N6-methyladenosine modification enables viral RNA to escape recognition by RNA sensor RIG-I

Abstract

Internal N⁶-methyladenosine (m⁶A) modification is one of the most common and abundant modifications of RNA. However, the biological roles of viral RNA m⁶A remain elusive. Here, using human metapneumovirus (HMPV) as a model, we demonstrate that m⁶A serves as a molecular marker for innate immune discrimination of self from non-self RNAs. We show that HMPV RNAs are m⁶A methylated and that viral m⁶A methylation promotes HMPV replication and gene expression. Inactivating m⁶A addition sites with synonymous mutations or demethylase resulted in m⁶A-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, m⁶A-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, m⁶A-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 m⁶A in their RNA as a means of mimicking cellular RNA to avoid detection by innate immunity and (2) viral RNA m⁶A can serve as a target to attenuate HMPV for vaccine purposes.