Influenza NS1 drives N ⁶ -methyladenosine (m ⁶ A)-mediated autoregulation of viral mRNA splicing

Influenza A virus replication requires precise balance between unspliced and spliced viral mRNAs. However, the regulatory mechanisms governing viral mRNA splicing remain poorly understood. In this study, we uncover an epigenetic strategy whereby the viral NS1 protein autoregulates its own mRNA splicing via <i>N⁶</i>-methyladenosine (m⁶A) modification. Specifically, m⁶A modification at residue A385 on NS mRNA recruits the m⁶A reader YTHDC1, which competitively inhibits the splicing factor SRSF3 from binding proximal sites. Importantly, we demonstrate that the A385 m⁶A site is conserved and essential for splicing regulation in the NS segment across human and avian influenza strains. Our findings suggest that, by dynamically modulating m⁶A levels, NS1 fine-tunes viral mRNA processing to enhance replication efficiency. This work elucidates NS1 as an m⁶A-driven splicing regulator and identifies this conserved m⁶A site as a potential target for broad-spectrum antiviral development.