INHIBITION OF HOST CELL TRANSCRIPTION BY POLIOVIRUS

本提案的长期目标是确定脊髓灰质炎病毒（一组具有医学重要性的病毒（小核糖核酸病毒）的原型因子）如何抑制细胞RNA聚合酶I、II和III启动宿主细胞RNA合成。 先前的研究已经鉴定了四种序列特异性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）、一种Pol III因子、与Pol III启动子相互作用的TFIIIC和一种Pol I因子SL-1，所有这些因子在病毒感染的细胞中转录失活。 生物化学和遗传学证据表明，病毒编码的蛋白酶3Cpro在体内和体外切割这些因子，并直接负责宿主细胞转录关闭。生物化学、血清学和遗传学方法将用于确定病毒感染细胞中转录因子失活的机制。 具体而言，如何TBP和Oct-1切割3Cpro有助于一般Pol II和小核RNA（snRNA）转录将得到解决。 由于TBP也需要的Pol III和Pol I转录（除了Pol II转录），实验提出了阐明TBP切割Pol III催化的snRNA转录和Pol I转录的作用。 此外，将讨论Pol I因子SL-1的四个亚基之一（TAF 110，TBP相关因子）的裂解导致Pol I关闭的机制。 将使用生物化学和遗传学方法研究关闭宿主细胞转录所需的病毒蛋白酶（3Cpro）进入核的机制。 最后，将研究病毒蛋白（2C）特异性刺激Pol I体外转录的机制。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理解病毒-宿主相互作用以及真核细胞中的转录调控。

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.