Green Fluorescent Protein

Abstract

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 ...

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