Abstract: The human ESCRT protein machinery is required for membrane remodeling events including multivesicular body biogenesis, cellular abscission, and viral budding. Specifically, the Pro-Thr-Ala-Pro (PTAP) motif of viral Gag proteins targets the ESCRT-I complex via a direct interaction with Tsg101 (tumor susceptibility gene 101). This interaction is necessary for the viral Gag proteins to be recruited to the membrane. Naturally this interaction site has been the target for designing anti-viral drugs by mimicking the PTAP motif. Recently we identified a small molecule inhibitor of HIV budding, which we expected to bind to the PTAP recognition site of Tsg101. This molecule belongs to a family of proton pump inhibitors that are clinically used to treat acid reflux. Initial characterization using solution NMR indicated that the inhibitor interacts with Tsg101 outside of the PTAP recognition site. The structure of Tsg101 and a small molecule inhibitor complex that we solved reveals a covalent interaction occurring at the ubiquitin (Ub) binding site of Tsg101. Tsg101’s main contribution to ESCRT-I function is in recognition of and binding to Ub-modified cargo. The fact that the inhibitor targeted ubiquitin-Tsg101 binding was significant, since this interaction was previously thought to have little influence on the HIV-1 life cycle. Using our new inhibitor as a tool, we uncovered the essential role of Ub-Tsg101 interaction to promote degradation of HIV-1 Gag protein in the cell and to block co-localization of Tsg101 and HIV-1 Gag at the plasma membrane required for budding. In addition, we also showed that Tsg101 has another binding site that can contact the second Ub moiety in K48 or K63 linked di-Ub molecules. This second Ub binding site on Tsg101 has a weaker affinity compared to the first site and its observation required the use of novel NMR methodology. Our recent results show the potential for development of broad spectrum antiviral inhibitor based on clinically approved proton pump inhibitors targeting Tsg101 and we also provided the first evidence for the important role of Tsg101 and di-Ub interaction in viral replication.