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.

Many cellular mRNAs contain the modified base m⁶A, and recent studies have suggested that various stimuli can lead to changes in m⁶A. The most common method to map m⁶A and to predict changes in m⁶A between conditions is methylated RNA immunoprecipitation sequencing (MeRIP-seq), through which methylated regions are detected as peaks in transcript coverage from immunoprecipitated RNA relative to input RNA. Here, we generated replicate controls and reanalyzed published MeRIP-seq data to estimate reproducibility across experiments. We found that m⁶A peak overlap in mRNAs varies from ~30 to 60% between studies, even in the same cell type. We then assessed statistical methods to detect changes in m⁶A peaks as distinct from changes in gene expression. However, from these published data sets, we detected few changes under most conditions and were unable to detect consistent changes across studies of similar stimuli. Overall, our work identifies limits to MeRIP-seq reproducibility in the detection both of peaks and of peak changes and proposes improved approaches for analysis of peak changes.

Significance: N6 -methyladenosine (m ⁶ A) has recently been found to regulate numerous aspects of RNA biology. Similar to methylation of cytosine residues in DNA, eukaryotic RNA is modified by enzymatic addition of methyl groups at adenosines. m ⁶ A modification of RNA affects a wide variety of RNA functions, including mRNA stability, translation, and in the case of viruses, viral replication and production. Our investigation revealed that the adenosine residues present in the known m ⁶ A consensus motif within the 5′ epsilon stem loop of pregenomic RNA and the 3′ ends of all the hepatitis B virus (HBV) transcripts are m ⁶ A modified. We demonstrate here that m ⁶ A modification differentially modulates HBV RNA stability and reverse transcription, thereby playing two distinct regulatory roles in the HBV life cycle.