N6-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 roles of viral RNA m 6 A remain elusive. Here, 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 non-self 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 increased expression of type I interferon, which was dependent on the cytoplasmic RNA sensor RIG-I, and not on melanoma differentiation-associated protein 5 (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 interferon expression. Furthermore, m 6 A-deficient recombinant HMPVs triggered increased interferon in vivo and were attenuated in cotton rats but retained high immunogenicity. Collectively, our results highlight that (1) viruses acquire m 6 A in their RNA as a means of mimicking cellular RNA to avoid detection by innate immunity and (2) viral RNA m 6 A can serve as a target to attenuate HMPV for vaccine purposes. This study reports a novel function for the N 6 -methyladenosine RNA modification in allowing RIG-I to discriminate self from non-self RNA and shows that human metapneumovirus induces this modification of its RNA to evade recognition in vivo.