Structural features of nectin-2 (HveB) required for herpes simplex virus entry

One step in the process of herpes simplex virus (HSV) entry into cells is the binding of viral glycoprotein D (gD) to a cellular receptor. Human nectin-2 (also known as HveB and Prr2), a member of the immunoglobulin (Ig) superfamily, serves as a gD receptor for the entry of HSV-2, variant forms of HSV-1 that have amino acid substitutions at position 25 or 27 of gD (for example, HSV-1/Rid), and porcine pseudorabies virus (PRV). The gD binding region of nectin-2 is believed to be localized to the N-terminal variable-like (V) Ig domain. In order to identify specific amino acid sequences in nectin-2 that are important for HSV entry activity, chimeric molecules were constructed by exchange of sequences between human nectin-2 and its mouse homolog, mouse nectin-2, which mediates entry of PRV but not HSV-1 or HSV-2. The nectin-2 chimeric molecules were expressed in Chinese hamster ovary cells, which normally lack a gD receptor, and tested for cell surface expression and viral entry activity. As expected, chimeric molecules containing the V domain of human nectin-2 exhibited HSV entry activity. Replacement of either of two small regions in the V domain of mouse nectin-2 with amino acids from the equivalent positions in human nectin-2 (amino acids 75 to 81 or 89) transferred HSV-1/Rid entry activity to mouse nectin-2. The resulting chimeras also exhibited enhanced HSV-2 entry activity and gained the ability to mediate wild-type HSV-1 entry. Replacement of amino acid 89 of human nectin-2 with the corresponding mouse amino acid (M89F) eliminated HSV entry activity. These results identify two different amino acid sequences, predicted to lie adjacent to the C' and C" beta-strands of the V domain, that are critical for HSV entry activity. This region is homologous to the human immunodeficiency virus binding region of CD4 and to the poliovirus binding region of CD155.     

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.     

Antigenic structure of soluble herpes simplex virus (HSV) glycoprotein D correlates with inhibition of HSV infection.

Soluble forms of herpes simplex virus (HSV) glycoprotein D (gD) block viral penetration. Likewise, most HSV strains are sensitive to gD-mediated interference by cells expressing gD. The mechanism of both forms of gD-mediated inhibition is thought to be at the receptor level. We analyzed the ability of different forms of soluble, truncated gD (gDt) to inhibit infection by different strains of HSV-1 and HSV-2. Strains that were resistant to gD-mediated interference were also resistant to inhibition by gDt, thereby suggesting a link between these two phenomena. Virion gD was the major viral determinant for resistance to inhibition by gDt. An insertion-deletion mutant, gD-1(delta 290-299t), had an enhanced inhibitory activity against most strains tested. The structure and function of gDt proteins derived from the inhibition-resistant viruses rid1 and ANG were analyzed. gD-1(ridlt) and gD-1(ANGt) had a potent inhibitory effect on plaque formation by wild-type strains of HSV but, surprisingly, little or no effect on their parental strains. As measured by quantitative enzyme-linked immunosorbent assay with a diverse panel of monoclonal antibodies, the antigenic structures of gD-1(rid1t) and gD-1(ANGt) were divergent from that of the wild type yet were similar to each other and to that of gD-1 (delta 290-299t). Thus, three different forms of gD have common antigenic changes that correlate with enhanced inhibitory activity against HSV. We conclude that inhibition of HSV infectivity by soluble gD is influenced by the antigenic conformation of the blocking gDt as well as the form of gD in the target virus.     

In vivo role of nectin-1 in entry of herpes simplex virus type 1 (HSV-1) and HSV-2 through the vaginal mucosa

Herpes simplex virus type 2 (HSV-2) is transmitted through the genital mucosa during sexual encounters. In recent years, HSV-1 has also become commonly associated with primary genital herpes. The mechanism of viral entry of HSV-1 and HSV-2 in the female genital tract is unknown. In order to understand the molecular interactions required for HSV entry into the vaginal epithelium, we examined the expression of herpesvirus entry mediator nectin-1 in the vagina of human and mouse at different stages of their hormonal cycle. Nectin-1 was highly expressed in the epithelium of human vagina throughout the menstrual cycle, whereas the mouse vaginal epithelium expressed nectin-1 only during the stages of the estrous cycle in which mice are susceptible to vaginal HSV infection. Furthermore, the ability of nectin-1 to mediate viral entry following intravaginal inoculation was examined in a mouse model of genital herpes. Vaginal infection with either HSV-1 or HSV-2 was blocked by preincubation of the virus with soluble recombinant nectin-1. Viral entry through the vaginal mucosa was also inhibited by preincubation of HSV-2 with antibody against gD. Together, these results suggest the importance of nectin-1 in mediating viral entry for both HSV-1 and HSV-2 in the genital mucosa in female hosts.     

