A Recombinant Vesicular Stomatitis Virus Bearing a Lethal Mutation in the Glycoprotein Gene Uncovers a Second Site Suppressor That Restores Fusion

Abstract

Vesicular stomatitis virus (VSV), a prototype of the Rhabdoviridae family, contains a single surface glycoprotein (G) that is responsible for attachment to cells and mediates membrane fusion. Working with the Indiana serotype of VSV, we employed a reverse genetic approach to produce fully authentic recombinant viral particles bearing lethal mutations in the G gene. By altering the hydrophobicity of the two fusion loops within G, we produced a panel of mutants, W72A, Y73A, Y116A, and A117F, that were nonfusogenic. Propagation of viruses bearing those lethal mutations in G completely depended on complementation by expression of the glycoprotein from the heterologous New Jersey serotype of VSV. The nonfusogenic G proteins oligomerize and are transported normally to the cell surface but fail to mediate acid pH-triggered membrane fusion. The nonfusogenic G proteins also interfered with the ability of wild-type G to mediate fusion, either by formation of mixed trimers or by inhibition of trimer function during fusion. Passage of one recombinant virus, A117F, identified a second site suppressor of the fusion block, E76K. When analyzed in the absence of the A117F substitution, E76K rendered G more sensitive to acid pH-triggered fusion, suggesting that this compensatory mutation is destabilizing. Our work provides a set of authentic recombinant VSV particles bearing lethal mutations in G, confirms that the hydrophobic fusion loops of VSV G protein are critical for membrane fusion, and underscores the importance of the sequence elements surrounding the hydrophobic tips of the fusion loops in driving fusion. This study has implications for understanding dominant targets for inhibition of G-mediated fusion. Moreover, the recombinant viral particles generated here will likely be useful in dissecting the mechanism of G-catalyzed fusion as well as study steps of viral assembly.Enveloped viral fusion machines fall into one of three classes: class I, II, or III. These classes are respectively exemplified by influenza A virus hemagglutinin (HA), flavivirus envelope (E) proteins, and the rhabdovirus glycoproteins (G) (18). All three classes of fusion proteins share basic characteristics. In response to specific triggers, conformational rearrangements in the protein lead to the insertion of a hydrophobic region(s) into the target cell membrane, followed by the refolding of the protein to bring the viral and cellular membrane in close proximity. Class I and II viral fusion machines undergo a priming cleavage event, and the conformational changes that occur on exposure to the appropriate trigger are irreversible. By contrast, the class III viral fusion machines do not undergo such a priming cleavage (as reviewed in reference 18). For rhabdoviruses, the trigger for conformational change in G is exposure to acidic pH. Uniquely, the pH-triggered conformational changes in class III fusion proteins are fully reversible, such that reexposure of viral particles to neutral pH results in G refolding into the prefusion conformation (4, 13, 31)...

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.…”

“…We used a previously described forward genetics approach (23,33), and we isolated a compensatory mutation in position 271 (L271P) which is also located in the segment that refolds at low pH to elongate the central helix (Fig. 10).…”

“…The polypeptide chain of the G ectodomain folds into three distinct domains. The fusion domain (FD) is made of an extended ␤-sheet structure at the tip of which are located two loops that constitute the membrane-interacting motif of the G ectodomain (22,23). FD is inserted in a loop of a pleckstrin homology domain (PHD) that .…”

Mechanism of membrane fusion induced by vesicular stomatitis virus G protein

“…4A). Virions incorporating this mutant G are noninfectious and, when expressed on the cell surface, the mutant does not mediate cell-cell fusion (16). We incubated G th , both wild-type (G th -WT) and mutant (G th -W72A), with liposomes at several pH values, separated the liposome-bound from free protein by sucrose-density centrifugation, and detected G th in each fraction of the gradient by immunoblotting with a conformation-specific monoclonal antibody, IE2 (17).…”

Oncolytic viruses: From bench to bedside with a focus on safety

“…Also, VSVs expressing attenuating transgenes like hIFNb can acquire mutations in this transgene, which has been shown in several studies. [96][97][98] Furthermore, oncolytic VSVs have been shown to optimize targeting to glycoproteins upon passaging in tumor cells, 99 and to mutate expressed transgenes to optimize replication. 100 These examples have strangely not been perceived as a safety problem, but should be taken into account in future (pre)clinical trials.…”