The ectodomain of a novel member of the immunoglobulin subfamily related to the poliovirus receptor has the attributes of a bona fide receptor for herpes simplex virus types 1 and 2 in human cells

We report on the functional cloning of a hitherto unknown member of the immunoglobulin (Ig) superfamily selected for its ability to confer susceptibility to herpes simplex virus (HSV) infection on a highly resistant cell line (J1.1-2 cells), derived by exposure of BHKtk- cells to a recombinant HSV-1 expressing tumor necrosis factor alpha (TNF-alpha). The sequence of herpesvirus Ig-like receptor (HIgR) predicts a transmembrane protein with an ectodomain consisting of three cysteine-bracketed domains, one V-like and two C-like. HIgR shares its ectodomain with and appears to be an alternative splice variant of the previously described protein PRR-1 (poliovirus receptor-related protein). Both HIgR and PRR-1 conferred on J1.1-2 cells susceptibility to HSV-1, HSV-2, and bovine herpesvirus 1. The viral ligand of HIgR and PRR-1 is glycoprotein D, a constituent of the virion envelope long known to mediate viral entry into cells through interaction with cellular receptor molecules. Recently, PRR-1, renamed HveC (herpesvirus entry mediator C), and the related PRR-2, renamed HveB, were reported to mediate the entry of HSV-1, HSV-2, and bovine herpesvirus 1, and the homologous poliovirus receptor was reported to mediate the entry of pseudorabies virus (R. J. Geraghty, C. Krummenacher, G. H. Cohen, R. J. Eisenberg, and P. G. Spear, Science 280:1618-1620, 1998; M. S. Warner, R. J. Geraghty, W. M. Martinez, R. I. Montgomery, J. C. Whitbeck, R. Xu, R. J. Eisenberg, G. H. Cohen, and P. G. Spear, Virology 246:179-189, 1998). Here we further show that HIgR or PRR-1 proteins detected by using a monoclonal antibody to PRR-1 are widely distributed among human cell lines susceptible to HSV infection and commonly used for HSV studies. The monoclonal antibody neutralized virion infectivity in cells transfected with HIgR or PRR-1 cDNA, as well as in the human cell lines, indicating a direct interaction of virions with the receptor molecule, and preliminarily mapping this function to the ectodomain of HIgR and PRR-1. Northern blot analysis showed that HIgR or PRR-1 mRNAs were expressed in human tissues, with the highest expression being detected in nervous system samples. HIgR adds a novel member to the cluster of Ig superfamily members able to mediate the entry of alphaherpesviruses into cells. The wide distribution of HIgR or PRR-1 proteins among human cell lines susceptible to HSV infection, coupled with the neutralizing activity of the antibody in the same cells, provides direct demonstration of the actual use of this cluster of molecules as HSV-1 and HSV-2 entry receptors in human cell lines. The high level of expression in samples from nervous system makes the use of these proteins in human tissues very likely. This cluster of molecules may therefore be considered to constitute bona fide receptors for HSV-1 and HSV-2.     

Herpes simplex virus glycoprotein D can bind to poliovirus receptor-related protein 1 or herpesvirus entry mediator, two structurally unrelated mediators of virus entry

Several cell membrane proteins have been identified as herpes simplex virus (HSV) entry mediators (Hve). HveA (formerly HVEM) is a member of the tumor necrosis factor receptor family, whereas the poliovirus receptor-related proteins 1 and 2 (PRR1 and PRR2, renamed HveC and HveB) belong to the immunoglobulin superfamily. Here we show that a truncated form of HveC directly binds to HSV glycoprotein D (gD) in solution and at the surface of virions. This interaction is dependent on the native conformation of gD but independent of its N-linked glycosylation. Complex formation between soluble gD and HveC appears to involve one or two gD molecules for one HveC protein. Since HveA also mediates HSV entry by interacting with gD, we compared both structurally unrelated receptors for their binding to gD. Analyses of several gD variants indicated that structure and accessibility of the N-terminal domain of gD, essential for HveA binding, was not necessary for HveC interaction. Mutations in functional regions II, III, and IV of gD had similar effects on binding to either HveC or HveA. Competition assays with neutralizing anti-gD monoclonal antibodies (MAbs) showed that MAbs from group Ib prevented HveC and HveA binding to virions. However, group Ia MAbs blocked HveC but not HveA binding, and conversely, group VII MAbs blocked HveA but not HveC binding. Thus, we propose that HSV entry can be mediated by two structurally unrelated gD receptors through related but not identical binding with gD.     

