Analysis of HIV-1 Nef protein for an apoptotic motif(s). (A) A set of 20-mer peptides, each with a 10-aa overlap, spanning the 205 aa of the KNFS Nef protein were obtained from the AIDS Reagent Program. (B) Jurkat cell cultures were untreated (UT) or treated with a 10-ng/ml (5 to 6 nM) concentration of each of the Nef peptides (N20 to N205) or 100 ng (4.3 nM; Nef) of the Nef protein/ml for 24 h at 37°C. The cultures were then fixed and analyzed for apoptosis by a TUNEL assay. The y axis denotes percentages of the total cells that were TUNEL positive, and the x axis denotes the full Nef protein, untreated cells, or the peptides, starting with aa 1 to 20 (N20) and ending with aa 190 to 205 (N205). The error bars show the standard errors of the measurements, and the results are a compilation of at least three independent experiments. The peaks of apoptosis are denoted motif 1 and motif 2. The corresponding sequence region is denoted by the underlined sequences in panel A.

Specificity of Nef peptide-induced apoptosis to CXCR4. (A) MDA-MB-468 cultures were either untransfected or transiently transfected with a vector, CCR5, or CXCR4 cDNA clone. Bars 1 to 5 represent experiments performed on untransfected MDA-MB-468 cells, bars 6 to 10 represent experiments performed on Pc-Fusin (CXCR4)-transfected MDA-MB-468 cells, bars 11 to 14 represent experiments performed on pCR3.1-transfected MDA-MB-468 cells, and bars 15 to 18 represent experiments performed on CCR5-transfected MDA-MB-468 cells. At 48 h posttransfection, the cultures were treated for 24 h as follows: for bars 1, 6, 11, and 15, cells were treated with medium (UT); for bars 2, 7, 12, and 16, cells were treated with Nef protein (N; 4.3 nM); for bars 3, 8, 13, and 17, cells were treated with M1 (4.2 nM); for bars 4 and 9, cells were treated with the 20-mer peptide N130 (5.7 nM); and for bars 5, 10, 14, and 18, cells were treated with M2 (4.3 nM). In the graph, the y axis is shown on a log scale to allow viewing of the treatments that did not induce apoptosis. (B) Jurkat cell cultures were pretreated with medium (bars 1 to 4), SDF-1α (bars 5 to 8; 4.7 nM), CXCR4 antibody (bars 9 to 12; 5 μg/ml), CD4 antibody (bars 13 to 16; 10 ng/ml), CCR5 antibody (bars 17 to 20; 5 μg/ml), or vMIP-II (bars 21 to 24; 5.5 nM). Subsequently, a subset of those cultures were treated with medium (UT; bars 1, 5, 9, 13, 17, and 21), Nef protein (N; bars 2, 6, 10, 14, 18, and 22; 4.3 nM), M1 (bars 3, 7, 11, 15, 19, and 23; 4.2 nM), or M2 (bars 4, 8, 12, 16, 20, and 24; 4.3 nM). The error bars show the standard errors of the measurements, and the results are a compilation of at least three independent experiments.

Relationship between Nef modifications and changes in Nef-induced apoptosis. (A) The changes in Nef-induced apoptosis are not the results of instability of the resultant Nef protein. Virions were transcomplemented with mutant Nef proteins, and the resultant viruses were analyzed for infectivity and the ability of the Nef protein to enhance infectivity. Bar 1 represents the infectivity of the wild-type HIV-1 virus in a MAGI assay; bar 2 represents the infectivity of a virus with a deletion of Nef; bars 3, 4, and 5 represent the infectivities of viruses with a deletion of Nef that were transcomplemented with normal Nef (bar 3), NefΔM1 (bar 4), or NefΔM1M2 (bar 5). The error bars show the standard errors of the measurements, and the results are a compilation of at least three independent experiments. (B) The changes in Nef-induced apoptosis are not the results of variations in Nef protein expression. A Western blot analysis with 40 μl of HEK 293 conditioned medium was performed by using an anti-Nef antibody. Lane 1: untransfected HEK 293 cell conditioned medium; lane 2: HIV Nef protein (1 μg); lane 3: HIV-1 Nef-transfected HEK 293 cell conditioned medium; lane 4: HIV-1 NefΔM1-transfected HEK 293 cell conditioned medium; lane 5: HIV-1 NefΔM1M2-transfected HEK 293 cell conditioned medium. (C) Deletion of apoptosis motifs in the Nef protein eliminates Nef-induced apoptosis. Jurkat cell cultures were treated with conditioned cell supernatants from HEK 293 cells expressing various Nef variants to analyze the effect of the changes in Nef on Nef-induced apoptosis. Bar 1 represents the results for Jurkat cells treated with medium (UT); bar 2 represents treatment with Nef protein (N; 4.3 nM); bar 3 represents treatment with conditioned medium from untransfected HEK 293 cells; bar 4 represents treatment with 40 μl of conditioned medium from pNL4-3 KFS-transfected HEK 293 cells; bar 5 represents treatment with 40 μl of conditioned medium from pNefΔM1-transfected HEK 293 cells; and bar 6 represents treatment with 40 μl of conditioned medium from pNefΔM1M2-transfected HEK 293 cells. The error bars show the standard errors of the measurements, and the results are a compilation of at least three independent experiments.

