Dynamic interplay between RNA N6-methyladenosine modification and porcine reproductive and respiratory syndrome virus infection

N6-methyladenosine (m6A) has attracted significant attention for its role in various biological processes, including RNA stability, translation, and the immune response. Understanding the role of m6A 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 m6A peaks in the PRRSV genome by m6A RNA immunoprecipitation sequencing (m6A-seq). Seven m6A-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 m6A peak, spanning approximately 178 nucleotides. Functional analyses demonstrated a positive correlation between m6A modification levels and PRRSV replication in porcine alveolar macrophages (PAMs), as modulating the expression of m6A 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 m6A in PRRSV infection, we analyzed the m6A landscape in PAMs infected with pandemic and highly pathogenic PRRSV strains. Among the 4,677 transcripts exhibiting altered m6A modification levels, the MAPK14 gene as well as other genes in p38/MAPK signaling pathway potentially emerged as the preliminary targets of m6A-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. IMPORTANCEThe involvement of m6A in viral genome has broader implications for our understanding of virus evolution and pathogenicity. The ability of viruses to adapt to their hosts and exploit host cellular mechanisms is a critical factor in their success as pathogens. As m6A is a modification that can be manipulated by both host and virus, studying its role in viral infections may reveal new insights into viral evolution. Understanding how different viruses utilize m6A could inform strategies for developing antiviral therapies that target these interactions. Such therapies could disrupt the ability of viruses to exploit m6A modifications, potentially reducing their virulence and enhancing host defenses.