Kamoshita N, Tsukiyama-Kohara K, Kohara M, Nomoto A
Department of Microbiology, University of Tokyo, Japan.
Virology. 1997 Jun 23;233(1):9-18. doi: 10.1006/viro.1997.8600.
Hepatitis C virus (HCV) carries an internal ribosomal entry site (IRES) within the 5' portion of the RNA. To identify structures that influence efficiency of the translation initiation, relative activities of modified IRESs were examined by using engineered bicistronic mRNAs, between the two cistrons of which various mutant IRESs were inserted. An IRES derived from genotype 2b is at least two times more efficient than one from genotype 1b in cultured cells. Activity ratios of genotype 2b IRES to 1b IRES differ in magnification among cultured cells, suggesting the difference in assortment of IRES-related host factors among individual cell types. Recombinant IRESs between the genotypes show similar or higher activities compared with 2b IRES in cell-free systems and show intermediate activities in cultured cells. Patterns of relative activities of those IRESs indicate that the IRES activity is not regulated by defined structure(s), although a cluster of different nucleotides is observed in the genome region of nucleotides 176-224 between the two alleles. The results suggest that a highly ordered structure formed by the entire 5' portion of the RNA is important for the IRES activity. The 5' border of HCV IRES was examined by using a series of deletion RNAs in various systems. The results strongly suggest that the border resides between nucleotide positions 28 and 45. Patterns of relative activities of the deletion IRESs differ in translation systems or cell types. These results imply that interactions of HCV RNA with the related transacting factor(s) may differ in the translation systems or cell types.
丙型肝炎病毒（HCV）在RNA的5'部分携带一个内部核糖体进入位点（IRES）。为了确定影响翻译起始效率的结构，通过使用工程化的双顺反子mRNA来检测修饰IRES的相对活性，在两个顺反子之间插入了各种突变IRES。在培养细胞中，源自2b基因型的IRES的效率至少是1b基因型的两倍。2b基因型IRES与1b基因型IRES的活性比在不同培养细胞中的放大倍数不同，这表明不同细胞类型中IRES相关宿主因子的种类存在差异。基因型之间的重组IRES在无细胞系统中与2b IRES相比表现出相似或更高的活性，在培养细胞中表现出中等活性。这些IRES的相对活性模式表明，IRES活性不受特定结构的调节，尽管在两个等位基因之间的176 - 224核苷酸的基因组区域中观察到了不同核苷酸的簇。结果表明，由RNA的整个5'部分形成的高度有序结构对IRES活性很重要。通过在各种系统中使用一系列缺失RNA来检测HCV IRES的5'边界。结果强烈表明该边界位于核苷酸位置28和45之间。缺失IRES的相对活性模式在翻译系统或细胞类型中有所不同。这些结果意味着HCV RNA与相关反式作用因子的相互作用在翻译系统或细胞类型中可能不同。
