N6-Methyladenosine Positively Regulates Coxsackievirus B3 Replication

Enteroviruses, particularly coxsackievirus B3 (CVB3), are recognized as a prevalent cause of viral myocarditis, yet the mechanisms underlying their replication and pathogenesis remain largely unclear. Many viral genomes, including that of CVB3, are known to contain N6-methyladenosine (m6A), a modification that plays a crucial role in viral replication. In this study, we utilized online bioinformatics tools such as SRAMP and conducted indirect immunofluorescence assays (IFA) to predict the presence of m6A sites within the CVB3 genome. Our findings indicated that CVB3 infection affects both the expression and cellular localization of m6A-related proteins. Notably, we demonstrated that 3-deazaadenosine (3-DAA), an inhibitor of m6A modification, significantly reduced CVB3 replication. Additionally, we found that m6A methyltransferases, specifically methyltransferase-like protein 3 (METTL3) and METTL14, positively influence CVB3 replication, while m6A demethylases fat mass and obesity-associated protein (FTO) and AlkB homolog 5 (ALKBH5) exert opposing effects. Silencing the m6A binding proteins YTH domain family proteins 1 (YTHDF1), 2 (YTHDF2), and 3 (YTHDF3) led to a marked decrease in CVB3 replication. Lastly, mutating the m6A site in the CVB3 genome resulted in reduced replication compared to the wild-type (WT) strain. Overall, our results suggest that CVB3 leverages m6A modification to enhance its replication, offering new insights into the interactions between CVB3 and its host.