Non-segmented negative-sense RNA viruses utilize N6-methyladenosine (m6A) as a common strategy to evade host innate immunity.

N 6-methyladenosine (m6A) is the most abundant internal RNA modification catalyzed by host RNA methyltransferases. As obligate intracellular parasites, many viruses acquire m6A methylation in their RNAs. However, the biological functions of viral m6A methylation are poorly understood. Here, we found that viral m6A methylation serves as a molecular marker for host innate immunity to discriminate self from nonself RNA and that this novel biological function of viral m6A methylation is universally conserved in several families in non-segmented negative-sense (NNS) RNA viruses. Using m6A methyltransferase (METTL3)-knockout cells, we produced m6A-deficient virion RNA from the representative members of the families Pneumoviridae, Paramyxoviridae, and Rhabdoviridae and found that these m6A-deficient viral RNAs triggered significantly higher levels of type I interferon compared to the m6A-sufficient viral RNAs, in a RIG-I dependent manner. Reconstitution of the RIG-I pathway revealed that m6A-deficient virion RNA induced higher expression of RIG-I, bound to RIG-I more efficiently, enhanced RIG-I ubiquitination, and facilitated RIG-I conformational rearrangement and oligomerization. Furthermore, the m6A binding protein YTHDF2 is essential for suppression of type I interferon signaling pathway included by virion RNA. Collectively, our results suggest that several families in NNS RNA viruses acquire m6A in viral RNA as a common strategy to evade host innate immunity.IMPORTANCE The non-segmented negative-sense (NNS) RNA viruses share many common replication and gene expression strategies. There is no vaccine or antiviral drugs for many of these viruses. We found that representative members in the families of Pneumoviridae, Paramyxoviridae, and Rhabdoviridae in NNS RNA viruses acquire m6A methylation in their genome and antigenome as a means to escape the recognition by host innate immunity via a RIG-I dependent signaling pathway. Viral RNA lacking m6A methylation induces a significantly higher type I interferon compared to m6A sufficient viral RNA. In addition to uncovering m6A methylation as a common mechanism for many NNS RNA viruses to evade host innate immunity, this study discovered a novel strategy to enhance type I interferon responses, which may have important applications in vaccine development, as a robust innate immunity will likely promote the subsequent adaptive immunity.