The dsRNA response is a critical defense pathway used by cells to guard against viral infection. Criticalfor the function of this pathway is the production of IFN-p, which requires the activation of the latenttranscription factor IRF-3. IRF-3 is normally found in the cytoplasm, but upon exposure to dsRNA or viralinfection IRF-3 is phosphorylated, dimerizes and translocates into the nucleus where it activates transcriptionof IFN-p. Recently, we found that infection of HeLa cells with rhinovirus type 14 (RV14) prevents dimerizationof IRF-3 and production of IFN-b, and thus attenuates this response. The overall goal of this proposal is todetermine the underlying mechanisms responsible for inhibition of the dsRNA response by rhinovirus anddetermine if other picornaviruses utilize a similar strategy to evade the host innate immune response.
The first aim of this proposal will examine the mechanism responsible for the lack of IFN-p mRNA and IRF-3dimerization in rhinovirus-infected cells. This will be accomplished by determining the rate of transcriptionfrom the IFN-p promoter using nuclear run-on experiments and by determining if phosphorylation of IRF-3 isdisrupted in infected cells.
The second aim of this proposal will identify the viral factors responsible forinhibition of the dsRNA response by examining induction of the dsRNA response in cell lines that expressindividual viral proteins. Mutants of identified proteins will be generated and analyzed to determine thefunctional domains responsible for inhibition of the dsRNA response. Ultimately, these mutations will beintroduced into an infectious rhinovirus cDNA to determine the role that inhibition of the dsRNA response hasin viral replication.
The third aim will determine if inhibition of the dsRNA response extends to other cellularpromoters and other picornaviruses. Full genome Affymetrix microarrays will be used to determine the globalhost transcriptional response to dsRNA in mock or RV14-infected cells. Subsequently, we will determine ifactivation and attenuation of the dsRNA response is unique to rhinoviruses or extends to otherpicornaviruses. This will be accomplished by examining IRF-3 and NF-icB activation and the globaltranscriptional response in cells infected with other picornaviruses.These studies will enhance our understanding of how rhinovirus evades host immune defensemechanisms and may lead to the development of better therapeutics and strategies to combat theseprevalent human pathogens. In addition, these experiments will also provide us with general insights into thebasic cellular processes of signal transduction and transcriptional regulation. With this understanding it maybe possible to counteract the effect of infectious agents and reduce disease and mortality in humans,animals and plants.
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| Kotla, Swathi; Major, Stephanie C; Gustin, Kurt E (2009) Rapid detection and quantitation of poliovirus and rhinovirus sequences in viral stocks and infected cells. J Virol Methods 157:32-9 |
