ABSTRACT RNA modifications play diverse roles in regulating viral infection. The RNA modification N6-methyladenosine (m6A) regulates infection by the positive-strand RNA viruses in the Flaviviridae family, through its effect on both viral and cellular RNA. m6A regulates key aspects of RNA biology, including RNA stability, translation, and localization, as well as interactions with m6A-binding proteins. While the basic proteins that add or bind m6A on mRNA are known, little is known about how the m6A-methyltransferase complex or m6A-binding proteins are specifically regulated or targeted to viral and cellular RNA during infection. Our previous work has revealed that the RNA genomes of viruses in the Flaviviridae family are m6A-modified at specific sites, and that m6A at one site in the hepatitis C virus (HCV) genome negatively regulates infectious particle production by inhibiting interaction of the HCV RNA genome with the viral packaging protein. We also found that Flaviviridae-induced cellular signaling pathways alter m6A changes in host mRNAs important for infection. Finally, our preliminary data identify novel m6A-methyltransferase interacting proteins and a novel m6A-reader with a described role in the antiviral interferon (IFN) response. The goal of this proposal is to elucidate the molecular mechanisms of how m6A-targeting to viral and cellular RNA regulates infection by specific viruses in the Flaviviridae family. Based on our preliminary data, the central hypothesis of this proposal is that the specific m6A regulatory pathways co-opted and induced by viral infection can be determinants of viral replication. Guided by our preliminary data, this hypothesis will be tested by pursuing the following three specific aims: 1) Define how the m6A- methyltransferase complex targets HCV RNA to regulate infection; 2) Determine how Flaviviridae infection alters m6A regulation of specific host mRNAs; 3) Characterize an IFN-induced m6A-reader that restricts Flaviviridae infection. In Aim 1, we will define how the nuclear m6A-methyltransferase is recruited to the cytoplasmic-localized RNA genome of HCV. In Aim 2, we will investigate how the m6A-methyltransferase complex is functionalized during viral infection to differentially target cellular mRNAs for methylation. In Aim 3, we will define the molecular determinants of how an IFN-induced m6A reader targets viral and host m6A-modified RNA to inhibit Flaviviridae infection. The proposed work will be significant and innovative because it will define cellular pathways and mechanisms that alter m6A-methyltransferase composition and function during Flaviviridae infection, as well as define new IFN-induced regulators of m6A, thus uncovering new host pathways exploited by viruses or utilized by the host to inhibit infection. Importantly, this work will also reveal novel aspects of how gene expression is regulated by m6A, including the fact that specific cellular signaling pathways alter how m6A is placed on spec...