Cytoplasmic N6-Methyladenosine Deposition on Hepatitis C Viral RNA

Abstract

Hepatitis C virus (HCV) exploits nearly all aspects of cellular RNA biology to regulate its viral RNA genome during infection. However, the molecular mechanisms by which HCV exploits one aspect of RNA regulation, RNA modification with N6-methyladenosine (m6A), are still emerging. This is because the current understanding of how RNA becomes m6A modified involves a variety of nuclear localized mechanisms that are incompatible with the cytoplasmic lifecycle of HCV. Thus, we set out to bridge the gap between our current understanding of m6A biology and how HCV RNA becomes m6A modified.In this work, we find that m6A modification of HCV RNA by the m6A-methyltransferase proteins METTL3 and METTL14 is regulated by WTAP. WTAP, a predominantly nuclear protein, is an essential member of the cellular mRNA m6A-methyltransferase complex and known to target METTL3 to mRNA. We found that HCV infection induces localization of WTAP to the cytoplasm. Importantly, we found that WTAP is required for both METTL3 interaction with HCV RNA and for m6A modification across the viral RNA genome. Further, we found that WTAP, like METTL3 and METTL14, negatively regulates the production of infectious HCV virions, a process that we have previously shown is regulated by m6A. Excitingly, WTAP regulation of both HCV RNA m6A modification and virion production were independent of its ability to localize to the nucleus. Together, these results reveal that WTAP is critical for HCV RNA m6A modification by METTL3 and METTL14 in the cytoplasm.