PROJECT DESCRIPTION The APOBEC3 (A3) family of proteins are cellular cytidine deaminases that suppress human immunodeficiency virus type 1 (HIV-1) infection by hypermutation of viral reverse transcripts and physically blocking reverse transcription. To evade this host defense mechanism, HIV-1 expresses the virion infectivity factor (Vif), which hijacks a cellular E3 ubiquitin ligase complex and targets A3 proteins (A3F/G/H/D) for proteasome-mediated degradation. Besides degrading A3s, HIV-1 Vif also causes G2 cell cycle arrest by targeting multiple protein phosphatase 2A (PP2A) regulators (PPP2R5 proteins) for degradation. Adding to the complexity of Vif-host protein interactions, HIV-1 Vif utilizes the transcription factor CBFβ as a non-canonical cofactor, while maedi-visna virus (MVV) Vif co-opts the prolyl isomerase cyclophilin A (CypA) instead. Our goal is to establish the biochemical and structural principles for the multifaceted activities of lentiviral Vif molecules that recruit cellular factors to degrade host proteins via ubiquitin-proteasome pathways. To achieve our goal, we will use a combination of biochemical, biophysical, structural biology, and cellular functional techniques. To establish the mechanisms by which A3 proteins are targeted by the HIV-1 Vif (Aim1), we will determine high- resolution structures of the A3-Vif-E3 interaction complexes, validate these structures by structure-guided mutagenesis experiments in vitro and in vivo, and interrogate the molecular determinants of Vif/A3/E3 ligase assembly and activation. In addition, we will also study the degradation-independent mode of Vif inhibition of A3 deamination and antiviral activities. To better understand CypA-mediated formation of MVV Vif-E3 ubiquitin ligase (Aim2), we will assemble MVV Vif/CypA/E3 ligase complexes with or without A3 substrates, determine their high-resolution structures, and perform biochemical and functional validations of our structural observations. The influences of capsid proteins on the assembly and activation of MVV Vif-E3 ligase will also be investigated. To delineate the mechanisms of PPP2R5/PP2A recruitment by lentiviral Vif-E3 ubiquitin ligases (Aim3), we will investigate the effects of PPP2R5 proteins on the assemblies of the CBFβ-mediated HIV-1 Vif and CypA-mediated MVV Vif-E3 ligases, obtain high-resolution structures, and perform structure- guided validations. Our comprehensive research design provides a robust approach that will generate unprecedented insights into the diverse functions of lentiviral Vif molecules.