Partial resistance to gD-mediated interference conferred by mutations affecting herpes simplex virus type 1 gC and gK.

Cells expressing herpes simplex virus (HSV) gD can be resistant to HSV entry as a result of gD-mediated interference. HSV strains differ in sensitivity to this interference, which blocks viral penetration but not binding. Previous studies have shown that mutations or variations in virion-associated gD can confer resistance to gD-mediated interference. Here we show that HSV-1 mutants selected for enhanced ability to bind and penetrate in the presence of inhibitory concentrations of heparin were partially resistant to gD-mediated interference. The resistance was largely due to the presence of two mutations: one in gC (the major heparin-binding glycoprotein) resulting in the absence of gC expression and the other in gK resulting in a syncytial phenotype. The results imply that heparin selected for mutants with altered postbinding requirements for entry. Resistance to gD-mediated interference conferred by mutations affecting gC and gK has not been previously described.     

Functional interaction between herpes simplex virus type 2 gD and HVEM transiently dampens local chemokine production after murine mucosal infection

Herpes virus entry mediator (HVEM) is one of two principal receptors mediating herpes simplex virus (HSV) entry into murine and human cells. It functions naturally as an immune signaling co-receptor, and may participate in enhancing or repressing immune responses depending on the natural ligand used. To investigate whether engagement of HVEM by HSV affects the in vivo response to HSV infection, we generated recombinants of HSV-2(333) that expressed wild-type gD (HSV-2/gD) or mutant gD able to bind to nectin-1 (the other principal entry receptor) but not HVEM. Replication kinetics and yields of the recombinant strains on Vero cells were indistinguishable from those of wild-type HSV-2(333). After intravaginal inoculation with mutant or wild-type virus, adult female C57BL/6 mice developed vaginal lesions and mortality in similar proportions, and mucosal viral titers were similar or lower for mutant strains at different times. Relative to HSV-2/gD, percentages of HSV-specific CD8(+) T-cells were similar or only slightly reduced after infection with the mutant strain HSV-2/gD-Δ7-15, in all tissues up to 9 days after infection. Levels of HSV-specific CD4(+) T-cells five days after infection also did not differ after infection with either strain. Levels of the cytokine IL-6 and of the chemokines CXCL9, CXCL10, and CCL4 were significantly lower in vaginal washes one day after infection with HSV-2/gD compared with HSV-2/gD-Δ7-15. We conclude that the interaction of HSV gD with HVEM may alter early innate events in the murine immune response to infection, without significantly affecting acute mortality, morbidity, or initial T-cell responses after lethal challenge.     

Amino acid substitutions in the V domain of nectin-1 (HveC) that impair entry activity for herpes simplex virus types 1 and 2 but not for Pseudorabies virus or bovine herpesvirus 1

The entry of herpes simplex virus (HSV) into cells requires the interaction of viral glycoprotein D (gD) with a cellular gD receptor to trigger the fusion of viral and cellular membranes. Nectin-1, a member of the immunoglobulin superfamily, can serve as a gD receptor for HSV types 1 and 2 (HSV-1 and HSV-2, respectively) as well as for the animal herpesviruses porcine pseudorabies virus (PRV) and bovine herpesvirus 1 (BHV-1). The HSV-1 gD binding domain of nectin-1 is hypothesized to overlap amino acids 64 to 104 of the N-terminal variable domain-like immunoglobulin domain. Moreover, the HSV-1 and PRV gDs compete for binding to nectin-1. Here we report that two amino acids within this region, at positions 77 and 85, are critical for HSV-1 and HSV-2 entry but not for the entry of PRV or BHV-1. Replacement of either amino acid 77 or amino acid 85 reduced HSV-1 and HSV-2 gD binding but had a lesser effect on HSV entry activity, suggesting that weak interactions between gD and nectin-1 are sufficient to trigger the mechanism of HSV entry. Substitution of both amino acid 77 and amino acid 85 in nectin-1 significantly impaired entry activity for HSV-1 and HSV-2 and eliminated binding to soluble forms of HSV-1 and HSV-2 gDs but did not impair the entry of PRV and BHV-1. Thus, amino acids 77 and 85 of nectin-1 form part of the interface with HSV gD or influence the conformation of that interface. Moreover, the binding sites for HSV and PRV or BHV-1 gDs on nectin-1 may overlap but are not identical.     

