While the importance of chemical modifications of DNA and proteins is well established, relatively little is known about how the post-transcriptional modification of mRNA transcripts affects their function. The most common modified base seen on cellular mRNAs in mammals is N6-methyladenosine (m6A), and recent data demonstrate that the total loss of m6A formation can have severe deleterious consequences, for example, blocking the differentiation of pluripotent stem cells. However, how m6A regulates mRNA expression remains to be established. Moreover, previous work has revealed that several pathogenic human viruses, including influenza A virus (IAV), encode RNAs that undergo m6A editing, suggesting that such modifications might exert a positive effect on virus replication. Indeed, recent data from this laboratory, and others, demonstrate that m6A editing can significantly enhance HIV-1 gene expression and replication in cultured T cells. This grant proposal seeks to fully define the sites of m6A modification on IAV transcripts and to test the hypothesis that m6A editing enhances IAV gene expression and, hence, replication. Using the PAR-CLIP technique, we have identified several specific binding sites for the human YTHDF m6A reader proteins on the IAV genome on both the viral mRNA/cRNA and vRNA transcripts. Consistent with the hypothesis that m6A editing can positively regulate IAV gene expression, we have observed that overexpression of the human YTHDF2 reader protein, but not of the related YTHDF1 protein, in human A549 cells greatly enhances IAV replication. This grant first aims to define the precise locations of m6A modifications on IAV transcripts at single nucleotide resolution and to then quantify the degree of editing at each site. We will then use targeted mutagenesis to determine precisely how specific m6A modifications on different segments of the IAV genome affect IAV gene expression and replication. In parallel, we will examine how the overexpression of key cellular proteins involved in ?writing?, ?reading? and ?erasing? m6A marks, or their elimination by genome editing using CRISPR/Cas, affects IAV gene expression and replication in A549 cells. Together, these data will reveal how m6A editing affects IAV replication and may lead to new approaches to the enhancement of IAV replication in culture, for use for example in IAV vaccine production, or the inhibition of IAV replication in vivo, for example by transient inhibition of m6A editing.