Franco David, Pathak Harsh B, Cameron Craig E, Rombaut Bart, Wimmer Eckard, Paul Aniko V
Department of Molecular Genetics and Microbiology, School of Medicine, Stony Brook University, Stony Brook, NY 11790, USA.
J Virol. 2005 May;79(10):6358-67. doi: 10.1128/JVI.79.10.6358-6367.2005.
The plus-strand RNA genome of poliovirus serves three distinct functions in the life cycle of the virus. The RNA is translated and then replicated, and finally the progeny RNAs are encapsidated. These processes can be faithfully reproduced in a HeLa cell-free in vitro translation-RNA replication system that produces viable poliovirus. We have previously observed a stimulation of virus synthesis when an mRNA, encoding protein 3CD(pro), is added to the translation-RNA replication reactions of poliovirus RNA. Our aim in these experiments was to further define the factors that affect the stimulatory activity of 3CD(pro) in virus synthesis. We observed that purified 3CD(pro) protein also enhances virus synthesis by about 100-fold but has no effect on the translation of the polyprotein. Optimal stimulation is observed only when 3CD(pro) is present early in the incubation period. The stimulation, however, is abolished by a mutation either in the RNA binding domain of 3CD(pro), 3C(pro)R84S/I86A, or by each of two groups of complementary mutations R455A/R456A and D339A/S341A/D349A at interface I in the 3D(pol) domain of 3CD(pro). Surprisingly, virus synthesis is strongly inhibited by the addition of both 3C(pro) and 3CD(pro) at the beginning of incubation. We also examined the effect of other viral or cellular proteins on virus synthesis in the in vitro system. No enhancement of virus synthesis occurred with viral proteins 3BC, 3ABC, 3BCD, 3D(pol), and 3C(pro) or with cellular protein PCBP2. These results suggest that 3CD(pro) has to be present in the reaction at the time the replication complexes are assembled and that both the 3C(pro) and 3D(pol) domains of the protein are required for its activity that stimulates virus production.
脊髓灰质炎病毒的正链RNA基因组在病毒生命周期中发挥三种不同功能。该RNA先被翻译，然后进行复制，最后子代RNA被衣壳化。这些过程可以在无细胞的HeLa体外翻译-RNA复制系统中如实地重现，该系统能产生有活力的脊髓灰质炎病毒。我们之前观察到，当将编码蛋白3CD(pro)的mRNA添加到脊髓灰质炎病毒RNA的翻译-RNA复制反应中时，病毒合成会受到刺激。我们在这些实验中的目的是进一步确定影响3CD(pro)在病毒合成中刺激活性的因素。我们观察到纯化的3CD(pro)蛋白也能将病毒合成增强约100倍，但对多聚蛋白的翻译没有影响。只有在潜伏期早期存在3CD(pro)时才能观察到最佳刺激效果。然而，3CD(pro) RNA结合结构域中的突变3C(pro)R84S/I86A，或3CD(pro) 3D(pol)结构域中界面I处的两组互补突变R455A/R456A和D339A/S341A/D349A中的任何一组，都会消除这种刺激作用。令人惊讶的是，在孵育开始时同时添加3C(pro)和3CD(pro)会强烈抑制病毒合成。我们还研究了其他病毒或细胞蛋白对体外系统中病毒合成的影响。病毒蛋白3BC、3ABC、3BCD、3D(pol)和3C(pro)以及细胞蛋白PCBP2均未增强病毒合成。这些结果表明，在复制复合物组装时反应中必须存在3CD(pro)，并且该蛋白的3C(pro)和3D(pol)结构域对于其刺激病毒产生的活性都是必需的。
