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 exhibit high levels of viral RNA m5C modification. We identified an m5C site at C7525 in the NS5A gene of the HCV RNA genome. HCV infection upregulates the expression of the host m5C methyltransferase NSUN2 via the transcription factor E2F1. NSUN2 deficiency decreases HCV RNA m5C methylation levels, which further reduces viral RNA stability, replication, and viral assembly and budding. A C7525-specific m5C-abrogating mutation in the HCV RNA genome similarly reduces viral replication, assembly, and budding by decreasing viral RNA stability. Notably, NSUN2 deficiency also reduces host global messenger RNA (mRNA) m5C levels during HCV infection, which upregulates the expression of antiviral innate immune response genes and further suppresses HCV RNA replication. Supported by both cellular and mouse infection models, our findings reveal that NSUN2-mediated m5C methylation of HCV RNA and host mRNAs facilitates viral RNA replication. HCV infection promotes host NSUN2 expression to facilitate HCV replication, suggesting a positive feedback loop. NSUN2 could be a potential therapeutic target for flavivirus therapeutics.