N6-Methyladenosine in Flaviviridae Viral RNA Genomes Regulates Infection

The RNA modification N6-methyladenosine (m(6)A) post-transcriptionally regulates RNA function. The cellular machinery that controls m(6)A includes methyl-transferases and demethylases that add or remove this modification, as well as m(6)A-binding YTHDF proteins that promote the translation or degradation of m(6)A-modified mRNA. We demonstrate that m(6)A modulates infection by hepatitis C virus (HCV). Depletion of m(6)A methyltransferases or an m(6)A 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 m(6)A sites across the HCV genome and determined that inactivating m(6)A in one viral genomic region increases viral titer without affecting RNA replication. Additional mapping of m(6)A on the RNA genomes of other Flaviviridae, including dengue, Zika, yellow fever, and West Nile virus, identifies conserved regions modified by m(6)A. Altogether, this work identifies m(6)A as a conserved regulatory mark across Flaviviridae genomes.