Epitranscriptomic Enhancement of Influenza A Virus Gene Expression and Replication

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 they also resulted in reduced IAV pathogenicity in mice. Thus, m⁶A residues in IAV transcripts enhance viral gene expression.