SUMMARY: Antiretroviral drugs are critical for the survival of the ~38 million people who are currently living with HIV-1 infection. These drugs have also been used as pre-exposure prophylaxis to prevent HIV-1 infection; in addition, they are a mainstay of possible HIV-1 cure strategies. However, development of drug resistance threatens to undermine these successes that are critical to the health of many millions of people worldwide. For these reasons, discovery of new antiretroviral targets and compounds that inhibit these targets is critical. Since key HIV-1 enzymes are already targeted by drugs in current use, targets of the future will need to come from poorly understood aspects of the viral life cycle, such as intracellular late events in the viral life cycle, which are critical for virus production. Two decades of studying these events led to identification of a host- catalyzed pathway of putative capsid assembly intermediates that contain the HIV-1 Gag protein and host enzymes. A drug screen that reconstituted this assembly pathway led to discovery of PAV117, an antiretroviral small molecule, and subsequently a more potent analog, PAV206. Preliminary data in the current proposal demonstrate that PAV206 blocks virus production at nanomolar concentrations in T cell lines and HIV-1 infected PBMCs, making it the first potent and selective inhibitor of intracellular late events. Imaging experiments demonstrate that PAV206 colocalizes with the viral protein Gag and also with a host enzyme present in HIV-1 capsid assembly intermediates suggesting that this small molecule targets one or more components of HIV-1 capsid assembly intermediates. This proposal seeks to 1) determine which retroviruses are inhibited by PAV206, 2) identify mutations in HIV-1 that confer PAV206 resistance, and 3) use imaging experiments to determine if PAV206 localizes to a distinct subcellular complex, and 4) use biochemical approaches to probe for PAV206 binding partners. The subcellular localization and binding partner studies will take advantage of PAV206 analogs that are available to us, including one that maintains antiviral activity but contains a biotin tag for antibody recognition and a chemical group for photo-crosslinking. We provide extensive data demonstrating how this analog can be used to great advantage for in situ imaging using the proximity ligation assay (PLA). We also propose to extend these imaging studies using a cutting-edge variation of this technique termed multiplex PLA. Finally, we describe a second analog of PAV206 that allows other tags to be added through click chemistry, thereby expanding options for affinity purification of target proteins and mass spectrometry analysis. With this impressive array of tools, our likelihood of successfully identifying the PAV206 target is high. In conclusion, by advancing our understanding of a novel small molecule inhibitor of HIV-1 late events, studies proposed here will provide an exciting tool for studying HIV-1 assembly and a springboard for generating compounds that could be advanced in the future.