N6-methyladenosine (m6A) at the RNA virus-host interface

Abstract

RNA is a versatile and tractable biomolecule that serves as a critical component of life, whether as a script for protein production, a carrier of genetic information, a scaffold, or an enzyme. The fate and function of RNA can be influenced by chemical modifications such as N6-methyladenosine (m6A). Here we sought to identify the role of m6A during infection by positive-sense RNA viruses in the Flaviviridae family. First, we investigated the role of m6A on viral RNA. We mapped m6A on the viral RNA genomes of hepatitis C virus (HCV), dengue virus (DENV), Zika virus (ZIKV), West Nile virus (WNV), and yellow fever virus (YFV). We then studied HCV as a model RNA and virus to understand the function of m6A on Flaviviridae RNA genomes. We found that the m6A methyltransferases METTL3 and METTL14 reduced HCV infectious particle production without affection viral RNA replication, while the m6A demethylase FTO had the opposite effect. Similarly, the m6A-binding YTHDF1-3 proteins also inhibited HCV particle production. Furthermore, the YTHDF proteins relocalized to cytoplasmic lipid droplets, the sites of HCV particle assembly, during infection. We then identified that m6A in a specific region of viral RNA was responsible for the role of m6A in viral particle production. Abrogation of m6A modification increased viral RNA binding to the capsid protein Core, an important step of HCV assembly, and also increased HCV particle production. These data suggest that m6A inhibits HCV particle production and that m6A modification of viral RNA can have a functional consequence for infection. We then investigated how m6A on cellular mRNA can impact Flaviviridae infection. Working in collaboration with Dr. Chris Mason’s lab, we developed stringent analytical tools for detecting m6A changes. When we applied these tools, we found that a subset of cellular transcripts had altered m6A modification following infection by DENV, ZIKV, WNV, and HCV. We identified that innate immune signaling and ER stress, cellular pathways which are activated during Flaviviridae infection, contribute to altered m6A modification of two model transcripts RIOK3 and CIRBP. The gain of m6A on RIOK3 promotes the translation of this transcript, while loss of m6A on CIRBP influences its alternative splicing. Importantly, the RIOK3, CIRBP, and other transcripts with altered m6A modification can promote or inhibit Flaviviridae infection. Taken together, these results highlight the important role of m6A on both viral and cellular RNA in regulating infection.