Nef is an HIV-1 virulence factor that promotes viral pathogenicity by altering host cell signaling pathways. Nef binds several members of the Src kinase family, and these interactions have been implicated in the pathogenesis of HIV/AIDS. However, the direct effect of Nef interaction on Src family kinase (SFK) regulation and activity has not been systematically addressed. We explored this issue using Saccharomyces cerevisiae, a well defined model system for the study of SFK regulation. Previous studies have shown that ectopic expression of c-Src arrests yeast cell growth in a kinase-dependent manner. We expressed Fgr, Fyn, Hck, Lck, Lyn, and Yes as well as c-Src in yeast and found that each kinase was active and induced growth suppression. Co-expression of the negative regulatory kinase Csk suppressed SFK activity and reversed the growth-inhibitory effect. We then co-expressed each SFK with HIV-1 Nef in the presence of Csk. Nef strongly activated Hck, Lyn, and c-Src but did not detectably affect Fgr, Fyn, Lck, or Yes. Mutagenesis of the Nef PXXP motif essential for SH3 domain binding greatly reduced the effect of Nef on Hck, Lyn, and c-Src, suggesting that Nef activates these Src family members through allosteric displacement of intramolecular SH3-linker interactions. These data show that Nef selectively activates Hck, Lyn, and c-Src among SFKs, identifying these kinases as proximal effectors of Nef signaling and potential targets for anti-HIV drug discovery.Nef is an accessory protein encoded by the human (HIV-1 and HIV-2) 2 and simian immunodeficiency viruses and is an essential mediator of viral pathogenicity (1-3). Experimental deletion within the simian immunodeficiency virus nef gene reduces viral load, delays the onset of AIDS-like disease, and offers immune protection against challenge with pathogenic simian immunodeficiency virus in rhesus macaques (4, 5). Strong selective pressure has been demonstrated for a functional nef gene, because some animals infected with non-pathogenic, nef-mutant simian immunodeficiency virus show in vivo repair of the mutation and progression to AIDS-like disease (5-7). In addition, some HIV-positive individuals that fail to develop AIDS exhibit nef mutations or deletions (8 -12), supporting the hypothesis that nef is essential for efficient disease progression.Nef has no known catalytic function and is believed to promote viral pathogenicity by altering signaling pathways in infected cells through its interactions with cellular proteins. Nef affects several distinct classes of host cell proteins, including immune receptors, protein kinases, trafficking proteins, and guanine nucleotide exchange factors (13-15). Through interactions with these and other signaling proteins, Nef can affect multiple cellular processes leading to enhancement of viral replication, immune evasion, and enhanced survival in T-cells and macrophages (1, 16 -18).Protein kinases are a major class of Nef effector proteins, and members of the Src family of non-receptor protein-tyrosine kinases have been str...

HIV-1 Nef, a critical AIDS progression factor, represents an important target protein for antiretroviral drug discovery. Because Nef lacks intrinsic enzymatic activity, we developed an assay that couples Nef to the activation of Hck, a Src-family member and Nef effector protein. Using this assay, we screened a large, diverse chemical library and identified small molecules that block Nef-dependent Hck activity with low micromolar potency. Of these, a diphenylpyrazolo compound demonstrated sub-micromolar potency in HIV-1 replication assays against a broad range of primary Nef variants. This compound binds directly to Nef via a pocket formed by the Nef dimerization interface and disrupts Nef dimerization in cells. Coupling of non-enzymatic viral accessory factors to host cell effector proteins amenable to high-throughput screening may represent a general strategy for the discovery of new antimicrobial agents.

Nef assembles a multi-kinase complex triggering MHC-I down-regulation. We identify an inhibitor that blocks MHC-I down-regulation, identifying a temporally regulated switch in Nef action from directing MHC-I endocytosis to blocking cell surface delivery. These findings challenge current dogma and reveal a regulated immune evasion program.

Nef is an HIV-1 accessory protein essential for AIDS progression and an attractive target for drug discovery. Lack of a catalytic function makes Nef difficult to assay in chemical library screens. We developed a high-throughput screening assay for inhibitors of Nef function by coupling it to one of its host cell binding partners, the Src-family kinase Hck. Hck activation is dependent upon Nef in this assay, providing a direct readout of Nef activity in vitro. Using this screen, a unique diphenylfuropyrimidine was identified as a strong inhibitor of Nef-dependent Hck activation. This compound also exhibited remarkable antiretroviral effects, blocking Nef-dependent HIV replication in cell culture. Structurally related analogs were synthesized and shown to exhibit similar Nef-dependent anti-viral activity, identifying the diphenylfuropyrimidine substructure as a new lead for antiretroviral drug development. This study demonstrates that coupling non-catalytic HIV accessory factors with host cell target proteins addressable by high-throughput assays may afford new avenues for the discovery of anti-HIV agents.

