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

N6-methyladenosine (m⁶A) is the most abundant internal modification in eukaryotic RNA and plays diverse roles in RNA metabolism. Increasing evidence indicates that m⁶A is also present in viral RNAs, where it exerts virus-specific effects. While several studies have shown that m⁶A 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 m⁶A in viral infection. We demonstrated that m⁶A modification is present in the TGEV genome and suppresses viral replication. The m⁶A reader proteins bind to viral RNA and reduce the stability of m⁶A-modified transcripts. Notably, TGEV infection increased global m⁶A levels in host RNA, particularly in interferon (IFN)-associated genes. Inhibition of m⁶A methylation significantly diminished IFN gene expression. Furthermore, compared to other viruses, TGEV genomic RNA displayed an abnormally higher m⁶A ratio, which can be distinguished by RIG-I to promote an immune response. Collectively, our findings reveal that high m⁶A 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 m⁶A during viral infection and host antiviral defense.IMPORTANCEN6-methyladenosine (m⁶A) is one of the most prevalent RNA modifications in viral genomes, but its functional impact varies widely across viruses. While m⁶A often promotes viral replication, it can exert inhibitory effects in certain viruses, particularly within the <i>Flaviviridae</i> and <i>Coronaviridae</i> 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 m⁶A. We show that the TGEV genome harbors a relatively high density of m⁶A 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 m⁶A modification can be sensed to activate antiviral immunity and provide deeper insight into the multifaceted role of m⁶A in host-virus interactions.