INHIBITION OF HOST CELL TRANSCRIPTION BY POLIOVIRUS

这一建议的长期目标是确定脊髓灰质炎病毒是如何抑制由细胞RNA聚合酶I、II和III启动的宿主细胞RNA合成的。以前的研究已经确定了四种序列特异的DNA结合Pol II转录因子(TBP、TATA结合蛋白、CREB、环状AMP反应元件结合蛋白、Oct-1、八聚体结合因子和转录激活因子P53)、一个Pol III因子TFIIIC和SL-1，它们都在病毒感染的细胞中转录失活。生化和遗传证据都表明，病毒编码的蛋白酶3Cpro在体内和体外都能裂解这些因子，并直接导致宿主细胞转录的关闭。将使用生化、血清学和遗传学方法来确定病毒感染细胞中转录因子失活的机制(S)。具体地说，3CPro对TBP和OCT-1的切割如何促进一般POL II和小核RNA(SnRNA)的转录。由于TBP也是Pol III和Pol I转录所必需的(除了Pol II转录之外)，因此建议通过实验来阐明TBP切割在Pol III催化的SnRNA转录和Pol I转录中的作用。此外，将讨论Pol I因子SL-1的四个亚基(TAF110，TBP相关因子)中的一个亚基的切割导致Pol I关闭的机制。关闭宿主细胞转录所需的病毒蛋白酶(3Cpro)的核进入机制将使用生化和遗传学方法进行研究。最后，我们将研究病毒蛋白(2C)在体外特异性刺激Pol I转录的机制。阐明脊髓灰质炎病毒对细胞转录因子活性产生负面影响的机制，无疑将有助于更好地了解病毒与宿主的相互作用以及真核细胞中转录的调控。

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.