Honda M, Brown E A, Lemon S M
Department of Medicine, The University of North Carolina, Chapel Hill 27599-7030, USA.
RNA. 1996 Oct;2(10):955-68.
The initiation of translation on the positive-sense RNA genome of hepatitis C virus (HCV) is directed by an internal ribosomal entry site (IRES) that occupies most of the 341-nt 5' nontranslated RNA (5'NTR). Previous studies indicate that this IRES differs from picornaviral IRESs in that its activity is dependent upon RNA sequence downstream of the initiator AUG. Here, we demonstrate that the initiator AUG of HCV is located within a stem-loop (stem-loop IV) involving nt -12 to +12 (with reference to the AUG). This structure is conserved among HCV strains, and is present in the 5'NTR of the phylogenetically distant GB virus B. Mutant, nearly genome-length RNAs containing nucleotide substitutions predicted to enhance the stability of stem-loop IV were generally deficient in cap-independent translation both in vitro and in vivo. Additional mutations that destabilize the stem-loop restored translation to normal. Thus, the stability of the stem-loop is strongly but inversely correlated with the efficiency of internal initiation of translation. In contrast, mutations that stabilize this stem-loop had comparatively little effect on translation of 5' truncated RNAs by scanning ribosomes, suggesting that internal initiation of translation follows binding of the 40S ribosome directly at the site of stem-loop IV. Because stem-loop IV is not required for internal entry of ribosomes but is able to regulate this process, we speculate that it may be stabilized by interactions with a viral protein, providing a mechanism for feedback regulation of translation, which may be important for viral persistence.
丙型肝炎病毒（HCV）正链RNA基因组上的翻译起始由一个内部核糖体进入位点（IRES）引导，该位点占据了341个核苷酸的5'非翻译RNA（5'NTR）的大部分。先前的研究表明，这种IRES与微小RNA病毒的IRES不同，其活性依赖于起始密码子AUG下游的RNA序列。在这里，我们证明HCV的起始密码子AUG位于一个茎环结构（茎环IV）内，该结构涉及核苷酸-12至+12（相对于AUG）。这种结构在HCV毒株中保守，并且存在于系统发育关系较远的GB病毒B的5'NTR中。预测可增强茎环IV稳定性的含核苷酸取代的突变型近基因组长度RNA在体外和体内通常缺乏不依赖帽子的翻译。使茎环不稳定的其他突变可将翻译恢复正常。因此，茎环的稳定性与内部翻译起始效率呈强烈的负相关。相反，稳定该茎环的突变对通过扫描核糖体对5'截短RNA的翻译影响相对较小，这表明内部翻译起始是40S核糖体直接结合在茎环IV位点之后发生的。因为茎环IV不是核糖体内部进入所必需的，但能够调节这一过程，我们推测它可能通过与病毒蛋白的相互作用而稳定，从而提供一种翻译反馈调节机制，这可能对病毒持续存在很重要。
