Yalamanchili P, Harris K, Wimmer E, Dasgupta A
Department of Microbiology and Immunology, University of California, Los Angeles, School of Medicine, 90024-1747, USA.
J Virol. 1996 May;70(5):2922-9. doi: 10.1128/JVI.70.5.2922-2929.1996.
Host cell RNA polymerase II (pol II)-mediated transcription is inhibited by poliovirus infection. We demonstrate here that both TATA- and initiator-mediated basal transcription is inhibited in extracts prepared from poliovirus-infected HeLa cells. This inhibition can be reproduced by incubation of uninfected HeLa cell extracts with purified, recombinant poliovirus protease, 3Cpro. Transient-transfection assays demonstrate that 3Cpro, in the absence of other viral proteins, is able to inhibit cellular pol II-mediated transcription in vivo. Three lines of evidence suggest that inactivation of TATA-binding protein (TBP) is the major cause of inhibition of basal transcription by poliovirus. First, RNA pol II transcription in poliovirus-infected cell extract is fully restored by bacterially expressed TBP. Second, addition of purified TBP restores transcription in heat-treated nuclear extracts from mock- and virus-infected cells to identical levels. Finally, using a gel mobility shift assay, we demonstrate that incubation of TBP with the viral protease (3Cpro) inhibits its ability to bind TATA sequence in vitro. These results suggest that inhibition of pol II transcription in mammalian cells infected with poliovirus is, at least in part, due to the inability of modified TBP to bind pol II promoter sequences.
脊髓灰质炎病毒感染会抑制宿主细胞RNA聚合酶II（pol II）介导的转录。我们在此证明，在从脊髓灰质炎病毒感染的HeLa细胞制备的提取物中，TATA盒和起始子介导的基础转录均受到抑制。通过将未感染的HeLa细胞提取物与纯化的重组脊髓灰质炎病毒蛋白酶3Cpro一起孵育，可以重现这种抑制作用。瞬时转染试验表明，在没有其他病毒蛋白的情况下，3Cpro能够在体内抑制细胞pol II介导的转录。三条证据表明，TATA结合蛋白（TBP）的失活是脊髓灰质炎病毒抑制基础转录的主要原因。首先，细菌表达的TBP可完全恢复脊髓灰质炎病毒感染的细胞提取物中的RNA pol II转录。其次，添加纯化的TBP可将模拟感染和病毒感染细胞的热处理核提取物中的转录恢复到相同水平。最后，使用凝胶迁移率变动分析，我们证明TBP与病毒蛋白酶（3Cpro）孵育会抑制其在体外结合TATA序列的能力。这些结果表明，脊髓灰质炎病毒感染的哺乳动物细胞中pol II转录的抑制至少部分是由于修饰后的TBP无法结合pol II启动子序列。
