N6 -Methyladenosine in Flaviviridae Viral RNA Genomes Regulates Infection

The RNA modification N6-methyladenosine (m⁶A) post-transcriptionally regulates RNA function. The cellular machinery that controls m⁶A includes methyltransferases and demethylases that add or remove this modification, as well as m⁶A-binding YTHDF proteins that promote the translation or degradation of m⁶A-modified mRNA. We demonstrate that m⁶A modulates infection by hepatitis C virus (HCV). Depletion of m⁶A methyltransferases or an m⁶A demethylase, respectively, increases or decreases infectious HCV particle production. During HCV infection, YTHDF proteins relocalize to lipid droplets, sites of viral assembly, and their depletion increases infectious viral particles. We further mapped m⁶A sites across the HCV genome and determined that inactivating m⁶A in one viral genomic region increases viral titer without affecting RNA replication. Additional mapping of m⁶A on the RNA genomes of other Flaviviridae, including dengue, Zika, yellow fever, and West Nile virus, identifies conserved regions modified by m⁶A. Altogether, this work identifies m⁶A as a conserved regulatory mark across Flaviviridae genomes.