Hepatitis C virus (HCV) infections are associated with the risk of progression to fibrosis, cirrhosis, and hepatocellular carcinoma. The HCV RNA genome is translated by an internal ribosome entry site (IRES)-dependent mechanism. The structure and function of the HCV IRES have been investigated by both biological and biophysical criteria. Recently, the role of N6-methyladenosine (m⁶A) in cellular RNA and viral transcripts has been intensely investigated. The HCV RNA genome is m⁶A-methylated, and this modification regulates the viral life cycle. In this study, we investigated the role of m⁶A modification of the HCV genome in the IRES-dependent translation function by mutating m⁶A consensus motifs (DRACH) within the IRES element in stem–loop III and IV regions and studied their effect on translation initiation. There are several DRACH motifs within the IRES element. Of these, the DRACH motif at nucleotide (nt) 329-333, located about 7 nt upstream of initiator AUG (iAUG) codon, regulates IRES-mediated translation initiation. Mutational analysis showed that m⁶A methylation of the adenosine at nt 331 is essential for the IRES-dependent translation. m⁶A reader protein YTHDC2, containing the RNA helicase domain, recognizes m⁶A-methylated adenosine at nt 331 and, in concert with the cellular La antigen, supports HCV IRES-dependent translation. The RNA helicase dead YTHDC2 (E332Q) mutant failed to stimulate HCV translation initiation. This report highlights the functional roles of m⁶A modification and YTHDC2 in the HCV IRES-dependent translation initiation, thus offering alternative therapeutic avenues to interfere with the infectious process.

丙型肝炎病毒 (HCV) 感染与进展为纤维化、肝硬化和肝细胞癌的风险相关。HCV RNA 基因组通过内部核糖体进入位点 (IRES) 依赖性机制进行翻译。HCV IRES 的结构和功能已通过生物学和生物物理标准进行了研究。最近，已经深入研究了 N6-甲基腺苷 (m ^{6 A) 在细胞 RNA 和病毒转录物中的作用。}HCV RNA基因组是m ⁶ A-甲基化的，这种修饰调节病毒生命周期。在这项研究中，我们通过突变 m ^{6研究了 HCV 基因组的 m 6} A 修饰在 IRES 依赖性翻译功能中的作用。茎环 III 和 IV 区域 IRES 元件内的共有基序 (DRACH) 并研究了它们对翻译起始的影响。IRES 元素中有几个 DRACH 基序。其中，位于起始 AUG (iAUG) 密码子上游约 7 nt 处的核苷酸 (nt) 329-333 处的 DRACH 基序调节 IRES 介导的翻译起始。突变分析表明，在 nt 331 处腺苷的 m ⁶ A 甲基化对于 IRES 依赖性翻译是必不可少的。m ⁶包含 RNA 解旋酶结构域的阅读器蛋白 YTHDC2 可识别 m ⁶nt 331 处的 A-甲基化腺苷与细胞 La 抗原一起支持 HCV IRES 依赖性翻译。RNA 解旋酶死亡 YTHDC2 (E332Q) 突变体未能刺激 HCV 翻译起始。本报告强调了 m ⁶ A 修饰和 YTHDC2 在 HCV IRES 依赖性翻译起始中的功能作用，从而提供了干扰感染过程的替代治疗途径。