m 6 A coordinates mumps virus replication and innate immune sensing

In this study, we test the hypothesis that N6-methyladenosine (m 6 A) coordinates a functional trade-off between viral replication and innate immune sensing during mumps virus (MuV) infection. Using single-base resolution GLORI-seq, we identified widespread and non-canonically distributed m 6 A sites across MuV genomic, antigenomic, and messenger RNAs. Functional analyses revealed that depletion of the m 6 A methyltransferase METTL3 enhances viral RNA synthesis and nucleocapsid encapsidation in interferon-deficient cells, indicating an intrinsic suppressive role of m 6 A in viral replication. In contrast, in immune-competent epithelial and dendritic cells, host m 6 A enhances antiviral signaling, as METTL3 deficiency reduces infection-induced interferon and cytokine responses. Mechanistically, m 6 A-deficient MuV RNA exhibited increased binding to multiple pattern recognition receptors, including RIG-I-like and toll-like receptors, resulting in enhanced innate immune activation. Additionally, MuV infection extensively remodels the host m 6 A epitranscriptome in a cell type-dependent manner, with m 6 A enrichment positively correlating with the expression of immune-related transcripts. Together, these findings demonstrate that m 6 A acts as a bidirectional regulator during MuV infection by simultaneously restricting viral replication and modulating host immune recognition, providing new insight into epitranscriptomic control of paramyxovirus-host interactions. IMPORTANCE N6-methyladenosine (m 6 A) 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 m 6 A landscape of a recombinant MuV and demonstrate that viral m 6 A restricts genome encapsidation and replication while attenuating innate immune sensing. MuV infection extensively remodels host m 6 A profiles in epithelial and dendritic cells, preferentially enhancing methylation of innate immune transcripts. Strikingly, m 6 A-deficient MuV RNA exhibits increased affinity for RIG-I-like and toll-like receptors, eliciting stronger interferon and proinflammatory responses. Our findings identify m 6 A as a bidirectional regulator of MuV replication and immunity and suggest an epitranscriptomic strategy to optimize live-attenuated vaccine design.