Human immunodeficiency virus type 1 virological synapse formation in T cells requires lipid raft integrity - PubMed (original) (raw)

Human immunodeficiency virus type 1 virological synapse formation in T cells requires lipid raft integrity

Clare Jolly et al. J Virol. 2005 Sep.

Abstract

Human immunodeficiency virus type 1 (HIV-1) can spread directly between T cells by forming a supramolecular structure termed a virological synapse (VS). HIV-1 envelope glycoproteins (Env) are required for VS assembly, but their mode of recruitment is unclear. We investigated the distribution of GM1-rich lipid rafts in HIV-1-infected (effector) T cells and observed Env colocalization with polarized raft markers GM1 and CD59 but not with the transferrin receptor that is excluded from lipid rafts. In conjugates of effector T cells and target CD4+ T cells, GM1, Env, and Gag relocated to the cell-cell interface. The depletion of cholesterol in the infected cell dispersed Env and GM1 within the plasma membrane, eliminated Gag clustering at the site of cell-cell contact, and abolished assembly of the VS. Raft integrity is therefore critical for Env and Gag co-clustering and VS assembly in T-cell conjugates.

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Figures

FIG. 1.

FIG. 1.

GM1 and Env expression in uninfected and infected T cells. (A) Jurkat CE6.1 T cells (top panels), naïve CD4+ T cells negatively enriched from peripheral blood mononuclear cells (PBMC) (middle panels), or 3-day PHA (5 μg/ml)- and interleukin-2 (10 IU)-activated primary CD4+ T cells (bottom panels) were washed and resuspended in RPMI-1% fetal calf serum (WB), and 5 × 105 cells were allowed to settle onto poly-

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-lysine-coated coverslips, fixed in ice-cold 4% paraformaldehyde in phosphate-buffered saline-1% bovine serum albumin, and stained for GM1-containing lipid rafts (green) with 10 μg/ml biotinylated B-Ctx (Sigma, United Kingdom) at 4°C followed by streptavidin-FITC (Jackson Immunoresearch). All staining with B-Ctx was performed on fixed cells to prevent Ctx-induced cross-linking and GM1 capping. (B) HIV-1LAI-infected (JurkatLAI) effector cells were washed, allowed to settle onto poly-

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-lysine-coated coverslips, and stained for surface Env (red) and GM1 (green) with 10 μg/ml of the human Env-specific antibody 50-69 (CFAR, United Kingdom) and 10 μg/ml B-Ctx followed by anti-human-TRITC and streptavidin-FITC, respectively. (C) HIV-1LAI-infected 3-day PHA- and interleukin-2-activated primary CD4+ T-cells negatively enriched from PBMC by magnetic selection (Miltenyi Biotech, United Kingdom) were stained for Env (red) and GM1 (green) as described above. All images represent single _x_-y sections through the center of a cell and were acquired using a Bio-Rad MicroRadiance laser scanning confocal microscope and subsequently processed using Metamorph v6 (Universal Imaging Corporation) and Photoshop 7.0 (Adobe Inc.). Fluorescence images are shown next to the corresponding Nomarski images, and the colocalization of red and green gives yellow staining.

FIG. 2.

FIG. 2.

HIV-1 Env colocalizes with the raft markers GM1 and CD59 but not with the nonraft marker TfR. JurkatLAI cells were incubated for 1 h at 12°C with B-Ctx, the human Env-specific antibody 50-69, and either a mouse CD59-specific monoclonal antibody (Serotec) or a mouse anti-human TfR-specific antibody (from T. Harder, University of Oxford). Cells were washed at 4°C and incubated for a further 1 h at 12°C with anti-human-TRITC, streptavidin-Cy5, and anti-mouse-FITC to promote antibody-induced patching of CD59 and TfR as described previously (14). Cells were then washed, fixed in paraformaldehyde, and analyzed by confocal microscopy. (A) CD59 (green) colocalizes with HIV-1 Env (red) and GM1 (blue). (B) TfR (green) does not colocalize with HIV-1 Env (red) and GM1 (blue). (C) Incorporation of GM1 into a budding HIV-1 virion. JurkatLAI cells were fixed sequentially in 4% and 8% paraformaldehyde, stained for Env with the human MAb 2G12 (20 μg/ml) and for GM1 with B-Ctx (20 μg/ml), washed, and labeled with anti-human immunoglobulin G and anti-biotin conjugated with 5-nm and 10-nm gold colloids, respectively (Agar, United Kingdom). Cells were then fixed in a mixture of glutaraldehyde and paraformaldehyde, washed, and postfixed in 1% osmium tetroxide in cacodylate buffer. After extensive washing, the cells were incubated in 0.5% magnesium uranyl acetate, dehydrated in ethanol and propylene oxide, and embedded in Epon resin. Ultrathin sections were examined using a Phillips FEI Technai 12 transmission electron microscope, and digital images were captured using Soft imaging software and processed using Photoshop. Bar, 100 nm.

