N(6)-methyladenosine modification enables viral RNA to escape recognition by RNA sensor RIG-I

Internal N(6)-methyladenosine (m(6)A) modification is one of the most common and abundant modifications of RNA. However, the biological role(s) of viral RNA m(6)A remains elusive. Using human metapneumovirus (hMPV) as a model, we demonstrate that m(6)A serves as a molecular marker for innate immune discrimination of self from nonself RNAs. We show that hMPV RNAs are m(6)A methylated and that viral m(6)A methylation promotes hMPV replication and gene expression. Inactivating m(6)A addition sites with synonymous mutations or demethylase resulted in m(6)A deficient recombinant hMPVs and virion RNAs that induced significantly higher expression of type I interferon (IFN) which was dependent on the cytoplasmic RNA sensor RIG-I, not MDA5. Mechanistically, m(6)A-deficient virion RNA induces higher expression of RIG-I, binds more efficiently to RIG-I, and facilitates the conformational change of RIG-I, leading to enhanced IFN expression. Furthermore, m(6)A-deficient rhMPVs triggered higher IFN in vivo and were significantly attenuated in cotton rats yet retained high immunogenicity. Collectively, our results highlight that (i) virus acquires m(6)A in their RNAs as a means of mimicking cellular RNA to avoid detection by innate immunity; and (ii) viral RNA m(6)A can serve as a target to attenuate hMPV for vaccine purposes.