Objective(s) Primary human trophoblasts were previously shown to be resistant to viral infection, and able to confer this resistance to non-trophoblast cells. Can trophoblasts protect non-trophoblastic cells from infection by viruses or other intracellular pathogens that are implicated in perinatal infection? Study Design Isolated primary term human trophoblasts were cultured for 72 h. Diverse non-placental human cell lines (U2OS, HFF, TZM-bl, MeWo, and Caco-2) were pre-exposed to either trophoblast conditioned, non-conditioned medium, or miR-517-3p for 24 h. Cells were infected with several viral and non-viral pathogens known to be associated with perinatal infections. Cellular infection was defined and quantified by plaque assays, luciferase assays, microscopy, and/or colonization assays. Differences in infection were assessed by Student's t-test or ANOVA with Bonferroni's correction. Results Infection by rubella and other togaviruses, HIV-1, and varicella zoster, was attenuated in cells pre-exposed to trophoblast conditioned medium (p <0.05), and a partial effect by the Ch.19 microRNA miR-517-3p on specific pathogens. The conditioned medium had no effect on infection by Toxoplasma gondii or Listeria monocytogenes. Conclusion Our findings indicate that medium conditioned by primary human trophoblasts attenuate viral infection in non-trophoblastic cells. Our data point to a trophoblast-specific antiviral effect that may be exploited therapeutically.

Treatment of cells with chemotherapy drugs activates the intrinsic mitochondrial pathway of apoptosis and the caspase protease cascade. Recently, the lysosomal protease cathepsin D has been implicated in apoptosis caused by oxidative stress, inhibition of protein kinase C, and stimulation of the TNFR1 and Fas death receptors.

The immune receptor signaling pathway is used by nonimmune cells, but the molecular adaptations that underlie its functional diversification are not known. Circulating platelets use the immune receptor homologue glycoprotein VI (GPVI) to respond to collagen exposed at sites of vessel injury. In contrast to immune cell responses, platelet activation must take place within seconds to successfully form thrombi in flowing blood. Here, we show that the GPVI receptor utilizes a unique intracellular proline-rich domain (PRD) to accelerate platelet activation, a requirement for efficient platelet adhesion to collagen under flow. The GPVI PRD specifically binds the Src-family kinase Lyn and directly activates it, presumably through SH3 displacement. In resting platelets, Lyn is constitutively bound to GPVI in an activated state and platelets lacking Lyn exhibit defective collagen adhesion like that of platelets with GPVI receptors lacking the PRD. These findings define a molecular priming mechanism that enables an immune-type receptor to adopt a hemostatic function. These studies also demonstrate that active kinases can constitutively associate with immune-type receptors without initiating signal transduction before receptor ligation, consistent with a recent molecular model of immune receptor signaling in which receptor ligation is required to bring active kinases to their receptor substrates. The response of platelets to vessel injury is essential to prevent posttraumatic blood loss but also underlies the pathophysiology of cardiovascular diseases, such as heart attack and stroke (1). Platelet activation requires the generation of rapid and coordinated intracellular signals that culminate in cell-matrix and cell-cell adhesion and thrombus formation within seconds (2). Collagen, the most abundant and thrombogenic subendothelial matrix protein exposed by vessel injury, provides both a primary activating stimulus and an adhesive surface for the initiation of platelet thrombi in the arterial system. Understanding these events has therapeutic potential, as blocking this early step may prevent the formation of pathologic thrombi associated with the coronary and cerebral arteries (3).Molecular and genetic studies have established that collagen-induced platelet activation is triggered by the platelet-specific surface receptor glycoprotein VI (GPVI). GPVI is an Ig (Ig)-like domain-containing receptor that is structurally and functionally homologous to immune receptors but is expressed exclusively on platelets and megakaryocytes (4). Extensive pharmacologic and genetic studies have demonstrated that GPVI signaling in response to collagen is highly analogous to that of the related multisubunit T-cell receptor and the IgE and IgA receptors (5). GPVI ligands, such as collagen or the snake-venom toxin convulxin, induce receptor clustering that facilitates the phosphorylation of the tandem tyrosines found in the immunotyrosine activating motif (ITAM) of the noncovalently associated FcR␥ chain adaptor by Src-family tyrosine kinases (S...

Activation of Src family kinases by HIV-1 Nef may play an important role in the pathogenesis of HIV/AIDS. Here we investigated whether diverse Nef sequences universally activate Hck, a Src family member expressed in macrophages and other HIV-1 target cells. In general, we observed that Hck activation is a highly conserved Nef function. However, we identified an unusual Nef variant from an HIV-positive individual that did not develop AIDS which failed to activate Hck despite the presence of conserved residues linked to Hck SH3 domain binding and kinase activation. Amino acid sequence alignment with active Nef proteins revealed differences in regions not previously implicated in Hck activation, including a large internal flexible loop absent from available Nef structures. Substitution of these residues in active Nef compromised Hck activation without affecting SH3 domain binding. These findings show that residues at a distance from the SH3 domain binding site allosterically influence Nef interactions with a key effector protein linked to AIDS progression.