Herpes simplex virus with highly reduced gD levels can efficiently enter and spread between human keratinocytes.

The rapid spread of herpes simplex virus type 1 (HSV-1) in mucosal epithelia and neuronal tissue depends primarily on the ability of the virus to navigate within polarized cells and the tissues they constitute. To understand HSV entry and the spread of virus across cell junctions, we have previously characterized a human keratinocyte cell line, HaCaT. These cells appear to reflect cells infected in vivo more accurately than many of the cultured cells used to propagate HSV. HSV mutants lacking gE/gI are highly compromised in spread within epithelial and neuronal tissues and also show defects in cell-to-cell spread in HaCaT cells, but not in other, nonpolarized cells. HSV gD is normally considered absolutely essential for entry and cell-to-cell spread, both in cultured cells and in vivo. Here, an HSV-1 gD mutant virus, F-US6kan, was found to efficiently enter HaCaT cells and normal human keratinocytes and could spread from cell to cell without gD provided by complementing cells. By contrast, entry and spread into other cells, especially highly transformed cells commonly used to propagate HSV, were extremely inefficient. Further analyses of F-US6kan indicated that this mutant expressed extraordinarily low (1/500 wild-type) levels of gD. Neutralizing anti-gD monoclonal antibodies inhibited entry of F-US6kan, suggesting F-US6kan utilized this small amount of gD to enter cells. HaCaT cells expressed high levels of an HSV gD receptor, HveC, and entry of F-US6kan into HaCaT cells could also be inhibited with antibodies specific for HveC. Interestingly, anti-HveC antibodies were not fully able to inhibit entry of wild-type HSV-1 into HaCaT cells. These results help to uncover important properties of HSV and human keratinocytes. HSV, with exceedingly low levels of a crucial receptor-binding glycoprotein, can enter cells expressing high levels of receptor. In this case, surplus gD may be useful to avoid neutralization by anti-gD antibodies.

Herpes simplex virus glycoprotein D bound to the human receptor HveA.

Herpes simplex virus (HSV) infection requires binding of the viral envelope glycoprotein D (gD) to cell surface receptors. We report the X-ray structures of a soluble, truncated ectodomain of gD both alone and in complex with the ectodomain of its cellular receptor HveA. Two bound anions suggest possible binding sites for another gD receptor, a 3-O-sulfonated heparan sulfate. Unexpectedly, the structures reveal a V-like immunoglobulin (Ig) fold at the core of gD that is closely related to cellular adhesion molecules and flanked by large N- and C-terminal extensions. The receptor binding segment of gD, an N-terminal hairpin, appears conformationally flexible, suggesting that a conformational change accompanying binding might be part of the viral entry mechanism.

Use of chimeric nectin-1(HveC)-related receptors to demonstrate that ability to bind alphaherpesvirus gD is not necessarily sufficient for viral entry.

Human nectin-1 (HveC, Prr1), a member of the immunoglobulin superfamily and a receptor for the entry of herpes simplex viruses 1 and 2 (HSV-1, HSV-2), pseudorabies virus (PRV), and bovine herpesvirus 1 (BHV-1), binds to viral gD. For HSV-1, HSV-2, and PRV, the gD-binding region of nectin-1 has been localized to the N-terminal V-like domain. To determine whether the two C-like domains of nectin-1 influenced gD binding and entry activity, genes encoding chimeric proteins were constructed. Portions of nectin-1 were replaced with homologous regions from nectin-2 (HveB, Prr2), a related protein with ability to mediate the entry of PRV, HSV-2, and Rid mutants of HSV-1, but not HSV-1 or BHV-1. Also, one or more domains of nectin-1 were fused to the two membrane-proximal Ig domains of CD4, a protein with no herpesvirus entry or gD-binding activity. The chimeric proteins were expressed in Chinese hamster ovary cells, which normally lack alphaherpesvirus entry receptors, and detected on the cell surface by one or more anti-nectin-1 monoclonal antibodies. One chimeric protein (nectin-1 amino acids 1-124 fused to CD4) failed to bind to soluble forms of HSV-1, HSV-2, PRV, and BHV-1 gD and, as expected, also failed to mediate entry of the viruses from which these gDs were derived. The other chimeric receptors bound all forms of gD. Some mediated the entry of all the viruses tested but others mediated entry of some but not all the viruses. We conclude that binding of gD to the nectin-1 V domain is not sufficient for entry activity, that there are structural requirements for entry activity independent of gD binding, and that these requirements are different for the several alphaherpesviruses that can use nectin-1 as a receptor.

