Blyn L B, Chen R, Semler B L, Ehrenfeld E
Department of Molecular Biology and Biochemistry, School of Biological Sciences, University of California, Irvine 92717, USA.
J Virol. 1995 Jul;69(7):4381-9. doi: 10.1128/JVI.69.7.4381-4389.1995.
Translation of poliovirus RNA occurs by the binding of ribosomes to an internal segment of RNA sequence within the 5' untranslated region of the viral RNA. This region is predicted to consist of six domains (I to VI) that possess complex secondary and tertiary structures. Domain IV is a large region in which alterations in the sequence or structure markedly reduce translational efficiency. In this study, we employed RNA mobility shift assays to demonstrate that a protein(s) from uninfected HeLa cell extracts, as well as from neuroblastoma extracts, interacts with the domain IV structure. A mutation in domain IV caused reduced binding of HeLa cell proteins and reduced translation both in vitro and in vivo, suggesting that the binding of at least one of these proteins plays a role in the mechanism of viral translation. UV cross-linking indicated that a protein(s) with a size of approximately 40 kDa interacted directly with the RNA. Using streptavidin beads to capture biotinylated RNA bound to proteins, we were able to visualize a number of HeLa and neuroblastoma cell proteins that interact with domain IV. These proteins have molecular masses of approximately 39, approximately 40, and approximately 42 kDa.
脊髓灰质炎病毒RNA的翻译是通过核糖体与病毒RNA 5'非翻译区内RNA序列的一个内部片段结合来实现的。该区域预计由六个结构域（I至VI）组成，这些结构域具有复杂的二级和三级结构。结构域IV是一个大区域，其中序列或结构的改变会显著降低翻译效率。在本研究中，我们采用RNA迁移率变动分析来证明，来自未感染的HeLa细胞提取物以及神经母细胞瘤提取物中的一种或多种蛋白质与结构域IV的结构相互作用。结构域IV中的一个突变导致HeLa细胞蛋白质的结合减少，并且在体外和体内的翻译均减少，这表明这些蛋白质中至少有一种的结合在病毒翻译机制中起作用。紫外线交联表明，一种大小约为40 kDa的蛋白质直接与RNA相互作用。使用链霉亲和素磁珠捕获与蛋白质结合的生物素化RNA，我们能够观察到许多与结构域IV相互作用的HeLa和神经母细胞瘤细胞蛋白质。这些蛋白质的分子量约为39 kDa、约40 kDa和约42 kDa。
