The 5-methylcytosine methylation in PB2 gene of influenza virus interferes viral RNA replication and pathogenicity

As a pivotal epigenetic modification, 5-methylcytosine (m5C) plays significant roles in RNA stability, translation regulation, and cellular metabolism. Growing evidence suggests that m5C methylation is involved in the regulation of virus-host interactions. However, its function during influenza virus infections remains elusive. Here, we demonstrate for the first time that m5C methylation of the influenza virus PB2 gene, encoding a core polymerase subunit, acts as a negative regulator of viral infection. Loss of m5C modification in complementary PB2 RNA enhances its transcription and translation efficiency, facilitates polymerase binding to RNA, and promotes viral genomic RNA synthesis. The subsequent accumulation of RNA and polymerase proteins accelerates the nuclear export of viral ribonucleoprotein complexes, enhances progeny virion assembly and maturation, and ultimately leads to enhanced viral pathogenicity. Our findings reveal a novel mechanism by which m5C methylation fine-tunes influenza virus replication by suppressing viral RNA synthesis, and provide evidence for a previously unrecognized innate antiviral strategy centered on m5C-mediated epitranscriptomic regulation.