FIG. 3.

FIG. 3.

GM1 colocalizes with Gag and Env and is polarized to the VS. (A) JurkatLAI cells were stained with MAb 50-69 for surface-expressed Env (red) and with B-Ctx for GM1 (green), fixed, permeabilized with 0.1% Triton X-100, and incubated with rabbit antisera against HIV-1 Gagp17/p24 (blue) obtained from CFAR, United Kingdom, followed by anti-human-TRITC, streptavidin-FITC, and anti-rabbit-Cy5. Merging of the three colors illustrates colocalization, which is superimposed in white on the corresponding Nomarski image. (B) Conjugates between equal numbers of JurkatLAI effector cells (lower cell in conjugate) and freshly isolated resting primary CD4+ T target cells negatively enriched from normal PBMC (upper cell) were made on poly-

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-lysine-treated coverslips for 1 h at 37°C, during which cells were stained for CD4 with L120 (blue; CFAR, United Kingdom) and for Env with 50-69 (red). Conjugate evolution was arresting by fixing with paraformaldehyde prior to staining for GM1 with B-Ctx (green), followed by anti-mouse-Cy5, anti-human-TRITC, and streptavidin-FITC. Merging of the three colors illustrates colocalization, which is superimposed in white on the corresponding Nomarski image. Arrows point to regions of GM1 and Env in the target cell membrane. (C) Conjugates between JurkatLAI effector and primary CD4+ T target cells were made as described above, during which cells were stained for Env with 50-69 (red). Conjugate evolution was arrested by fixing with paraformaldehyde prior to staining with B-Ctx (green). Conjugates were then permeabilized in 0.1% Triton X-100 and labeled with rabbit antisera against Gagp17/p24 (blue), followed by anti-human-TRITC, streptavidin-FITC, and anti-rabbit-Cy5. Merging of the three colors illustrates colocalization, which is superimposed in white on the corresponding Nomarski image.

FIG. 4.

FIG. 4.

Depletion of cholesterol eliminates Env detection and prevents VS assembly. JurkatLAI effector cells were treated with 10 mM β-CD in RPMI for 30 min at 37°C to extract membrane cholesterol. Red, green, and blue staining was converted to white in all images by using Photoshop to increase contrast. (A) β-CD-treated JurkatLAI cells (lower cell in conjugate) were washed, mixed with an equal number of primary CD4+ target T cells (labeled with an asterisk), and incubated for 1 h at 37°C with the Env-specific MAb 50-69 (top right panel) and the CD4-specific MAb L120 (lower left panel). Conjugate evolution was arrested by fixing with paraformaldehyde prior to staining with B-Ctx to detect GM1 on the effector cell (top left panel). (B) JurkatLAI effector cells were either left untreated (top panel) or treated with 5 mM (middle panel) or 10 mM (bottom panel) β-CD and stained with the HIV-1 Env-specific MAb 50-69 and an appropriate conjugate. (C) ELISA for soluble gp120. JurkatLAI cells (1 × 107) were treated with 10 mM β-CD or left untreated, and supernatants were collected and assayed for their Env content by ELISA. Supernatants were serially diluted and captured on plates coated with the sheep anti-gp120 antibody D7320, and bound gp120 was detected using MAb 2G12 followed by anti-human-horseradish peroxidase and developed with Ultra-TMB ELISA substrate (Pierce). The data show the means ± 1 standard deviation for untreated (▪) and β-CD-treated (▴) samples from two independent experiments performed in triplicate. (D) Flow cytometric analysis of surface-expressed Env. JurkatLAI cells were left untreated (solid line) or treated with 10 mM β-CD (dotted line) and then were stained for surface-expressed Env using the MAb 2G12 followed by anti-human-phycoerythrin. Mean fluorescence intensities relative to the unstained control (shaded) are shown. (E) β-CD-treated JurkatLAI effector cells were mixed with an equal number of primary CD4+ target T cells, incubated for 1 h at 37°C, fixed, and stained for GM1 (top left panel) and Env (top right panel). Conjugates were permeabilized and labeled with rabbit antisera against HIV Gagp17/p24 (lower left panel).

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