NSUN2-mediated HCV RNA m⁵C Methylation Facilitates Viral RNA Stability and Replication

RNA modifications have emerged as new efficient targets against viruses. However, little is known about 5-methylcytosine (m⁵C) modification in the genomes of Flaviviruses. Herein, we demonstrate that hepatitis C virus (HCV), dengue virus, and Zika virus contain high levels of viral RNA m⁵C modification. We find that m⁵C modification in HCV RNA genome is located at C7525 site in the viral NS5A gene. HCV infection increases host m⁵C machinery NSUN2 expression via transcription factor E2F1. Deficiency of NSUN2 decreases HCV RNA m⁵C methylation levels, which further reduces viral RNA stability, replication as well as viral assembly and budding. HCV RNA genomic m⁵C specific abrogating mutation at C7525 site reduces viral replication, assembly, and budding through decreasing viral RNA stability. Deficiency of NSUN2 also reduces host global messenger RNA (mRNA) m⁵C modification levels during HCV infection, which upregulates antiviral innate immune response genes expression and further suppresses HCV RNA replication. Supported by both cellular and mouse infection models, our findings reveal that NSUN2-mediated HCV RNA and host mRNA m⁵C methylations facilitate viral RNA replication. HCV infection promotes host NSUN2 expression to facilitate HCV replication, which implies a positive feedback loop. NSUN2 could be a potential new target for Flavivirus therapeutics.