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

Abstract

Abstract Internal N6-methyladenosine (m6A) modification of RNA is one of the most abundant modifications in eukaryotic cells as well as in viruses. However, the biological role(s) of RNA m6A in virus-host interaction remains elusive. Using human metapneumovirus (hMPV), a medically important non-segmented negative-sense RNA virus as a model, we demonstrate that m6A serves as a molecular marker for innate immune discrimination of self from nonself RNAs. We show that hMPV RNAs are m6A methylated and that viral m6A methylation promotes hMPV replication and gene expression. Inactivating these m6A sites with synonymous mutations resulted in m6A deficient recombinant hMPVs that induced significantly higher expression of type I interferon (IFN) that restricted viral replication. Notably, the induction of type I IFN by m6A-deficient rhMPVs and virion RNA was dependent on the cytoplasmic RNA sensor RIG-I, not MDA5. Using RIG-I pull down and RIG-I limited digestion assay, we mechanistically demonstrated that m6A-deficient virion RNA exposed to cytoplasm has much high accessibility to the binding of RIG-I, which allows RIG-I’s translocation along the RNA strand and subsequential conformational change, oligomerization, activation of IFN signaling cascade, and then even higher RIG-I expression during anti-virus status. Furthermore, m6A-deficient rhMPVs triggered higher type I IFN in vivo and were significantly attenuated in the lower respiratory tract yet retained high immunogenicity in cotton rats. Collectively, our results highlight that (i) virus acquires m6A in their RNAs as a means of mimicking cellular RNA to avoid the detection by innate immunity; and (ii) viral m6A RNA can serve as a novel target to attenuate hMPV for vaccine purposes.