The V domain of herpesvirus Ig-like receptor (HIgR) contains a major functional region in herpes simplex virus-1 entry into cells and interacts physically with the viral glycoprotein D

The herpesvirus entry mediator C (HveC), previously known as poliovirus receptor-related protein 1 (PRR1), and the herpesvirus Ig-like receptor (HIgR) are the bona fide receptors employed by herpes simplex virus-1 and -2 (HSV-1 and -2) for entry into the human cell lines most frequently used in HSV studies. They share an identical ectodomain made of one V and two C2 domains and differ in transmembrane and cytoplasmic regions. Expression of their mRNA in the human nervous system suggests possible usage of these receptors in humans in the path of neuron infection by HSV. Glycoprotein D (gD) is the virion component that mediates HSV-1 entry into cells by interaction with cellular receptors. We report on the identification of the V domain of HIgR/PRR1 as a major functional region in HSV-1 entry by several approaches. First, the epitope recognized by mAb R1. 302 to HIgR/PRR1, capable of inhibiting infection, was mapped to the V domain. Second, a soluble form of HIgR/PRR1 consisting of the single V domain competed with cell-bound full-length receptor and blocked virion infectivity. Third, the V domain was sufficient to mediate HSV entry, as an engineered form of PRR1 in which the two C2 domains were deleted and the V domain was retained and fused to its transmembrane and cytoplasmic regions was still able to confer susceptibility, although at reduced efficiency relative to full-length receptor. Consistently, transfer of the V domain of HIgR/PRR1 to a functionally inactive structural homologue generated a chimeric receptor with virus-entry activity. Finally, the single V domain was sufficient for in vitro physical interaction with gD. The in vitro binding was specific as it was competed both by antibodies to the receptor and by a mAb to gD with potent neutralizing activity for HSV-1 infectivity.     

Protective immunity of bovine herpesvirus-1 (BHV-1) recombinants which express pseudorabies virus (PRV) glycoproteins gB, gC, gD and gE

We have constructed recombinants of bovine herpesvirus-1 (BHV-1) which express pseudorabies virus (PRV) gB, gC, gD or gE either individually or in combination. To test the protective immunity, mice were inoculated with these BHV-1 recombinants and challenged three weeks later with virulent PRV. A BHV-1 recombinant, BHV-1/TF7-1, which express PRV gC, gD, gE and gI but not PRV gB, protected all 7 mice from the challenge with 20 LD50 virulent PRV and 6 out of 7 from the challenge with 100 LD50 PRV, while one dead mice survived for 5 days after the challenge. All the control mice died in 3 days. BHV-1 recombinants which express PRV gB, gC and gD individually also gave some protection but not so effective as BHV/TF7-1. Before challenging with virulent PRV, sera were collected from immunized mice and antibody against PRV was assayed. Western blot analyses indicated that all the recombinants induced antibody in mice against PRV gB, gC or gD individually or in combination. Virus neutralizing (VN) titer against PRV was highest in mice which were inoculated with BHV-1/TF6-1, which expresses PRV gB. BHV-1/TF7-1, which was more effective to protect mice from the challenge of virulent PRV, induced lower VN titer.     

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.     

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)     

Evidence that deficient IFN-gamma production is a biological basis of herpes simplex virus type-2 neurovirulence

Although immune response control of herpes simplex virus (HSV) has been well demonstrated, numerous HSV-2 strains are neurovirulent in immunocompetent mice. Using an RNase protection assay and an ELISA, we found that HSV-2-infected mice exhibited a deficient IFN-gamma response, an inability to clear virus, and eventual death. An HSV-based amplicon vector expressing mouse IFN-gamma was constructed and packaged into HSV-1-helper virus (HSV(pIFN-gamma)). In mice treated with HSV(pIFN-gamma), (i) the LD50 of HSV-2(G) increased 5000-fold, (ii) intracerebral IFN-gamma expression increased 10-fold, and (iii) HSV titer rapidly decreased. We suggest that the deficient IFN-gamma response is a basis for HSV-2 neurovirulence in mice.     

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 (ICP10DeltaPK). ICP10DeltaPK 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 ICP10DeltaPK 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 ICP10DeltaPK 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.     

