Summary. Existing antiretroviral drugs do not clear HIV-1 latent reservoirs, underscoring the urgent need for new therapeutic strategies. The HIV-1 Nef accessory factor is an attractive target for drug development because of its critical roles in the HIV-1 life cycle and immune system escape. Our group has discovered novel small molecules that bind directly to Nef and block many of its functions, including enhancement of viral infectivity and replication in donor PBMCs. Importantly, our Nef inhibitors rescue cell-surface MHC-I expression in latently infected, patient-derived CD4+ T-cells, enabling recognition and killing by autologous CTLs. Thus, Nef inhibitors represent an innovative approach to antiretroviral therapy that may provide a path to eradication of viral reservoirs. Our most promising class of inhibitors (hydroxypyrazoles) bind tightly to their Nef protein target in vitro and are active against multiple Nef functions in cell-based systems without cytotoxicity. Experiments proposed here will leverage these compounds as chemical probes to shed new light on Nef functions while unraveling their mechanism of action with the following Specific Aims: Aim 1. Map the binding site for hydroxy- pyrazole Nef inhibitors by X-ray crystallography. Preliminary and published data strongly suggest that hydroxy- pyrazole Nef inhibitors, which disrupt multiple Nef functions, may perturb the structure of functional Nef-effector complexes. X-ray crystallography of inhibitors with Nef alone and in complexes with host cell effector proteins will be used to test this idea and identify inhibitor binding sites. Aim 2. Identification of Nef residues essential for inhibitor action through in vitro selection. Using PCR-based saturation mutagenesis, we have replaced every codon in the Nef core region with each of the 64 nucleotide triplets in the context of HIV-1. CD4 T cells will be infected with the Nef mutant viral ‘library’ in the presence or absence of Nef inhibitors, and viral supernatants analyzed by deep sequencing to identify mutations enriched by inhibitor treatment. This method has the potential to identify Nef regions that allosterically influence inhibitor action in addition to residues directly involved in ligand binding. Aim 3. Explore the mechanisms by which Nef inhibitors suppress HIV-1 infectivity. Hydroxypyrazole Nef inhibitors reduce HIV-1 infectivity in TZM-bl reporter cells to the same extent as Nef-deleted viruses. This Aim will explore the whether Nef inhibitors restore virion incorporation of SERINC proteins and Ezrin, two host cell restriction factors linked to Nef. We will also pursue Nef inhibitor effects on overall HIV-1 protein composition by whole-virus proteomics, which has the potential to identify host cell factors that are uniquely incorporated (or excluded) by Nef inhibition. Aim 4. Investigate the mechanism of Nef inhibitor action on MHC-I downregulation. This Aim will explore the effect of Nef inhibitors on crystal structures o...