Dual Split Protein-Based Fusion Assay Reveals that Mutations to Herpes Simplex Virus (HSV) Glycoprotein gB Alter the Kinetics of Cell-Cell Fusion Induced by HSV Entry Glycoproteins

Abstract

e Herpes simplex virus (HSV) entry and cell-cell fusion require glycoproteins gD, gH/gL, and gB. We propose that receptor-activated changes to gD cause it to activate gH/gL, which then triggers gB into an active form. We employed a dual split-protein (DSP) assay to monitor the kinetics of HSV glycoprotein-induced cell-cell fusion. This assay measures content mixing between two cells, i.e., fusion, within the same cell population in real time (minutes to hours). Titration experiments suggest that both gD and gH/gL act in a catalytic fashion to trigger gB. In fact, fusion rates are governed by the amount of gB on the cell surface. We then used the DSP assay to focus on mutants in two functional regions (FRs) of gB, FR1 and FR3. FR1 contains the fusion loops (FL1 and FL2), and FR3 encompasses the crown at the trimer top. All FL mutants initiated fusion very slowly, if at all. However, the fusion rates caused by some FL2 mutants increased over time, so that total fusion by 8 h looked much like that of the WT. Two distinct kinetic patterns, "slow and fast," emerged for mutants in the crown of gB (FR3), again showing differences in initiation and ongoing fusion. Of note are the fusion kinetics of the gB syn mutant (LL871/872AA). Although this mutant was originally included as an ongoing high-rate-of-fusion control, its initiation of fusion is so rapid that it appears to be on a "hair trigger." Thus, the DSP assay affords a unique way to examine the dynamics of HSV glycoprotein-induced cell fusion. Our goal is to understand the earliest events in herpes simplex virus (HSV) entry into susceptible host cells and the related process of cell-cell fusion. To gain entry, HSV must fuse its lipid envelope with a host membrane, either at the cell surface or in an endosome (reviewed in reference 1), thereby delivering the capsid and tegument to the target cell. HSV uses four glycoproteins for this process, gD, gB, and gH/gL, and a cell receptor, either herpesvirus entry mediator (HVEM) or nectin-1 (2). These complex events, also common to HSV glycoprotein-induced cell-cell fusion, occur in a stepwise temporal process that requires receptor activation of gD to a form that can activate the gH/gL heterodimer. Once gH/gL is activated, it upregulates the fusogenic activity of gB, a class III fusion protein. Solution of the 3-dimensional (3-D) structures of the entry glycoproteins and receptors for HSV has provided significant insights into the entry-fusion process, but major issues remain unresolved.First, although HSV gB and Epstein-Barr virus (EBV) gB look remarkably alike and have the structural features of a fusion protein (3, 4), neither works in the absence of gH/gL. This additional requirement is ubiquitous among herpesviruses but distinct from the mechanisms of other class III fusion proteins, including baculovirus gp64 and vesicular stomatitis virus glycoprotein G (VSV G) (5, 6). Second, the solved structures of HSV gB (4) and EBV gB (3) resemble the postfusion form of VSV G (6). Although a prefusion form of VSV G is known (...

Functional Characterization of Glycoprotein H Chimeras Composed of Conserved Domains of the Pseudorabies Virus and Herpes Simplex Virus 1 Homologs

“…In most alphaherpesviruses four conserved viral envelope glycoproteins are relevant for entry: the core fusion machinery consisting of glycoprotein B (gB), the gH/gL heterodimer, and the receptor-binding gD (1)(2)(3). Recent publications suggest that gB represents the actual fusion protein and that gD and gH/gL are required for activation of gB via direct gDgH/gL and gH/gL-gB interactions (4)(5)(6)(7)(8).…”

Regulation of Herpes Simplex Virus Glycoprotein-Induced Cascade of Events Governing Cell-Cell Fusion

“…In support of this, the overall levels of cell-cell fusion were proportional to the amount of gB expressed on the cell surface (38). However, to further evaluate the specific role of each protein in the process, we undertook a comparative analysis of the gD, gH/gL, and gB glycoproteins from HSV-1 (referred to as gD 1 , gH 1 /gL 1 , and gB 1 or type 1 glycoproteins below) and HSV-2 (referred to as gD 2 , gH 2 /gL 2 , and gB 2 or type 2 glycoproteins below).…”

“…The split luciferase assay has been described in detail elsewhere (38,45). Briefly, amounts of 5 ϫ 10 4 B78 cells (effector cells) were seeded on white 96-well luciferase plates treated for cell culture (Corning).…”

“…Because certain mutations convert gB of HSV-1 (gB 1 ) into a hypo-or a hyperfusogenic form (38)(39)(40), we assumed that the major regulation of the fusion process would be inherent to gB. In support of this, the overall levels of cell-cell fusion were proportional to the amount of gB expressed on the cell surface (38).…”

“…Key to this study is the fact that the HSV-2 forms of each of these glycoproteins can replace the HSV-1 forms with no apparent qualitative effect on cell fusion, as estimated by the formation of syncytia (11,41,42). To understand the dynamics of fusion, we extended our previous studies using a dual split luciferase-based cell fusion assay (43,44) to monitor content mixing in real time (38,45). By mixing and matching different forms of each member of the fusion complex in the presence and absence of receptor, we defined the contribution of each to the overall process.…”