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.

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.

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.

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.

Two distinct binding affinities of poliovirus for its cellular receptor.

To study the kinetics and equilibrium of poliovirus binding to the poliovirus receptor, we used surface plasmon resonance to examine the interaction of a soluble form of the receptor with poliovirus. Soluble receptor purified from mammalian cells is able to bind poliovirus, neutralize viral infectivity, and induce structural changes in the virus particle. Binding studies revealed that there are two binding sites for the receptor on the poliovirus type 1 capsid, with affinity constants at 20 degrees C of K(D)(1) = 0.67 microm and K(D)(2) = 0.11 microm. The relative abundance of the two binding sites varies with temperature. At 20 degrees C, the K(D)(2) site constitutes approximately 46% of the total binding sites on the sensor chip, and its relative abundance decreased with decreasing temperature such that at 5 degrees C, the relative abundance of the K(D)(2) site is only 12% of the total binding sites. Absolute levels of the K(D)(1) site remained relatively constant at all temperatures tested. The two binding sites may correspond to docking sites for domain 1 of the receptor on the viral capsid, as predicted by a model of the poliovirus-receptor complex. Alternatively, the binding sites may be a consequence of structural breathing, or could result from receptor-induced conformational changes in the virus.

The three HveA receptor ligands, gD, LT-alpha and LIGHT bind to distinct sites on HveA.

The herpes virus entry mediator A (HveA), a member of the tumor necrosis factor receptor (TNFR) superfamily, interacts with three different protein ligands; lymphotoxin-alpha (LT-alpha) and LIGHT (LIGHT stands for lymphotoxin homolog, which exhibits inducible expression and competes with HSV glycoprotein D for HveA and is expressed on T-lymphocytes) from the host and the herpes simplex virus (HSV) surface glycoprotein gD. It has been reported that the gD binding site on HveA is located within the receptor's two N-terminal CRP domains, and that gD and LIGHT compete for their binding to HveA. However, whether these ligands interact with the same or different sites on the receptor is unclear. We analyzed and compared the sites of interaction between HveA and its TNF ligands, by using two recombinant forms of the receptor, comprising the full-receptor ectodomain (HveA (200t)) and its two first CRP domains (HveA (120t)), as well as several monoclonal antibodies recognizing HveA. Two HveA peptide ligands (BP-1 and BP-2) that differentially inhibit binding of soluble gD and LT-alpha to the receptor were also used to demonstrate that gD, LIGHT and LT-alpha bind to distinct sites on the receptor. Our results suggest that binding of a ligand to HveA may alter the conformation of this receptor, thereby affecting its interaction with its other ligands.

Structure of the HIV-1 integrase catalytic domain complexed with an inhibitor: a platform for antiviral drug design.

HIV integrase, the enzyme that inserts the viral DNA into the host chromosome, has no mammalian counterpart, making it an attractive target for antiviral drug design. As one of the three enzymes produced by HIV, it can be expected that inhibitors of this enzyme will complement the therapeutic use of HIV protease and reverse transcriptase inhibitors. We have determined the structure of a complex of the HIV-1 integrase core domain with a novel inhibitor, 5ClTEP, 1-(5-chloroindol-3-yl)-3-hydroxy-3-(2H-tetrazol-5-yl)-pro penone, to 2.1-A resolution. The inhibitor binds centrally in the active site of the integrase and makes a number of close contacts with the protein. Only minor changes in the protein accompany inhibitor binding. This inhibitor complex will provide a platform for structure-based design of an additional class of inhibitors for antiviral therapy.

The major neutralizing antigenic site on herpes simplex virus glycoprotein D overlaps a receptor-binding domain.

