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. 2005 Sep;79(18):12088–12094. doi: 10.1128/JVI.79.18.12088-12094.2005

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).