NSUN2-dependent 5-methylcytosine Modification Regulates Influenza A virus Gene Expression and Genomic Packaging

Emerging evidence indicates that methyltransferase NSUN2 catalyzes 5-methylcytosine (m ⁵ C) modifications on various viral RNAs and plays important roles in viral biology. However, the regulatory roles of NSUN2 in influenza A virus (IAV) replication have not been elucidated. Here, we revealed that NSUN2 negatively regulated the viral RNA transcription and protein production by knocking out and over-expressing NSUN2 . By m ⁵ C MeRIP-seq and RNA-BisSeq, NSUN2-dependent m ⁵ C sites were identified on both the plus and minus viral RNA strands. In NSUN2- KO cells, the m ⁵ C modification on vRNAs was reduced, resulting in the production of a large number of deficient interfering particles (DIPs) which had lower vRNA content, imbalanced genome fragments, abnormal morphology and reduced pathogenicity. Mutation of m ⁵ C sites at the 5’ and 3’ ends of PB2 vRNA interfered with the selective packaging of the 8 vRNA segments into virus particles, resulting in the formation of a variety of abnormally packaged virus particles. PB2-vRNA mutants also had reduced replication ability and pathogenicity in mice. Overall, these data demonstrate that the m ⁵ C residues catalyzed by NSUN2 are required on vRNAs for the proper assembly of infectious viral particles, suggesting the depletion of m ⁵ C modification as a potential strategy that can be utilized to attenuate IAV strains.