Nef is an HIV-1 accessory protein that facilitates virus infectivity, replication, and immune evasion [1
]. In non-human primate models of AIDS, high-titer viral replication and development of AIDS-like disease requires an intact Nef
]. Long-term non-progressive HIV infection in humans is also associated with Nef-defective HIV isolates in some cases [5
]. Complementary in vivo studies have shown that directed expression of Nef
alone to HIV target cells induces an AIDS-like syndrome in transgenic mice [7
]. Taken together, these studies underscore the importance of HIV-1 Nef in AIDS pathogenesis.
Nef is not known to exhibit any intrinsic enzymatic activity. Instead, Nef interacts with multiple host cell signaling pathways to enhance HIV-1 replication and promote AIDS progression [10
]. Previous work from our group has identified members of the Src kinase family as direct Nef effectors [11
]. This kinase family includes Hck, a Src-family member expressed in macrophages, which are a critical HIV target cell type and viral reservoir. Nef interacts with the Hck SH3 domain, leading to constitutive Hck activation that may contribute to macrophage survival, MHC-I downregulation and M-tropic HIV replication [11
]. Nef has also been shown to bind and activate the Src-family kinases Lyn and c-Src, which exhibit a broader expression pattern including other HIV target cell types [14
]. Thus, Nef-dependent activation of Src family kinases is likely to occur in most HIV-infected cells.
Hck shares a similar domain organization and structural architecture with other members of the Src kinase family [19
]. Key structural features include an N-terminal unique domain with sites for lipid attachment that drive membrane association, followed by the regulatory SH3 and SH2 domains, an SH2-kinase linker, the kinase domain, and a C-terminal negative regulatory tail. Nef binds to the Hck SH3 domain through a bipartite mechanism revealed in structural analyses of Nef:SH3 complexes [22
]. Nef:SH3 interaction is dependent in part on a highly conserved PxxPxR motif, which forms a polyproline type II helix typical of most SH3 ligands. In addition, the αA and αB helices of Nef form a hydrophobic pocket that interacts with an Ile residue in the RT loop of the SH3 domain. Nef binding displaces the SH3 domain from its negative regulatory position on the back of the kinase domain, leading to kinase activation. Interestingly, mutation of the Nef PxxPxR motif completely abolished development of the AIDS-like phenotype in Nef-transgenic mice [8
]. Furthermore, crossing Nef transgenic mice into a hck-
null background increased the latency for AIDS-like disease onset and decreased mortality [8
]. These data provide strong evidence that Src-family kinase activation by Nef is important for AIDS pathogenesis, and identify this signaling pathway as a target for therapeutic intervention.
Recently, we developed a chemical library screening assay based on Nef-dependent activation of Hck in vitro [15
]. Using this assay, we identified a series of diphenylfuropyrimidine (DFP) analogs that preferentially inhibit Hck in the presence of Nef. These compounds also potently blocked HIV-1 replication in a Nef-dependent manner [15
], validating inhibitors of Nef-SFK signaling as potential antiretroviral agents. Our observation that DFP-based kinase inhibitors selectively inhibit the Nef:Hck complex suggested that Nef binding to the Hck SH3 domain induces structural changes in the kinase domain that favor inhibitor association. In the present study, we developed a system to test this hypothesis directly using a "gatekeeper" mutant of Hck with engineered sensitivity to the pyrazolopyrimidine analog, NaPP1 [26
]. This mutation involves substitution of the gatekeeper threonine (Thr338; numbering as per c-Src crystal structure [28
]) with a much smaller alanine residue (Hck-TA mutant), providing access for NaPP1 to the hydrophobic cavity adjacent to the ATP binding site. This combination of mutant kinase and NaPP1 results in a high degree of inhibitor selectivity and potency both in vitro and in cell-based assays [27
]. Because NaPP1 binds to the Hck-TA active site in a specific location, it serves as a chemical probe for conformational changes that may occur in response to Nef binding. In addition to the gatekeeper mutation, we modified the SH3 domain to enhance interaction with Nef [29
]. This modification enabled stable association of Hck with Nef in solution-based kinase assays, thus mimicking the stable association that is likely to occur between Hck and Nef at cellular membranes [17
]. Use of this modified form of Hck combined with the selective inhibitor enabled us to demonstrate that Nef binding results in changes in the Km
for ATP as well as inhibitor potency. These observations support the idea that Nef binding induces a unique active conformation of the Hck active site that can be targeted with selective inhibitors.