Differential mechanism of cytostatic effect of (E)-5-(2-bromovinyl)-2'-deoxyuridine, 9-(1,3-dihydroxy-2-propoxymethyl)guanine, and other antiherpetic drugs on tumor cells transfected by the thymidine kinase gene of herpes simplex virus type 1 or type 2

After they have been transfected with the herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) thymidine kinase (TK) gene murine mammary carcinoma (FM3A) cells become highly sensitive to the growth inhibitory properties of the antiherpetic agents (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), 9(-)[(2-hydroxyethoxy)methyl]guanine (acyclovir, ACV), 9(-)[(1,3-dihydroxy-2-propoxy)methyl]guanine (DHPG, ganciclovir), and 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-methyluracil (FMAU). BVDU was 100-fold more potent an inhibitor of HSV TK gene-transfected tumor cell growth (50% inhibitory concentration (IC50), 0.0020-0.0047 microM) than FMAU or DHPG (IC50, 0.051-0.277 microM) and 1000-fold more potent than ACV (IC50, 0.42-4.9 microM). As a rule, the test compounds were more cytostatic to HSV-2 TK than HSV-1 TK gene-transfected FM3A cells. This may be ascribed to the higher phosphorylating capacity (Vmax/Km) of HSV-2 TK than HSV-1 TK and/or to the higher TK enzyme levels of the HSV-2 TK gene-transfected FM3A cells than the HSV-1 TK gene-transfected FM3A cells. Thymidylate synthase of the HSV TK gene-transfected FM3A cells appears to be the target enzyme for the cytostatic action of BVDU, but not FMAU, DHPG, or ACV. Instead, the cytostatic activity of DHPG seems to be correlated with its conversion to the triphosphate form and subsequent incorporation into the DNA of HSV TK gene-transfected FM3A cells.     

Exchangeability of Qsr1p, a large ribosomal subunit protein required for subunit joining, suggests a novel translational regulatory mechanism

The incidence of herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) in herpes simplex encephalitis (HSE) was investigated using cerebrospinal fluid (CSF) samples from sixty-four cases of HSE. A polymerase chain reaction (PCR) employing primers flanking a region of the HSV thymidine kinase gene common to both HSV-1 and HSV-2 was used to detect HSV in the CSF. HSV-1 and HSV-2 were differentiated by digestion with restriction enzymes. Two enzymes were employed; Aval which cleaved only the HSV-2 gene product and Avall which cleaved only the HSV-1 gene product. Sixty-three cases of HSE were found to be due to HSV-1; one case due to HSV-2. These data confirm previous observations that HSV-2 is a rare cause of post-neonatal herpes encephalitis but indicates that a PCR procedure capable of detection of both viruses is essential for efficient diagnosis of HSE.     

Immunization with a single major histocompatibility complex class I-restricted cytotoxic T-lymphocyte recognition epitope of herpes simplex virus type 2 confers protective immunity

We have evaluated the potential of conferring protective immunity to herpes simplex virus type 2 (HSV-2) by selectively inducing an HSV-specific CD8(+) cytotoxic T-lymphocyte (CTL) response directed against a single major histocompatibility complex class I-restricted CTL recognition epitope. We generated a recombinant vaccinia virus (rVV-ES-gB498-505) which expresses the H-2Kb-restricted, HSV-1/2-cross-reactive CTL recognition epitope, HSV glycoprotein B residues 498 to 505 (SSIEFARL) (gB498-505), fused to the adenovirus type 5 E3/19K endoplasmic reticulum insertion sequence (ES). Mucosal immunization of C57BL/6 mice with this recombinant vaccinia virus induced both a primary CTL response in the draining lymph nodes and a splenic memory CTL response directed against HSV gB498-505. To determine the ability of the gB498-505-specific memory CTL response to provide protection from HSV infection, immunized mice were challenged with a lethal dose of HSV-2 strain 186 by the intranasal (i.n.) route. Development of the gB498-505-specific CTL response conferred resistance in 60 to 75% of mice challenged with a lethal dose of HSV-2 and significantly reduced the levels of infectious virus in the brains and trigeminal ganglia of challenged mice. Finally, i.n. immunization of C57BL/6 mice with either a recombinant influenza virus or a recombinant vaccinia virus expressing HSV gB498-505 without the ES was also demonstrated to induce an HSV-specific CTL response and provide protection from HSV infection. This finding confirms that the induction of an HSV-specific CTL response directed against a single epitope is sufficient for conferring protective immunity to HSV. Our findings support the role of CD8(+) T cells in the control of HSV infection of the central nervous system and suggest the potential importance of eliciting HSV-specific mucosal CD8(+) CTL in HSV vaccine design.     

