Ventoso I, Carrasco L
Centro de Biología Molecular (CSIC-UAM), Universidad Autónoma, Canto Blanco, Madrid, Spain.
J Virol. 1995 Oct;69(10):6280-8. doi: 10.1128/JVI.69.10.6280-6288.1995.
Four poliovirus mutants with modifications of tyrosine 88 in 2A(pro) were generated and introduced into the cloned poliovirus genome. Mutants Y88P and Y88L were nonviable, mutant Y88F showed a wild-type (WT) phenotype, and mutant Y88S showed a delayed cytopathic effect and formed small plaques in HeLa cells. Growth of Y88S in HeLa cells was restricted, giving rise to about 20% of the PFU production of the WT poliovirus. The 2A (Y88S) mutant synthesized significantly lower levels of viral proteins in HeLa cells than did the WT poliovirus, while the kinetics of p220 cleavage were identical for both viruses. Strikingly, the 2A (Y88S) mutant was unable to cleave 3CD, as shown by analysis of poliovirus proteins labeled with [35S]methionine or immunoblotted with a specific anti-3C serum. The ability of the Y88S mutant to form infectious virus and cleave 3CD can be complemented by the WT poliovirus. Synthesis of viral RNA was diminished in the Y88S mutant but less than the inhibition of translation of viral RNA. Experiments in which guanidine was used to inhibit poliovirus RNA synthesis suggest that the primary defect of the Y88S mutant virus is at the level of poliovirus RNA translation, while viral genome replication is much less affected. Transfection of HeLa cells infected with the WT poliovirus with a luciferase mRNA containing the poliovirus 5' untranslated sequence gives rise to a severalfold increase in luciferase activity. This enhanced translation of leader-luc mRNA was not observed when the transfected cells were infected with the 2A (Y88S) mutant. Moreover, cotransfection with mRNA encoding WT poliovirus 2A(pro) enhanced translation of leader-luc mRNA. This enhancement was much lower upon transfection with mRNA encoding 2A(Y88S), 2A(Y88L), or 2A(Y88P). These findings support the view that 2A(pro) itself, rather than the 3C' and/or 3D' products, is necessary for efficient translation of poliovirus RNA in HeLa cells.
构建了4种在2A蛋白酶（2A(pro)）中酪氨酸88位点发生修饰的脊髓灰质炎病毒突变体，并将其导入克隆的脊髓灰质炎病毒基因组中。突变体Y88P和Y88L无法存活，突变体Y88F表现出野生型（WT）表型，而突变体Y88S在HeLa细胞中表现出延迟的细胞病变效应并形成小斑块。Y88S在HeLa细胞中的生长受到限制，其产生的空斑形成单位（PFU）约为野生型脊髓灰质炎病毒的20%。与野生型脊髓灰质炎病毒相比，2A（Y88S）突变体在HeLa细胞中合成的病毒蛋白水平显著降低，而两种病毒的p220切割动力学相同。引人注目的是，如用[35S]甲硫氨酸标记或用特异性抗3C血清进行免疫印迹分析脊髓灰质炎病毒蛋白所示，2A（Y88S）突变体无法切割3CD。Y88S突变体形成感染性病毒和切割3CD的能力可被野生型脊髓灰质炎病毒互补。Y88S突变体中病毒RNA的合成减少，但低于病毒RNA翻译的抑制程度。使用胍抑制脊髓灰质炎病毒RNA合成的实验表明，Y88S突变体病毒的主要缺陷在于脊髓灰质炎病毒RNA翻译水平，而病毒基因组复制受影响较小。用含有脊髓灰质炎病毒5'非翻译序列的荧光素酶mRNA转染感染野生型脊髓灰质炎病毒的HeLa细胞，可使荧光素酶活性增加数倍。当用2A（Y88S）突变体感染转染细胞时，未观察到前导-荧光素酶mRNA的这种增强翻译。此外，与编码野生型脊髓灰质炎病毒2A(pro)的mRNA共转染可增强前导-荧光素酶mRNA的翻译。用编码2A(Y88S)、2A(Y88L)或2A(Y88P)的mRNA转染时，这种增强作用要低得多。这些发现支持了这样一种观点，即2A(pro)本身而非3C'和/或3D'产物对于HeLa细胞中脊髓灰质炎病毒RNA的有效翻译是必需的。
