WTAP Targets the METTL3 m6A-Methyltransferase Complex to Cytoplasmic Hepatitis C Virus RNA to Regulate Infection

Positive-strand RNA viruses such as HCV represent a significant global health burden. Previous work has described that HCV RNA contains the RNA modification m(6)A and how this modification regulates viral infection. Modification of the hepatitis C virus (HCV) positive-strand RNA genome by N6-methyladenosine (m(6)A) regulates the viral life cycle. This life cycle 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 methyltransferase-like 3 and 14 (METTL3 and METTL14) is regulated by Wilms' tumor 1-associating protein (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 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 was 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. IMPORTANCE Positive-strand RNA viruses such as HCV represent a significant global health burden. Previous work has described that 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 life cycle. 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 life cycles such as enterovirus 71 and SARS-CoV-2 and provides an exciting new target for potential antiviral therapies.