Selective Activation of Src Family Kinases by the HIV-1 Nef Protein

Nef is a critical HIV-1 accessory factor shown to promote viral pathogenesis by altering host cell signaling pathways. Nef has been shown to bind several members of the Src family of protein-tyrosine kinases, and these interactions have been implicated in the pathogenesis of HIV/AIDS. The studies summarized below investigated this key interaction between virus and host cell proteins. We explored the direct effect of Nef interaction on Src family kinases (SFKs) using Saccharomyces cerevisiae, a well-defined system in which c-Src expression arrests yeast cell growth in a kinase-dependent manner. The seven SFKs found in HIV target cells wre expressed in yeast; each was found to be active alone, but repressed by co-expression of the negative regulatory kinase Csk. We then co-expressed each SFK with both Csk and HIV-1 Nef and found that Nef selectively activated Hck, Lyn, and c-Src among SFKs.We then used our yeast-based system to identify small molecule inhibitors of the active Nef:Hck complex using the auto-dowregulated Hck-YEEI molecule. Yeast expressing the Nef:Hck-YEEI complex were used to screen a library of small heterocyclic compounds based on their ability to rescue growth inhibition. Two compounds identified in this screen potently blocked Nef-dependent HIV replication, indicating Nef:SFK complexes as valid targets for anti-HIV drug therapy. Finally, we used the yeast assay to identify novel mechanisms of Nef:SFK interactions. We screened a panel of primary Nef alleles containing the known SH3-binding elements and discovered four alleles whose proteins demonstrated altered activation of SFKs. Sequence examination revealed the existence of amino acid changes in regions not previously suspected to be involved in SH3-mediated interaction. Particularly intriguing are residues in a large unstructured loop that projects from the Nef core. These findings suggest that critical residues outside of the known SH3-binding motifs may affect SFK binding and activation. Together, the results presented here advance the field of HIV research by furthering our understanding of the interaction between the HIV-1 Nef virulence factor and the Src kinase family, as well as validating this virus:host cell interaction as a rational target for anti-HIV drug discovery