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-transcriptional regulation of cellular processes including RNA stability and translation. Although advances in analytical methods have improved detection sensitivity, the precise mapping of RNA modifications at single-base resolution remains challenging. Especially for low abundant viral RNAs extracted from infected cells, requirements for sensitivity and purity limit accuracy and reproducibility. Here we report the two-step method ViREn for the enrichment of the genomic RNA (gRNA) of dengue virus (DENV), a positive-sense single-stranded RNA virus. This approach enabled the preparation of gRNA with significantly increased purity and led to the identification of a high-confidence 5-methylcytosine (m 5 C) site in DENV gRNA, orthogonally validated by Illumina-based bisulfite sequencing and direct RNA sequencing by Nanopore Oxford Technologies. Strikingly, this m 5 C modification was exclusively detected in gRNA extracted from infected cells but not in gRNA extracted from viral particles. We identified NSUN6 as the host methyltransferase catalyzing this modification and demonstrated a role for m 5 C in regulating DENV gRNA turnover. ViREn thus enables the mapping of m 5 C on low abundance viral gRNA with unprecedented precision and sensitivity and facilitates mechanistic studies into the role of RNA modification in virus replication.