Molecular mechanisms of HIV Nef-induced Src kinase activation and survival signaling in myeloid cells

The Nef protein unique to the primate lentiviruses HIV and SIV is essential for high-titer viral replication and AIDS progression. Despite its essential role, the molecular mechanisms by which Nef functions in HIV pathogenesis are not fully understood. Since Nef lacks intrinsic catalytic activity, research has been focused on analyzing interactions between Nef and cellular proteins in an attempt to understand the many functions attributed to Nef. Nef binds to the macrophage-specific Src family member Hck through its SH3 domain with the highest affinity known for an SH3-mediated protein-protein interaction. Previous studies from our laboratory have shown that Nef-Hck interaction results in constitutive Hck kinase activation capable of transforming Rat-2 fibroblasts. Nef-Hck interaction may be crucial to M-tropic HIV replication and AIDS pathogenesis, identifying this virus-host protein complex as a rational target for anti-HIV drug discovery.; To investigate whether interaction with Hck is a common feature of Nef alleles from different strains of HIV-l, we compared the ability of four different HIV-1 Nef alleles to induce Hck activation and transformation in our Rat-2 fibroblast model. We found that not all HIV-1 Nef alleles have a similar affinity for Hck, despite strong conservation of the PxxPxR motif and hydrophobic pocket residues identified in the crystal structure as part of the SH3 interface. Further characterization of the interface of the Hck SH3-Nef complex revealed additional critical residues in the Nef hydrophobic pocket responsible for the differential interaction of HIV-1 Nef alleles to Hck. This study provides the first evidence that the HIV-1 Nef hydrophobic pocket is critical for SH3-mediated Hck activation in vivo and identifies the pocket as a rational target for drug design to selectively disrupt Nef-Hck signaling in HIV infected cells.; Suppression of cell death of HIV-infected cells allows time for viral replication prior to cell lysis, facilitating productive viral replication indirectly. Recently Nef has been demonstrated to be an important factor in promoting the survival of HIV-infected T cells by affecting mediators of apoptosis. Since macrophages serve as HIV viral reservoirs and play a critical role in persistent virus infection, we were interested in the role of Nef in survival of macrophages. Previous work from our laboratory has shown that Nef promotes cytokine independent proliferation of the macrophage precursor cell line, TF-1, through a mechanism that requires the Stat3 transcription factor. Studies presented in this dissertation demonstrate that Nef suppresses apoptosis in this cell line by selectively upregulating the anti-apoptosis gene, Bcl-X L. This signaling induction by Nef is dependent on Erk MAPK activation but not Stat3. This Nef-induced survival signal is the first to show that Nef generates anti-apoptosis signals in cells of the myelomonocytic lineage and adds important evidence to the hypothesis that Nef may contribute to the establishment and maintenance of an HIV reservoir by conferring a survival advantage on HIV-infected macrophages.