Deletion of the S component inverted repeat sequence c' and the nonessential genes U(S)1 through U(S)5 from the herpes simplex virus type 1 genome substantially impairs productive viral infection in cell culture and pathogenesis in the rat central nervous system

A distinctive feature of the genetic make-up of herpes simplex virus type 1 (HSV-1), a human neurotropic virus, is that approximately half of the 81 known viral genes are not absolutely required for productive infection in Vero cells, and most can be individually deleted without substantially impairing viral replication in cell culture. If large blocks of contiguous viral genes could be replaced with foreign DNA sequences, it would be possible to engineer highly attenuated recombinant HSV-1 gene transfer vectors capable of carrying large cellular genes or multiple genes having related functions. We report the isolation and characterization of an HSV-1 mutant, designated d311, containing a 12 kb deletion of viral DNA located between the L-S Junction a sequence and the U(S)6 gene, spanning the S component inverted repeat sequence c' and the nonessential genes U(S)1 through U(S)5. Replication of d311 was totally inhibited in rat B103 and mouse Neuro-2A neuroblastoma cell lines, and was reduced by over three orders of magnitude in human SK-N-SH neuroblastoma cells compared to wild-type (wt) HSV-1 KOS. This suggested that the deleted genes, while nonessential for replication in Vero cells, play an important role in HSV replication in neuronal cells, particularly those of rodent origin. Unlike wt KOS which replicated locally and spread to other regions of brain following stereotactic inoculation into rat hippocampus, d311 was unable to replicate and spread within the brain, and did not cause any apparent local neuronal cell damage. These results demonstrate that d311 is highly attenuated for the rat central nervous system. d311 and other mutants of HSV containing major deletions of the nonessential genes within U(S) have the potential to serve as useful tools for gene transfer applications to brain.     

Vectorial release of poliovirus from polarized human intestinal epithelial cells

Polarized epithelial cells represent the primary barrier to virus infection of the host, which must also be traversed prior to virus dissemination from the infected organism. Although there is considerable information available concerning the release of enveloped viruses from such cells, relatively little is known about the processes involved in the dissemination of nonenveloped viruses. We have used two polarized epithelial cell lines, Vero C1008 (African green monkey kidney epithelial cells) and Caco-2 (human intestinal epithelial cells), infected with poliovirus and investigated the process of virus release. Release of poliovirus was observed to occur almost exclusively from the apical cell surface in Caco-2 cells, whereas infected Vero C1008 cells exhibited nondirectional release. Structures consistent with the vectorial transport of virus contained within vesicles or viral aggregates were observed by electron microscopy. Treatment with monensin or ammonium chloride partially inhibited virus release from Caco-2 cells. No significant cell lysis was observed at the times postinfection when extracellular virus was initially detected, and transepithelial resistance and vital dye uptake measurements showed only a moderate decrease. Brefeldin A was found to significantly and specifically inhibit poliovirus biosynthetic processes by an as yet uncharacterized mechanism. The vectorial release of poliovirus from the apical (or luminal) surface of human intestinal epithelial cells has significant implications for viral pathogenesis in the human gut.     

Protective vaccination against primary and recurrent disease caused by herpes simplex virus (HSV) type 2 using a genetically disabled HSV-1

The vaccine potential of a mutant herpes simplex virus (HSV) type 1, with a deletion in the glycoprotein H (gH) gene, was evaluated. The virus requires a gH-expressing cell line for multi-cycle growth but can complete a single cycle of infection in noncomplementing cells. Such viruses, termed DISC (disabled infectious single cycle) viruses, should be safe, yet still able to stimulate humoral and cell-mediated responses against a broad range of virus antigens in vaccinated hosts. Prophylactic vaccination of guinea pigs with DISC HSV-1, by ear scarification or direct infection of the vaginal mucosa, afforded a high degree of protection against HSV-2-induced primary genital disease and reduced significantly the frequency of subsequent disease recurrence. There was also a trend toward reduced recurrence following therapeutic vaccination of animals already infected with HSV-2. DISC HSV vaccination, therefore, offers an effective route for control of HSV disease.     

