Nucleoprotein S69 phosphorylation regulates influenza A virus replication

Abstract Phosphorylation of the influenza A virus (IAV) nucleoprotein (NP) is crucial for the viral life cycle, as it encapsidates the viral genomic RNA. Here, we employed mass spectrometry to identify serine 69 (S69) as a novel phosphorylation site of NP in IAV. Sequence homology alignment revealed that NP S69 is highly conserved across various subtypes of IAV. The S69A mutation, which is designed to mimic constant dephosphorylation, had minimal effects on viral replication and pathogenicity in both cellular and murine models. In contrast, the S69E mutation, which mimics constant phosphorylation, was lethal. Mechanistically, the phosphorylation of NP S69 reduces the polymerase activity of viral ribonucleoprotein (vRNP) by impairing the interaction between NP and PB2. Additionally, NP S69 phosphorylation inhibited NP nuclear import by disrupting its interaction with importin-α3. These findings identify NP S69 phosphorylation as a critical regulatory mechanism in IAV replication, providing valuable insights into the role of NP phosphorylation in the life cycle of influenza virus.