Development of a syngenic murine B16 cell line-derived melanoma susceptible to destruction by neuroattenuated HSV-1.

HSV-1 ICP34.5 mutants can slow progression of preformed tumors in rodent models. However, the current models available for study are limited due to the lack of a syngenic, low-immunogenic tumor model susceptible to HSV-1. Thus we have developed a new model to determine the role of the immune response in viral-mediated tumor destruction. The human herpesvirus entry (Hve) receptors (HveA, HveB, and HveC) and a control plasmid were transfected into B78H1 murine melanoma cells. Transfection of HveA and HveC conferred sensitivity to HSV-1 to these cells. A10 (HveA), C10 (HveC), and control cells were able to form tumors reproducibly in vivo. The transfection of the receptors into B78H1 cells did not induce a detectable in vivo immunogenicity to the tumors. Finally, A10 and C10 tumor-bearing mice treated with HSV-1 1716 had significant prolongation of survival compared to mock-treated mice. These data suggest that A10 and C10 will be useful as in vivo models for studying the role of the immune response in viral-mediated tumor destruction.

Porcine HveC, a member of the highly conserved HveC/nectin 1 family, is a functional alphaherpesvirus receptor.

Human herpesvirus entry mediator C (HveC) is an alphaherpesvirus receptor which binds to virion glycoprotein D (gD). We identified porcine HveC and studied its interaction with pseudorabies virus (PrV) and herpes simplex virus type 1 (HSV-1) gD. Porcine and human HveC have 96% amino acid identity and HveC from African green monkey, mouse, hamster, and cow are similarly conserved. Porcine HveC mediates entry of HSV-1, HSV-2, PrV, and bovine herpesvirus type 1. Truncated soluble forms of HSV-1 and PrV gD bind competitively to porcine HveC. Biosensor analysis shows that PrV gD binds with a 10-fold higher affinity than HSV-1 gD. Monoclonal antibodies against human HveC recognize the porcine homologue and can block gD binding and entry of HSV-1 and PrV. Porcine HveC is functionally indistinguishable from human HveC. Our results are consistent with the suggestion that HveC is a pan-alphaherpesvirus receptor that interacts with a conserved structural domain of gD.

Glycoprotein D homologs in herpes simplex virus type 1, pseudorabies virus, and bovine herpes virus type 1 bind directly to human HveC(nectin-1) with different affinities.

Distinct subsets of human receptors for alphaherpesviruses mediate the entry of herpes simplex virus (HSV), pseudorabies virus (PrV), or bovine herpes virus type 1 (BHV-1) into cells. Glycoprotein D (gD) is essential for receptor-mediated entry of all three viruses into cells. However, the gD homologs of these viruses share only 22-33% amino acid identity. Several entry receptors for HSV have been identified. Two of these, HveA (HVEM) and HveC (nectin-1), mediate entry of most HSV-1 and HSV-2 strains and are bound directly by HSV gD. A third receptor, HveB (nectin-2), mediates entry of HSV-2 and only a limited number of HSV-1 strains. HveB and HveC can also serve as entry receptors for PrV, whereas only HveC can serve this function for BHV-1. We show here that gD from PrV and BHV-1 binds directly to the human receptors that mediate PrV and BHV-1 entry. We expressed soluble forms of PrV gD and BHV-1 gD using recombinant baculoviruses and purified each protein. Using ELISA, we detected direct binding of PrV gD to HveB and HveC and direct binding of BHV-1 gD to HveC. Biosensor analysis revealed that PrV gD had a 10-fold higher affinity than HSV-1 gD for human HveC. In contrast, the binding of BHV-1 gD to HveC was weak. PrV gD and HSV-1 gD competed for binding to the V domain of HveC and both inhibited entry of the homologous and heterologous viruses. These data suggest that the two forms of gD bind to a common region on human HveC despite their low amino acid similarity. Based on affinities for human HveC, we predict a porcine HveC homolog may be important for PrV infection in its natural host, whereas a BHV-1 infection in its natural host may be mediated by a receptor other than a bovine HveC homolog.

Localization of the gD-binding region of the human herpes simplex virus receptor, HveA.

