The Membrane-Proximal Region of Vesicular Stomatitis Virus Glycoprotein G Ectodomain Is Critical for Fusion and Virus Infectivity

Abstract

The glycoprotein (G) of vesicular stomatitis virus (VSV) is responsible for binding of virus to cells and for mediating virus entry following endocytosis by inducing fusion of the viral envelope with the endosomal membrane. The fusion peptide of G is internal (residues 116 to 137) and exhibits characteristics similar to those of other internal fusion peptides, but recent studies have implicated the region adjacent to the transmembrane domain as also being important for G-mediated membrane fusion. Sequence alignment of the membrane-proximal region of G from several different vesiculoviruses revealed that this domain is highly conserved, suggesting that it is important for G function. Mutational analysis was used to show that this region is not essential for G protein oligomerization, transport to the cell surface, or incorporation into virus particles but that it is essential for acid-induced membrane fusion activity and for virus infectivity. Deletion of the 13 membrane-proximal amino acids (N449 to W461) dramatically reduced cell-cell fusion activity and reduced virus infectivity approximately 100-fold, but mutation of conserved aromatic residues (W457, F458, and W461) either singly or together had only modest effects on cell-cell fusion activity; recombinant virus encoding these mutants replicated as efficiently as wild-type (WT) VSV. Insertion of heterologous sequences in the juxtamembrane region completely abolished membrane fusion activity and virus infectivity, as did deletion of residues F440 to N449. The insertion mutants showed some changes in pH-dependent conformational changes and in virus binding, which could partially explain the defects in membrane fusion activity, but all the other mutants were similar to WT G with respect to conformational changes and virus binding. These data support the hypothesis that the membrane-proximal domain contributes to G-mediated membrane fusion activity, yet the conserved aromatic residues are not essential for membrane fusion or virus infectivity.

Characterization of pH-Sensitive Molecular Switches That Trigger the Structural Transition of Vesicular Stomatitis Virus Glycoprotein from the Postfusion State toward the Prefusion State

“…As the other mutants (VSV G.D268L, VSV G.D274N, and VSV G.D393N) were not generated spontaneously, we employed a complementation strategy to support their growth (23,33). Briefly, the recovery of the recombinant VSV was supported by expression of functional WT VSV G protein in trans from a transfected plasmid.…”

Conserved baculoviral ORFs 10 and 14 from Bombyx mori multiple nucleopolyhedrovirus

“…Likely, the vesicular stomatitis virus has a region, called glycoprotein G, that acts as a receptor, promoting the viral envelop fusion with the host cell membrane through a low pH, followed by the endocytosis process (Jeetendra et al, 2003). This region is also rich in hydrophobic amino acids, but when mutations (insertions and deletions) are detected, there is also a reduction in host cell fusion activity.…”

“…All these repeated sequences of hydrophobic amino acids are related to viral fusion in cell membranes. Part of this glycoprotein G (residues from 421 to 461), which includes ectodomain and transmembrane regions, could potentiate the fusion activities between the membranes (Jeetendra et al, 2003).…”

Fusion-active glycoprotein G mediates the cytotoxicity of vesicular stomatitis virus M mutants lacking host shut-off activity

“…This results in the release of the RNP complex into the cytosol, where transcription and replication take place. In addition to its role in virus entry, the G protein is important for efficient virus budding and release (Jeetendra et al, 2003).…”

“…This results in the release of the RNP complex into the cytosol, where transcription and replication take place. In addition to its role in virus entry, the G protein is important for efficient virus budding and release (Jeetendra et al, 2003).VSV shows a very broad cell tropism and replicates rapidly in various cell lines that are derived from different species and tissues. Infected cells normally release high numbers of (Faria et al, 2005;Lyles, 2000;Petersen et al, 2000;von Kobbe et al, 2000).…”