Development of a glycoprotein D-expressing dominant-negative and replication-defective herpes simplex virus 2 (HSV-2) recombinant viral vaccine against HSV-2 infection in mice

Using the T-REx (Invitrogen, California) gene switch technology and a dominant-negative mutant polypeptide of herpes simplex virus 1 (HSV-1)-origin binding protein UL9, we previously constructed a glycoprotein D-expressing replication-defective and dominant-negative HSV-1 recombinant viral vaccine, CJ9-gD, for protection against HSV infection and disease. It was demonstrated that CJ9-gD is avirulent following intracerebral inoculation in mice, cannot establish detectable latent infection following different routes of infection, and offers highly effective protective immunity against primary HSV-1 and HSV-2 infection and disease in mouse and guinea pig models of HSV infections. Given these favorable safety and immunological profiles of CJ9-gD, aiming to maximize levels of HSV-2 glycoprotein D (gD2) expression, we have constructed an ICP0 null mutant-based dominant-negative and replication-defective HSV-2 recombinant, CJ2-gD2, that contains 2 copies of the gD2 gene driven by the tetracycline operator (tetO)-bearing HSV-1 major immediate-early ICP4 promoter. CJ2-gD2 expresses gD2 as efficiently as wild-type HSV-2 infection and can lead to a 150-fold reduction in wild-type HSV-2 viral replication in cells coinfected with CJ2-gD2 and wild-type HSV-2 at the same multiplicity of infection. CJ2-gD2 is avirulent following intracerebral injection and cannot establish a detectable latent infection following subcutaneous (s.c.) immunization. CJ2-gD2 is a more effective vaccine than HSV-1 CJ9-gD and a non-gD2-expressing dominant-negative and replication-defective HSV-2 recombinant in protection against wild-type HSV-2 genital disease. Using recall response, we showed that immunization with CJ2-gD2 elicited strong HSV-2-specific memory CD4(+) and CD8(+) T-cell responses. Collectively, given the demonstrated preclinical immunogenicity and its unique safety profiles, CJ2-gD2 represents a new class of HSV-2 replication-defective recombinant viral vaccines in protection against HSV-2 genital infection and disease.     

Glycoprotein D-independent spread of pseudorabies virus infection in cultured peripheral nervous system neurons in a compartmented system

The molecular mechanisms underlying the directional neuron-to-epithelial cell transport of herpesvirus particles during infection are poorly understood. To study the role of the viral glycoprotein D (gD) in the directional spread of herpes simplex virus (HSV) and pseudorabies virus (PRV) infection, a culture system consisting of sympathetic neurons or epithelial cells in different compartments was employed. We discovered that PRV infection could spread efficiently from neurons to cells and back to neurons in the absence of gD, the viral ligand required for entry of extracellular particles. Unexpectedly, PRV infection can also spread transneuronally via axo-axonal contacts. We show that this form of interaxonal spread between neurons is gD independent and is not mediated by extracellular virions. We also found that unlike PRV gD, HSV-1 gD is required for neuron-to-cell spread of infection. Neither of the host cell gD receptors (HVEM and nectin-1) is required in target primary fibroblasts for neuron-to-cell spread of HSV-1 or PRV infection.     

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.     

Insertion mutants of herpes simplex virus have a duplication of the glycoprotein D gene and express two different forms of glycoprotein D.

