首页> 美国卫生研究院文献>Journal of Virology >Defective RNA replication by poliovirus mutants deficient in 2A protease cleavage activity.
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Defective RNA replication by poliovirus mutants deficient in 2A protease cleavage activity.

机译:脊髓灰质炎病毒突变体的2A蛋白酶切割活性不足的RNA复制缺陷。

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摘要

2A protease (2Apro) catalyzes the initial cleavage of the poliovirus polyprotein which separates the P1 structural protein precursor from the P2-P3 nonstructural protein precursor. In addition, 2Apro indirectly induces cleavage of the p220 component of eukaryotic initiation factor 4F, which is thought to contribute to the specific inhibition of host cell protein synthesis observed in virus-infected HeLa cells. However, it is unclear whether the trans function of 2Apro which induces host cell shutoff is essential or merely facilitates efficient poliovirus replication. In this study, three point mutations in 2Apro (D38E, Y88L, and Y89L [S. F. Yu and R. E. Lloyd, Virology 182:615-625, 1991]) which cause specific loss of trans but not cis cleavage function were independently introduced into the full-length poliovirus cDNA. In addition, mutations which caused only partial loss of both cis and trans cleavage activities (Y88S) or resulted in a wild-type phenotype (Y88F) were individually introduced. When each of these mutant poliovirus cDNAs was transcribed and translated in vitro, normal proteolytic processing of the viral polyprotein was observed, and p220 was not cleaved in those reactions containing proteases defective in trans function, as expected. Surprisingly, Northern (RNA) blot analysis and reverse transcriptase-PCRs performed after transfection of COS-7 or HeLa cells with these viral RNAs revealed that Y88S and Y88L RNAs replicated at only very low levels. RNA replication could not be detected at all in cells transfected with D38E and Y89L RNAs. Taken together, the results suggest a correlation between the function of 2Apro and productive poliovirus RNA replication in vivo that may be independent of the ability to cause p220 cleavage.
机译:2A蛋白酶(2Apro)催化脊髓灰质炎病毒多蛋白的初始裂解,该裂解将P1结构蛋白前体与P2-P3非结构蛋白前体分离。此外,2Apro间接诱导真核起始因子4F的p220组分裂解,这被认为有助于特异性抑制在病毒感染的HeLa细胞中观察到的宿主细胞蛋白质合成。然而,尚不清楚诱导宿主细胞关闭的2Apro的反式功能是否必不可少或仅促进有效的脊髓灰质炎病毒复制。在这项研究中,将2Apro的三个点突变(D38E,Y88L和Y89L [SF Yu and RE Lloyd,Virology 182:615-625,1991])独立引入了完整的反式中,但没有引起顺式切割功能的丧失。长脊髓灰质炎病毒cDNA。另外,单独引入仅引起顺式和反式切割活性的部分丧失(Y88S)或导致野生型表型(Y88F)的突变。当在体外转录和翻译这些突变脊髓灰质炎病毒的每个cDNA时,观察到病毒多蛋白的正常蛋白水解过程,并且如预期的那样,在那些含有反式功能缺陷蛋白酶的反应中,p220没有被切割。出人意料的是,用这些病毒RNA转染COS-7或HeLa细胞后进行的RNA印迹分析和逆转录酶PCR显示Y88S和Y88L RNA仅以非常低的水平复制。在用D38E和Y89L RNA转染的细胞中根本检测不到RNA复制。两者合计,结果表明2Apro的功能与体内生产性脊髓灰质炎病毒RNA复制之间的相关性可能与引起p220裂解的能力无关。

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