The HIV-1 Nef protein is essential for high-titer viral replication and AIDS progression. Nef functions by interacting with multiple host cell signaling proteins, including the members of the Src protein-tyrosine kinase family. Work completed during the previous grant period shows that Nef binds and activates Hck, Lyn, and c-Src, all of which are expressed in HIV target cells. This renewal application will test the overall hypothesis that these Src family members are important for Nef and HIV-1 function with the following Specific Aims: Aim 1. Determine whether Hck, Lyn and c-Src are interchangeable in support of Net- dependent HIV-1 replication. RNAi and pharmacological approaches will be used to selectively suppress Hck, Lyn and c-Src activity in U87MG cells, followed by analysis of HIV infectivity and replication. Phosphoproteomics will be applied to identify signaling partners for Nef-Src family kinase complexes related to HIV replication. These experiments will address whether Hck, Lyn and c-Src serve unique or redundant functions in terms of Nef signaling and HIV replication. Aim 2. Investigate the role of Nef oligomerization in Src family kinase activation, MHC and CD4 down-regulation, and HIV replication. We will map the Nef dimerization interface using a combination of molecular modeling, site-directed mutagenesis, and bimolecular fluorescence complementation (BiFC), which allows direct visualization of Nef:Nef interaction and subcellular localization in live cells. Oligomerization-defective Nef mutants will be tested for their ability to activate Src family kinases, induce MHC and CD4 down-regulation and support HIV infectivity and replication. Aim 3. Determine whether Src family kinase activation and oligomerization are general properties of Nef alleles from a wide range of HIV-1 strains. Experiments in this Aim will address the generality of Nef-induced Src family kinase activation using Nef alleles representative of all major HIV-1 subgroups and a high-throughput yeast-based screen that faithfully models physiological Src family kinase regulation. We will also determine whether oligomerization is a general property of these HIV-1 Nef alleles using the BiFC approach described in Aim 2. Finally, we will test the hypothesis that primary Nef alleles from long-term non-progressors exhibit defects in oligomerization and/or Src family kinase activation. Systematic, large-scale analysis of Nef oligomerization and interactions with Src family kinases is an important prerequisite for future drug discovery efforts aimed at this Nef signaling pathway. Public Health Relevance: These studies are focused on a unique HIV-1 protein (Nef) that is essential for the progression of AIDS. Our work is focused on the interaction of Nef with protein kinases in infected cells. Completion of these experiments will validate these Nef-kinase complexes as targets for the discovery of new anti-HIV therapeutics.