The long term objective of this proposal is to determine how poliovirus inhibits initiation of host cell RNA synthesis (transcription)by RNA polymerases II and III. In contrast with uninfected extracts, poliovirus-infected cell extracts are unable to transcribe specifically DNA templates containing polII-and polIII-promoters. Supplementation of the infected cell extracts, with partially purified polII-transcription factor TFIIIC (which interacts with internal promoter elements of tRNA and VA1 genes) specifically restore transcription from polIII-and polIII- promoters, respectively in these transcription-restoration factors with known polIII and polIII -transcription factors. The mechanism(s) of inhibition of transcription factor activities by virus-infection will be examined by comparing the quantity, specific activity and posttranslational modifications of these factors in virus-infected vs. mock-infected cells using biochemical and serological techniques. Specific promoter-binding abilities of these factors isolated form mock-and virus-infected cells will be analyzed by footprinting and gel-retardation assays. We will isolate virus-encoded or -induced inhibitor(S)from infected cell extracts, capable of inhibiting in vitro transcription in mock-infected extracts using various biochemical techniques and examine how the inhibitor affects activities of transcription factors in vitro. Mutant polioviruses which may be defective in host cell transcription-inhibition, will be used to further examine the mechanism of inhibition of host cell transcription. Elucidation of the mechanism by which poliovirus negatively affects cellular transcription factor activities would undoubtedly facilitate a better understanding of the regulation of transcription in eukaryotic cells.
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