Herpes simplex virus (HSV) entry is dependent on the interaction of virion glycoprotein D (gD) with one of several cellular receptors. We previously showed that gD binds specifically to two structurally dissimilar receptors, HveA and HveC. We have continued our studies by using (i) a panel of baculovirus-produced gD molecules with various C-terminal truncations and (ii) a series of gD mutants with nonoverlapping 3-amino-acid deletions between residues 222 and 254. Binding of the potent neutralizing monoclonal antibody (MAb) DL11 (group Ib) was unaffected in forms of gD containing residues 1 to 250 but was greatly diminished in molecules truncated at residue 240 or 234. Both receptor binding and blocking of HSV infection were also affected by these C-terminal truncations. gD-1(234t) bound weakly to both HveA and HveC as determined by enzyme-linked immunosorbent assay (ELISA) and failed to block infection. Interestingly, gD-1(240t) bound well to both receptors but blocked infection poorly, indicating that receptor binding as measured by ELISA is not the only gD function required for blocking. Optical biosensor studies showed that while gD-1(240t) bound HveC with an affinity similar to that of gD-1(306t), the rates of complex formation and dissociation were significantly faster than for gD-1(306t). Complementation analysis showed that any 3-amino-acid deletion between residues 222 and 251 of gD resulted in a nonfunctional protein. Among this set of proteins, three had lost DL11 reactivity (those with deletions between residues 222 and 230). One of these proteins (deletion 222-224) was expressed as a soluble form in the baculovirus system. This protein did not react with DL11, bound to both HveA and HveC poorly as shown by ELISA, and failed to block HSV infection. Since this protein was bound by several other MAbs that recognize discontinuous epitopes, we conclude that residues 222 to 224 are critical for gD function. We propose that the potent virus-neutralizing activity of DL11 (and other group Ib MAbs) likely reflects an overlap between its epitope and a receptor-binding domain of gD.

A novel role for 3-O-sulfated heparan sulfate in herpes simplex virus 1 entry.

Herpes simplex virus type 1 (HSV-1) binds to cells through interactions of viral glycoproteins gB and gC with heparan sulfate chains on cell surface proteoglycans. This binding is not sufficient for viral entry, which requires fusion between the viral envelope and cell membrane. Here, we show that heparan sulfate modified by a subset of the multiple D-glucosaminyl 3-O-sulfotransferase isoforms provides sites for the binding of a third viral glycoprotein, gD, and for initiation of HSV-1 entry. We conclude that susceptibility of cells to HSV-1 entry depends on (1) presence of heparan sulfate chains to which virus can bind and (2) 3-O-sulfation of specific glucosamine residues in heparan sulfate to generate gD-binding sites or the expression of other previously identified gD-binding receptors.

The first immunoglobulin-like domain of HveC is sufficient to bind herpes simplex virus gD with full affinity, while the third domain is involved in oligomerization of HveC.

The human herpesvirus entry mediator C (HveC/PRR1) is a member of the immunoglobulin family used as a cellular receptor by the alphaherpesviruses herpes simplex virus (HSV), pseudorabies virus, and bovine herpesvirus type 1. We previously demonstrated direct binding of the purified HveC ectodomain to purified HSV type 1 (HSV-1) and HSV-2 glycoprotein D (gD). Here, using a baculovirus expression system, we constructed and purified truncated forms of the receptor containing one [HveC(143t)], two [HveC(245t)], or all three immunoglobulin-like domains [HveC(346t)] of the extracellular region. All three constructs were equally able to compete with HveC(346t) for gD binding. The variable domain bound to virions and blocked HSV infection as well as HveC(346t). Thus, all of the binding to the receptor occurs within the first immunoglobulin-like domain, or V-domain, of HveC. These data confirm and extend those of Cocchi et al. (F. Cocchi, M. Lopez, L. Menotti, M. Aoubala, P. Dubreuil, and G. Campadelli-Fiume, Proc. Natl. Acad. Sci. USA 95:15700, 1998). Using biosensor analysis, we measured the affinity of binding of gD from HSV strains KOS and rid1 to two forms of HveC. Soluble gDs from the KOS strain of HSV-1 had the same affinity for HveC(346t) and HveC(143t). The mutant gD(rid1t) had an increased affinity for HveC(346t) and HveC(143t) due to a faster rate of complex formation. Interestingly, we found that HveC(346t) was a tetramer in solution, whereas HveC(143t) and HveC(245t) formed dimers, suggesting a role for the third immunoglobulin-like domain of HveC in oligomerization. In addition, the stoichiometry between gD and HveC appeared to be influenced by the level of HveC oligomerization.

