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. Among the 11 envelope glycoproteins of herpes simplex virus (HSV), glycoprotein D (gD) plays an essential role during viral entry into mammalian cells (14). gD binds specifically to one of several cell surface receptors during the pH-independent process that leads to fusion of the HSV envelope with the cell plasma membrane (13). Other essential glycoproteins such as gB and the gH-gL heterodimer also participate in the fusion event in ways that remain to be elucidated (9,35,38).Several HSV gD receptors have been identified. Herpesvirus entry mediator A (HveA; also known as HVEM and TNFRSF14) is a member of the tumor necrosis factor receptor family which binds gD and allows the entry of most HSV-1 and HSV-2 strains (25, 41). HveB (nectin-2) and HveC (nectin-1) are members of the immunoglobulin (Ig) superfamily that are closely related to the poliovirus receptor (PVR; also known as CD155) and to the newly discovered nectin-3 (8, 21, 22, 33). Whereas the activity of HveB is limited to certain HSV-2 strains and some laboratory strains of HSV-1 (rid1 and ANG) and pseudorabies virus (PRV) (20, 39), HveC allows the entry of all the HSV-1 and HSV-2 strains tested as well as PRV and bovine herpesvirus 1 (10). Poliovirus receptor does not function as an HSV receptor but can be used by PRV and bovine herpesvirus 1 (10). A specific type of heparan sulfate modified by D-glucosaminyl-3-O-sulfotransferase 3 can substitute for HveA or HveC and binds to gD to allow the entry of HSV-1 KOS into cells (34).HveB and HveC appear to be involved in cell-cell interaction and were named nectin-2 and nectin-1, respectively, according to their newly discovered function (1,19,37). In this paper, we will refer to them according to their viral usage (i.e., HveB and HveC).Recently, mutations in the HveC gene (named PVRL1 in that study) were linked to a form of cleft lip/palate-ectodermal dysplasia in humans (36).Although they have different structures, HveA and HveC bound to HSV-1 gD with similar affinity (17, 42). Using antibody competition and mutagenesis, the binding sites for HveC and HveA were mapped to common and distinct regions of gD (16,28,40). Reciprocally, the gD binding site on HveC has been localized to the first and most distal of the three Ig-like domains (or V domain) of its extr...

The herpesvirus entry mediator A (HVEM/HveA) and nectin-1 (HveC/CD111) are two major receptors for herpes simplex virus (HSV). Although structurally unrelated, both receptors can independently mediate entry of wild-type (wt) HSV-1 and HSV-2 by interacting with the viral envelope glycoprotein D (gD). Laboratory strains with defined mutations in gD (e.g. rid1) do not use HVEM but use nectin-2 (HveB/CD112) for entry. The relative usage of HVEM and nectin-1 during HSV infection in vivo is not known. In the absence of a defined in vivo model, we used in vitro approaches to address this question. First, we screened HSV clinical isolates from various origins for receptor tropism and found that all used both HVEM and nectin-1. Second, we determined the numbers of surface receptors on various susceptible and resistant cell lines as well as on primary fibroblasts derived from an individual with cleft lip/palate ectodermal dysplasia (CLPED1). Although CLPED1 cells can only express a defective form of nectin-1, they allowed entry of wild type and mutant HSV strains by usage of either HVEM or nectin-2. Finally, we compared the ability of HVEM and nectin-1 to mediate entry when expressed at varying cell surface densities. Both receptors showed a direct relationship between the number of receptors and HSV susceptibility. Direct comparison of receptors suggests that nectin-1 is more efficient at promoting entry than HVEM. Overall, our data suggest that both receptors play a role during HSV infection in vivo and that both are highly efficient even at low levels of expression.

