Fukushi S, Katayama K, Kurihara C, Ishiyama N, Hoshino F B, Ando T, Oya A
Basic Research Division, BioMedical Laboratories, Inc., Saitama, Japan.
Biochem Biophys Res Commun. 1994 Mar 15;199(2):425-32. doi: 10.1006/bbrc.1994.1246.
The mechanism of translational initiation by the 5' noncoding region (5'NCR) of hepatitis C virus (HCV) genome was analyzed. Using an in vitro translation system with artificial RNA containing a modified 5' NCR of HCV under the various KCl conditions, nucleotides (nt.) 62 to 341 of the HCV 5'NCR were not functional as an internal ribosome entry site (IRES). However, the full-length 5'NCR (nt. 1 to 341) produced an efficient internal initiation. To identify the essential region of the HCV-IRES, various mutants were produced in which stem-loops, predicted by secondary structure analysis of the HCV 5'NCR, were deleted. These constructs were analyzed by in vitro translation. Comparison of translation efficiency among these mutants suggested that the alpha- or both alpha- and beta-branches of domain II are essential for efficient translation. Moreover, the formation of correct secondary structure of IRES seems to be stabilized by the presence of domain I in 5'NCR. Furthermore, the uncapped 5'NCR of HCV promotes translation more efficiently than capped truncated 5'NCR constructs. Our results strongly suggested that complete 5'NCR containing all stem-loop structures is necessary for initiation by HCV-IRES.
对丙型肝炎病毒（HCV）基因组5'非编码区（5'NCR）介导的翻译起始机制进行了分析。在不同氯化钾条件下，使用含有经修饰的HCV 5'NCR的人工RNA体外翻译系统，HCV 5'NCR的核苷酸（nt.）62至341不能作为内部核糖体进入位点（IRES）发挥作用。然而，全长5'NCR（nt. 1至341）能产生有效的内部起始。为了确定HCV-IRES的关键区域，构建了多种突变体，这些突变体删除了通过HCV 5'NCR二级结构分析预测的茎环结构。通过体外翻译对这些构建体进行分析。这些突变体之间翻译效率的比较表明，结构域II的α-分支或α-和β-分支对于有效翻译至关重要。此外，5'NCR中结构域I的存在似乎稳定了IRES正确二级结构的形成。此外，HCV的无帽5'NCR比有帽截短的5'NCR构建体更有效地促进翻译。我们的结果强烈表明，包含所有茎环结构的完整5'NCR对于HCV-IRES起始是必需的。
