Abstract: The long-term goal of this application is to characterize host-virus interaction interface as a novel drug target and to develop inhibitors to disrupt intracellular protein-protein interactions (PPI) between the host and the virus to curb HIV-1 replication. It has been established that perturbing IN without affecting its enzymatic activity can inhibit late stages of HIV-1 replication such as assembly, particle production and/or particle morphogenesis. Such class II IN mutations and allosteric inhibitors of IN (ALLINI), inhibit late events and they do so by perturbing IN/IN multimerization, IN/host factor interaction or IN/RNA interactions. We have observed that such defects in particle morphogenesis can also be observed in IN mutants defective for interaction with a host factor, INI1/hSNF5, an IN-binding host factor, that is selectively incorporated into HIV-1 virions. INI1 is required for HIV- 1 late events. Expression of a minimal-IN-binding domain of INI1 (INI1183-292) termed S6, disrupts IN/INI1 interaction in vivo and potently inhibits HIV-1 particle production. Knocking down INI1 and use of INI1-/- cell lines inhibit HIV-1 particle production. INI1-binding defective IN mutants lead to the production of morphologically defective particles indicating that targeting IN/INI1 interaction is an effective strategy to inhibit HIV-1 particle production. Lack of structure of INI1 and IN/INI1 complex have precluded our ability to develop inhibitors to target this interaction. New research from our laboratory including the NMR structure of the IN-binding Repeat 1 (Rpt1) domain of INI1, and molecular docking of IN/INI1 interaction have helped overcome this knowledge gap. We found that IN-binding domain of INI1, termed Rpt1, and Trans Activating Response element (TAR) of HIV-1 genomic RNA structurally mimic each other, a novel finding. Both Rpt1 and TAR bind to same surface of IN C-terminal domain (CTD) and compete for binding to IN with an identical IC50 value (0.005 µM). Furthermore, INI1-interaction-defective mutants of IN cause impairment of particle morphogenesis. We hypothesize that peptidomimetics and small molecules derived from Rpt1 have dual activity and inhibit both IN/INI1 and IN/TAR interactions. As a proof of principle, we have developed a stapled peptide derived from interface a-1 helix of Rpt1, that potently disrupts IN/INI1 and IN/RNA interactions, inhibits particle morphogenesis and in vivo HIV-1 replication. In this proposal, we will characterize IN/INI1 interface as an outstanding drug target by carrying out: i) Genetic analysis to understand the mechanism of INI1 influence on assembly/particle production via trans-complementation and “synthethic rescue” experiments; ii) Development of a novel class of stapled peptides and small molecules with dual activity in targeting IN/INI1 and IN/RNA interactions by SAR and virtual-ligand screening; and determine the NMR structure of INI1-stapled peptide complexes with IN-C-terminal do...