Rijnbrand R C, Abbink T E, Haasnoot P C, Spaan W J, Bredenbeek P J
Department of Virology, Leiden University, The Netherlands.
Virology. 1996 Dec 1;226(1):47-56. doi: 10.1006/viro.1996.0626.
The initiation of translation of hepatitis C virus (HCV) is cap-independent and mediated by an internal ribosome entry site (IRES) that is located in the 5' nontranslated region (5' NTR) of the viral genome. This 5' NTR is relatively long and folds into a complex structure involving multiple hairpins and a pseudoknot. Within the sequence encompassing the IRES there are several AUG triplets. Some of these AUG codons are conserved between HCV genotypes and the related pestiviruses. In this study the 5 AUG codons (positions 13, 32, 85, 96, and 215) that are present in the 5' NTR of the HCV H-strain have been mutagenized to determine their influence on HCV cap-independent translation. The effect of these mutations on the expression of a chloramphenicol acetyl transferase (CAT) gene was tested in vaccinia virus. vTF7-3 infected Hep2 cells transfected with plasmids for the expression of a monocistronic HCV 5' NTR-CAT mRNA. Mutating the AUG codons at positions 13, 32, and 215 does not have a significant effect on CAT expression, inactivating the AUG codons at either position 85 or position 96 severely impaired IRES function. To determine whether ribosomes scan the RNA to select the initiation site, AUG codons were inserted up- and downstream of the authentic HCV polyprotein translation initiation codon (position 342). Analysis of these mutants has revealed that the ribosome is unable to use an AUG codon that is placed either 7 nucleotides upstream or 8 nucleotides downstream of the inactivated AUG at position 342. These results indicate that when scanning is involved in the recognition of the translation initiating AUG, it is limited to a narrow region between nucleotides 335 and 350.
丙型肝炎病毒（HCV）的翻译起始不依赖于帽子结构，而是由位于病毒基因组5'非翻译区（5'NTR）的内部核糖体进入位点（IRES）介导。这个5'NTR相对较长，折叠成一个包含多个发夹和一个假结的复杂结构。在包含IRES的序列中有几个AUG三联体。其中一些AUG密码子在HCV基因型和相关瘟病毒之间是保守的。在本研究中，对HCV H株5'NTR中存在的5个AUG密码子（第13、32、85、96和215位）进行了诱变，以确定它们对HCV不依赖帽子结构翻译的影响。在痘苗病毒中测试了这些突变对氯霉素乙酰转移酶（CAT）基因表达的影响。vTF7-3感染用表达单顺反子HCV 5'NTR-CAT mRNA的质粒转染的Hep2细胞。突变第13、32和215位的AUG密码子对CAT表达没有显著影响，而使第85位或第96位的AUG密码子失活则严重损害IRES功能。为了确定核糖体是否扫描RNA以选择起始位点，在真实的HCV多蛋白翻译起始密码子（第342位）的上下游插入了AUG密码子。对这些突变体的分析表明，核糖体无法使用位于第342位失活AUG上游7个核苷酸或下游8个核苷酸处的AUG密码子。这些结果表明，当扫描参与翻译起始AUG的识别时，它仅限于核苷酸335和350之间的狭窄区域。
