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...

A number of sentinels sense incoming herpes simplex virus (HSV) virions and initiate an immediate innate response. The first line of defense at the cell surface is TLR2 (Toll-like receptor 2), whose signature signaling activity leads to activation of the key transcription factor NF-κB. We report that the HSV pathogen-associated molecular patterns for TLR2 are the virion glycoproteins gH/gL and gB, which constitute the conserved fusion core apparatus across the members of the Herpesviridae family. Specifically, virions devoid singly of one of essential fusion glycoproteins (gD, gB, or gH null), able to attach to cells but defective in fusion/entry, were sufficient to elicit the first wave of NF-κB response to HSV. The most effective were the gD-null virions, positive for gH/gL and gB. A soluble form of gB, truncated upstream of the transmembrane sequence (gB 730t-st ), was produced in human cells and purified by means of a Strep tag. gH/gL and gB were each able to physically interact with TLR2 in coimmunoprecipitation assays, one independently of the other, yet gH t-st /gL, but not gB 730t-st , elicited an NF-κB response. Thus, whereas both HSV gH/gL and gB are ligands to TLR2, only gH/gL is sufficient to initiate a signaling cascade which leads to NF-κB activation.

Herpes simplex virus (HSV) - and herpesviruses in general - encode for a multipartite entry/fusion apparatus. In HSV it consists of the HSV-specific glycoprotein D (gD), and three additional glycoproteins, gH/gL and gB, conserved across the Herpesviridae family and responsible for the execution of fusion. According to the current model, upon receptor binding, gD propagates the activation to gH/gL and to gB in a cascade fashion. Questions remain about how the cascade of activation is controlled and how it is synchronized with virion endocytosis, to avoid premature activation and exhaustion of the glycoproteins. We considered the possibility that such control might be carried out by as yet unknown receptors. Indeed, receptors for HSV gB, but not for gH/gL, have been described. In other members of the Herpesviridae family, such as Epstein-Barr virus, integrin receptors bind gH/gL and trigger conformational changes in the glycoproteins. We report that αvβ6- and αvβ8-integrins serve as receptors for HSV entry into experimental models of keratinocytes and other epithelial and neuronal cells. Evidence rests on loss of function experiments, in which integrins were blocked by antibodies or silenced, and gain of function experiments in which αvβ6-integrin was expressed in integrin-negative cells. αvβ6- and αvβ8-integrins acted independently and are thus interchangeable. Both bind gH/gL with high affinity. The interaction profoundly affects the route of HSV entry and directs the virus to acidic endosomes. In the case of αvβ8, but not αvβ6-integrin, the portal of entry is located at lipid microdomains and requires dynamin 2. Thus, a major role of αvβ6- or αvβ8-integrin in HSV infection appears to be to function as gH/gL receptors and to promote virus endocytosis. We propose that placing the gH/gL activation under the integrin trigger point enables HSV to synchronize virion endocytosis with the cascade of glycoprotein activation that culminates in execution of fusion.

The multipartite entry-fusion system of herpes simplex virus is made of a quartet of glycoproteins-gD, gB, gH.gL-and three alternative gD receptors, herpesvirus entry mediator (HVEM), nectin1 and modified sites on heparan sulphate. This multipartite system recapitulates the basic steps of virus-cell fusion, i.e. receptor recognition, triggering of fusion and fusion execution. Specifically, in addition to serving as the receptor-binding glycoprotein, gD triggers fusion through a specialised domain, named pro-fusion domain (PFD), located C-terminally in the ectodomain. In the unliganded gD the C-terminal region folds around the N-terminal region, such that gD adopts a closed autoinhibited conformation. In HVEM- and nectin1-bound gD the C-terminal region is displaced (opened conformation). gD is the tool for modification of HSV tropism, through insertion of ligands to heterologous tumour-specific receptors. It is discussed whether gD responds to the interaction with the natural and the heterologous receptors by adopting similar conformations, and whether the closed-to-open switch in conformation is a generalised mechanism of activation. A peculiar recombinant highlighted that the central Ig-folded core of gD may not encode executable functions for entry and that the 219-314 aa segment may be sufficient to trigger fusion. With respect to fusion execution, gB appears to be a prospective fusogen based on its coiled-coil trimeric structure, similar to that of another fusion glycoprotein. On the other hand, gH exhibits molecular elements typical of class 1 fusion glycoproteins, in particular heptad repeats and strong tendency to interact with lipids. Whether fusion execution is carried out by gB or gH.gL, or both glycoproteins in complex or sequentially remains to be determined.