Dynamic interplay between RNA N ⁶ -methyladenosine modification and porcine reproductive and respiratory syndrome virus infection

N ⁶ -methyladenosine (m ⁶ A) has attracted significant attention for its role in various biological processes, including RNA stability, translation, and the immune response. Understanding the role of m ⁶ A in viral infections is crucial to deep the complex interaction between virus and host cells. Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is a significant pathogen affecting swine health worldwide. Here, we firstly identified the m ⁶ A peaks in the PRRSV genome by m ⁶ A RNA immunoprecipitation sequencing (m ⁶ A-seq). Seven m ⁶ A-enriched regions within the PRRSV genome were detected, with one located in the N protein-coding region and the others distributed across nonstructural protein-coding regions. Notably, the Nsp2-coding region contained the highest m ⁶ A peak, spanning approximately 178 nucleotides. Functional analyses demonstrated a positive correlation between m ⁶ A modification levels and PRRSV replication in porcine alveolar macrophages (PAMs), as modulating the expression of m ⁶ A methyltransferases and demethylases significantly influenced viral replication. Moreover, treatment with the universal methylation inhibitor 3-deazaadenosine (3-DAA) effectively suppressed PRRSV replication, suggesting its potential as a novel anti-PRRSV therapeutic. To further elucidate the role of m ⁶ A in PRRSV infection, we analyzed the m ⁶ A landscape in PAMs infected with pandemic and highly pathogenic PRRSV strains. Among the 4,677 transcripts exhibiting altered m ⁶ A modification levels, the MAPK14 gene as well as other genes in p38/MAPK signaling pathway potentially emerged as the preliminary targets of m ⁶ A-mediated epigenetic regulation during PRRSV infection. These findings provide new insights into the epigenetic mechanisms underlying PRRSV infection and may facilitate the development of targeted anti-PRRSV strategies and therapeutics.