The N6-methyladenosine (m6A) modification of RNA has been reported to affect viral infections. Studies have confirmed the role of m6A in replication of several vector-borne flaviviruses, including dengue virus (DENV), in mammalian cells. Here, we explored the role of m6A in DENV replication in the mosquito Aedes aegypti Aag2 cell line. We first determined the presence of m6A on the RNAs from mosquito cells and using methylated RNA immunoprecipitation and sequencing (MeRIP-Seq) identified m6A modification of the mosquito transcriptome and those that changed upon DENV infection. Depletion of m6A methyltransferases and the m6A binding protein YTHDF3 RNAs decreased the replication of DENV. In particular, we found that the Ae. aegypti ubiquitin carrier protein 9 (Ubc9) is m6A modified and its expression increases after DENV infection. Silencing of the gene and ectopic expression of Ubc9 led to reduced and increased DENV replication, respectively. The abundance of Ubc9 mRNA and its stability were reduced with the inhibition of m6A modification, implying that m6A modification of Ubc9 might enhance expression of the gene. We also show that the genome of DENV is m6A modified at five sites in mosquito cells. Altogether, this work reveals the involvement of m6A modification in Ae. aegypti-DENV interaction.

The N6‐methyladenosine (m6A) machinery functions through three groups of proteins in eukaryotic cells, including m6A writers, erasers and readers. The m6A cellular machinery has mostly been characterised in mammalian species, and the relevant literature on insects is currently scant. While homologues of m6A writers and readers have been reported from insects, no erasers have been described so far. Here, using BLAST search, we searched for potential erasers in insects. While we found homologues of human m6A eraser ALKBH5 in termites, beetles and true bugs, they could not be found in representative dipteran and lepidopteran species. However, a potential m6A eraser, ALKBH8, was identified and experimentally investigated. Our results showed that ALKBH8 can reduce the m6A levels of Aedes aegypti and Drosophila melanogaster RNAs, suggesting that AeALKBH8 could be a candidate m6A eraser in insects.