The cellular anti-viral response to infection can be an important determinant of the outcome of an infection, viral tropism, and viral pathogenesis. Infection of most cells with the paramyxovirus Simian Virus 5 (SV5) results in minimal cytopathic effect, cells remain persistently infected and high titers of virus are produced for long periods. Thus, SV5 has very potent mechanisms to avoid activation of host anti-viral pathways that can lead to cell death. By contrast to rSV5-WT, our published data have shown that host apoptotic pathways are activated by infection with a recombinant SV5 (rSV5-P/V-CPI-) containing naturally occurring substitutions in the P/V gene. The basic features of the apoptotic pathways activated by this rSV5- P/V-CPI- have not been determined. The long-term goal of this research project is to determine the mechanisms by which SV5-WT prevents activation of cellular death pathways. In this short term R03 pilot project we will determine the pathways activated by the cytopathic SV5 P/V mutant and test the hypothesis that infection with WT rSV5 leads to inactivation of caspase-3.
In aim 1, we will identify the apoptotic pathways that are activated by infection with the highly cytopathic SV5 variant rSV5-P/V-CPI-. Time course studies of apoptotic events in human lung cells infected with the rSV5-P/V mutant will determine if this cytopathic mutant induces apoptosis primarily through the extrinsic caspase-8 pathway or the intrinsic caspase-9 pathway. Remarkably, our preliminary results have demonstrated that human lung cells infected with WT SV5 show a time-dependent loss of immuno-staining for active caspase-3 enzyme. We hypothesize that WT SV5 infection results in either a loss of caspase-3 polypeptide, a change in subcellular location of caspase-3 or an increase in expression of cellular inhibitors of apoptosis (IAP).
In aim 2, we will distinguish between these hypotheses through immunofluorescence, western blotting, and RNAse protection assays. There is intense interest in defining mechanisms employed by viruses in their battle against host antiviral responses. Our preliminary data have shown that SV5 has potent mechanisms to counteract activation of host apoptotic pathways, and we have in hand a set of defined mutants that are defective in blocking these steps. At the completion of this project, we will have established a firm baseline from which we can pursue detailed mechanistic questions concerning interactions of paramyxoviruses with host antiviral pathways. ? ?
