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 (PV)通过RNA聚合酶I抑制宿主细胞RNA合成的起始， 二和三。与TATA盒相互作用的pol Il因子，TFIID 启动子，pol I因子，与内部相互作用的TFIIIC tRNA基因的启动子元件，以及启动子元件中的poll因子 识别（上游结合因子，UBF）在PV后不久失活 感染生物化学和遗传学证据表明， 蛋白酶，3CPro直接参与宿主细胞转录关闭 3CPro特异性地切割TATA结合蛋白TBP（TATA的一种组分）。 TFIID），TFIIIC和一个尚未确定的民意调查因素。此外，一些 因子如TFIIIC和pol II因子CREB（环AMP反应性 元件结合蛋白）在感染的细胞中被去磷酸化。因此两 蛋白水解和去磷酸化有助于宿主细胞转录 关闭。 将采用生物化学、血清学和遗传学方法来确定 病毒中TBP、TFIIIC、UBF和CREB的失活机制 被感染的细胞 具体地，我们将确定3CPro如何切割TBP 导致TBP失活。我们将确定截短的TBP是否 与其他一般转录因子或TBP相关的 蛋白质（TAF）。是否突变的TBP，其对切割具有抗性， 3CPro对转录关闭敏感。 我们将 还确定是否存在3CPro功能缺陷的突变脊髓灰质炎病毒， 在关闭pol II转录和3CPro表达方面有缺陷 HeLa细胞中的基因导致转录关闭。的机理 CREB的去磷酸化将通过使用生物化学和 血清学技术 生化和血清学技术将用于检查性质 在TFIIIC中被3CPro切割的多肽的量。遗传、生物化学和 血清学技术将用于鉴定受影响的pol I因子 并研究其失活机制 转录活性由于TATA结合蛋白最近被 显示参与pol I和pol III转录，我们将确定 3CPro切割TBP是否有助于pol III和pol I 转录关闭使用遗传和生物化学手段。 阐明脊髓灰质炎病毒负面影响 细胞转录因子的活动无疑会促进 更好地理解真核细胞中的转录调控， 以及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.