Three-dimensional structure of herpes simplex virus type 1 glycoprotein D at 2.4-nanometer resolution.

Herpes simplex virus type 1 glycoprotein D (gD) is essential for virus infectivity and is responsible for binding to cellular membrane proteins and subsequently promoting fusion between the virus envelope and the cell. No structural data are available for gD or for any other herpesvirus envelope protein. Here we present a three-dimensional model for the baculovirus-expressed truncated protein gD1(306t) based on electron microscopic data. We demonstrate that gD1(306t) appears as a homotetramer containing a pronounced pocket in the center of the molecule. Monoclonal antibody binding demonstrates that the molecule is oriented such that the pocket protrudes away from the virus envelope.

Inhibition of herpes simplex virus gD and lymphotoxin-alpha binding to HveA by peptide antagonists.

The herpesvirus entry mediator A (HveA) is a recently characterized member of the tumor necrosis factor receptor family that mediates the entry of most herpes simplex virus type 1 (HSV-1) strains into mammalian cells. Studies on the interaction of HSV-1 with HveA have shown that of all the viral proteins involved in uptake, only gD has been shown to bind directly to HveA, and this binding mediates viral entry into cells. In addition to gD binding to HveA, the latter has been shown to interact with proteins of tumor necrosis factor receptor-associated factor family, lymphotoxin-alpha (LT-alpha), and a membrane-associated protein referred to as LIGHT. To study the relationship between HveA, its natural ligands, and the viral proteins involved in HSV entry into cells, we have screened two phage-displayed combinatorial peptide libraries for peptide ligands of a recombinant form of HveA. Affinity selection experiments yielded two peptide ligands, BP-1 and BP-2, which could block the interaction between gD and HveA. Of the two peptides, only BP-2 inhibited HSV entry into CHO cells transfected with an HveA-expressing plasmid. When we analyzed these peptides for the ability to interfere with HveA binding to its natural ligand LT-alpha, we found that BP-1 inhibited the interaction of cellular LT-alpha with HveA. Thus, we have dissected the sites of interaction between the cell receptor, its natural ligand LT-alpha and gD, the virus-specific protein involved in HSV entry into cells.

Herpes simplex virus type 1 glycoprotein gC mediates immune evasion in vivo.

Many microorganisms encode proteins that interact with molecules involved in host immunity; however, few of these molecules have been proven to promote immune evasion in vivo. Herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) binds complement component C3 and inhibits complement-mediated virus neutralization and lysis of infected cells in vitro. To investigate the importance of the interaction between gC and C3 in vivo, we studied the virulence of a gC-null strain in complement-intact and C3-deficient animals. Using a vaginal infection model in complement-intact guinea pigs, we showed that gC-null virus grows to lower titers and produces less severe vaginitis than wild-type or gC rescued virus, indicating a role for gC in virulence. To determine the importance of complement, studies were performed with C3-deficient guinea pigs; the results demonstrated significant increases in vaginal titers of gC-null virus, while wild-type and gC rescued viruses showed nonsignificant changes in titers. Similar findings were observed for mice where gC null virus produced significantly less disease than gC rescued virus at the skin inoculation site. Proof that C3 is important was provided by studies of C3 knockout mice, where disease scores of gC-null virus were significantly higher than in complement-intact mice. The results indicate that gC-null virus is approximately 100-fold (2 log10) less virulent that wild-type virus in animals and that gC-C3 interactions are involved in pathogenesis.

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.