Entry of herpes simplex virus (HSV) 1 into cells requires the interaction of HSV gD with herpesvirus entry mediator or nectin1receptors, and fusion with cell membrane mediated by the fusion glycoproteins gB, gH, and gL. We report that the gD ectodomain in soluble form (amino acids 1-305) was sufficient to rescue the infectivity of a gD-null HSV mutant, indicating that gD does not need to be anchored to the virion envelope to mediate entry. Entry mediated by soluble gD required, in addition to the receptorbinding sites contained within residues 1-250, a discrete downstream portion (amino acids 261-305), located proximal to the transmembrane segment in full-length gD. We named it as profusion domain. The pro-fusion domain was required for entry mediated by virion-bound gD, because its substitution with the corresponding region of CD8 failed to complement the infectivity of gD ؊/؉ HSV. Furthermore, a receptor-negative gD (gD⌬6-259) inhibited virus infectivity when coexpressed with wild-type gD; i.e., it acted as a dominant-negative gD mutant. The pro-fusion domain is proline-rich, which is characteristic of regions involved in protein-protein interactions. P291L-P292A substitutions diminished the gD capacity to complement gD ؊/؉ HSV infectivity. We propose that gD forms a tripartite complex with its receptor and, by way of the proline-rich pro-fusion domain, with the fusion glycoproteins, or with one of them. The tripartite complex would serve to recruit͞activate the fusion glycoproteins and bring them from a fusion-inactive to a fusion-active state, such that they execute fusion of the virion envelope with cell membrane. H erpes simplex virus (HSV) enters cells through the coordinated action of four essential glycoproteins; gD, gB, gH, and gL, that act after the binding of gC and gB to the glycosaminoglycans of cell-surface proteoglycans (1, 2). Of the four glycoproteins required for entry, gD is the receptor-binding glycoprotein. It interacts with two alternative protein receptors, HVEM (herpesvirus entry mediator) and nectin1, that belong to the tumor necrosis factor receptor family (3), and to a growing family of intercellular adhesion molecules with Ig structure, respectively (4-8). The four essential glycoproteins required for HSV entry are required and are also sufficient to induce fusion of cells that express a gD receptor (9). The gD-binding site on HVEM maps mainly to the N-terminal cysteine-rich domain 1, with a hot spot at Y23 (10, 11). For nectin1, the N-terminal V domain, in particular its CCЈCЉ ridge (amino acids 64-104) is sufficient to mediate HSV entry (12-15). Critical residues were located in the 69-75 region and at positions 77 and 85 (16, 17). Insertion and deletion mutants in gD were the first mutants used to define functional regions (18). Subsequently, the x-ray crystal structure of the first 259 residues of gD [of the 315 that compose the ectodomain] was solved (19). The gD ectodomain is composed of an Ig-folded core (residues 56-184), with N-and C-terminal extensions. The latter folds bac...

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The herpesvirus entry mediator C (HveC), previously known as poliovirus receptor-related protein 1 (PRR1), and the herpesvirus Ig-like receptor (HIgR) are the bona fide receptors employed by herpes simplex virus-1 and -2 (HSV-1 and -2) for entry into the human cell lines most frequently used in HSV studies. They share an identical ectodomain made of one V and two C2 domains and differ in transmembrane and cytoplasmic regions. Expression of their mRNA in the human nervous system suggests possible usage of these receptors in humans in the path of neuron infection by HSV. Glycoprotein D (gD) is the virion component that mediates HSV-1 entry into cells by interaction with cellular receptors. We report on the identification of the V domain of HIgR͞PRR1 as a major functional region in HSV-1 entry by several approaches. First, the epitope recognized by mAb R1.302 to HIgR͞PRR1, capable of inhibiting infection, was mapped to the V domain. Second, a soluble form of HIgR͞ PRR1 consisting of the single V domain competed with cell-bound full-length receptor and blocked virion infectivity. Third, the V domain was sufficient to mediate HSV entry, as an engineered form of PRR1 in which the two C2 domains were deleted and the V domain was retained and fused to its transmembrane and cytoplasmic regions was still able to confer susceptibility, although at reduced efficiency relative to full-length receptor. Consistently, transfer of the V domain of HIgR͞PRR1 to a functionally inactive structural homologue generated a chimeric receptor with virus-entry activity. Finally, the single V domain was sufficient for in vitro physical interaction with gD. The in vitro binding was specific as it was competed both by antibodies to the receptor and by a mAb to gD with potent neutralizing activity for HSV-1 infectivity.

The receptors for entry of herpes simplex viruses 1 and 2 (HSV-1 and -2), widely expressed in human cell lines, are members of a subset of the immunoglobulin superfamily exemplified by herpesvirus entry mediator C (HveC) and the herpesvirus immunoglobulin-like receptor (HIgR). This report focuses on two members of this subset, herpesvirus entry mediator B (HveB), recently designated nectin2/PRR2␣, and its splice variant isoform, nectin2/PRR2␦. Nectin2␣ and -␦ share the ectodomain but differ in the transmembrane and cytoplasmic regions. HveB was reported to enable entry of HSV-1 carrying mutations in glycoprotein D (gD) and of HSV-2, but not of wild-type (wt) HSV-1. We report that (i) both nectin2␣ and -␦ served as receptors for the entry of HSV-1 mutant viruses HSV-1(U10) and -(U21) and AP7 r that carry the Leu25Pro substitution in gD but not for HSV-1 mutants U30 and R5000 that carry the Ser140 or Ala185 substitution in gD. All of these mutants were able to overcome the block to entry mediated by expression of wt gD.