The soluble ectodomain of herpes simplex virus gD contains a membrane-proximal pro-fusion domain and suffices to mediate virus entry

Abstract

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...

Herpes simplex virus type 1 mediates fusion through a hemifusion intermediate by sequential activity of glycoproteins D, H, L, and B

“…Here we report evidence for a sequential model for fusion, whereby each glycoprotein is required for a phase of the fusion process. It would appear that gD has Phase I function to bring membranes into proximity and activate gHL and gB for fusion (8,10,12). Receptor binding may cause a conformational change in gD that induces gHL to initiate lipid mixing to the hemifusion intermediate (Phase II).…”

“…The current paradigm for HSV-1 fusion is that the required glycoproteins form a fusion complex (7,8), where gD binds a cell surface receptor (herpesvirus entry mediator, nectin-1, or modified heparin sulfate), inducing a conformational change that leads to fusion mediated by gB and/or gHL (8)(9)(10)(11). Because it is hypothesized that the main function of gD resides in Phase I (8,12) and gL functions solely as a gH chaperone (13), gB and gH are the most likely candidates to be involved in Phases II and III of the fusion process. However, their exact role in the fusion mechanism has yet to be elucidated.…”

Use of herpes simplex virus and pseudorabies virus chimeric glycoprotein D molecules to identify regions critical for membrane fusion

“…It seems unlikely that gD is an actual fusogen. All known viral fusogens must be anchored to the viral envelope as a transmembrane protein, whereas a glycosylphosphatidylinositol-linked gD ectodomain is functional for cell fusion (5) and soluble forms of the gD ectodomain can complement the entry defect of a gD-negative HSV (6). It has been proposed that interactions of gD with one of its receptors causes conformational changes in gD that enable it to activate the fusogenic activity of gB, a homooligomer, and͞or gH-gL, a heterodimer (6)(7)(8).…”

A strategy for systemic delivery of the oncolytic herpes virus HSV1716: redirected tropism by antibody-binding sites incorporated on the virion surface as a glycoprotein D fusion protein

“…Possibly, the fusion proteins can function as the sole receptor-binding protein to mediate infection of targeted cells as the scFv will mimic the specific interaction required to tether the virus closely to the cellular membrane and membrane fusion will be elicited by the remaining membrane-proximal residues of gD that form a profusion domain required for recruitment of gB and gH/gL. 46 Currently, we are attempting to create a gD-negative HSV1716 variant but, as gD is an essential protein, such a virus will be severely attenuated or even replication incompetent and it is interesting to speculate whether a gD-negative virus is mandatory to achieve optimal tumour targeting. Certainly, HSV1716 variants displaying both the targeting moiety and native gD will still be able to infect offtarget cells expressing HSV-1 receptors but, as demonstrated by HSV1716EGFR, the targeting moiety enhanced tumour uptake and destruction compared to unmodified HSV1716 suggesting that adsorption by non-tumour cells of systemically injected HSV1716 variants is not a major issue.…”