<i>N</i> 6-methyladnosine of vRNA facilitates influenza A virus replication by promoting the interaction of vRNA with polymerase proteins

<i>N</i>6-methyladnosine (m⁶A) 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 m⁶A in IAV RNA. In the present study, we identified the m⁶A methylation of the viral RNA of different IAV subtypes and confirmed that m⁶A modification promotes the polymerase activity and replication of IAV. By mutating m⁶A motifs on the multiple viral RNAs (vRNAs) of IAV, we revealed that m⁶A deficiency in vRNA suppresses the expression of viral genes and the replication of the virus in vitro. In addition, m⁶A deficiency in vRNA reduced the pathogenicity of IAV in a mouse model. Mechanistically, m⁶A deficiency in vRNA suppresses the assembly of the viral ribonucleoprotein (vRNP) complex by impairing the interaction between vRNA and vRNP proteins in an m⁶A methyltransferase-dependent manner, but not the m⁶A reader proteins. Together, our findings reveal an important role for m⁶A 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.