ABSTRACT, CORE 3 Biophysical methods, such as single-molecule techniques, quantitative imaging/spectroscopy and native mass spectrometry (nMS) are powerful tools to gain mechanistic insights into key dynamic interactions and processes occurring during the early and late steps of the HIV-1 lifecycle. The goal of Core 3 (Biophysics Core) is to provide requisite instrumentation, expertise and training to augment the B-HIVE capabilities across all three center projects by enabling cutting-edge studies of protein-protein, protein-nucleic acid and protein-membrane interactions in living cells and in model systems. To accomplish these goals we will: (1) Provide advanced fluorescence microscopy capabilities to visualize early and late events of the HIV life cycle (Melikian); (2) provide advanced capabilities in single-molecule biophysics to probe dynamics of proteins, nucleic acids and their complexes (Millar & Musier-Forsyth); and (3) provide methods for biophysical characterization of macromolecular complexes (Musier-Forsyth and Wysocki). A critical part of the Core 3 mission is training the users remotely or in person, through visits to the labs of the Core co-leads, workshops and videoconferences. To accomplish this goal, the core will provide basic imaging, single-molecule and nMS services using established pipelines to all B-HIVE projects, as needed, implement advanced techniques and engage in long-term projects through collaborative efforts with the B-HIVE investigators, and develop novel technologies in response to the needs of Projects 1-3. 1