Cascade of Events Governing Cell-Cell Fusion Induced by Herpes Simplex Virus Glycoproteins gD, gH/gL, and gB

Abstract

Herpesviruses minimally require the envelope proteins gB and gH/gL for virus entry and cell-cell fusion; herpes simplex virus (HSV) additionally requires the receptor-binding protein gD. Although gB is a class III fusion protein, gH/gL does not resemble any documented viral fusion protein at a structural level. Based on those data, we proposed that gH/gL does not function as a cofusogen with gB but instead regulates the fusogenic activity of gB. Here, we present data to support that hypothesis. First, receptor-positive B78H1-C10 cells expressing gH/gL fused with receptor-negative B78H1 cells expressing gB and gD (fusion in trans). Second, fusion occurred when gH/gL-expressing C10 cells preexposed to soluble gD were subsequently cocultured with gB-expressing B78 cells. In contrast, prior exposure of gB-expressing C10 cells to soluble gD did not promote subsequent fusion with gH/gL-expressing B78 cells. These data suggest that fusion involves activation of gH/gL by receptor-bound gD. Most importantly, soluble gH/gL triggered a low level of fusion of C10 cells expressing gD and gB; a much higher level was achieved when gB-expressing C10 cells were exposed to a combination of soluble gH/gL and gD. These data clearly show that gB acts as the HSV fusogen following activation by gD and gH/gL. We suggest the following steps leading to fusion: (i) conformational changes to gD upon receptor binding, (ii) alteration of gH/gL by receptor-activated gD, and (iii) upregulation of the fusogenic potential of gB following its interaction with activated gH/gL. The third step may be common to other herpesviruses.Herpesviruses enter cells by fusing their envelopes with host cell membranes either by direct fusion at the plasma membrane or by pH-dependent or -independent endocytosis, depending on the target cell (27,29,39). Although the entry pathways of other enveloped viruses are similarly diverse (8), all systems for which molecular details have been obtained rely on a single fusion protein (43); herpesviruses are unique in their use of gB and the gH/gL heterodimer as their core fusion machinery (17, 37). Some herpesviruses employ additional receptor-binding glycoproteins, e.g., herpex simplex virus (HSV) gD, and others require gH/gL-associated proteins, e.g., UL128-131 of cytomegalovirus (CMV) (34) or gp42 of Epstein-Barr virus (EBV) (42). This complexity has made it difficult to unravel the mechanism of herpesvirus entry.Ultrastructural and biochemical studies have shown that for HSV entry, binding of gD to one of its receptors, either HVEM or nectin-1 (36), activates the downstream events that drive gB-and gH/gL-dependent fusion (17). The structure of the gB ectodomain (18) bears striking structural homology to the postfusion form of the single fusion protein G of vesicular stomatitis virus (VSV) (33). However, unlike the other class III viral fusion proteins, VSV G and baculovirus gp64 (5), gB requires gH/gL to function in virus-cell and cell-cell fusion (17). A number of investigations support the concept that gH/gL might also...

Novel Mutations in gB and gH Circumvent the Requirement for Known gD Receptors in Herpes Simplex Virus 1 Entry and Cell-to-Cell Spread

“…Given our rudimentary understanding of how the 4 essential glycoproteins interact, it is premature to speculate how this potentially common trigger promotes one event but not the other depending on the particular mutant glycoprotein. While recent reports have suggested that gB is the fusogen in HSV entry and spread, whereas gH serves a regulatory function (30), much of the evidence comes from virus-free systems that do not clearly distinguish between entry and spread (30,34,35). In fact, the virus-free cell fusion assay in our study reflected the activities of gB:NT and gH:KV in spread but not in entry, supporting the view that this assay is limited as a model for HSV entry.…”

“…Previous evidence indicated that gH/gL could carry out hemifusion, an intermediate state in which the outer membrane leaflets of the target and viral membranes mix, whereas both gB and gH/gL were required for complete fusion (32), but these findings were recently called into question (33). Recent studies suggested that gH/gL activates the dormant fusogenic activity of gB in response to signaling by receptor-bound gD (30,34,35), indicating that gH/gL performs an intermediate regulatory function in the steps leading to membrane fusion. However, since these studies were performed in virus-free cell fusion systems, it is unclear whether they apply equally to virus entry and spread.…”

Dissociation of HSV gL from gH by αvβ6- or αvβ8-integrin promotes gH activation and virus entry

“…Soluble gD (gD t ) rescued the infection of gD −/− virions, providing indirect evidence that gD undergoes conformational changes upon interaction with receptor-positive cells (16). gH t /gL can promote cell-cell fusion in cells expressing gD (or exposed to gD t ), gB, plus gD receptor (19). gH t /gL rescues the infection of gH −/− EBV virions (34).…”

Structural and biochemical studies of HCMV gH/gL/gO and Pentamer reveal mutually exclusive cell entry complexes

“…The role of gH/gL in fusion is less clear because crystal structures of herpes simplex virus 2 (HSV-2), pseudo-rabies virus (PrV), and Epstein-Barr virus (EBV) gH/gL did not reveal any similarity to known viral fusion proteins (9)(10)(11). It has been proposed that gH/gL is involved in the entry process through activation of gB (12). In addition to gB and gH/gL, most herpesviruses encode additional glycoproteins that are able to interact with gH/gL and are capable of either mediating binding to specific cellular receptors or regulating the activity of the gH/gL-gB complex (5,6).…”