The long term objective of this proposal is to determine how poliovirus, the prototype agent of a medically important group of viruses (picornaviruses), inhibits initiation of host cell RNA synthesis by cellular RNA polymerases I, II and III. Previous studies have identified four sequence specific DNA binding Pol II transcription factors (TBP, the TATA binding protein, CREB, the cyclic AMP-responsive element binding protein, Oct-1, the octamer binding factor, and transcriptional activator p53), one Pol III factor, TFIIIC, which interacts with Pol III promoter, and SL-1, a Pol I factor, all of which are transcriptionally inactivated in virus-infected cells. Both biochemical and genetic evidence suggests that the virus-encoded protease, 3Cpro, cleaves these factors in vivo and in vitro and is directly responsible for host cell transcription shut-off. Biochemical, serological and genetic approaches will be used to determine the mechanism(s) of inactivation of transcription factors in virus-infected cells. Specifically, how TBP and Oct-1 cleavage by 3Cpro contribute to general Pol II and small nuclear RNA (snRNA) transcription will be addressed. Because TBP is also required of Pol III and Pol I transcription (in addition to Pol II transcription), experiments are proposed to elucidate the role of TBP cleavage in Pol III-catalyzed snRNA transcription and Pol I transcription. Additionally, the mechanism by which cleavage of one of the four subunits (TAF110, TBP associated factor) of the Pol I factor SL-1, contribute to Pol I shut-off, will be addressed. The mechanism of nuclear entry of the viral protease (3Cpro) required to shut-off host cell transcription will be studied using biochemical and genetic approaches. Finally, the mechanism by which a viral protein (2C) specifically stimulates Pol I transcription in vitro will be studied. Elucidation of the mechanism by which poliovirus negatively affects cellular transcription factor activities would undoubtedly facilitate a better understanding of virus-host interaction as well as regulation of transcription in eukaryotic cells.
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