INHIBITION OF HOST-CELL TRANSCRIPTION BY POLIOVIRUS

本提案的长期目标是确定如何 脊髓灰质炎病毒抑制宿主细胞RNA合成的起始 （转录）通过RNA聚合酶II和III。相比 未感染的提取物，脊髓灰质炎病毒感染的细胞提取物不能 特异性地转录含有polII-和 polIII启动子。 补充感染细胞提取物， 用部分纯化的polII-转录因子TFIIIC（其 与tRNA和VA 1基因的内部启动子元件相互作用） 特异性恢复polIII-和polIII-转录， 启动子，分别在这些转录恢复因子 与已知的polIII和polIII -转录因子。 抑制转录因子活性的机制 将通过比较数量， 它们的特异性活性和翻译后修饰 病毒感染与模拟感染细胞中的因子， 和血清学技术。 特异性启动子结合能力 从模拟和病毒感染的细胞中分离出的这些因子将 通过足迹法和凝胶阻滞试验进行分析。 我们将从感染的细胞中分离出病毒编码或诱导的抑制剂（S）， 细胞提取物，能够抑制体外转录， 使用各种生化技术模拟感染的提取物， 研究抑制剂如何影响转录活性 体外因素。 突变型脊髓灰质炎病毒可能在 宿主细胞转录抑制，将用于进一步检查 抑制宿主细胞转录的机制。 阐明脊髓灰质炎病毒负面影响 细胞转录因子的活动无疑 有助于更好地了解 真核细胞中的转录。

The long term objective of this proposal is to determine how poliovirus inhibits initiation of host cell RNA synthesis (transcription)by RNA polymerases II and III. In contrast with uninfected extracts, poliovirus-infected cell extracts are unable to transcribe specifically DNA templates containing polII-and polIII-promoters. Supplementation of the infected cell extracts, with partially purified polII-transcription factor TFIIIC (which interacts with internal promoter elements of tRNA and VA1 genes) specifically restore transcription from polIII-and polIII- promoters, respectively in these transcription-restoration factors with known polIII and polIII -transcription factors. The mechanism(s) of inhibition of transcription factor activities by virus-infection will be examined by comparing the quantity, specific activity and posttranslational modifications of these factors in virus-infected vs. mock-infected cells using biochemical and serological techniques. Specific promoter-binding abilities of these factors isolated form mock-and virus-infected cells will be analyzed by footprinting and gel-retardation assays. We will isolate virus-encoded or -induced inhibitor(S)from infected cell extracts, capable of inhibiting in vitro transcription in mock-infected extracts using various biochemical techniques and examine how the inhibitor affects activities of transcription factors in vitro. Mutant polioviruses which may be defective in host cell transcription-inhibition, will be used to further examine the mechanism of inhibition of host cell transcription. Elucidation of the mechanism by which poliovirus negatively affects cellular transcription factor activities would undoubtedly facilitate a better understanding of the regulation of transcription in eukaryotic cells.