Abstract. Existing antiretroviral drugs do not clear HIV-1 from infected individuals, requiring life-long administra- tion. The HIV-1 Nef accessory factor is an attractive target for drug development because it supports HIV repli- cation in vivo and promotes immune escape of HIV-infected cells. Our laboratory has discovered small molecules that bind directly to Nef and inhibit its ability to enhance HIV-1 replication in cell culture. These inhibitors interfere with Nef-mediated MHC-I downregulation in latently infected CD4 T cells, enabling recognition and killing by CTLs. Thus, Nef inhibitors represent a new approach to antiretroviral therapy that may promote eradication of viral reservoirs. During the previous funding cycle, we developed Proteolysis Targeting Chimeras (PROTACs) for the targeted destruction of the Nef protein in HIV-infected cells. These PROTAC molecules induce ternary complexes between Nef and the Cereblon E3 ubiquitin ligase resulting in Nef ubiquitylation and proteolytic deg- radation. Nef PROTACs efficiently reverse Nef-mediated downregulation of MHC-I and CD4 from the T cell sur- face and inhibit Nef-mediated enhancement of HIV-1 replication in donor PBMCs. PROTAC-mediated degra- dation is likely to reverse all Nef functions, suppressing viral rebound while enhancing antiviral immunity and reservoir reduction. Here we propose to explore the mechanism of action and on-target selectivity of our Nef-directed PROTACs, their propensity for inducing acquired resistance, and their ability to enhance cellular antiviral immunity. Our proposal is responsive to NOT-AI-23-049, Using Targeted Degradation of Protein and non-Protein Targets for the Development of Novel Anti-Infectives, and PAR-23-297, Opportunities for HIV Cure Strategies at the Time of ART Initiation. The following Specific Aims are proposed: 1) Determine the mecha- nism of action of Nef-directed PROTACs. This Aim will identify the Nef lysine residues required for the initiation of ubiquitylation by the PROTACs. In addition, PROTAC selectivity for Nef in T cells will be evaluated by prote- omics. 2) Test the hypothesis the Nef PROTACs are unlikely to induce drug resistance in HIV-infected cells. PBMCs will be infected with HIV-1 carrying a Nef mutant library in the presence or absence of ‘occupancy- based’ Nef inhibitors vs. PROTACs. Resistance mutations will be identified in emergent viruses by RNAseq. Targeted degraders may be less likely to generate resistance compared to their occupancy-based counterparts, because elimination of Nef may make infected cells susceptible to apoptosis. 3) Test the hypothesis that tar- geted degradation of HIV-1 Nef affects latent viral reservoirs. Nef-mediated downregulation of MHC-I and induction of PD-1 both may serve to promote and stabilize HIV reservoirs. This Aim will examine reservoir cells from HIV-infected donors to determine whether targeted degradation of Nef influences latency reversal and in- creases the susceptibility of re...