N6-methyladenosine modification enables viral RNA to escape recognition by RNA sensor RIG-I.

Abstract

Internal N6-methyladenosine (m6A) modification is one of the most common and abundant modifications of RNA. However, the biological role(s) of viral RNA m6A remains elusive. Using human metapneumovirus (hMPV) as a model, we demonstrate that m6A serves as a molecular marker for innate immune discrimination of self from nonself RNAs. We show that hMPV RNAs are m6A methylated and that viral m6A methylation promotes hMPV replication and gene expression. Inactivating m6A addition sites with synonymous mutations or demethylase resulted in m6A 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, m6A-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, m6A-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 m6A in their RNAs as a means of mimicking cellular RNA to avoid detection by innate immunity; and (ii) viral RNA m6A can serve as a target to attenuate hMPV for vaccine purposes.