Haller A A, Nguyen J H, Semler B L
Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine 92717.
J Virol. 1993 Dec;67(12):7461-71. doi: 10.1128/JVI.67.12.7461-7471.1993.
Translation initiation by internal ribosome binding is a recently discovered mechanism of eukaryotic viral and cellular protein synthesis in which ribosome subunits interact with the mRNAs at internal sites in the 5' untranslated RNA sequences and not with the 5' methylguanosine cap structure present at the extreme 5' ends of mRNA molecules. Uncapped poliovirus mRNAs harbor internal ribosome entry sites (IRES) in their long and highly structured 5' noncoding regions. Such IRES sequences are required for viral protein synthesis. In this study, a novel poliovirus was isolated whose genomic RNA contains two gross deletions removing approximately 100 nucleotides from the predicted IRES sequences within the 5' noncoding region. The deletions originated from previously in vivo-selected viral revertants displaying non-temperature-sensitive phenotypes. Each revertant had a different predicted stem-loop structure within the 5' noncoding region of their genomic RNAs deleted. The mutant poliovirus (Se1-5NC-delta DG) described in this study contains both stem-loop deletions in a single RNA genome, thereby creating a minimum IRES. Se1-5NC-delta DG exhibited slow growth and a pinpoint plaque phenotype following infection of HeLa cells, delayed onset of protein synthesis in vivo, and defective initiation during in vitro translation of the mutated poliovirus mRNAs. Interestingly, the peak levels of viral RNA synthesis in cells infected with Se1-5NC-delta DG occurred at slightly later times in infection than those achieved by wild-type poliovirus, but these mutant virus RNAs accumulated in the host cells during the late phases of virus infection. UV cross-linking assays with the 5' noncoding regions of wild-type and mutated RNAs were carried out in cytoplasmic extracts from HeLa cells and neuronal cells and in reticulocyte lysates to identify the cellular factors that interact with the putative IRES elements. The cellular proteins that were cross-linked to the minimum IRES may represent factors playing an essential role in internal translation initiation of poliovirus mRNAs.
通过内部核糖体结合进行的翻译起始是一种最近发现的真核病毒和细胞蛋白质合成机制，在该机制中，核糖体亚基与5'非翻译RNA序列内部位点的mRNA相互作用，而不与mRNA分子5'末端的5'甲基鸟苷帽结构相互作用。无帽脊髓灰质炎病毒mRNA在其长且高度结构化的5'非编码区含有内部核糖体进入位点（IRES）。此类IRES序列是病毒蛋白质合成所必需的。在本研究中，分离出一种新型脊髓灰质炎病毒，其基因组RNA包含两个大的缺失，从5'非编码区内预测的IRES序列中去除了约100个核苷酸。这些缺失源自先前在体内选择的表现出非温度敏感表型的病毒回复株。每个回复株在其基因组RNA缺失的5'非编码区内具有不同的预测茎环结构。本研究中描述的突变脊髓灰质炎病毒（Se1-5NC-δDG）在单个RNA基因组中包含两个茎环缺失，从而形成了最小IRES。感染HeLa细胞后，Se1-5NC-δDG表现出生长缓慢和针尖状噬斑表型，体内蛋白质合成起始延迟，并且在突变脊髓灰质炎病毒mRNA的体外翻译过程中起始存在缺陷。有趣的是，感染Se1-5NC-δDG的细胞中病毒RNA合成的峰值水平在感染过程中出现的时间比野生型脊髓灰质炎病毒稍晚，但这些突变病毒RNA在病毒感染后期在宿主细胞中积累。使用野生型和突变RNA的5'非编码区进行紫外线交联试验，在HeLa细胞和神经元细胞的细胞质提取物以及网织红细胞裂解物中进行，以鉴定与假定的IRES元件相互作用的细胞因子。与最小IRES交联的细胞蛋白质可能代表在脊髓灰质炎病毒mRNA内部翻译起始中起关键作用的因子。
