PROJECT SUMMARY/ABSTRACT HIV has a complex expression pattern of RNA with three different classes of transcripts: unspliced, singly spliced, and multiply spliced. The unspliced transcripts encode the Gag and Pol proteins, and the singly spliced transcripts encode the Env, Vif, and Vpu proteins. The multiply spliced transcripts encode the Tat, Rev, and Nef proteins. The multiply spliced transcripts are translated early on following infection due to their more efficient export from the nucleus. The unspliced and singly spliced transcripts, on the other hand, are retained in the nucleus due to cis-acting elements including the Rev Response Element (RRE). However, the post- transcriptional processing of these three classes of transcripts beyond their differential splicing is an understudied area of HIV research. Past research has revealed that alongside the virally encoded Rev protein, multiple host factors are also necessary for the export of unspliced and singly spliced HIV transcripts. Among these are RNA binding proteins including proteins that can recognize the RNA modification N6-methyladenosine (m6A). The role of m6A modification of HIV transcripts has been studied in active infection of cell lines but has yielded differing results. Some reports suggest that m6A modification of HIV transcripts enhances HIV replication while others suggest that it inhibits replication. Current maps of m6A sites on HIV transcripts cannot discern how the different transcript classes are differentially modified. Additionally, how m6A mechanistically impacts post- transcriptional processing of HIV transcripts and their nuclear trafficking is unknown. Preliminary data suggests that the HIV RNA species are, in fact, differentially modified and the presence or absence of m6A at these sites affects RNA expression levels. Therefore, I hypothesize that the presence or absence of m6A on HIV transcripts impacts the interactions of HIV RNA with nuclear RNA binding proteins, and this, in turn, affects the nuclear trafficking and stability of the RNA. To test this hypothesis, I will use nanopore direct RNA sequencing, knockdowns of the m6A machinery proteins and m6A reader proteins in actively infected and latently infected primary T cells, and HITS-CLIP to address the following questions: (i) where does m6A modification occur on HIV transcripts from primary T cell models of HIV latency and HIV+ donor cells? (ii) how does perturbation of the m6A machinery proteins and m6A readers affect the nuclear localization and distribution of the different HIV RNA species during active infection and following latency reversal? Ultimately, the results of this project will provide a better understanding of the post-transcriptional processing of HIV RNA in primary T cells which is paramount to the design of new therapeutics for eradicating the latent HIV reservoir.