INHIBITION OF HOST CELL TRANSCRIPTION BY POLIOVIRUS

这项提案的长期目标是确定脊髓灰质炎病毒如何 (Pv)抑制由RNA聚合酶I启动的宿主细胞RNA合成， II和III.与TATA盒相互作用的POLIL因子，TFIID 推动者，PolI因子，TFIIIC，与内部 TRNA基因的启动子元件和参与启动子的轮询因子 识别(上游结合因子，UBF)在PV后不久失活 感染。生化和遗传证据表明，一种编码的病毒 蛋白水解酶3CPRO直接参与宿主细胞转录关闭 3CPro特异性切割TATA结合蛋白TBP(TBP的一种成分 TFIID)、TFIIIC和一个尚未确定的民调因素。此外，还有一些 TFIIIC和POL II因子CREB(环磷酸腺苷反应因子 元件结合蛋白)在受感染的细胞中被去磷酸化。因此，两者都 蛋白分解和去磷酸化有助于宿主细胞的转录 关机。 将使用生化、血清学和遗传学方法来确定 病毒中TBP、TFIIIC、UBF和CREB失活的机制(S) 被感染的细胞。具体地说，我们将确定TBP如何被3CPro切割 导致TBP失活。我们将确定截断的TBP是否 与其他通用转录因子或TBP相关的转录因子相互作用 蛋白质(TAFs)。一种突变的TBP，它能抵抗被 3CPRO，是敏感的转录关闭将被检查。我们会 还要确定3CPro功能缺陷的脊髓灰质炎变异病毒是否 Pol II转录和3CPro的IF表达缺陷 HeLa细胞中的基因导致转录关闭。它的作用机制 CREB的去磷酸化将通过生化和 血清学技术。 将使用生化和血清学技术来检查性质 3CPro在TFIIIC中切割的多肽(S)。遗传、生化和 将使用血清学技术来鉴定受影响的poli因子。 并研究其失活机制。 转录活性。由于TATA结合蛋白最近被 被证明参与了POL I和POL III转录，我们将确定 3CPro对TBP的切割是否有助于POL III和POL I 使用遗传和生化手段关闭转录。 阐明脊髓灰质炎病毒的负面影响机制 细胞转录因子的活性无疑会促进 更好地了解真核细胞的转录调控 以及一般情况下病毒与宿主的相互作用。

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.