WTAP targets the METTL3 m6A-methyltransferase complex to cytoplasmic hepatitis C virus RNA to regulate infection

Modification of the hepatitis C virus (HCV) positive-strand RNA genome by N6-methyladenosine (m 6 A) regulates the viral lifecycle. This lifecycle takes place solely in the cytoplasm, while m 6 A addition on cellular mRNA takes place in the nucleus. Thus, the mechanisms by which m 6 A is deposited on the viral RNA have been unclear. In this work, we find that m 6 A modification of HCV RNA by the m 6 A-methyltransferase proteins METTL3 and METTL14 is regulated by WTAP. WTAP, a predominantly nuclear protein, is an essential member of the cellular mRNA m 6 A-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 m 6 A 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 m 6 A. Excitingly, WTAP regulation of both HCV RNA m 6 A 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 m 6 A modification by METTL3 and METTL14 in the cytoplasm.Positive-strand RNA viruses such as HCV represent a significant global health burden. Previous work has described how HCV RNA contains the RNA modification m 6 A and how this modification regulates viral infection. Yet, how this modification is targeted to HCV RNA has remained unclear due to the incompatibility of the nuclear cellular processes that drive m 6 A modification with the cytoplasmic HCV lifecycle. In this study, we present evidence for how m 6 A modification is targeted to HCV RNA in the cytoplasm by a mechanism in which WTAP recruits the m 6 A-methyltransferase METTL3 to HCV RNA. This targeting strategy for m 6 A modification of cytoplasmic RNA viruses is likely relevant for other m 6 A-modified positive-strand RNA viruses with cytoplasmic lifecycles such as enterovirus 71 and SARS-CoV-2 and provides an exciting new target for potential antiviral therapies.