Summary Many viral RNAs are modified by methylation of the N6 position of adenosine (m6A). m6A is thought to regulate RNA splicing, stability, translation and secondary structure. Influenza A virus (IAV) expresses m6A-modified RNAs but the effects of m6A on this segmented RNA virus remain unclear. We demonstrate that global inhibition of m6A addition inhibits IAV gene expression and replication. In contrast, overexpression of the cellular m6A “reader” protein YTHDF2 increases IAV gene expression and replication. To address whether m6A residues modulate IAV RNA function in cis, we mapped m6A residues on the IAV plus (mRNA) and minus (vRNA) strands and used synonymous mutations to ablate m6A on both strands of the hemagglutinin (HA) segment. These mutations inhibited HA mRNA and protein expression, while leaving other IAV mRNAs and proteins unaffected, and also resulted in reduced IAV pathogenicity in mice. Thus, m6A residues in IAV transcripts enhance viral gene expression.

Highlights d HIV-1 mRNA in infected cells are highly modified by addition of m 5 C d These m 5 C residues are added in the nucleus by the host NSUN2 methyltransferase d Loss of NSUN2, and hence loss of m 5 C addition, inhibits HIV-1 mRNA translation d NSUN2 deficiency perturbs ribosomal recruitment and HIV-1 RNA alternative splicing