Epitranscriptomic mC methylation of SARS-CoV-2 RNA regulates viral replication and the virulence of progeny viruses in the new infection.

While the significance of N6-methyladenosine (mA) in viral regulation has been extensively studied, the functions of 5-methylcytosine (mC) modification in viral biology remain largely unexplored. In this study, we demonstrate that mC is more abundant than mA in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and provide a comprehensive profile of the mC landscape of SARS-CoV-2 RNA. Knockout of NSUN2 reduces mC levels in SARS-CoV-2 virion RNA and enhances viral replication. deficiency mice exhibited higher viral burden and more severe lung tissue damages. Combined RNA-Bis-seq and mC-MeRIP-seq identified the NSUN2-dependent mC-methylated cytosines across the positive-sense genomic RNA of SARS-CoV-2, and the mutations of these cytosines enhance RNA stability. The progeny SARS-CoV-2 virions from deficiency mice with low levels of mC modification exhibited a stronger replication ability. Overall, our findings uncover the vital role played by NSUN2-mediated mC modification during SARS-CoV-2 replication and propose a host antiviral strategy via epitranscriptomic addition of mC methylation to SARS-CoV-2 RNA.