Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)


Molecular Sciences

Research Advisor

Michael A. Whitt, Ph.D.


Allen Portner, Ph.D. Charles J. Russell, Ph.D. James Patrick Ryan, Ph.D. Susan E. Senogles, Ph.D.


Vesicular Stomatitis Virus, Viral Entry, Viral Fusion, VSV G


Vesicular stomatitis virus (VSV) is an enveloped, nonsegmented, negative-sense, RNA virus belonging to the Rhabdoviridae family. VSV is considered the prototypic Rhabdovirus due to its simple genetic organization, broad host cell tropism, and ability to be easily grown in cell culture. Therefore, VSV has been used as the prototype to study viral entry, transcription, replication, and assembly. Viral entry, a critical step in the lifecycle of the virus, is mediated by the outer surface protein, G and will be the focus of this dissertation.

We hypothesize that the highly conserved residues in the membrane-proximal region of VSV G protein are critical to membrane fusion through participation in low pH-induced conformational changes and stability of theses structures. This hypothesis was tested initially through the use of site directed mutagenesis and then by studying rapidly acquired second site mutations. Residues that are conserved completely or conserved for their biochemical properties were either deleted or mutated to alanines. The mutated G proteins were examined through the use of transient transfection assays for their surface expression levels, fusion capacity, and ability to undergo pH dependent conformational changes. Mutation of a conserved HPH motif (H423/P424/H425) resulted in a dramatic decrease in surface expression. ΔH423/P424/H425 was completely fusion defective as assessed by syncytium formation assays that measured cell-cell fusion. Likewise, the cell-cell fusion activity for ΔH425 was affected in that the pH threshold required to trigger the fusion event was decreased. These findings support previous reports suggesting a requirement for histidine protonation in order for the pH dependent conformational changes needed for membrane fusion to occur. Recombinant viruses encoding the mutated G proteins were recovered and all of the viruses expressing G proteins with reduced cell-cell fusion capacity, as compared to wild type, grew to lower titers, with the exception of one mutant: D435A/D436A/E437A. Mutation of the DDE motif resulted in a G protein with a reduced capacity to fuse in transiently transfected cells. However, recombinant virus encoding D435A/D436A/E437A was recovered and unexpectedly grew to wild type titers, suggesting that the virus is overcoming the fusion defect caused by the mutation. Wild type VSV virions have been shown to express a truncated G protein on their surface, called G-stem (GS). GS was detected on the virion of this mutant at levels higher than that of wild type suggesting the truncated G protein was contributing to enhancing the infectivity of the virus.

Mutation of conserved phenylalanines (F440A/F441A) also resulted in a similar phenotype as the ΔH425 mutant. Reduced surface expression was observed when this mutated G protein was transiently expressed in cells and a decrease in the threshold pH required for fusion was also observed. Recombinant VSVs encoding the mutated G proteins were recovered to analyze the impact of the mutations on the ability of the viruses to grow and spread. Interestingly, viruses encoding the ΔH425 and the F440A/F441A mutant G proteins rapidly acquired additional mutations that made the viruses better able to grow and spread as indicated by increased titers and plaque sizes. Plaque isolates were obtained and subjected to sequence analysis revealing several variants: ΔH425/S438I, ΔHI426S/S438I, F440V/F441A, F440T/F441A, F440A/F441A/S438I, and F440A/F441A/CT9. To identify the advantage the additional mutations were providing the recombinant viruses (if any), the mutations were cloned into expression vectors and the mutant G proteins were examined for their surface expression levels and fusion capacity. The mutations appeared to enhance cell surface expression levels, with the exception of the ΔH425/S438I and F440A/F441A/CT9 mutants. The F440A/F441A mutants still were reduced in cell-cell fusion activity at pH 6.0 as compared to wild type G. However, the ΔH425 mutants’ fusion activity was partially restored at pH 6.0. Results from these experiments suggest that when both the ΔH425 and F440A/F441A mutant G acquire a S438I mutation the fusion capacity at pH 6.0 is restored. The findings in these studies indicate residues in the membrane-proximal region of VSV G are critical to the fusion process, suggesting a likely contribution to the stabilization of the pre- and postfusion structures.