Novel entry pathway of bovine herpesvirus 1 and 5

Herpesviruses enter cells by a yet poorly understood mechanism. We visualized the crucial steps of the entry pathway of bovine herpesvirus 1 (BHV-1) and BHV-5 by transmission and scanning electron microscopy, employing cryotechniques that include time monitoring, ultrarapid freezing, and freeze substitution of cultured cells inoculated with virus. A key step in the entry pathway of both BHV-1 and BHV-5 is a unique fusion of the outer phospholipid layer of the viral envelope with the inner layer of the plasma membrane and vice versa resulting in "crossing" of the fused membranes and in partial insertion of the viral envelope into the plasma membrane. The fusion area is proposed to function as an axis for driving the virus particle into an invagination that is concomitantly formed close to the fusion site. The virus particle enters the cytoplasm through the opened tip of the invagination, and the viral envelope defuses from the plasma membrane. There is strong evidence that the intact virus particle is then transported to the nuclear region.     

Prevalence of herpes simplex virus type 1- and 2- specific antibodies among the acute, recurrent, and provoked types of female genital herpes

Sixty-eight sera from the acute, recurrent, and provoked types of female genital herpes were compared for the seroprevalence of herpes simplex virus (HSV) types 1 and 2 by immunodot assay using HSV glycoprotein G. In the HSV-1-isolated patients, no HSV-2 antibodies were detected, whereas in the HSV-2-isolated patients, HSV-1 seroprevalence was 9% for the acute type, 89% for the provoked type (P < 0.005), and 55% for the recurrent type (P < 0.05). The natural history of female genital herpes and the possible protective role of pre-existing antibodies in preventing the acquisition or clinical manifestation of a subsequent HSV infection are discussed.     

Inhibition of major histocompatibility complex class I antigen presentation in pig and primate cells by herpes simplex virus type 1 and 2 ICP47

Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) express an immediate-early protein, ICP47, that effectively inhibits the human transporter associated with antigen presentation (TAP), blocking major histocompatibility complex (MHC) class I antigen presentation to CD8+ T cells. Previous work indicated that the mouse TAP is relatively resistant to inhibition by the HSV-1 and HSV-2 ICP47 proteins (ICP47-1 and ICP47-2) and that mouse cells infected with HSV-1 are lysed by anti-HSV CD8+ cytotoxic T lymphocytes (CTL). Therefore, mice are apparently not suitable animals in which to study the in vivo effects of ICP47. In order to find an animal model, we introduced ICP47-1 and ICP47-2 into cells from various animal species-mice, rats, guinea pigs, rabbits, dogs, pigs, cows, monkeys, and humans-and measured TAP activity in the cells. Both proteins were unable to inhibit TAP in mouse, rat, guinea pig, and rabbit cells. In contrast, ICP47-1 and ICP47-2 inhibited TAP in pig, dog, cow, and monkey cells, and the TAP in pig and dog fibroblasts was often more sensitive to both proteins than TAP in human fibroblasts. These results were extended by measuring CD8+-T-cell recognition (CTL lysis) of cells from various species. Cells were infected with recombinant HSV-1 constructed to express murine MHC class I proteins so that the cells would be recognized and lysed by well-characterized murine anti-HSV CTL unless antigen presentation was blocked by ICP47. Anti-HSV CD8+ CTL effectively lysed pig and primate cells infected with a recombinant HSV-1 ICP47- mutant but were unable to lyse pig or primate cells infected with a recombinant HSV-1 that expressed ICP47. Therefore, pigs, dogs, and monkeys may be useful animal models in which to test the effects of ICP47 on HSV pathogenesis or the use of ICP47 as a selective immunosuppressive agent.     

Evidence of latency and reactivation of both herpes simplex virus (HSV)-1 and HSV-2 in the genital region

While superinfection with different herpes simplex virus (HSV) types has been demonstrated in animals, the ability of the two HSV types to colonize and reactivate in the same anatomic region in humans has not been well demonstrated. In 6 patients, both HSV-1 and HSV-2 was recovered from genital lesions. In 4 of them, who initially acquired genital HSV-1 infection, subsequent HSV-2 infection presented as a prolonged episode of genital lesions and a marked increase in the frequency of genital recurrences. While most of the subsequent clinical reactivations were HSV-2, in 2 patients the recurrence rate of genital HSV-1 increased after the acquisition of HSV-2. These data demonstrate the ability of a second HSV type to infect the same anatomic region and illustrate the difference in reactivation frequency of the two types in the same person. Typing of HSV isolates may be useful in persons with recent alteration in recurrence rates of genital HSV.     

