The effects of soluble Nef protein on CD4؉ T cells were examined. CD4 ؉ -T-cell cultures exposed to soluble Nef were analyzed for apoptosis by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling and hallmarks of apoptosis including cytoplasmic shrinkage, nuclear fragmentation, DNA laddering, and caspase activation. We observed dose-and time-dependent inductions of apoptosis. DNA laddering and activated caspase 3 were also evident. Cells treated with Nef/protein kinase inhibitor complexes were protected from Nef-induced apoptosis, suggesting possible roles for protein kinases in the apoptosis pathway. Similarly, cells treated with Nef/anti-Nef antibody complexes were protected from Nef-induced apoptosis. The cellular receptor responsible for Nef-induced apoptosis was identified through antibody-and ligand-blocking experiments as a receptor commonly involved in viral entry. CXCR4 antibodies, as well as the endogenous ligand SDF-1␣, were effective in blocking Nef-induced apoptosis, while CCR5 and CD4 antibodies were ineffective. Moreover, a CXCR4-deficient cell line, MDA-MB-468, which was resistant to Nef-induced apoptosis, became sensitive upon transfection with a CXCR4-expressing vector. This study suggests that extracellular Nef protein could contribute to the decline of CD4 counts prior to and during the onset of AIDS in patients with human immunodeficiency virus type 1 infections.

The HIV-1 Nef protein was analyzed for apoptotic structural motifs that interact with the CXCR4 receptor and induce apoptosis in CD4 ؉ lymphocytes. Two apoptotic motifs were identified. One centered on Nef amino acids ( The reduction and killing of lymphocytes by retroviruses have traditionally been directly linked to the viral load, and the depletion process is induced by viral infectivity (21, 104). However, an alternative scenario (bystander effect) posits that lymphocyte killing leading to depletion is a result of apoptosis and that apoptosis predominantly occurs in uninfected, bystander cells, with a distinct lack of cell killing in the productively infected cells themselves (3,23,80). Many studies have contributed to the bystander effect premise that the longevity of infected cells is due to intracellularly expressed Nef protein (9,23,44,107,108). Alternatively, a second premise of the bystander effect scenario directly implicates viral proteins (i.e., Nef) and/or indirectly implicates virally stimulated cellular factors as mediators of bystander cell apoptosis (4, 10, 30-33, 40, 41, 45, 49, 50, 59, 61, 63, 72, 80-82, 93, 98, 109).We have previously shown that the Nef proteins expressed by human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIV) efficiently induce apoptosis in T-cell lines, peripheral blood mononuclear cells (PBMCs), and other cell lines (60). Receptor-ligand and antibody competition studies, as well as receptor insertion experiments with cell lines lacking CXCR4 expression, revealed that the chemokine receptor CXCR4 is the surface receptor involved in Nef-induced apoptosis. These studies and others (41, 47, 91; also our unpublished data) directly showed that exogenous Nef protein is secreted extracellularly at concentrations that could contribute to the CD4 ϩ lymphocyte depletion that occurs prior to and during the onset of AIDS. The body of evidence from patient, primate, and transgenic animal studies suggests that soluble Nef protein causes pathogenic effects, including T-cell depletion (3, 15, 28, 30-33, 36, 45, 49, 50, 63, 65, 68, 74, 88, 98). Thus, there is enough evidence to directly implicate the Nef protein in bystander cell death leading to CD4 ϩ -T-cell depletion, and we have identified the receptor through which Nef induces an apoptotic signal in T cells. The next step is to determine the mechanics of this receptor-ligand interaction that lead to programmed cell death. This is one step toward the development of therapeutics that can antagonize pathogenesis due to Nef and can prolong or possibly halt the progression toward AIDS.Chemokines are a superfamily of small, cytokine-like proteins that induce cytoskeletal rearrangement and firm adhesion to endothelial cells and that are involved in directional migration (chemotaxis) through interactions with G-protein-coupled receptors. The chemokine receptor-ligand pair CXCR4-SDF-1␣ is unique in that SDF-1␣ is the only known ligand for this receptor (70,76,97,112). The pair induces strong chemotactic...