Genomic RNA of hepatitis C virus (HCV) has an internal ribosome entry site (IRES), a specific highly structured fragment responsible for its non-canonical translation initiation. The HCV IRES contains a major part of the 5′-untranslated region of the viral RNA and a small portion of the open reading frame (ORF). At the first step of initiation, IRES directly binds to 40S ribosomal subunits so that the AUG start codon appears at the P site region without scanning and without involving initiation factors. However, it is still not entirely clear whether the IRES ORF is correctly loaded into the 40S ribosomal mRNA binding channel in the resulting binary complex. To address this issue, we applied site-directed cross-linking using HCV IRES derivatives bearing a perfluorophenyl azide cross-linker at nucleotides in definite positions relative to the adenine of the AUG start codon. We found that the modifier at the IRES position −3 cross-links to ribosomal proteins uS11 and eS26. These proteins have been identified together with uS7 as those interacting with the mRNA nucleotide in position −3 relative to the first nucleotide of the codon directed to the P site by a cognate tRNA. Thus, our results indicate a certain difference in the locations of the above parts of HCV IRES and canonical mRNAs on 40S subunits. The modifier at the IRES positions +4/5 was attached to uS19, which is specific for ribosomal complexes with the P site tRNA and similar derivatives of model canonical mRNAs when the modifier is in the same positions. However, the cross-linking efficiency of the IRES derivative was drastically lower than that previously observed with derivatives of model mRNAs. This implies that the IRES ORF portion is correctly loaded into the mRNA binding channel only in a tiny fraction of the binary complexes.

丙型肝炎病毒（HCV）的基因组RNA具有内部核糖体进入位点（IRES），这是负责其非规范翻译起始的特定高度结构化的片段。HCV IRES包含病毒RNA 5'-非翻译区的主要部分和开放阅读框（ORF）的一小部分。在起始的第一步，IRES直接与40S核糖体亚基结合，因此AUG起始密码子出现在P位点区域，而无需扫描且不涉及起始因子。然而，仍不清楚IRES ORF是否正确地加载到所得二元复合物中的40S核糖体mRNA结合通道中。为了解决这个问题，我们应用了带有HCF IRES衍生物的定点交联，该衍生物在相对于AUG起始密码子的腺嘌呤的确定位置的核苷酸处带有全氟苯基叠氮化物交联剂。我们发现，IRES位置-3处的修饰剂与核糖体蛋白uS11和eS26交联。这些蛋白质与uS7一起被鉴定为与同源tRNA相对于指向P位点的密码子的第一个核苷酸的-3位的mRNA核苷酸相互作用的蛋白。因此，我们的结果表明HCV IRES的上述部分的位置和40S亚基上的经典mRNA的位置存在一定差异。IRES位置+4/5处的修饰子与uS19相连，该修饰物对具有P位点tRNA的核糖体复合物和模型规范mRNA的类似衍生物具有特异性，当修饰子位于相同位置时。然而，IRES衍生物的交联效率大大低于以前使用模型mRNA衍生物观察到的交联效率。这意味着仅在二元复合物中的一小部分中，IRES ORF部分才被正确加载到mRNA结合通道中。