The interactions between herpes simplex virus gD and its nectin1 receptor or between gD, gB, and gH were analyzed by complementation of the N and C portions of split enhanced green fluorescent protein (EGFP) fused to the glycoproteins. The gD N -Nect C complex was readily detected; the gD N -gC C complex was undetectable, highlighting the specificity of the assay. Split EGFP complementation was detected between proteins designated gD N ؉gH C , gD N ؉gB C , and gH N ؉gB C ؉wtgD (gB was deleted of endocytosis motifs), both in cells transfected with two-tree glycoproteins and in syncytia. The in situ assay provides evidence that gD interacts with gH and gB independently of each other and supports a model whereby gH and gB in complex exert their activities to gD.The entry of herpes simples virus (HSV) into cells requires a multipartite fusion system made of a quartet of glycoproteins (3,24,28). The receptor-binding glycoprotein gD interacts with three alternative receptors, nectin1, herpesvirus entry mediator, and modified heparan sulfate (5,11,22,27). gD also encodes a profusion domain at the ectodomain C terminus, which is required to trigger fusion (4). In the unliganded gD, the ectodomain C terminus folds around the N terminus. At receptor binding, the C terminus is displaced, gD adopts an open conformation, and fusion is triggered (8,20). Three glycoproteins conserved across the Herpesviridae family, gB and gH ⅐ gL, execute fusion (2, 7, 25). The identity of the executor-whether it is gB, gH ⅐ gL, or the three glycoproteins together-remains unclear. Thus, gB exhibits a trimeric structure, properties typical of class I and II viral fusion proteins, and a candidate fusion loop (16,17). On the other hand, gH exhibits elements typical of class I fusion glycoproteins, including two heptad repeats able to form a coiled coil and a candidate fusion peptide, besides having additional hydrophobic regions (9,10,(12)(13)(14)(15). Hemifusion (the fusion of the outer layers of the virion and cell membranes) requires gD and gH ⅐ gL; complete fusion (the mixing of both outer and inner lipid layers) additionally requires gB (29). Fusion between perinuclear virions and the outer nuclear membranes, which culminates in capsid release into the cytoplasm, requires gD plus either gB or gH ⅐ gL, implying that, under particular conditions, either gB or gH ⅐ gL suffice for fusion execution (6).A key question in HSV entry/fusion centers on how gD signals the encounter with its receptor to the downstream glycoproteins and thus triggers fusion. The working model investigated in this laboratory envisions that the receptor-bound gD forms complexes with the downstream glycoproteins or with a subset of them (3). Indeed, by coimmunoprecipitation, gD was shown to be in a complex with gH (23).The aim of this work was to investigate, by means of a protein complementation assay (CA) (19,21), in intact cells, the molecular interactions that take place between gD and nectin1 and between the four glycoproteins. In the CA, proteins like enhanced green ...

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.

Signals involved in protection against apoptosis by herpes simplex virus 1 (HSV-1) were investigated. Using U937 monocytoid cells as an experimental model, we have demonstrated that HSV-1 rendered these cells resistant to Fas-induced apoptosis promptly after infection. UV-inactivated virus as well as the envelope glycoprotein D (gD) of HSV-1, by itself, exerted a protective effect on Fas-induced apoptosis. NF-B was activated by gD, and protection against Fas-mediated apoptosis by gD was abolished in cells stably transfected with a dominant negative mutant I-B␣, indicating that NF-B activation plays a role in the antiapoptotic activity of gD in our experimental model. Moreover, NF-B-dependent protection against Fas-mediated apoptosis was associated with decreased levels of caspase-8 activity and with the up-regulation of intracellular antiapoptotic proteins.Interest in the understanding of mechanisms by which viruses belonging to a variety of families regulate cell apoptosis has grown rapidly in recent years (1-3). Herpesviruses, due to the relatively large quantity of information contained in their genomes, seem particularly well equipped to exert a fine control over cell apoptosis (4). This occurs through various interactions among viral and cell products acting at different levels (5).Among herpesviruses, herpes simplex viruses have been shown to regulate apoptosis of infected cells both positively and negatively, according to the presence or absence of specific genes, experimental conditions, or specificity of target cells (6 -21).Glycoprotein D (gD) 1 is a main component of the external structure of HSV-1, and its function is essential for HSV-1 spread. Interaction between gD and cell receptors allows virion entry into cells to be infected (22)(23)(24)(25). At least one of the cell receptors for gD, namely herpesvirus entry mediator A (HveA; also known as HVEM, TNFRSF14), belongs to the family of tumor necrosis factor receptors, which play a central role in mediating signal transduction leading to death receptor-associated apoptosis (26 -28). Recent results have shown that gD delivered in trans blocks the apoptotic cascade triggered by HSV-1 mutants lacking the gene encoding gD in SK-N-SH cells (29,30). Cellular signals involved in the antiapoptotic action exerted by HSV-1-gD remain to be elucidated. Interestingly, overexpression of the gD receptor HveA has been shown to cause activation of the transcription factor, NF-B (28). Furthermore, it has been reported that engagement with HveA receptor of its natural ligand, LIGHT, can stimulate the activation of NF-B in different cellular systems (31,32). This suggests the possibility that also engagement of gD with HveA could lead to NF-B activation. The transcription factor NF-B consists of a homodimeric or heterodimeric complex of two subunits belonging to the highly conserved family of Rel-related proteins (33). The most important complex is that formed by two proteins with molecular masses of 50 kDa (p50) and 65 kDa (p65), respectively. This heterodimer is pre...

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.

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