PHYSICAL AND FUNCTIONAL INTERACTION OF NEF WITH LCK - HIV-1 NEF-INDUCED T-CELL SIGNALING DEFECTS

The nef gene is unique to the primate lentiviruses and encodes a cytop lasmic membrane-associated protein that affects T-cell signaling and i s essential for both maintenance of a high virus load in vivo and for disease progression. Here we investigated the perturbation of cell sig naling by Nef in T-cells and found that Nef interacts with the T-cell restricted Lck tyrosine kinase both in vitro and in vivo. The molecula r basis for this interaction was analyzed. We show that cell-derived N ef is precipitated in a synergistic manner by the recombinant Src homo logy 2 (SH2) and SH3 domains from Lck. A functional proline-rich motif and the tyrosine phosphorylation of Nef were evidenced as likely part icipants in this interaction, The precipitation of Nef by the Lck reco mbinant proteins was specific, since neither Fyn, Csk, p85 phosphatidy linositol 3-kinase nor phospholipase C gamma SH2 domains coprecipitate d Nef from T-cells, Finally, depressed Lck kinase activity resulted fr om the presence of Nef, both in vitro and in intact cells, and nef exp ression resulted in impairment of both proximal and distal Lck-mediate d signaling events, These results provide a molecular basis for the Ne f-induced T-cell signaling defect and its role in AIDS pathogenesis.