N6-methyladenosine within transmissible gastroenteritis virus genomic RNA inhibits its replication via efficient recognition by RNA sensor RIG-I.

N6-methyladenosine (m(6)A) is the most abundant internal modification in eukaryotic RNA and plays diverse roles in RNA metabolism. Increasing evidence indicates that m(6)A is also present in viral RNAs, where it exerts virus-specific effects. While several studies have shown that m(6)A can facilitate viral replication, its antiviral mechanisms remain less understood. In this study, we used transmissible gastroenteritis virus (TGEV) as a model to investigate the inhibitory role of m(6)A in viral infection. We demonstrated that m(6)A modification is present in the TGEV genome and suppresses viral replication. The m(6)A reader proteins bind to viral RNA and reduce the stability of m(6)A-modified transcripts. Notably, TGEV infection increased global m(6)A levels in host RNA, particularly in interferon (IFN)-associated genes. Inhibition of m(6)A methylation significantly diminished IFN gene expression. Furthermore, compared to other viruses, TGEV genomic RNA displayed an abnormally higher m(6)A ratio, which can be distinguished by RIG-I to promote an immune response. Collectively, our findings reveal that high m(6)A modification enhances RIG-I-mediated sensing of TGEV RNA, leading to the activation of IFN responses and inhibition of viral replication. This study provides new insights into the complex regulatory functions of m(6)A during viral infection and host antiviral defense.IMPORTANCEN6-methyladenosine (m(6)A) is one of the most prevalent RNA modifications in viral genomes, but its functional impact varies widely across viruses. While m(6)A often promotes viral replication, it can exert inhibitory effects in certain viruses, particularly within the Flaviviridae and Coronaviridae families. Despite growing evidence of this antiviral role, the underlying mechanisms remain largely unclear. Here, we used transmissible gastroenteritis virus (TGEV), a swine coronavirus, as a model to explore the inhibitory function of m(6)A. We show that the TGEV genome harbors a relatively high density of m(6)A modification compared to other viruses and host mRNA, which are efficiently detected by the host pattern recognition receptor RIG-I. This interaction enhances innate immune activation and restricts viral replication. Our findings uncover the mechanism by which abnormal m(6)A modification can be sensed to activate antiviral immunity and provide deeper insight into the multifaceted role of m(6)A in host-virus interactions.