Epitranscriptomic m5C 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 (m⁶A) in viral regulation has been extensively studied, the functions of 5-methylcytosine (m⁵C) modification in viral biology remain largely unexplored. In this study, we demonstrate that m⁵C is more abundant than m⁶A in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and provide a comprehensive profile of the m⁵C landscape of SARS-CoV-2 RNA. Knockout of NSUN2 reduces m⁵C levels in SARS-CoV-2 virion RNA and enhances viral replication. Nsun2 deficiency mice exhibited higher viral burden and more severe lung tissue damages. Combined RNA-Bis-seq and m⁵C-MeRIP-seq identified the NSUN2-dependent m⁵C-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 Nsun2 deficiency mice with low levels of m⁵C modification exhibited a stronger replication ability. Overall, our findings uncover the vital role played by NSUN2-mediated m⁵C modification during SARS-CoV-2 replication and propose a host Antiviral strategy via epitranscriptomic addition of m⁵C methylation to SARS-CoV-2 RNA.