5-methylcytosine (m(5)C) RNA modification controls the innate immune response to virus infection by regulating type I interferons

5-methylcytosine (m(5)C) is one of the most prevalent modifications of RNA, playing important roles in RNA metabolism, nuclear export, and translation. However, the potential role of RNA m(5)C methylation in innate immunity remains elusive. Here, we show that depletion of NSUN2, an m(5)C methyltransferase, significantly inhibits the replication and gene expression of a wide range of RNA and DNA viruses. Notably, we found that this antiviral effect is largely driven by an enhanced type I interferon (IFN) response. The antiviral signaling pathway is dependent on the cytosolic RNA sensor RIG-I but not MDA5. Transcriptome-wide mapping of m(5)C following NSUN2 depletion in human A549 cells revealed a marked reduction in the m(5)C methylation of several abundant noncoding RNAs (ncRNAs). However, m(5)C methylation of viral RNA was not noticeably altered by NSUN2 depletion. In NSUN2-depleted cells, the host RNA polymerase (Pol) III transcribed ncRNAs, in particular RPPH1 and 7SL RNAs, were substantially up-regulated, leading to an increase of unshielded 7SL RNA in cytoplasm, which served as a direct ligand for the RIG-I–mediated IFN response. In NSUN2-depleted cells, inhibition of Pol III transcription or silencing of RPPH1 and 7SL RNA dampened IFN signaling, partially rescuing viral replication and gene expression. Finally, depletion of NSUN2 in an ex vivo human lung model and a mouse model inhibits viral replication and reduces pathogenesis, which is accompanied by enhanced type I IFN responses. Collectively, our data demonstrate that RNA m(5)C methylation controls antiviral innate immunity through modulating the m(5)C methylome of ncRNAs and their expression.