INTERNAL RIBOSOME ENTRY SITE WITHIN HEPATITIS-C VIRUS-RNA
INTERNAL RIBOSOME ENTRY SITE WITHIN HEPATITIS-C VIRUS-RNA
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DOI：
10.1128/jvi.66.3.1476-1483.1992
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发表时间：
1992-03-01
影响因子：
5.4
通讯作者：
NOMOTO, A
中科院分区：
文献类型：
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作者：
TSUKIYAMAKOHARA, K;IIZUKA, N;NOMOTO, A
研究了丙型肝炎病毒（HCV）RNA体外翻译起始的机制。 通过使用噬菌体T7 RNA聚合酶从对应于基因组的核苷酸位置9至1772的cDNA转录HCV RNA。 这样转录的加帽和未加帽RNA在具有从HeLa S3细胞或兔网织红细胞制备的裂解物的无细胞蛋白质合成系统中作为mRNA是活性的，并且通过抗gp 35抗体检测翻译产物。 数据表明，蛋白质合成开始于第四个AUG，这是本研究中使用的HCV RNA的333位的起始AUG。 加帽的甲基化RNA的翻译效率似乎与加帽的未甲基化RNA的翻译效率相似。 然而，加帽的甲基化RNA显示出比加帽的未甲基化RNA在兔网织红细胞裂解物中的mRNA高得多的活性，当RNA缺乏位置267上游的核苷酸序列时。 这些结果有力地表明，HCV RNA携带内部核糖体进入位点（IRES）。 制备人工单顺反子和双顺反子mRNA，并用于鉴定携带IRES的区域。 结果表明，HCVRNA 5'端非翻译区101 ~ 332位的序列在高效翻译中起重要作用。 我们的数据表明IRES位于RNA的这一区域。 此外，IRES在组II HCV RNA中被发现比在组I HCV RNA中更有效。
The mechanism of initiation of translation on hepatitis C virus (HCV) RNA was investigated in vitro. HCV RNA was transcribed from the cDNA that corresponded to nucleotide positions 9 to 1772 of the genome by using phage T7 RNA polymerase. Both capped and uncapped RNAs thus transcribed were active as mRNAs in a cell-free protein synthesis system with lysates prepared from HeLa S3 cells or rabbit reticulocytes, and the translation products were detected by anti-gp35 antibodies. The data indicate that protein synthesis starts at the fourth AUG, which was the initiator AUG at position 333 of the HCV RNA used in this study. Efficiency of translation of the capped methylated RNA appeared to be similar to that of the capped unmethylated RNA. However, a capped methylated RNA showed a much higher activity as mRNA than did the capped unmethylated RNA in rabbit reticulocyte lysates when the RNA lacked a nucleotide sequence upstream of position 267. The results strongly suggest that HCV RNA carries an internal ribosome entry site (IRES). Artificial mono- and dicistronic mRNAs were prepared and used to identify the region that carried the IRES. The results indicate that the sequence between nucleotide positions 101 and 332 in the 5' untranslated region of HCV RNA plays an important role in efficient translation. Our data suggest that the IRES resides in this region of the RNA. Furthermore, an IRES in the group II HCV RNA was found to be more efficient than that in the group I HCV RNA.