Analysis of host gene products that contribute to the induction of apoptosis by oncolytic vesicular stomatitis virus

The goal of my project was to analyze the role of host gene products in apoptosis induced by a matrix (M) protein mutant of vesicular stomatitis virus (VSV) that is a promising agent for oncolytic virus therapy. VSV induces apoptosis by at least two mechanisms. The viral matrix (M) protein induces apoptosis via the caspase-9 pathway due to the inhibition of host gene expression. However, in some cell types, the inhibition of host gene expression by VSV expressing wild-type (wt) M protein delays VSV-induced apoptosis, indicating that another mechanism is involved. In support of this, the rM51R-M virus, expressing a mutant M protein that is defective in its ability to inhibit host gene expression, induces apoptosis much more rapidly in L929 cells than do viruses expressing wt M protein. I have determined the caspase pathways by which rM51R-M virus induces apoptosis. Analysis of caspase activity using fluorometric caspase assays and Western blots indicated that each of the main initiator caspases, caspase-8, caspase-9, and caspase-12, were activated during infection with the rM51R-M virus. Over-expression of Bcl-2, an inhibitor of the caspase-9 apoptotic pathway, or MAGE-3, an inhibitor of caspase-12 activation, did not delay apoptosis induction in rM51R-M virus-infected L929 cells. However, an inhibitor of caspase-8 activity significantly delayed apoptosis induction. Furthermore, inhibition of caspase-8 activity prevented the activation of caspase-9, suggesting that caspase-9 is activated by cross-talk with caspase-8. These data indicate that VSV expressing the mutant M protein induces apoptosis via the death receptor apoptotic pathway, a mechanism distinct from that induced by VSV expressing the wt M protein.; rM51R-M virus is defective in its ability to inhibit host gene expression and, therefore, fails to suppress host interferon (IFN) responses, making this virus a particularly promising oncolytic agent for cancer therapy. Apoptosis induced by M protein mutant VSV involves induction of expression of many proapoptotic genes that are suppressed during infection with viruses containing wild-type M protein. Previous research has implicated Fas (CD95) and protein kinase R (PKR) in activation of death receptor pathways by other viruses. I show that apoptosis induced by rM51R-M virus was inhibited by expression of dominant-negative mutants of Fas and PKR. Most previous research has focused on the adapter protein FADD as a necessary transducer of Fas-mediated cell death. However, expression of dominant-negative mutant FADD had little effect on induction of apoptosis by rM51R-M virus. Instead, virus-induced apoptosis was inhibited by expression of a dominant-negative mutant of an alternative adapter protein Daxx. These data indicate that Daxx is more important than FADD for apoptosis induced by M protein mutant VSV. These results show that PKR- and Fas-mediated signaling play important roles in cell death during rM51R-M virus infection, and that Daxx has novel functions in the host response to virus infection by mediating virus-induced apoptosis.; I and others have found that naturally occurring VSVs are more potent apoptosis inducers than recombinant viruses expressing genes from divergent VSV strains. I demonstrate that substitution of P and M genes from the naturally occurring Orsay strain of VSV into a less-potent recombinant wt virus background enhances replication and apoptosis induction by the recombinant virus. This suggests a correlation between VSV growth and apoptosis induction, and indicates a role for the VSV P protein in the regulation of virus-induced apoptosis.