The long term objective of this proposal is to determine how poliovirus (PV) inhibits initiation of host cell RNA synthesis by RNA polymerases I, II and III. The pol Il factor, TFIID which interacts with the TATA box promotors, the pol I factor, TFIIIC which interacts with the internal promotor elements of tRNA genes, and a poll factor involved in promotor recognition (upstream binding factor, UBF) are inactivated soon after PV infection. Biochemical and genetic evidence suggests that a virus-encoded proteinase, 3CPro is directly involved in host cell transcription shut-off 3CPro specifically cleaves the TATA-binding protein, TBP (a component of TFIID), TFIIIC and an yet unidentified poll factor. Additionally, some factors such as TFIIIC and a pol II factor CREB (cyclic AMP-responsive element binding protein) are dephosphorylated in infected cells. Thus both proteolysis and dephosphorylation contribute to host cell transcription shut-off. Biochemical, serological and genetic approaches will be used to determine the mechanism(s) of inactivation of TBP, TFIIIC, UBF and CREB in virus- infected cells. Specifically, we will determine how TBP cleavage by 3CPro leads to inactivation of TBP. We will determine whether truncated TBP interacts with other general transcription factors or TBP-associated proteins (TAFs). Whether a mutant TBP, which is resistant to cleavage by 3CPro, is susceptible to transcription shut-off will be examined. We will also determine whether a mutant poliovirus defective in 3CPro function is defective in shutting-off pol II transcription and if expression of 3CPro gene in HeLa cells leads to transcription shut-off. The mechanism of dephosphorylation of CREB will be examined by using both biochemical and serological techniques. Biochemical and serological techniques will be used to examine the nature of the polypeptide(s) cleaved by 3CPro in TFIIIC. Genetic, biochemical and serological techniques will be used to identify the pol I factor affected in virus-infected cells and to study the mechanism of inactivation of its transcriptional activity. Since the TATA binding protein has recently been shown to be involved in pol I and pol III transcription, we will determine whether cleavage of TBP by 3CPro contributes to pol III and pol I transcription shut-off using genetic and biochemical means. Elucidation of the mechanism by which poliovirus negatively affects cellular transcription factor activities would undoubtedly facilitate a better understanding of regulation of transcription in eukaryotic cells as well as virus-host interactions in general.
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