PROJECT SUMMARY Antiretroviral therapy (ART) has transformed HIV into a manageable non-fatal chronic disease. However, significant challenges remain with increasing drug resistance and adverse drug reactions. Therefore, there is an urgent need to identify novel therapeutic targets with unique mechanisms of action to sustain the gains of ART. HIV transcription represents a vital part of the viral life cycle that has not been successfully targeted for therapy. We recently discovered that an interaction between the essential viral protein Tat and splicing factor 3B subunit 1 (SF3B1) is required for HIV transcription. The primary goal of this project is to define the role played by the interaction between Tat and SF3B1 in HIV transcription. The long term objective is to identify compounds that can block this interaction and therefore HIV replication. We hypothesize that Tat-mediated HIV transcription requires Tat to interacts with SF3B1 and that this interaction, when blocked will inhibit HIV replication. Our specific aims are: Aim 1: Identify the molecular domains of Tat-SF3B1 interactions required for HIV-1 transcription. We have shown that Tat and SF3B1 interact, and that this interaction requires the C-terminal portion of Tat. However, the specific domains of Tat or SF3B1 required for this interaction are unknown. First, we will use mutagenesis, along with in-vitro and in-vivo protein binding studies to narrow down the residues of Tat and the domain of SF3B1 required for the interaction. Second, we will perform in-vivo alanine scanning to better define the interaction surfaces for the two binding domains. The effect of alanine substitutions will be quantified with the NanoLuc® Binary Technology (NanoBiT) split luciferase complementation protein: protein interaction system. We will also perform Tat-SF3B1 immunoprecipitations and transcription assays in primary CD4+ T cells and monocyte-derived macrophages (MDMs) to ascertain the physiological relevance of our findings. Aim 2: Define the specific role of SF3B1 in Tat-mediated HIV-1 transcription. We showed that depletion of SF3B1 abrogates Tat-mediated HIV transcription, but the exact mechanism for SF3B1 actions on HIV transcription is unknown. SF3B1 interacts with Tat, the nuclear inhibitor of PP1 (NIPP1), and is also known to methylate HIV transcription-activating histone H3K36me3. Therefore, we hypothesize that SF3B1 may control HIV transcription through one or more of these pathways in primary CD4+T cells, MDMs and HIV latency models. First, we will use RNA Immunoprecipitation (RNAIP) and fluorescent electrophoretic mobility shift assay (fEMSA) to quantify the effects of SF3B1 on Tat/TAR interactions. Second, we will determine how SF3B1 influences Tat methylation as well as the occupancy of the HIV promoter by H3K36me3. Finally we will determine if Tat-SF3B1 interactions plays a role in NIPP1-mediated inhibition of HIV transcription. This work will be highly significant to further the understan...