Project Abstract This project aims to better understand the molecular mechanisms of HIV spread through cell-cell contact across a virological synapse (VS), a specialized structure formed between HIV infected cells and target cells. We have developed a novel flow cytometry assay for quantifying and purifying infected cell-target cell pairs. We have generated infected producer cells that stably express ascorbate peroxidase (APEX) enzymes localized to different subcellular compartments. Upon addition of hydrogen peroxide and biotin-phenol, APEX generates short-lived BP radicals that covalently biotinylate proteins and RNAs within a few nanometers of the enzyme. We will use APEX localized to nucleus, cytoplasm, ER, plasma membrane, and mitochrondria to provide a robust analysis of how host and viral proteins and RNAs are regulated by VS formation. The successful completion of this grant will identify determinants of the VS formation and its composition, identify changes to host cell proteins following VS formation with an emphasis on RNA binding proteins, and determine how host and viral RNAs are regulated by cell-cell contact. Aim 1: Identify determinants and composition of virological synapse (VS) formation. Aim 2: Determine how VS formation affects protein abundance and localization within infected cells, with an emphasis on cellular RNA binding proteins. Aim 3: Determine how cell-cell signaling during VS formation impacts the RNA regulatory landscape of infected cells.