During virus entry, herpes simplex virus (HSV) glycoprotein D (gD) binds to one of several human cellular receptors. One of these, herpesvirus entry mediator A (HveA), is a member of the tumor necrosis factor receptor (TNFR) superfamily, and its ectodomain contains four characteristic cysteine-rich pseudorepeat (CRP) elements. We previously showed that gD binds the ectodomain of HveA expressed as a truncated, soluble protein [HveA(200t)]. To localize the gD-binding domain of HveA, we expressed three additional soluble forms of HveA consisting of the first CRP [HveA(76t)], the second CRP [HveA(77-120t)], or the first and second CRPs [HveA(120t)]. Biosensor and enzyme-linked immunosorbent assay studies showed that gD bound to HveA(120t) and HveA(200t) with the same affinity. However, gD did not bind to HveA(76t) or HveA(77-120t). Furthermore, HveA(200t) and HveA(120t), but not HveA(76t) or HveA(77-120t), blocked herpes simplex virus (HSV) entry into CHO cells expressing HveA. We also generated six monoclonal antibodies (MAbs) against HveA(200t). MAbs CW1, -2, and -4 bound linear epitopes within the second CRP, while CW7 and -8 bound linear epitopes within the third or fourth CRPs. None of these MAbs blocked the binding of gD to HveA. In contrast, MAb CW3 recognized a discontinuous epitope within the first CRP of HveA, blocked the binding of gD to HveA, and exhibited a limited ability to block virus entry into cells expressing HveA, suggesting that the first domain of HveA contains at least a portion of the gD binding site. The inability of gD to bind HveA(76t) suggests that additional amino acid residues of the gD binding site may reside within the second CRP.

Localization of a binding site for herpes simplex virus glycoprotein D on herpesvirus entry mediator C by using antireceptor monoclonal antibodies.

The human herpesvirus entry mediator C (HveC), also known as the poliovirus receptor-related protein 1 (PRR1) and as nectin-1, allows the entry of herpes simplex virus type 1 (HSV-1) and HSV-2 into mammalian cells. The interaction of virus envelope glycoprotein D (gD) with such a receptor is an essential step in the process leading to membrane fusion. HveC is a member of the immunoglobulin (Ig) superfamily and contains three Ig-like domains in its extracellular portion. The gD binding site is located within the first Ig-like domain (V domain) of HveC. We generated a panel of monoclonal antibodies (MAbs) against the ectodomain of HveC. Eleven of these, which detect linear or conformational epitopes within the V domain, were used to map a gD binding site. They allowed the detection of HveC by enzyme-linked immunosorbent assay, Western blotting, and biosensor analysis or directly on the surface of HeLa cells and human neuroblastoma cell lines, as well as simian Vero cells. The anti-HveC V-domain MAbs CK6, CK8, and CK41, as well as the previously described MAb R1.302, blocked HSV entry. Their binding to soluble HveC was blocked by the association of gD with the receptor, indicating that their epitopes overlap a gD binding site. Competition assays on an optical biosensor showed that CK6 and CK8 (linear epitopes) inhibited the binding of CK41 and R1.302 (conformational epitopes) to HveC and vice versa. Epitope mapping showed that CK6 and CK8 bound between residues 80 and 104 of HveC, suggesting that part of the gD binding site colocalizes in the same region.

The lymphotoxin-beta receptor is necessary and sufficient for LIGHT-mediated apoptosis of tumor cells.

LIGHT is a tumor necrosis factor (TNF) ligand superfamily member, which binds two known cellular receptors, lymphotoxin-beta receptor (LTbetaR) and the herpesvirus entry mediator (HveA). LIGHT is a homotrimer that activates proapoptotic and integrin-inducing pathways. Receptor binding residues via LIGHT were identified by introducing point mutations in the A' --> A" and D --> E loops of LIGHT, which altered binding to LTbetaR and HveA. One mutant of LIGHT exhibits selective binding to HveA and is inactive triggering cell death in HT29.14s cells or induction of ICAM-1 in fibroblasts. Studies with HveA- or LTbetaR-specific antibodies further indicated that HveA does not contribute, either cooperatively or by direct signaling, to the death pathway activated by LIGHT. LTbetaR, not HveA, recruits TNF receptor-associated factor-3 (TRAF3), and LIGHT-induced death is blocked by a dominant negative TRAF3 mutant. Together, these results indicate that TRAF3 recruitment propagates death signals initiated by LIGHT-LTbetaR interaction and implicates a distinct biological role for LIGHT-HveA system.