The Transmembrane Domain and Cytoplasmic Tail of Herpes Simplex Virus Type 1 Glycoprotein H Play a Role in Membrane Fusion

Abstract

Herpes simplex virus glycoprotein H (gH) is one of the four virion envelope proteins which are required for virus entry and for cell-cell fusion in a transient system. In this report, the role of the transmembrane and cytoplasmic tail domains of gH in membrane fusion was investigated by generating chimeric constructs in which these regions were replaced with analogous domains from other molecules and by introducing amino acid substitutions within the membrane-spanning sequence. gH molecules which lack the authentic transmembrane domain or cytoplasmic tail were unable to mediate cell-cell fusion when coexpressed with gB, gD, and gL and were unable to rescue the infectivity of a gH-null virus as efficiently as a wild-type gH molecule. Many amino acid substitutions of specific amino acid residues within the transmembrane domain also affected cell-cell fusion, in particular, those introduced at a conserved glycine residue. Some gH mutants that were impaired in cell-cell fusion were nevertheless able to rescue the infectivity of a gH-negative virus, but these pseudotyped virions entered cells more slowly than wild-type virions. These results indicate that the fusion event mediated by the coexpression of gHL, gB, and gD in cells shares common features with the fusion of the virus envelope with the plasma membrane, they point to a likely role for the membrane-spanning and cytoplasmic tail domains of gH in both processes, and they suggest that a conserved glycine residue in the membrane-spanning sequence is crucial for efficient fusion.Herpes simplex viruses (HSVs) enter cells by fusion of the virus envelope with the host cell plasma membrane at a neutral pH (reviewed by Spear [37]), and four of the HSV envelope glycoproteins, gB, gD, gH, and gL, are not only essential for virus entry (7, 13, 24) but can also induce the fusion of cellular membranes when coexpressed from plasmid vectors in the absence of any other virus components (30,40). Efficient fusion induction with this system is dependent on the presence of a gD receptor on the plasma membrane of recipient cells (4, 34), but the means by which gB, gD, gH, and gL interact with each other or with other components of the plasma membrane to induce polykaryocyte formation and virus entry remain unclear. It is also uncertain whether the cell-cell fusion induced by the coexpression of gB, gD, gH, and gL mirrors the events which take place when the virion envelope fuses with the plasma membrane during virus entry.Homologues of HSV type 1 (HSV-1) gB and the gHL heterodimer have been identified in herpesviruses of all subfamilies and are likely to play a direct role in the fusion process. Indeed, studies with pseudorabies virus and human herpesvirus 8 have shown that the expression of gB and gHL is sufficient to induce cell fusion (17, 33) and, in varicella-zoster virus, the expression of gHL alone or gB in combination with gE has been reported to induce fusion (11,25). Despite the compelling evidence for the activity of gB and gHL as fusion proteins, these molecules have n...

Analysis of the role of the membrane-spanning and cytoplasmic tail domains of herpes simplex virus type 1 glycoprotein D in membrane fusion

“…In agreement with the results of cell fusion assays, a gD molecule containing the CD8 TM and cytoplasmic tail domains conferred entry kinetics that were equivalent to those mediated by the wildtype molecule, while virions containing gDDAF entered cells more slowly. These data confirm that there is a correlation between the phenotypes of mutated glycoprotein molecules in transient cell fusion assays and their ability to mediate virus entry (as has been reported previously by Harman et al, 2002), and that there are no requirements for specific sequences in the TM domain and tail of gD for either process to take place. They also imply that receptor binding by gD is not the only event required for efficient fusion to occur, as both chimeric molecules contain the receptor-binding regions of gD and yet the gDDAF molecule is compromised in its ability to mediate fusion.…”

“…To test whether these molecules could mediate cell fusion, we co-expressed them together with wild-type gHL and gB in 293T cells, as described by Harman et al (2002), and scored the number of nuclei that were recruited into polykaryocytes after overlaying the transfectants with Vero cells. Under these conditions, no background syncytia were detected in untransfected controls and only polykaryocytes containing more than one nucleus were scored as positive.…”

Function of Herpes Simplex Virus Type 1 gD Mutants with Different Receptor-Binding Affinities in Virus Entry and Fusion

“…We also wanted to test whether the rate of entry assay revealed a correlation between receptor binding affinity and entry for gD(ٌ126) and gD(⌬290-299). The complementation and fusion assays had not revealed any convincing association; however, since neither assay is rate limited (20), functional differences between mutants that are dependent on variations in affinity might be missed. Figure 7 shows the rate of entry of KOSgD␤ complemented with WT gD or with gD(ٌ243).…”

Analysis of a Membrane Interacting Region of Herpes Simplex Virus Type 1 Glycoprotein H

“…For example, certain mutations in the transmembrane region and cytoplasmic tail affect fusion (27,28), as do mutations in the region preceding the transmembrane (24). Furthermore, peptides matching a number of regions of the gH ectodomain have been shown to interact with membranes and have been proposed to play a role in the fusion process (20,21).…”