Metals pollution in marine sediments of the United Arab Emirates creeks along the Arabian Gulf shoreline

In order to evaluate the association between seropositivity for herpes simplex virus (type 1 and type 2) and cervical intraepithelial neoplacia (CIN), we analysed data from a population-based case-control study of CIN grade II-III which included Norwegian women aged 20 to 44 years, 94 cases and 228 controls. Our objectives were to determine if HSV-1 and/or HSV-2 seropositivity were independent risk factors for CIN, taking human papillomavirus exposure into account, and to elucidate the combined effect of HPV positivity and seropositivity for HSV In logistic regression analyses, the association between HSV-2 or HSV-1 seropositivity and CIN II-III was not explained by HPV (adjusted OR 3.0; 95%, CI 1.3-7.2 and adjusted OR 3.3; 95% CI 1.3-8.4, respectively). In analyses restricted to HPV-16 DNA-positive individuals, seropositivity for HSV-2 increased the risk of CIN (OR 11.1; 95% CI 1.2-105.7), whereas HSV-1 seropositivity was not significantly associated with CIN. In women positive for other HPV types, only HSV-1 seropositivity was associated with CIN (OR 8.5; 95% CI 1.3-55.8). In analyses of the HPV-16-seropositive individuals, neither HSV-1 nor HSV-2 seropositivity was associated with CIN. Compared to the reference group of jointly unexposed subjects, the HPV-16 DNA-positive women who were anti-HSV-2 negative had an increased risk of CIN (OR 29; 95% CI 12-67), whereas the risk in women who were both HPV-16 DNA-positive and HSV-2 was OR=247 (95% CI 31-1996). The estimate of interaction was strong, but did not reach significance, and our findings may suggest that the combined effect of the two viruses is of aetiological importance in cervical carcinogenesis. Furthermore, the results indicate that HPV DNA positivity is not sufficient to explain the sexual behaviour-associated risk of cervical neoplasia and that further studies on the role of genital HSV (type 1 as well as type 2) and other STDs are warranted.     

The N-7-substituted acyclic nucleoside analog 2-amino-7-[(1,3-dihydroxy-2-propoxy)methyl]purine is a potent and selective inhibitor of herpesvirus replication

2-Amino-7-[(1,3-dihydroxy-2-propoxy)methyl]purine (compound S2242) represents the first antivirally active nucleoside analog with the side chain attached to the N-7 position of the purine ring. Compound S2242 strongly inhibits the in vitro replication of both herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) (50% effective concentration [EC50], 0.1 to 0.2 microgram/ml), varicella-zoster virus (EC50, 0.01 to 0.02 microgram/ml) and thymidine kinase (TK)-deficient strains of HSV (EC50, 0.4 microgram/ml) and varicella-zoster virus (EC50, 0.2 to 0.5 microgram/ml). Potent activity was also observed against murine cytomegalovirus (EC50, 1 microgram/ml), human cytomegalovirus (HCMV) (EC50, 0.04 to 0.1 microgram/ml), and human herpesvirus 6 (EC50, 0.0005 microgram/ml). Compound S2242 (i) was not cytotoxic to confluent Vero, HeLa, or human fibroblast cells at concentrations of > 100 micrograms/ml, (ii) proved somewhat more cytostatic to Vero, HEL, HeLa, and C127I cells than ganciclovir, and (iii) was markedly more cytostatic than ganciclovir to the growth of the human lymphocytic cell lines HSB-2 and CEM degrees. In contrast to ganciclovir, (i) compound S2242 proved not to be cytocidal to murine mammary carcinoma (FM3A) cells transfected with the HSV-1 or HSV-2 TK gene, (ii) exogenously added thymidine had only a limited effect on its anti-HSV-1 activity, and (iii) the compound was not phosphorylated by HSV-1-encoded TK derived from HSV-1 TK-transfected FM3A cells, indicating that the compound is not activated by a virally encoded TK. Compound S2242 inhibited (i) the expression of late HCHV antigens at an EC50 of 0.07 microgram/ml (0.6 microgram/ml for ganciclovir) and (ii) HCMV DNA synthesis at an EC50 of 0.1 microgram/ml (0.32 microgram/ml for ganciclovir), i.e., values that are close to the EC50S for inhibition of HCMV-induced cytopathogenicity. Neither ganciclovir nor S2242 had any effect on the expression of immediate-early HCMV antigens, which occurs before viral DNA synthesis. In time-of-addition experiments, S2242 behaved like ganciclovir and acyclovir; i.e., the addition of the drugs could be delayed until the onset of viral DNA synthesis.