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 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.