We produced insertion mutants of herpes simplex virus (HSV) that contain two functional copies of genes encoding different forms of glycoprotein D (gD). These viruses have the gene for HSV type 2 (HSV-2) gD at the normal locus and the gene for HSV-1 gD inserted into the thymidine kinase locus. Results of immunoprecipitation experiments done with monoclonal antibodies revealed that both gD genes were expressed by these viruses, regardless of orientation of the inserted HSV-1 gD gene, and that maximal synthesis of both glycoproteins depended on viral DNA replication. This apparently normal expression of the inserted HSV-1 gD gene was from a DNA fragment (SacI fragment, 0.906 to 0.924 map units) containing nucleotide sequences extending from approximately 400 base pairs upstream of the 5' end of the gD mRNA to about 200 base pairs upstream of the 3' end. The glycoproteins expressed from both genes were incorporated into the surfaces of infected cells. Electrophoretic analyses of purified virions and neutralization studies suggest that both glycoproteins were also incorporated into virions. This nonpreferential utilization of both gene products makes these viruses ideal strains for the generation and characterization of a variety of mutations.     

Disruption of adherens junctions liberates nectin-1 to serve as receptor for herpes simplex virus and pseudorabies virus entry

Nectin-1, a cell adhesion molecule belonging to the immunoglobulin superfamily, can bind to virion glycoprotein D (gD) to mediate entry of herpes simplex viruses (HSV) and pseudorabies virus (PRV). Nectin-1 colocalizes with E-cadherin at adherens junctions in epithelial cells. The disruption of cell junctions can result in the redistribution of nectin-1. To determine whether disruption of junctions by calcium depletion influenced the susceptibility of epithelial cells to viral entry, Madin-Darby canine kidney cells expressing endogenous nectin-1 or transfected human nectin-1 were tested for the ability to bind soluble forms of viral gD and to be infected by HSV and PRV, before and after calcium depletion. Confocal microscopy revealed that binding of HSV and PRV gD was localized to adherens junctions in cells maintained in normal medium but was distributed, along with nectin-1, over the entire cell surface after calcium depletion. Both the binding of gD and the fraction of cells that could be infected by HSV-1 and PRV were enhanced by calcium depletion. Taken together, these results provide evidence that nectin-1 confined to adherens junctions in epithelial cells is not very accessible to virus, whereas dissociation of cell junctions releases nectin-1 to serve more efficiently as an entry receptor.     

Herpesvirus entry mediator and nectin-1 mediate herpes simplex virus 1 infection of the murine cornea

Herpes simplex virus 1 (HSV-1) is a ubiquitous human pathogen that enters cells by the receptor-mediated fusion of the viral envelope with a host cell membrane. The envelope glycoprotein gD of HSV must bind to one of its receptors for entry to take place. Recent studies using knockout (KO) mice demonstrated that the gD receptors herpesvirus entry mediator (HVEM) and nectin-1 are the primary entry receptors for HSV-2 in the mouse vagina and brain. Nectin-1 was most crucial for the neuronal spread of HSV-2, particularly in the brain. HVEM was dispensable for infection in these models, but when both HVEM and nectin-1 were absent, infection was completely prevented. We sought to determine the receptor requirements of HSV-1 in an ocular model of infection using knockout mice. Wild-type, HVEM KO, nectin-1 KO, and HVEM/nectin-1 double-KO mice were infected via corneal scarification and monitored for clinical signs of infection and viral replication in various tissues. We report that either HVEM or nectin-1 must be present for HSV-1 infection of the cornea. Additionally, we observed that the infection was attenuated in both HVEM KO and nectin-1 KO mice. This is in contrast to what was reported for studies of HSV-2 in vagina and brain and suggests that receptor requirements for HSV vary depending on the route of inoculation and/or serotype.     

Neutralizing Antibodies Inhibit Axonal Spread of Herpes Simplex Virus Type 1 to Epidermal Cells In Vitro

