The m6A Landscapes on the RNA of Mumps Virus and the Host 3 Modulate the Viral Replication and Antiviral Innate Immunity

N6-methyladenosine (m6A) is the most prevalent internal modification in eukaryotic mRNA and has emerged as a critical regulator of RNA virus infection. However, its role in the mumps virus (MuV), a non-segmented negative-strand (NNS) RNA virus, remains undefined. Here, we comprehensively characterize the m6A epitranscriptome of MuV JL2 strain and its functional impact on viral replication and host innate immunity. Using single-base resolution GLORI-seq, we identified abundant m6A modifications on MuV genomic, antigenomic and messenger RNAs, with uneven distribution and non-canonical motif enrichment. Genetic depletion of METTL3 enhanced viral replication by facilitating RNA synthesis and nucleocapsid encapsidation in Vero-E6 cells. In epithelial A549 and monocytoid THP1-iDC cells, MuV infection triggered robust type I/III interferon and proinflammatory responses by activating multiple pattern recognition receptors in a m6A-dependent manner and induced incomplete iDC maturation characterized by downregulated HLA class II; however, the latter molecule can be partially restored by METTL3-knockdown. The MuV infection reshaped the host m6A landscape with a positive correlation between m6A enrichment and transcripts enhancement of numerous innate immune genes in a cell type-specific manner. Functional analyses revealed that host m6A machinery modulates antiviral signaling, which varies between viral infection and viral RNA-transfection due to the infection-induced host m6A machinery fluctuation. Collectively, our findings demonstrate that m6A serves as a bidirectional regulator during MuV infection, simultaneously constraining viral replication and modulating host immune recognition, thereby highlighting RNA methylation as a pivotal determinant of MuV pathogenesis and a potential target for optimized immunogenicity. ImportanceN6-methyladenosine (m6A) modification has emerged as a key regulator of RNA virus infection, yet its role in mumps virus (MuV) remains undefined. Here we map the single-base resolution m6A landscape of a recombinant MuV and demonstrate that viral m6A restricts genome encapsidation and replication while attenuating innate immune sensing. MuV infection extensively remodels host m6A profiles in epithelial and dendritic cells, preferentially enhancing methylation of innate immune transcripts. Strikingly, m6A-deficient MuV RNA exhibits increased affinity for RIG-I-like and Toll-like receptors, eliciting stronger interferon and proinflammatory responses. Our findings identify m6A as a bidirectional regulator of MuV replication and immunity and suggest an epitranscriptomic strategy to optimize live-attenuated vaccine design.