell-to-cell spread, suggesting that the same cellular factor participates in both types of entry.     

Herpes simplex virus virion host shutoff (vhs) activity alters periocular disease in mice

During lytic infection, the virion host shutoff (vhs) protein of herpes simplex virus (HSV) mediates the rapid degradation of RNA and shutoff of host protein synthesis. In mice, HSV type 1 (HSV-1) mutants lacking vhs activity are profoundly attenuated. HSV-2 has significantly higher vhs activity than HSV-1, eliciting a faster and more complete shutoff. To examine further the role of vhs activity in pathogenesis, we generated an intertypic recombinant virus (KOSV2) in which the vhs open reading frame of HSV-1 strain KOS was replaced with that of HSV-2 strain 333. KOSV2 and a marker-rescued virus, KOSV2R, were characterized in cell culture and tested in an in vivo mouse eye model of latency and pathogenesis. The RNA degradation kinetics of KOSV2 was identical to that of HSV-2 333, and both showed vhs activity significantly higher than that of KOS. This demonstrated that the fast vhs-mediated degradation phenotype of 333 had been conferred upon KOS. The growth of KOSV2 was comparable to that of KOS, 333, and KOSV2R in cell culture, murine corneas, and trigeminal ganglia and had a reactivation frequency similar to those of KOS and KOSV2R from explanted latently infected trigeminal ganglia. There was, however, significantly reduced blepharitis and viral replication within the periocular skin of KOSV2-infected mice compared to mice infected with either KOS or KOSV2R. Taken together, these data demonstrate that heightened vhs activity, in the context of HSV-1 infection, leads to increased viral clearance from the skin of mice and that the replication of virus in the skin is a determining factor for blepharitis. These data also suggest a role for vhs in modulating host responses to HSV infection.     

Specificities of monoclonal and polyclonal antibodies that inhibit adsorption of herpes simplex virus to cells and lack of inhibition by potent neutralizing antibodies.

Polyclonal and monoclonal antibodies to individual herpes simplex virus (HSV) glycoproteins were tested for ability to inhibit adsorption of radiolabeled HSV type 1 (HSV-1) strain HFEMsyn [HSV-1(HFEM)syn] to HEp-2 cell monolayers. Polyclonal rabbit antibodies specific for glycoprotein D (gD) or gC and three monoclonal mouse antibodies specific for gD-1 or gC-1 most effectively inhibited HSV-1 adsorption. Antibodies of other specificities had less or no inhibitory activity despite demonstrable binding of the antibodies to virions. Nonimmune rabbit immunoglobulin G and Fc fragments partially inhibited adsorption when used at relatively high concentrations. These results suggest involvement of gD, gC, and perhaps gE (the Fc-binding glycoprotein) in adsorption. The monoclonal anti-gD antibodies that were most effective at inhibiting HSV-1 adsorption had only weak neutralizing activity. The most potent anti-gD neutralizing antibodies had little effect on adsorption at concentrations significantly higher than those required for neutralization. This suggests that, although some anti-gD antibodies can neutralize virus by blocking adsorption, a more important mechanism of neutralization by anti-gD antibodies may be interference with a step subsequent to adsorption, possibly penetration.