Extracellular Nef protein targets CD4+ T cells for apoptosis by interacting with CXCR4 surface receptors

Abstract

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-1alpha, 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.

FIG. 1.

Nef-induced apoptosis as measured by dose response in lymphocytic cells. (A) Jurkat or H9 cell cultures were untreated or were treated with 1, 10, 100, or 500 ng of Nef/ml of medium. After 24 h, cells were assayed for apoptosis by TUNEL labeling, and then percent apoptosis was determined by fluorescent microscopic analysis of 10 fields per slide. Percent apoptosis was calculated as the number of FITC-labeled cells per total cell count. 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 (error bars). UT represents untreated cells. (B) PBMCs were either untreated or exposed to soluble Nef protein for 24 h. Apoptotic cells were detected by TUNEL, and then percent apoptosis in treated cells was compared to 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 (error bars). (C and D) Representative images of Nef-treated PBMCs described for panel B. Images were taken via phase and fluorescence microscopy and arranged via Adobe Photoshop software (version 5.0.2; Adobe Systems). (C) Matched-phase image for panel D, which is the combined fluorescent images for TUNEL (FITC, green) and CD4 staining (Texas Red). Cells fluorescing yellow were simultaneously stained for TUNEL and CD4. Magnification, ×240.

FIG. 2.

Nef-induced apoptosis as a function of time. (A) Jurkat cell cultures were either untreated (▪) or exposed to soluble HIV-1 Nef (□) for various times. Cells were then harvested, assayed for apoptosis by TUNEL, and analyzed by fluorescence microscopy. Data from two experiments performed in triplicate with six individually treated cell sets were pooled to generate average values and were used to determine standard errors. (B) Jurkat cell cultures were exposed to 100 ng of Nef/ml of medium for 12, 24, and 36 h and subsequently harvested and assayed by TUNEL. Images were taken via fluorescence microscopy and arranged via Adobe Photoshop software (version 5.0.2; Adobe Systems). Subpanels A, C, and E represent untreated Jurkat cell cultures at 12, 24, and 36 h, respectively, after initial medium replenishment. Subpanels B, D, and F depict Jurkat cell cultures after 12, 24, and 36 h exposure to soluble Nef protein (100 ng/ml). Magnification, ×280. The image was formatted using Adobe Photoshop 5.0.2 software.

FIG. 3.

Hallmarks of apoptosis. (A) DNA from Jurkat cell extracts was resolved by agarose gel electrophoresis, stained with ethidium bromide, and visualized by exposure to UV light. Pictures were taken using the DP12 Microscope Digital Camera System (Olympus Optical Co., Ltd., Melville, N.Y.), and the negative image was generated via Adobe Photoshop software (version 5.0.2). (B) Jurkat cell cultures were either untreated or exposed to extracellular Nef for various times. Caspase 3 activation was tested by Western blot analysis. The high-molecular-mass band is pro-caspase 3 (32 kDa), and the large catalytic subunit of active caspase 3 is 17 kDa. Tubulin (50 kDa) is the gel-loading control. Prestained SDS-polyacrylamide gel standard (broad range) (Bio-Rad Labs) was used as the molecular weight marker. The image was formatted using Adobe Photoshop software (version 5.0.2).

FIG. 4.

Inhibitory molecules suppress HIV-1 Nef-induced apoptosis in Jurkat cell cultures. Nef was preincubated with anti-Nef antibody (1:4,000 dilutions), fasudil (20 mM), or H7 (20 mM) for 2 h at 4°C. Then, Jurkat cells were left untreated or were treated with Nef protein, Nef anti-Nef antibody, Nef-fasudil, or Nef-H7 cocktail. After 24 h at 37°C, cells were harvested and then assayed by TUNEL. Ab-1 is the NIH polyclonal antibody, and Ab-2 is the polyclonal antibody from Warner Greene. Fasudil and H7 are protein kinase inhibitors. Data from two experiments performed in triplicate were pooled to generate average values and were used to determine standard errors (error bars). Abbreviations: UT, untransfected; NRS, normal rabbit serum.

FIG. 5.

Apoptotic effects of eukaryotic cell-expressed Nef protein. HEK 293 cells were either untransfected or transfected with plasmids containing HIV-1, HIV-2, or SIV nef reading frames. Cultures were incubated for 36 h, and the cell-conditioned supernatant was removed and stored. (A) The changes in Nef-induced apoptosis are not the result of variations in Nef protein expression. Western analysis of 40 μl of HEK 293-conditioned medium was performed using rabbit anti-Nef antibody. Lane 1, HIV Nef protein (1 μg); lane 2, untransfected HEK 293 cell-conditioned medium; lane 3, HIV-1 nef-transfected HEK 293 cell-conditioned medium; lane 4, HIV-2 nef-transfected HEK 293 cell-conditioned medium; lane 5, SIV nef-transfected HEK 293 cell-conditioned medium. (B) Jurkat cell cultures were exposed for 24 h to cell-conditioned supernatant from HIV-1, HIV-2, or SIV nef-transfected cells alone, or pretreated for 2 h with a 1:250 dilution of rabbit anti-Nef antibody. Cultures were then assayed for apoptosis by TUNEL. Bars: Media, unconditioned medium; bNef, bacterially expressed and purified Nef protein; UT, cell-conditioned medium from untransfected 293 cells; HIV-1, cell-conditioned medium from HIV-1 nef-transfected 293 cells; HIV-2, cell-conditioned medium from HIV-2 nef-transfected 293 cells; SIV, cell-conditioned medium from SIV nef-transfected 293 cells; αNef Ab, pretreated with Nef antibody. Error bars show the standard error of measurement. Data from at least two experiments performed in triplicate were pooled to generate average values and were used to determine standard errors (error bars).

FIG. 6.

Nef-induced apoptosis is mediated by CXCR4 molecules. Jurkat cultures were either untreated (UT) or pretreated with the natural ligand for CXCR4, SDF-1α, or antibodies to CXCR4 (anti-X4), CD4 (anti-D4), or CCR5 (anti-R5). Subsequently, cultures were treated with HIV-1 Nef for 24 h, and apoptosis was then determined for all cultures by TUNEL. Error bars show the standard error of measurement. Data from two experiments performed in triplicate were pooled to generate average values and were used to determine standard errors.

FIG. 7.

Surface expression of CXCR4 molecules in a CXCR4-deficient cell line restores sensitivity to Nef-induced apoptosis. MDA-MB-468 cultures were either untransfected or transiently transfected with 1 μg of a CXCR4 cDNA clone. (A) At 48 h posttransfection, half the CXCR4-transfected cultures were assayed for cell surface expression of CXCR4 receptor by immunocytochemical assay using an anti-CXCR4 antibody. Subpanels: a, phase image of untransfected MDA-MB-468 cells; b, anti-CXCR4 immunostained image of untransfected MDA-MB-468 cells; c, phase image of CXCR4-transfected MDA-MB-468 cells; d, anti-CXCR4 immunostained image of CXCR4-transfected MDA-MB-468 cells. Magnification, ×240. (B) MDA-MB-468 cultures were either untransfected (Media), transiently transfected with 1 μg of a CXCR4 cDNA clone, transiently transfected with 1 μg of a CCR5 cDNA clone, or transfected with 1 μg of the empty vector pCR3.1. At 48 h posttransfection, cultures were treated with HIV-1 Nef for 24 h and subsequently assayed for apoptosis by TUNEL. At 48 h posttransfection, cultures were treated with medium (Media) or with Nef protein. Error bars show the standard error of measurement. Data from two experiments performed in triplicate were pooled to generate average values and were used to determine standard errors.