The pro-fusion domain of herpes simplex virus glycoprotein D (gD) interacts with the gD N terminus and is displaced by soluble forms of viral receptors

Abstract

Entry of herpes simplex virus into the cell requires the interaction of gD with one of its receptors, herpesvirus entry mediator or nectin 1, and the intervention of gB, gH, or gL, required to execute fusion of the virion envelope with cell membranes. The gD ectodomain is organized in two structurally and functionally differentiated regions. The N terminus (residues 1-260) carries the receptor binding sites, and the C terminus (residues 260 -310) functions as the pro-fusion domain (PFD), which is required for viral infectivity and fusion but not for receptor binding. The objective of our studies is to elucidate how gD links receptor recognition to the triggering of fusion. Here, we show that PFD is made of subdomains 1 and 2 (amino acids 260 -285 and 285-310). Each one partially contributed to herpes simplex virus infectivity. By means of glutathione S-transferase (GST) fusion proteins, we show that PFD bound soluble forms of gD, truncated at residue 260 (gD 260t) or downstream. Both PFD subdomains bound gD 260t, highlighting multiple contact sites between the N and C termini of gD. When gD 260t was in complex with either receptor, it failed to bind GST-PFD. In turn, the receptors did not bind GST-PFD, irrespective of whether they were in complex with gD. Thus, gD 260t interacted with the C terminus only if unbound to the receptor. We propose that (i) before receptor binding, gD adopts a ''closed'' conformation in which the N and C termini interact; and (ii) on encounter with a receptor, gD modifies its conformation and the N and C termini are released from reciprocal interactions (''opened'' conformation) and enabled to trigger fusion.

Separation of receptor-binding and profusogenic domains of glycoprotein D of herpes simplex virus 1 into distinct interacting proteins

“…The interactions of gD with nectin-1 are abolished by mutations at residues 34, 38, 215, 222, and 223 (13,14,20). A profusion domain, essential for activation of fusogenic glycoproteins B, H, and L, has been mapped to residues 260-310 in close proximity to the transmembrane domain beginning at residue 314 (6). gD suppresses apoptosis induced by mutants lacking gD (17).…”

“…At least some of the interactions of gD with its receptors have been mapped to the N terminus of gD, whereas the fusogenic determinant maps in the C-terminal portion of the ectodomain (residues 260-310). These residues (260-310) become actively engaged in membrane fusion after the interaction of the N-terminal domain with one of its cognate receptors (6)(7)(8)(10)(11)(12)(13)(14). gD also blocks apoptosis resulting from endocytosis of virus particles lacking gD.…”

“…In the next step in the entry cascade, gD then binds a specific surface receptor such as nectin-1 or HveA (1,4,5). The interaction of glycoprotein D (gD) with the receptor alters the conformation of gD to enable the activation of gB, glycoprotein H, and glycoprotein L, a trio of glycoproteins that execute the fusion of the viral envelope with the plasma membrane (6). In this sequence of events, gD emerges as the surface protein that imparts to the virion the specificity of entry into cells.…”

“…In this sequence of events, gD emerges as the surface protein that imparts to the virion the specificity of entry into cells. This 369-residue glycoprotein has been the subject of numerous structural and functional studies (6)(7)(8). The protein appears to function as a dimer (9).…”

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

“…HSV-1 gH is not trafficked to the cell surface in the absence of gL (6). 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.…”