Glycoprotein B (gB), along with gD, gH, and gL, is essential for herpes simplex virus (HSV) entry. The crystal structure of the gB ectodomain revealed it to be an elongated multidomain trimer. We generated and characterized a panel of 67 monoclonal antibodies (MAbs). Eleven of the MAbs had virus-neutralizing activity. To organize gB into functional regions within these domains, we localized the epitopes recognized by the entire panel of MAbs and mapped them onto the crystal structure of gB. Most of the MAbs were directed to continuous or discontinuous epitopes, but several recognized discontinuous epitopes that showed some resistance to denaturation, and we refer to them as pseudo-continuous. Each category contained some MAbs with neutralizing activity. To map continuous epitopes, we used overlapping peptides that spanned the gB ectodomain and measured binding by enzyme-linked immunosorbent assay. To identify discontinuous and pseudocontinuous epitopes, a purified form of the ectodomain of gB, gB(730t), was cleaved by ␣-chymotrypsin into two major fragments comprising amino acids 98 to 472 (domains I and II) and amino acids 473 to 730 (major parts of domains III, IV, and V). We also constructed a series of gB truncations to augment the other mapping strategies. Finally, we used biosensor analysis to assign the MAbs to competition groups. Together, our results identified four functional regions: (i) one formed by residues within domain I and amino acids 697 to 725 of domain V; (ii) a second formed by residues 391 to 410, residues 454 to 475, and a less-defined region within domain II; (iii) a region containing residues of domain IV that lie close to domain III; and (iv) the first 12 residues of the N terminus that were not resolved in the crystal structure. Our data suggest that multiple domains are critical for gB function.Herpes simplex virus (HSV) is a neurotropic agent responsible for episodic cold sores and genital legions. After primary infection of mucosal epithelial cells, the virus establishes lifelong latency in sensory neurons, from which it periodically reactivates. After reactivation, the virus migrates along the axons and infects cells at the site of primary infection, causing painful blisters on the surface of the lips in the case of HSV type 1 (HSV-1) or of the genital mucosa for the closely related HSV-2 (48).A critical event in the life cycle resides in the entry of the virus into target cells. Recent progress has been made in understanding the mechanism governing this process (reviewed in references 21 and 38). Of the 12 different glycoproteins of the viral envelope, 4 have essential functions for entry, namely, glycoprotein B (gB), gD, gH, and gL. First, the virion attaches by interaction of gC and gB with cell surface heparan sulfate proteoglycans (42). Although not essential for entry, this step provides stable interactions between the virion and the cell that favor the next steps. These include the association of gD with one of its three identified receptors-HVEM, nectin-1, and 3-O-sulfated...

Herpes simplex virus (HSV) entry requires the interaction of glycoprotein D (gD) with a cellular receptor such as herpesvirus entry mediator (HVEM or HveA) or nectin-1 (HveC). However, the fusion mechanism is still not understood. Since cholesterol-enriched cell membrane lipid rafts are involved in the entry of other enveloped viruses such as human immunodeficiency virus and Ebola virus, we tested whether HSV entry proceeds similarly. Vero cells and cells expressing either HVEM or nectin-1 were treated with cholesterolsequestering drugs such as methyl-␤-cyclodextrin or nystatin and then exposed to virus. In all cases, virus entry was inhibited in a dose-dependent manner, and the inhibitory effect was fully reversible by replenishment of cholesterol. To examine the association of HVEM and nectin-1 with lipid rafts, we analyzed whether they partitioned into nonionic detergent-insoluble glycolipid-enriched membranes (DIG). There was no constitutive association of either receptor with DIG. Binding of soluble gD or virus to cells did not result in association of nectin-1 with the raft-containing fractions. However, during infection, a fraction of gB but not gC, gD, or gH associated with DIG. Similarly, when cells were incubated with truncated soluble glycoproteins, soluble gB but not gC was found associated with DIG. Together, these data favor a model in which HSV uses gB to rapidly mobilize lipid rafts that may serve as a platform for entry and cell signaling. It also suggests that gB may interact with a cellular molecule associated with lipid rafts.

Herpes simplex virus (HSV) entry into cells requires four membrane glycoproteins: gD is the receptor binding protein, and gB and gH/gL constitute the core fusion machinery. Crystal structures of gD and its receptors have provided a basis for understanding the initial triggering steps, but how the core fusion proteins function remains unknown. The gB crystal structure shows that it is a class III fusion protein, yet unlike other class members, gB itself does not cause fusion. Bimolecular complementation (BiMC) studies have shown that gD-receptor binding triggers an interaction between gB and gH/gL and concurrently triggers fusion. Left unanswered was whether BiMC led to fusion or was a by-product of it. We used gB monoclonal antibodies (MAbs) to block different aspects of these events. Non-virus-neutralizing MAbs to gB failed to block BiMC or fusion. In contrast, gB MAbs that neutralize virus blocked fusion. These MAbs map to three functional regions (FR) of gB. MAbs to FR1, which contains the fusion loops, and FR2 blocked both BiMC and fusion. In contrast, MAbs to FR3, a region involved in receptor binding, blocked fusion but not BiMC. Thus, FR3 MAbs separate the BiMC interaction from fusion, suggesting that BiMC occurs prior to fusion. When substituted for wild-type (wt) gB, fusion loop mutants blocked fusion and BiMC, suggesting that loop insertion precedes BiMC. Thus, we postulate that each of the gB FRs are involved in different aspects of the path leading to fusion. Upon triggering by gD, gB fusion loops are inserted into target lipid membranes. gB then interacts with gH/gL, and this interaction is eventually followed by fusion.

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...

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