During viral entry, herpes simplex virus (HSV) glycoprotein D (gD) interacts with a specific cellular receptor such as nectin-1 (PRR1/HveC/CD111) or the herpesvirus entry mediator A (HVEM/HveA). Nectin-1 is involved in cell-to-cell adhesion. It is located at adherens junctions, where it bridges cells through homophilic or heterophilic interactions with other nectins. Binding of HSV gD prevents nectin-1-mediated cell aggregation. Since HSV gD affects the natural function of nectin-1, we further investigated the effects of gD expression on nectin-1 during HSV infection or in transfected cells. We also studied the importance of the interaction between nectin-1 and the cytoplasmic protein afadin for HSV entry and spread as well as the effects of infection on this interaction. In these investigations, we used a panel of cells expressing nectin-1 or nectin-1-green fluorescent protein fusions as the only mediators of HSV entry. During HSV infection, nectin-1 localization at adherens junction was dramatically altered in a manner dependent on gD expression. Nectin-1 and gD colocalized at cell contact areas between infected and noninfected cells and at the edges of plaques. This specific accumulation of gD at junctions was driven by expression of nectin-1 in trans on the surface of adjacent cells. Reciprocally, nectin-1 was maintained at junctions by the trans expression of gD in the absence of a cellular natural ligand. Our observations indicate that newly synthesized gD substitutes for nectin-1 of infected cells at junctions with noninfected cells. We propose that gD attracts and maintains the receptor at junctions where it can be used for virus spread.Binding of a specific cell surface receptor to herpes simplex virus (HSV) envelope glycoprotein D (gD) is an essential step in the process of viral entry into mammalian cells (5, 62). This interaction is required but not sufficient for membrane fusion, an event which also requires gB and the gH/gL complex (48, 70). Cellular receptors for HSV gD include herpesvirus entry mediator A (HVEM/HveA) (47), nectin-1 (PRR1/HveC/ CD111) (23), and nectin-2 (PRR2/HveB/CD112) (72), as well as a specific type of heparan sulfate modified by 3-OST-3 (D-glucosaminyl 3-O-sulfotransferase 3) (60). HVEM is a lymphotoxin receptor that belongs to the tumor necrosis factor receptor family (6,41,47), whereas nectins form a subgroup of immunoglobulin (Ig)-like molecules (16,37,68).HVEM receptor activity is limited to wild-type HSV-1 and HSV-2, whereas nectin-1 acts as a receptor for many alphaherpesviruses (HSV-1, HSV-2, pseudorabies virus [PRV], and bovine herpesvirus 1) (13,23,43,45,47,73). The related nectin-2 can be used only by HSV strains carrying specific mutations in the N-terminal part of gD and by HSV-2 (36, 72). Nectin-3 and nectin-4 are not receptors for herpesviruses, but the related human poliovirus receptor (PVR/CD155) can be used by animal herpesviruses such as PRV and bovine herpesvirus 1 (23,55,56,59).Nectins are widely expressed in tissues and cells (10,16,23,25,37,40,57), where...

Herpes simplex virus (HSV) entry requires the interaction between the envelope glycoprotein D (gD) and a cellular receptor such as nectin-1 (also named herpesvirus entry mediator C [HveC]) or HveA/HVEM. Nectin-1 is a cell adhesion molecule found at adherens junctions associated with the cytoplasmic actin-binding protein afadin. Nectin-1 can act as its own ligand in a homotypic interaction to bridge cells together. We used a cell aggregation assay to map an adhesive functional site on nectin-1 and identify the effects of gD binding and HSV early infection on nectin-1 function. Soluble forms of nectin-1 and anti-nectin-1 monoclonal antibodies were used to map a functional adhesive site within the first immunoglobulin-like domain (V domain) of nectin-1. This domain also contains the gD-binding site, which appeared to overlap the adhesive site. Thus, soluble forms of gD were able to prevent nectin-1-mediated cell aggregation and to disrupt cell clumps in an affinity-dependent manner. HSV also prevented nectin-1-mediated cell aggregation by occupying the receptor. Early in infection, nectin-1 was not downregulated from the cell surface. Rather, detection of nectin-1 changed gradually over a 30-min period of infection, as reflected by a decrease in the CK41 epitope and an increase in the CK35 epitope. The level of detection of virion gD on the cell surface increased within 5 min of infection in a receptor-dependent manner. These observations suggest that cell surface nectin-1 and gD may undergo conformational changes