The overall goal of this application is to understand the molecular interactions between Nef and the cellular proteins that mediate its role in HIV- 1 pathogenesis. Nef is a multifunctional adaptor protein which exerts its effects by altering the expression of specific cell surface receptors and perturbing signal transduction pathways. These effects involve many independent interactions between Nef and the cellular proteins that are components of the protein sorting and signal transduction machineries. The overall goal of this current application is to identify the immediate downstream effectors of Nef, and to characterize the underlying protein-protein interactions. These studies will use a recently developed unbiased biochemical method to purify cellular proteins that are associated with Nef in T cells and identify them by microsequencing. This approach already identified a subset of Nef associated proteins and discovered several novel and biologically relevant interactions between Nef and cellular proteins, including N-myristoyl transferase, components of the cytoskeleton and proteins that regulate lymphocyte migration. Specifically, the first set of experiments will characterize the association of Nef with proteins important for lymphocyte chemotaxis. The second set of experiments will characterize the mechanisms of the Nef induced defect in lymphocyte migration in response to chemokines. The third set of experiments will continue to identify additional proteins that are associated with Nef in T cells and assess the relevance of these associations to Nef functions. Finally, the effect of mutations that disrupt selected molecular interactions of Nef on AIDS pathogenesis will be studied in rhesus monkeys Our studies will continue to provide a molecular framework for understanding the functions of Nef and the molecular interactions that underlie these functions, and for assessing their contribution to the pathogenic process. Results from these studies will have implications for the rational design of pharmacological agents that disrupt these functions of Nef that are required for AIDS pathogenesis.
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