Haller A A, Stewart S R, Semler B L
Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine 92717, USA.
J Virol. 1996 Mar;70(3):1467-74. doi: 10.1128/JVI.70.3.1467-1474.1996.
The nucleotide at position 480 in the 5' noncoding region of the viral RNA genome plays an important role in directing the attenuation phenotype of the Sabin vaccine strain of poliovirus type 1. In vitro translation studies have shown that the attenuated viral genomes of the Sabin strains direct levels of viral protein synthesis lower than those of their neurovirulent counterparts. We previously described the isolation of pseudorevertant polioviruses derived from transfections of HeLa cells with genome-length RNA harboring an eight-nucleotide lesion in a stem-loop structure (stem-loop V) that contains the attenuation determinant at position 480 (A. A. Haller and B. L. Semler, J. Virol. 66:5075-5086, 1992). This stem-loop structure is a major component of the poliovirus internal ribosome entry site required for initiation of viral protein synthesis. The eight-nucleotide lesion (X472) was lethal for virus growth and gave rise only to viruses which had partially reverted nucleotides within the original substituted sequences. In this study, we analyzed two of the poliovirus revertants (X472RI and X472R2) for cell-type-specific growth properties. The X472RI and X472R2 RNA templates directed protein synthesis to wild-type levels in in vitro translation reaction mixtures supplemented with crude cytoplasmic HeLa cell extracts. In contrast, the same X472 revertant RNAs displayed a decreased translation initiation efficiency when translated in a cell-free system supplemented with extracts from neuronal cells. This translation initiation defect of the X472R templates correlated with reduced yields of infectious virus particles in neuronal cells compared with those obtained from HeLa cells infected with the X472 poliovirus revertants. Our results underscore the important of RNA secondary structures within the poliovirus internal ribosome entry site in directing translation initiation and suggest that such structures interact with neuronal cell factors in a specific manner.
脊髓灰质炎病毒1型Sabin疫苗株的病毒RNA基因组5'非编码区第480位核苷酸在引导其减毒表型方面发挥着重要作用。体外翻译研究表明，Sabin株的减毒病毒基因组指导的病毒蛋白合成水平低于其神经毒力对应株。我们之前描述了从用在包含第480位衰减决定簇的茎环结构（茎环V）中具有八个核苷酸损伤的基因组长度RNA转染HeLa细胞中获得的假回复脊髓灰质炎病毒的分离（A. A. Haller和B. L. Semler，《病毒学杂志》66:5075 - 5086，1992年）。这种茎环结构是病毒蛋白合成起始所需的脊髓灰质炎病毒内部核糖体进入位点的主要组成部分。这八个核苷酸损伤（X472）对病毒生长是致死性的，并且只产生了在原始取代序列内部分回复核苷酸的病毒。在本研究中，我们分析了两种脊髓灰质炎病毒回复株（X472RI和X472R2）的细胞类型特异性生长特性。在补充有HeLa细胞粗细胞质提取物的体外翻译反应混合物中，X472RI和X472R2 RNA模板指导蛋白合成达到野生型水平。相比之下，当在补充有神经元细胞提取物的无细胞系统中翻译时，相同的X472回复株RNA显示出翻译起始效率降低。与用X472脊髓灰质炎病毒回复株感染的HeLa细胞相比，X472R模板的这种翻译起始缺陷与神经元细胞中感染性病毒颗粒产量降低相关。我们的结果强调了脊髓灰质炎病毒内部核糖体进入位点内的RNA二级结构在指导翻译起始方面的重要性，并表明这些结构以特定方式与神经元细胞因子相互作用。
