Our recent work has led to the overall hypothesis that following infection, paramyxoviruses delay abundant RNA synthesis to provide time for virus-mediated suppression of host anti-virus responses. This hypothesis emerged from our results with variants of the prototype paramyxovirus Simian Virus 5 (SV5) that have either accelerated or delayed kinetics of RNA synthesis. A remarkable result from this work was our finding that mutations in the P/V gene that accelerate viral RNA synthesis also convert SV5 from a noncytopathic virus into a virus that activates type I interferon (IFN) and kills cells by apoptosis. Thus, the overall goal of our work is to understand mechanisms that can accelerate or delay paramyxovirus RNA synthesis and how changes in the timing of viral gene expression affect key virus:host cell interactions. ? We have isolated a naturally-occurring SV5 variant (WF-CPIV) that shows a remarkable delay in viral gene expression compared to WT SV5.
In Aim 1, we will determine the basis for the delay in WF-CPIV gene expression. Real-time PCR assays will be used to test the hypotheses that delayed WF-CPIV gene expression is due to a defect in either primary or secondary transcription. Recombinant SV5 viruses containing exchanges of polymerase-associate genes will identify WF-CPIV component(s) responsible for delayed gene expression.
Aim 2 addresses our finding that an rSV5 with substitutions in the P/V gene expresses viral mRNA and proteins earlier and to higher levels than WT rSV5. Novel rSV5 viruses will be generated that express WT or mutant P and V proteins from separate transcription units. Biochemical and minigenome replication assays will be used to test the hypotheses that P/V substitutions either decrease the ability of V to inhibit SV5 genome replication or confer higher RNA synthesis activity on the P protein.
In Aim 3, we will test the hypothesis that the cytopathic rSV5 variant containing P/V substitutions induces interferon (IFN) synthesis and apoptosis due to premature expression of viral gene products. Cell lines with inducible expression of WT and mutant P and V proteins, and new rSV5 P/V mutants will be used to test a two-step model for the role of accelerated and delayed SV5 RNA synthesis in the induction of IFN and apoptosis. ? We hypothesize that paramyxoviruses have evolved to optimize the timing of the onset of viral gene expression to avoid host antiviral responses, and changes that accelerate or significantly delay RNA synthesis can activate IFN and/or apoptotic pathways. Our work will increase our understanding of important virus-host cell interactions, as well as provide new approaches to improve the safety and potency of vaccine vectors. ? ? ?
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