The ability of antibodies to interfere with anterograde transmission of herpes simplex virus (HSV) from neuronal axons to the epidermis was investigated in an in vitro model consisting of human fetal dorsal root ganglia innervating autologous skin explants in a dual-chamber tissue culture system. The number and size of viral cytopathic plaques in epidermal cells after axonal transmission from HSV type 1 (HSV-1)-infected dorsal root ganglionic neurons were significantly reduced by addition to the outer chamber of neutralizing polyclonal human sera to HSV-1, of a human recombinant monoclonal group Ib antibody to glycoprotein D (gD), and of rabbit sera to HSV-1 gB and gD but not by rabbit anti-gE or anti-gG. A similar pattern of inhibition of direct infection of epidermal cells by these antibodies was observed. High concentrations of the monoclonal anti-gD reduced transmission by 90%. Rabbit anti-gB was not taken up into neurons, and human anti-gD did not influence spread of HSV in the dorsal root ganglia or axonal transport of HSV antigens when applied to individual dissociated neurons. These results suggest that anti-gD and -gB antibodies interfere with axonal spread of HSV-1, possibly by neutralizing HSV during transmission across an intercellular gap between axonal termini and epidermal cells, and thus contribute to control of HSV spread and shedding. Therefore, selected human monoclonal antibodies to protective epitopes might even be effective in preventing epidermis-to-neuron transmission during primary HSV infection, especially neonatal infection.     

Cells expressing herpes simplex virus glycoprotein gC but not gB, gD, or gE are recognized by murine virus-specific cytotoxic T lymphocytes

To determine which viral molecule(s) is recognized by herpes simplex virus (HSV)-specific cytotoxic T lymphocytes (CTL), target cells were constructed which express individual HSV glycoproteins. A mouse L cell line, Z4/6, which constitutively expressed high levels of HSV type 2 (HSV-2) gD (gD-2) was isolated and characterized previously (D. C. Johnson and J. R. Smiley, J. Virol. 54:682-689, 1985). Despite the expression of gD on the surface of Z4/6 cells, these cells were not killed by anti-HSV-2 CTL generated following intravaginal infection of syngeneic mice. In contrast, parental Z4 or Z4/6 cells infected with HSV-2 were lysed. Furthermore, unlabeled Z4/6 cells were unable to block the lysis of HSV-2-infected labeled target cells. Cells which express HSV-1 gB (gB-1) were isolated by transfecting L cells with the recombinant plasmid pSV2gBneo, which contains the HSV-1 gB structural sequences and the neomycin resistance gene coupled to the simian virus 40 early promoter and selecting G418-resistant cell lines. One such cell line, Lta/gB15, expressed gB which was detected by immunoprecipitation and at the cell surface by immunofluorescence. Additionally, cells expressing HSV-1 gC (gC-1) or gE (gE-1) were isolated by transfecting Z4 cells, which are L cells expressing ICP4 and ICP47, with either the recombinant plasmid pGE15neo, which contains the gE structural sequences and the neomycin resistance gene, or pDC17, which contains the gC structural gene coupled to the gD-1 promoter. A number of G418-resistant cell lines were isolated which expressed gC-1 or gE-1 at the cell surface. Anti-HSV-1 CTL generated following footpad infection of syngeneic mice were unable to lyse target cells expressing gB-1 or gE-1. In contrast, target cells expressing very low levels of gC-1 were killed as well as HSV-1-infected target cells. Furthermore, infection of gC-1-transformed target cells with wild-type HSV-1 or a strain of HSV-1 that does not express gC did not result in a marked increase in susceptibility to lysis. These results suggest that murine class I major histocompatibility complex-restricted anti-HSV CTL recognize gC-1 but do not recognize gB, gD, or gE as these molecules are expressed in transfected syngeneic target cells. The results are discussed in terms of recent evidence concerning the specificity of antiviral CTL.     

Structure of herpes simplex virus glycoprotein D bound to the human receptor nectin-1

Binding of herpes simplex virus (HSV) glycoprotein D (gD) to a cell surface receptor is required to trigger membrane fusion during entry into host cells. Nectin-1 is a cell adhesion molecule and the main HSV receptor in neurons and epithelial cells. We report the structure of gD bound to nectin-1 determined by x-ray crystallography to 4.0 Å resolution. The structure reveals that the nectin-1 binding site on gD differs from the binding site of the HVEM receptor. A surface on the first Ig-domain of nectin-1, which mediates homophilic interactions of Ig-like cell adhesion molecules, buries an area composed by residues from both the gD N- and C-terminal extensions. Phenylalanine 129, at the tip of the loop connecting β-strands F and G of nectin-1, protrudes into a groove on gD, which is otherwise occupied by C-terminal residues in the unliganded gD and by N-terminal residues in the gD/HVEM complex. Notably, mutation of Phe129 to alanine prevents nectin-1 binding to gD and HSV entry. Together these data are consistent with previous studies showing that gD disrupts the normal nectin-1 homophilic interactions. Furthermore, the structure of the complex supports a model in which gD-receptor binding triggers HSV entry through receptor-mediated displacement of the gD C-terminal region.

