Distribution of tetraspanins in uninfected Jurkat T cells. (A) Uninfected Jurkat T cells were adhered to poly-l-lysine-coated coverslips for 1 h at 37°C, fixed with paraformaldehyde, permeabilized, and stained with MAbs against CD63, CD81, or CD9 (red). Images shown are single confocal sections through the middle of a cell. (B) Cell surface expression of CD63, CD81, and CD9 was measured on uninfected Jurkat T cells by flow cytometry. The mean fluorescence intensity of CD63, CD81, and CD9 staining (solid line) is shown compared to a control MAb (broken line), and the mean fluorescence values (insert) are shown for each plot. Levels of surface expression of CD63 (white bars), CD81 (gray bars), and CD9 (black bars) are compared between HIV-1 infected Jurkat cells and uninfected Jurkat cells (bottom panel). Data are the means of three independent experiments, and error bars represent the standard error of the mean. (C) CD63 does not colocalize with CD81 or CD9 in Jurkat T cells. Jurkat cells were adhered to coverslips, fixed, permeabilized, and stained for CD63 (green) and either CD81 (left panel, red) or CD9 (right panel, red). Primary MAbs were detected with anti-IgG2b (CD63) and anti-IgG1 (CD81 and CD9) isotype-specific conjugated secondary antibodies. The three-dimensional reconstructed z series (top) and a single optical section through the middle of the cell (center) are shown. The bottom panels each show a magnified image of a region from the single optical section selected for strong staining (boxed).

HIV-1 Env accumulates in CD63⁺ CD81⁺ TEMs at the plasma membrane of T cells. (A) Jurkat T cells and (B) primary CD4⁺ T cells were used uninfected (left panel) or infected with HIV-1 (right panel). Cells were adhered to coverslips for 1 h at 37°C in the presence of the human Env-specific antibody 2G12 and then incubated at 4°C for 30 min with MAbs against CD63 and CD81. Cells were fixed, and the surface staining of Env (blue), CD63 (green), and CD81 (red) was analyzed by LSCM. The top panels show a single section through the middle of the cell, and the bottom panels are magnified images of a region selected for strong staining from the single optical section (boxed). Regions of CD63 and CD81 colocalization are yellow, and regions of Env, CD63, and CD81 triple colocalization appear white.

Actin depolymerization disrupts TEM-HIV-1 polarized assembly platforms. Jurkat_LAI cells (5 × 10⁵) were washed, resuspended in RPMI 1640-1% FCS, and adhered to poly-l-lysine-coated coverslips at 37°C. The cells were either untreated (left panel) or treated with latrunculin A (right panels) and stained for HIV-1 Env with the MAb 50-69 (blue). Cells were fixed, permeabilized, and stained for HIV-1 Gag (green) and either CD63 (A [red]) or CD81 (B [red]). Images are single sections through the middle of the cell with the corresponding Nomarski image: areas of red/green colocalization appear yellow, and areas of red/green/blue colocalization appear white.

HIV-1 Env and Gag colocalize with tetraspanins in virus-infected T cells. HIV-1-infected Jurkat_LAI T cells (A) or primary_LAI cells (B) were surface stained for Env and tetraspanins. Cells were adhered to coverslips for 1 h at 37°C in the presence of the human Env-specific antibody 2G12 and then incubated at 4°C for 30 min with MAbs against CD63 (left panel), CD81 (center panel), or CD9 (right panel). Cells were fixed, and surface staining of Env (green) and tetraspanins (red) was analyzed by LSCM. Images are single sections through the middle of a cell, and areas of colocalization are yellow. Jurkat_LAI T cells (C) or primary_LAI cells (D) were permeabilized and stained for Gag and tetraspanins. Cells were adhered to coverslips, fixed and permeabilized, and incubated with rabbit antisera against Gag p17 and p24 and MAbs against CD63 (left panel), CD81 (center panel), or CD9 (right panel). Intracellular staining of Gag (green) and tetraspanins (red) was analyzed by LSCM. Images are single sections through the middle of the cell, and areas of colocalization are yellow. (E) Jurkat _LAI cells were adhered to coverslips, fixed, permeabilized, and stained for intracellular Gag and Lamp2. Images are single sections through the middle of the cell, and areas of colocalization are yellow.

CD63 and CD81 are incorporated into budding virions. Ultrathin cryosections of HIV-1-infected Jurkat_LAI cells were labeled with rabbit antiserum against HIV Gag p17 and a mouse MAb specific for either CD63 (A) or CD81 (B). Primary antibodies were labeled with mouse-specific 5-nm and rabbit-specific 10-nm gold colloids. CD63 and CD81 labeling (5-nm gold colloid) is highlighted with arrowheads. (C) Cryosections were labeled with a mouse MAb against Gag and rabbit antiserum specific for Lamp2. Here Gag was visualized with mouse-specific 5-nm gold colloids and Lamp2 with rabbit-specific 10-nm colloids. Lamp2 labeling is highlighted with arrowheads, and a budding virion (Lamp⁻ Gag⁺) is highlighted with an asterisk. Bar, 100 nm. (D) Virus precipitation with antibodies against HIV-1 proteins or tetraspanins. Cell-free viral supernatants were precipitated with Env-specific antibodies (MAbs 2G12 and IgG1b12), tetraspanin-specific MAbs, or a control antibody, and the Gag p24 content of the precipitated material was measured by ELISA. The nonspecific background signal was determined by incubating viral supernatants with preblocked Pansorbin cells without antibody, and these values were subtracted to calculate the concentration of p24 in ng/ml. Bars represent means of two independent experiments, and variability is represented by the standard error of the mean.

Tetraspanins are components of the T-cell VS. HIV-1-infected Jurkat_LAI cells (effectors) were mixed with an equal number of freshly-isolated primary CD4⁺ T cells (targets) and incubated on poly-l-lysine-treated coverslips in the presence of the Env-specific MAb 50-69 (blue) for 1 h at 37°C. Conjugate evolution was arrested by fixing, and cells were permeabilized and stained using rabbit antisera directed against Gag p17 and p24 (green) and mouse MAbs specific for either CD63 (red, left panel), CD81 (red, second panel from left), or CD9 (red, second panel from right). Alternatively, cells were stained with a mouse MAb against Gag (green) and rabbit antisera against Lamp2 (red, right panel). CD81 and CD63, and to a lesser extent CD9, but not Lamp2, are enriched in the effector cell at the conjugate interface and colocalize with HIV-1 Env and Gag. Images are single sections through the middle of a conjugate, and the target cell is labeled with an asterisk. Areas of colocalization appear white, and the corresponding Nomarski image is shown.