Analysis of CXCR4 binding. MDA-MB-468 cultures were transfected with Pc-Fusin or Pc-CCR5 and allowed to transiently express the specific receptor. (A) The cultures were then exposed to a fluorescently tagged (with Flc) ligand, either the Nef protein (4.3 nM) or M1 (4.2 nM) peptide, for 1 h at 37°C. The first supernatants, containing unbound fluorescently tagged ligand, were removed and assayed for fluorescence levels. The cultures were treated with proteinase K to strip off the cell membrane-bound ligand (fluorescently tagged), and these second supernatants were assayed for fluorescence levels. The resultant data were plotted for each condition to obtain a bound fluorescence (first supernatant)/unbound fluorescence (second supernatant) ratio. Bars 1 and 3 represent CXCR4-transfected MDA-MB-468 cells, and bars 2 and 4 represent CCR5-transfected MDA-MB-468 cells. Bars 1 and 2 represent cultures exposed to the fluorescently tagged motif 1 peptide; bars 3 and 4 represent cultures exposed to the fluorescently tagged Nef protein. (B) The cultures were exposed to tagged Nef protein (4.3 nM) alone or tagged Nef protein with one of the following competitive ligands: SDF-1α (4.7 nM), CXCR4 antibody (5 μg/ml), or CCR5 antibody (5 μg/ml). The supernatants were collected and assayed as described above. Bars 1 to 4 represent CXCR4-transfected MDA-MB-468 cells, and bars 5 to 8 represent CCR5-transfected MDA-MB-468 cells. Bars 1 and 5 represent cells exposed to tagged Nef alone, bars 2 and 6 represent cells exposed to tagged Nef and CCR5 antibody, bars 3 and 7 represent cells exposed to tagged Nef and CXCR4 antibody, and bars 4 and 8 represent cells exposed to tagged Nef and SDF-1α. The error bars show the standard errors of the measurements, and the results are a compilation of at least two independent experiments.

Identification of hallmarks of apoptosis. (A) Jurkat cell cultures were exposed to ceramide (lane 1; 23.4 μM), no treatment (lane 2), gp120 IIIb (lane 3; 0.22 nM), sM1 (lane 4; 4.2 nM), Nef protein (lane 5; 4.3 nM), N60 (lane 6; 5.8 nM), M1 (lane 7; 4.2 nM), or M2 (lane 8; 4.3 nM) for 24 h (A-24) or 48 h (A-48). Twenty micrograms of total protein was loaded per lane. Caspase 3 activation was tested by Western blot analysis. The high-molecular-mass band (32 kDa) was procaspase 3, and the large catalytic subunit of active caspase 3 was 17 kDa. Tubulin (50 kDa) was the gel loading control. Prestained SDS-PAGE standards (broad range) (Bio-Rad) were used as molecular weight markers. The image was formatted with Adobe Illustrator 8.0 software. (B) DNAs from Jurkat cell extracts were resolved by agarose gel electrophoresis, stained with ethidium bromide, and visualized by exposure to UV light. Pictures were taken with the DP12 microscope digital camera system (Olympus Optical Co., Ltd., Melville, N.Y.), and the image was generated with Adobe Photoshop 5.0 software and formatted with Adobe Illustrator 8.0 software. The results are typical representations of at least two independent experiments. The lanes are as follows: the One kb Plus DNA ladder marker (lanes M), ceramide (lane 1; 23.4 μM, 24 h), untreated cells (lane 2; 24 h), gp120IIIb (lane 3; 0.22 nM, 24 h), sM1 (lane 4; 4.2 nM, 24 h), Nef protein (lane 5; 4.3 nM, 24 h), N60 (lane 6; 5.8 nM, 24 h), M1 (lane 7; 4.2 nM, 24 h), and N180 (lane 8; 5.7 nM, 24 h). Note that the treatments shown at the top of panel A-24, corresponding to lanes 1 to 8, apply similarly for lanes 1 to 8 of panels A-48 and B.

Analysis of apoptotic motifs. (A) Specificity of Nef-induced apoptosis to the identified motif regions. Jurkat cell cultures were treated with the specified Nef peptides for 24 h and subsequently fixed and assayed for apoptosis by TUNEL. Bar 1 represents the results from Jurkat cells treated with medium (UT), bar 2 represents Jurkat cells treated with Nef protein (N; 4.3 nM), bar 3 represents Jurkat cells treated with the 20-mer peptide N60 (5.8 nM), bar 4 represents treatment with M1 (4.2 nM), and bar 5 represents treatment with sM1 (4.2 nM). (B) Dose-response analysis of apoptotic motifs. Jurkat cell cultures were treated with either the M1 peptide or M2 peptide at different concentrations and subsequently fixed and assayed for apoptosis by TUNEL. The peptide concentrations ranged from 1 fg/ml (0.849 fM) to 100 ng/ml (84.9 nM). The error bars show the standard errors of the measurements, and the results are a compilation of at least three independent experiments.

PBMCs were either untreated or exposed to Nef (4.3 nM), various Nef peptides (M1 and sM1, 4.2 nM; M2, 4.3 nM), or ΔNef proteins (40 μl) for 24 h. (A) Apoptotic cells were detected by TUNEL, and then percentages of apoptosis in treated cells were compared to the levels in untreated cells. Data from two experiments performed in triplicate with 12 individually treated cell sets were pooled to generate average values and were used to determine standard errors. (B) PBMCs processed for TUNEL (FITC; green) were then stained by using a CD4 primary antibody and a secondary antibody tagged with Texas Red (red). Typical combined FITC-Texas Red images of fields of PBMCs are shown. (a) Untreated; (b) bacterial Nef protein, 4.3 nM; (c) M1, 4.2 nM; (d) sM1, 4.2 nM; (e) M2, 4.3 nM; (f) NefΔM1M2, 40 μl. The images were taken via fluorescence microscopy and arranged with Adobe Photoshop 5.0.2 software. Magnification, ×400.