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

Abstract

Internal N ⁶-methyladenosine (m ⁶A) modification is one of the most common and abundant modifications of RNA. However, the biological role(s) of viral RNA m ⁶A remains elusive. Using human metapneumovirus (hMPV) as a model, we demonstrate that m ⁶A serves as a molecular marker for innate immune discrimination of self from nonself RNAs. We show that hMPV RNAs are m ⁶A methylated and that viral m ⁶A methylation promotes hMPV replication and gene expression. Inactivating m ⁶A addition sites with synonymous mutations or demethylase resulted in m ⁶A 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 ⁶A-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 ⁶A-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 ⁶A in their RNAs as a means of mimicking cellular RNA to avoid detection by innate immunity; and (ii) viral RNA m ⁶A can serve as a target to attenuate hMPV for vaccine purposes.