Nonsegmented Negative-Sense RNA Viruses Utilize N⁶-Methyladenosine (m⁶A) as a Common Strategy To Evade Host Innate...

N⁶-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 nonsegmented negative-sense (NNS) RNA viruses. Using m⁶A methyltransferase (METTL3) knockout cells, we produced m⁶A-deficient virion RNAs 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 the type I interferon signaling pathway, including 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 nonsegmented negative-sense (NNS) RNA viruses share many common replication and gene expression strategies. There are no vaccines or antiviral drugs for many of these viruses. We found that representative members of the families Pneumoviridae, Paramyxoviridae, and Rhabdoviridae among the NNS RNA viruses acquire m⁶A methylation in their genome and antigenome as a means to escape recognition by host innate immunity via a RIG-I-dependent signaling pathway. Viral RNA lacking m⁶A methylation induces a significantly higher type I interferon response than 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 robust innate immunity will likely promote the subsequent adaptive immunity.