N6-Methyladenosine in Flaviviridae Viral RNA Genomes Regulates Infection.

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