Authors Summary

Herpes simplex virus (HSV) is a widespread human pathogen. Four viral glycoproteins (gD, gB, gH/gL) are required for HSV entry into host cells. gD binding to a cell surface receptor triggers conformational changes in the other viral glycoproteins leading to membrane fusion and viral entry. Two structurally unrelated cellular protein receptors, nectin-1 and HVEM, can mediate HSV entry upon binding to gD. The structure presented here reveals the molecular basis for the stable interaction between HSV-1 gD and the receptor nectin-1. Comparison with the previously determined structures of the gD/HVEM complex and unliganded gD shows that, despite the fact that the two receptors interact with different binding sites, they both cause a similar conformational change in gD. Therefore, our data point to a conserved mechanism for receptor mediated activation of the HSV entry process. In addition, the gD/Nectin-1 structure reveals that the gD-binding site overlaps with a surface involved in nectin-1 homo-dimerization. This observation explains how gD interferes with the cell adhesion function of nectin-1. Finally, the gD/Nectin-1 complex displays similarities with other viral ligands bound to immunoglobulin-like receptors suggesting a convergent mechanism for receptors selection and usage.     

Striking similarity of murine nectin-1alpha to human nectin-1alpha (HveC) in sequence and activity as a glycoprotein D receptor for alphaherpesvirus entry

A cDNA encoding the murine homolog of human nectin-1alpha (also known as poliovirus receptor-related protein 1 [Prr1] and herpesvirus entry protein C [HveC]) was isolated. The protein encoded by this cDNA proved to be 95% identical in sequence to the human protein and to have similar herpesvirus entry activity. Upon expression of the murine cDNA in hamster cells resistant to alphaherpesvirus entry, the cells became susceptible to the entry of herpes simplex virus types 1 and 2 (HSV-1 and -2), pseudorabies virus, and bovine herpesvirus 1. HSV envelope glycoprotein D (gD), a viral ligand for human nectin-1alpha, is also a ligand for the murine homolog based on evidence that (i) a soluble hybrid protein composed in part of the murine nectin-1 ectodomain bound specifically to purified soluble forms of HSV-1 and HSV-2 gD as demonstrated by enzyme-linked immunosorbent assay, (ii) a soluble hybrid of HSV-1 gD bound to hamster cells expressing murine nectin-1alpha but not to control cells, and (iii) cells expressing both murine nectin-1alpha and one of the alphaherpesvirus gDs were resistant to entry of HSV-1, indicative of interference with entry resulting from interactions of cell-associated gD with the entry receptor. Northern blot analysis revealed that nectin-1 is expressed in most of the mouse tissues examined and at high levels in the brain, skin, and kidneys. Immunocytochemical localization demonstrated the presence of nectin-1 in epithelial cells of the mouse vagina and also in neuronal cells of the central nervous system, suggesting an expression pattern relevant to both infection at a portal of entry and spread of infection to the brain.     

A role for 3-O-sulfotransferase isoform-4 in assisting HSV-1 entry and spread

Many heparan sulfate (HS) 3-O-sulfotransferase (3-OST) isoforms generate cellular receptors for herpes simplex virus type-1 (HSV-1) glycoprotein D (gD). Interestingly, the ability of 3-OST-4 to mediate HSV-1 entry and cell-to-cell fusion has not been determined, although it is predominantly expressed in the brain, a primary target of HSV-1 infections. We report that expression of 3-OST-4 can render Chinese hamster ovary K1 (CHO-K1) cells susceptible to entry of wild-type and a mutant (Rid1) strain of HSV-1. Evidence for generation of gD receptors by 3-OST-4 was suggested by gD-mediated interference assay and the ability of 3-OST-4 expressing CHO-K1 cells to preferentially bind HSV-1 gD, which could be reversed by prior treatment of cells with HS lyases (heparinases-II/III). In addition, 3-OST-4 expressing CHO-K1 cells acquired the ability to fuse with cells-expressing HSV-1 glycoproteins. Demonstrating specificity, the cell fusion was inhibited by soluble 3-O-sulfated forms of HS, but not unmodified HS. Taken together our results suggest a role of 3-OST-4 in HSV-1 pathogenesis.     

