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

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.