The two-step purification method ViREn identifies a single NSUN6- mediated 5-methylcytosine modification promoting dengue virus RNA genome turnover
Chemical modifications on cellular and viral RNAs are new layers of post-transcriptionalregulation of cellular processes including RNA stability and translation. Although advances inanalytical methods have improved detection sensitivity, the precise mapping of RNAmodifications at single-base resolution remains challenging. Especially for low abundant viralRNAs extracted from infected cells, requirements for sensitivity and purity limit accuracy andreproducibility. Here we report the two-step method ViREn for the enrichment of the genomicRNA (gRNA) of dengue virus (DENV), a positive-sense single-stranded RNA virus. Thisapproach enabled the preparation of gRNA with significantly increased purity and led to theidentification of a high-confidence 5-methylcytosine (m5C) site in DENV gRNA, orthogonallyvalidated by Illumina-based bisulfite sequencing and direct RNA sequencing by NanoporeOxford Technologies. Strikingly, this m5C modification was exclusively detected in gRNAextracted from infected cells but not in gRNA extracted from viral particles. We identifiedNSUN6 as the host methyltransferase catalyzing this modification and demonstrated a role form5C in regulating DENV gRNA turnover. ViREn thus enables the mapping of m5C on lowabundance viral gRNA with unprecedented precision and sensitivity and facilitates mechanisticstudies into the role of RNA modification in virus replication.