Hellen C U, Pestova T V, Wimmer E
Department of Microbiology, School of Medicine, State University of New York at Stony Brook 11794-8621.
J Virol. 1994 Oct;68(10):6312-22. doi: 10.1128/JVI.68.10.6312-6322.1994.
Initiation of poliovirus translation is mediated by a large, structured segment of the 5' nontranslated region known as the internal ribosome entry site (IRES) and normally occurs 155 nucleotides (nt) downstream of the IRES at AUG743 (the AUG at nucleotide 743). Functional AUG codons introduced at nt 611 or 614 reduced initiation at AUG743 by 10 to 40% in vitro but had no effect on virus phenotype. To investigate the role of the nt 586-743 spacer in greater detail, four intervening termination codons were removed, and an additional AUG triplet at nt 683 was introduced by nucleotide substitution. Initiation at AUG743 was reduced by only 50 to 80%, depending on the number of upstream initiation codons. Initiation at AUG743 was also reduced following insertion of a stable hairpin at nt 630, but the reduction was modest in an ascites carcinoma cell extract. Initiation was more frequent at AUG743 than at AUG683 if mRNAs contained either an upstream initiation codon or the stable hairpin. These results suggested that not all initiation events at AUG743 can be accounted for by a scanning-dependent mechanism. Translation of bicistronic mRNAs in which the intercistronic spacer contained nt 630 to 742 of the poliovirus 5' nontranslated region indicated that these residues are not able to act as an entry point for ribosomes independently of the IRES. Insertion of increasingly longer sequences immediately downstream of the stable hairpin progressively reduced initiation at AUG743 without affecting initiation at AUG683. These results are discussed in terms of a model for initiation of poliovirus translation in which a complex RNA superstructure upstream of nt 586 promotes ribosome binding at an entry point determined by specific downstream cis-acting elements.
脊髓灰质炎病毒的翻译起始由5'非翻译区的一个大的结构化片段介导，该片段称为内部核糖体进入位点（IRES），通常发生在IRES下游155个核苷酸（nt）处的AUG743（核苷酸743处的AUG）。在nt 611或614处引入的功能性AUG密码子在体外使AUG743处的起始减少了10%至40%，但对病毒表型没有影响。为了更详细地研究nt 586 - 743间隔区的作用，去除了四个中间的终止密码子，并通过核苷酸替换在nt 683处引入了一个额外的AUG三联体。根据上游起始密码子的数量，AUG743处的起始仅减少了50%至80%。在nt 630处插入一个稳定的发夹后，AUG743处的起始也减少了，但在腹水癌细胞提取物中减少幅度不大。如果mRNA包含上游起始密码子或稳定发夹，则AUG743处的起始比AUG683处更频繁。这些结果表明，并非所有AUG743处的起始事件都可以用扫描依赖性机制来解释。双顺反子mRNA的翻译，其中顺反子间间隔区包含脊髓灰质炎病毒5'非翻译区的nt 630至742，表明这些残基不能独立于IRES作为核糖体的进入点。在稳定发夹的紧邻下游插入越来越长的序列逐渐减少了AUG743处的起始，而不影响AUG683处的起始。本文根据脊髓灰质炎病毒翻译起始模型对这些结果进行了讨论，在该模型中，nt 586上游的复杂RNA超结构促进核糖体在由特定下游顺式作用元件确定的进入点处结合。