during HSV entry as part of an evolving interaction between the viral envelope and the cell plasma membrane.The interaction between herpes simplex virus (HSV) envelope glycoprotein D (gD) and a specific cellular receptor is required for virus entry into mammalian cells (2, 48). This essential step follows an initial attachment mediated by HSV gC and gB bound to cell surface heparan sulfate proteoglycans (17,18,56). In addition, fusion of the envelope with the cell plasma membrane involves gB and the gH/gL complex (36,50,55).Receptors for gD belong to at least three unrelated families. The herpesvirus entry mediator A (HveA; also called HVEM and TNFRSF14) belongs to the tumor necrosis factor alpha (TNF) receptor family and mediates entry of most HSV-1 and HSV-2 strains (34, 53). Nectin-1 (HveC; also called PRR1 and CD111) (14, 26) and nectin-2 (HveB; also called PRR2 and CD112) (11, 51) are members of the immunoglobulin (Ig) superfamily. Nectin-1 allows entry of all the HSV-1 and HSV-2 strains tested as well as pseudorabies virus and bovine herpesvirus type 1 (9, 14, 32). In contrast, nectin-2 can be used only by HSV-2, some laboratory strains of HSV-1 (rid1 and ANG), and pseudorabies virus (25, 51). The related poliovirus receptor (PVR) CD155 does not function as an HSV receptor, but can be used by pseudorabies virus and bovine herpesvirus type 1 (14). Lastly, a specific type of heparan sulfate modified by D-glucosaminyl 3-O-sulfotransferase 3 can substitute for HveA or nectin-1 and binds to gD to allow e...

The gH/gL complex plays an essential role in virus entry and cell-cell spread of herpes simplex virus (HSV). Very few immunologic reagents were previously available to either identify important functional regions or gain information about structural features of this complex. Therefore, we generated and characterized a panel of 31 monoclonal antibodies (MAbs) against HSV type 2 (HSV-2) gH/gL. Fourteen MAbs bound to a conformation-dependent epitope of the gH2/gL2 complex, and all blocked virus spread. The other 17 MAbs recognized linear epitopes of gH (12) or gL (5). Interestingly, two of the gL MAbs and six of the gH MAbs were type common. Overlapping synthetic peptides were used to map MAbs against linear epitopes. These data, along with results of competition analyses and functional assays, assigned the MAbs to groups representing eight distinct antigenic sites on gH (I to VIII) and three sites on gL (A, B, and C). Of most importance, the MAbs with biological activity mapped either to site I of gH2 (amino acids 19 to 38) or to sites B and C of gL2 (residues 191 to 210). Thus, these MAbs constitute a novel set of reagents, including the first such reagents against gH2 and gL2 as well as some that recognize both serotypes of each protein. Several recognize important functional domains of gH2, gL2, or the complex. We suggest a common grouping scheme for all of the known MAbs against gH/gL of both HSV-1 and HSV-2.Four glycoproteins (gB, gD, and gH/gL) as well as a gD-binding cellular receptor are required for entry of most alphaherpesviruses, including herpes simplex virus (HSV), pseudorabies virus, and bovine herpesvirus type 1 (36). Although gL shares little amino acid identity across herpesviruses, the gH/gL heterodimer is highly conserved and essential for virus-cell and cell-cell fusion (20,32,34). It is not clear how herpesvirus glycoproteins, singly or in combination, mediate fusion. A recent report suggests that gH is the HSV fusion protein (16), but a variety of other studies have shown that gB, gL, and gD are also essential for both virus-cell and cell-cell fusion (27,33,38). Although the crystal structure of HSV gD in complex with one of its receptors (HVEM; also called HveA) has been solved (6, 33), the three-dimensional structure of gB is still in a rudimentary stage (E. Heldwein, personal communication) and that of gH/gL has not yet been solved.Using a genetic approach, a hypothetical disulfide bond structure for HSV type 2 (HSV-2) gH has been proposed (5). Cysteines at positions 2 (residue 258) and 4 (residue 429) are thought to constitute a disulfide-bonded pair (C2-C4), while gH2 C1 (residue 90) is unbound. HSV-1 gH contains one more cysteine in its ectodomain than gH2 (at position 3, residue 404); it is unclear if gH1 C1 remains free or is the disulfide bond partner of gH1 C3. Genetic studies have also suggested disulfide bonds between cysteines 5 and 6 (residues 554 and 589) and cysteines 7 and 8 (residues 652 and 706) (5, 32).The N-and C-terminal halves of gH comprise separate structural and fun...