N6-methyladenosine modification of HCV RNA genome regulates cap-independent IRES-mediated translation via YTHDC2 recognition

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 (m(6)A) in cellular RNA and viral transcripts has been intensely investigated. The HCV RNA genome is m(6)A-methylated, and this modification regulates the viral life cycle. In this study, we investigated the role of m(6)A modification of the HCV genome in the IRES-dependent translation function by mutating m(6)A 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 m(6)A methylation of the adenosine at nt 331 is essential for the IRES-dependent translation. m(6)A reader protein YTHDC2, containing the RNA helicase domain, recognizes m(6)A-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 m(6)A modification and YTHDC2 in the HCV IRES-dependent translation initiation, thus offering alternative therapeutic avenues to interfere with the infectious process.

Automated summary

The m(6)A modification of the HCV RNA genome plays a crucial role in IRES-dependent translation, with the m(6)A reader protein YTHDC2 being essential for recognizing and supporting HCV translation initiation. This study highlights the functional significance of m(6)A modification and YTHDC2 in HCV IRES-dependent translation initiation, providing potential therapeutic targets for interfering with the infectious process.