Identification of novel immunodominant CD4+ Th1-type T-cell peptide epitopes from herpes simplex virus glycoprotein D that confer protective immunity

The molecular characterization of the epitope repertoire on herpes simplex virus (HSV) antigens would greatly expand our knowledge of HSV immunity and improve immune interventions against herpesvirus infections. HSV glycoprotein D (gD) is an immunodominant viral coat protein and is considered an excellent vaccine candidate antigen. By using the TEPITOPE prediction algorithm, we have identified and characterized a total of 12 regions within the HSV type 1 (HSV-1) gD bearing potential CD4(+) T-cell epitopes, each 27 to 34 amino acids in length. Immunogenicity studies of the corresponding medium-sized peptides confirmed all previously known gD epitopes and additionally revealed four new immunodominant regions (gD(49-82), gD(146-179), gD(228-257), and gD(332-358)), each containing naturally processed epitopes. These epitopes elicited potent T-cell responses in mice of diverse major histocompatibility complex backgrounds. Each of the four new immunodominant peptide epitopes generated strong CD4(+) Th1 T cells that were biologically active against HSV-1-infected bone marrow-derived dendritic cells. Importantly, immunization of H-2(d) mice with the four newly identified CD4(+) Th1 peptide epitopes but not with four CD4(+) Th2 peptide epitopes induced a robust protective immunity against lethal ocular HSV-1 challenge. These peptide epitopes may prove to be important components of an effective immunoprophylactic strategy against herpes.     

Mutations in the N termini of herpes simplex virus type 1 and 2 gDs alter functional interactions with the entry/fusion receptors HVEM,nectin-2, and 3-O-sulfated heparan sulfate but not with nectin-1

Multiple cell surface molecules (herpesvirus entry mediator [HVEM], nectin-1, nectin-2, and 3-O-sulfated heparan sulfate) can serve as entry receptors for herpes simplex virus type 1 (HSV-1) or HSV-2 and also as receptors for virus-induced cell fusion. Viral glycoprotein D (gD) is the ligand for these receptors. A previous study showed that HVEM makes contact with HSV-1 gD at regions within amino acids 7 to 15 and 24 to 32 at the N terminus of gD. In the present study, amino acid substitutions and deletions were introduced into the N termini of HSV-1 and HSV-2 gDs to determine the effects on interactions with all of the known human and mouse entry/fusion receptors, including mouse HVEM, for which data on HSV entry or cell fusion were not previously reported. A cell fusion assay was used to assess functional activity of the gD mutants with each entry/fusion receptor. Soluble gD:Fc hybrids carrying each mutation were tested for the ability to bind to cells expressing the entry/fusion receptors. We found that deletions overlapping either or both of the HVEM contact regions, in either HSV-1 or HSV-2 gD, severely reduced cell fusion and binding activity with all of the human and mouse receptors except nectin-1. Amino acid substitutions described previously for HSV-1 (L25P, Q27P, and Q27R) were individually introduced into HSV-2 gD and, for both serotypes, were found to be without effect on cell fusion and the binding activity for nectin-1. Each of these three substitutions in HSV-1 gD enhanced fusion with cells expressing human nectin-2 (ordinarily low for wild-type HSV-1 gD), but the same substitutions in HSV-2 gD were without effect on the already high level of cell fusion observed with the wild-type protein. The Q27P or Q27R substitution in either HSV-1 and HSV-2 gD, but not the L25P substitution, significantly reduced cell fusion and binding activity for both human and mouse HVEM. Each of the three substitutions in HSV-1 gD, as well as the deletions mentioned above, reduced fusion with cells bearing 3-O-sulfated heparan sulfate. Thus, the N terminus of HSV-1 or HSV-2 gD is not necessary for functional interactions with nectin-1 but is necessary for all of the other receptors tested here. The sequence of the N terminus determines whether nectin-2 or 3-O-sulfated heparan sulfate, as well as HVEM, can serve as entry/fusion receptors.