6-methyladnosine (mA) modification is present in both positive- and negative-strand RNA of influenza A virus (IAV) and affects the replication and pathogenicity of IAV. However, little is known about the regulatory mechanism of mA in IAV RNA. In the present study, we identified the mA methylation of the viral RNA of different IAV subtypes and confirmed that mA modification promotes the polymerase activity and replication of IAV. By mutating mA motifs on the multiple viral RNAs (vRNAs) of IAV, we revealed that mA deficiency in vRNA suppresses the expression of viral genes and the replication of the virus in vitro. In addition, mA deficiency in vRNA reduced the pathogenicity of IAV in a mouse model. Mechanistically, mA deficiency in vRNA suppresses the assembly of the viral ribonucleoprotein (vRNP) complex by impairing the interaction between vRNA and vRNP proteins in an mA methyltransferase-dependent manner, but not the mA reader proteins. Together, our findings reveal an important role for mA on viral RNAs in facilitating the activity of the polymerase complex and the replication and pathogenicity of IAV, which provides insights for the development of novel anti-influenza strategies.
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