Cross Talk among the Glycoproteins Involved in Herpes Simplex Virus Entry and Fusion: the Interaction between gB and gH/gL Does Not Necessarily Require gD

The gD, gB, and gH/gL glycoprotein quartet constitutes the basic apparatus for herpes simplex virus (HSV) entry into the cell and fusion. gD serves as a receptor binding glycoprotein and trigger of fusion. The conserved gB and gH/gL execute fusion. Central to understanding HSV entry/fusion has become the dissection of how the four glycoproteins engage in cross talk. While the independent interactions of gD with gB and gD with gH/gL have been documented, less is known of the interaction of gB with gH/gL. So far, this interaction has been detected only in the presence of gD by means of a split green fluorescent protein complementation assay. Here, we show that gB interacts with gH/gL in the absence of gD. The gB-gH/gL complex was best detected with a form of gB in which the endocytosis and phosphorylation motif have been deleted; this form of gB persists in the membranes of the exocytic pathway and is not endocytosed. The gB-gH/gL interaction was detected both in whole transfected cells by means of a split yellow fluorescent protein complementation assay and, biochemically, by a pull-down assay. Results with a panel of chimeric forms of gB, in which portions of the glycoprotein bracketed by consecutive cysteines were replaced with the corresponding portions from human herpesvirus 8 gB, favor the view that gB carries multiple sites for interaction with gH/gL, and one of these sites is located in the pleckstrin-like domain 1 carrying the bipartite fusion loop.Entry of herpes simplex virus (HSV) into the cell requires a multipartite apparatus made of a quartet of viral glycoproteins, gD, gB, and the heterodimer gH/gL, and a multistep process that culminates in the fusion of the virion envelope with cell membranes (5, 6, 10, 25, 36, 41). gD serves as the receptor-binding glycoprotein, able to interact with alternative receptors, nectin1, herpesvirus entry mediator (HVEM) and, in some cells, modified heparan sulfate (9, 13, 30, 39). It can also be engineered to accept heterologous ligands able to interact with selected receptors present on tumor cells and thus represents a tool to redirect HSV tropism (21, 28, 29, 42). The heterodimer gH/gL and gB execute fusion and constitute the conserved fusion apparatus across the Herpesviridae family. gB structure in the postfusion conformation shows a trimer with a central coiled coil (19). gH shows elements typical of type 1 fusion glycoproteins, in particular, helices able to interact with membranes, and two heptad repeats potentially able to form a coiled coil (12, 15-18). The discovery that a soluble form of gD enables entry of gD-null virions revealed that gD serves the additional function of triggering fusion and led to the view that the major roles of gD are to sense that virus has reached a receptor-positive cell and to signal to gB and gH/gL that fusion is to be executed (8). Biochemical and structural analyses showed that the C-terminal region of the gD ectodomain, containing the profusion domain required for fusion but not for receptor binding, can undergo major conformational changes (11, 24). Specifically, it binds the gD core and masks or hinders the receptor binding sites, conferring upon the molecule a closed, auto-inhibited conformation (24). Alternatively, it may unfold, conferring upon gD an open conformation. It was proposed that the C terminus of gD unfolds from gD core at receptor binding and recruits gH/gL and gB to a quaternary complex. A key feature of the model was that complexes among the glycoprotein quartet were not preformed, but, rather, they would assemble at the onset of or at fusion execution.Central to understanding HSV entry/fusion has become the dissection of the interactions that occur among the members of the glycoprotein quartet and their significance to the process. A first evidence of a gD-gH/gL interaction was provided in coimmunoprecipitation studies (35). Interactions between gD and gH/gL and between gD and gB were subsequently detected by split green fluorescence protein (GFP) complementation assays, implying that gD can recruit gB and gH/gL independently of one another, a result that argues against a stepwise recruitment of the glycoproteins to gD. In agreement with the proposed model, the interaction between gH/gL and gB was detected in the presence of transfected or soluble gD (1, 2). However, further studies highlighted levels of complexity not foreseen in the initial model. Thus, pull-down analyses showed that the interaction sites in gD with gB and with gH/gL lie in part outside the C-terminal portion of the gD ectodomain, that resting virions contain small amounts of gD in complex with gB and with gH/gL prior to encountering cells, and that de novo gD-gB complexes were not detected at virus entry into the cell (14).A major objective of current studies was to analyze the interaction of gB with gH/gL. We documented the interaction by two independent assays, i.e., by a complementation assay of split yellow fluorescent protein Venus (herein indicated as YFP) (31) in whole cells and, biochemically, by a pull-down assay. The latter was applied recently in our laboratory and is based on the ability of One-Strep-tagged proteins (e.g., gH) to specifically absorb to Strep-Tactin resin and thus retain any protein in complex (14). To preliminarily search for gB regions critical for the interaction with gH/gL, we engineered chimeric forms of HSV-1 and human herpesvirus 8 (HHV-8) gB in which the cysteines were preserved. While none of the chimeras was completely defective in the interaction, the interactions in the chimeras carrying substitutions in the pleckstrin-like domain 1—the domain that carries the bipartite fusion loops—were hampered. Altogether, the results underscore the ability of gB to interact with gH/gL in the absence of gD and favor the view that sites in gB for interaction with gH/gL involve multiple contacts, one of which is located in the domain that carries the fusion loops.