Figure 1.
Figure 1. Rab27a is required for HIV replication in CD4+ T lymphocytes and macrophages. (A–D) Jurkat cells were transduced with lentivirus encoding different Rab27a shRNA sequences (A and B; open bars), or a control scrambled shRNA (black bars). (A) Rab27a mRNA levels were determined by qPCR. Means ± SD of a representative experiment (n = 3) performed in triplicates is shown. (B) Cells (3 × 104/0.1 ml) were infected with a VSV-G–pseudotyped HIV (20 ng p24/ml), and p24 production was evaluated in cell supernatants at day 5 p.i. Means ± SD of two independent experiments expressed as percentages of p24 production of control cells are shown. (C) Immunoblot of Rab27a protein levels. Actin was used as a loading control. (D) Cell viability was determined at day 7 after puromycin selection by annexin V staining and propidium iodide exclusion followed by FACS analysis. Dot plots from a representative experiment (n = 4) are shown. Percentage of death cells (annexin V+/propidium iodide+) is indicated. (E–G) Control (closed circles) and Rab27a-silenced Jurkat cells (open circles; 3 × 104/0.1 ml) were infected with HIV (IIIb strain; 50 ng p24/ml). The production of p24 (E) and the percentages of p24-positive cells (F and G) were determined by ELISA and FACS analysis, respectively. Representative dot plots (F) and the percentages of infected cells (G) from a representative experiment (n = 6) are shown. (H) Virus release assay. Cells were spinoculated with a high MOI of VSV-G–pseudotyped HIV strain. 48 h later, the amounts of cell-associated and released virus were analyzed by immunoblotting. (I and J) Control and Rab27a-silenced PBMCs (I) or PBMCs from a GS patient and an age-matched control (J) were infected with a VSV-G–pseudotyped HIV-1 (200 and 50 ng/ml p24, respectively). The production of p24 was evaluated at different days p.i. Results from a representative experiment (n = 3 and n = 2) performed in triplicates are expressed as the means ± SD. (K) Quantification of p24 antigen in cell supernatants from control (closed triangles) or Rab27a-silenced (open triangles) MDMs (5 × 104/0.1 ml) infected with HIV-1(BaL strain; 50 ng p24/ml). Kinetics of p24 production from a representative experiment are shown in the left graph. p24 production by cells from seven different blood donors at day 5 p.i. is shown in the right graph. Asterisks indicate statistically significant differences from the control: **, P < 0.01. KD, knockdown.

Rab27a is required for HIV replication in CD4+ T lymphocytes and macrophages. (A–D) Jurkat cells were transduced with lentivirus encoding different Rab27a shRNA sequences (A and B; open bars), or a control scrambled shRNA (black bars). (A) Rab27a mRNA levels were determined by qPCR. Means ± SD of a representative experiment (n = 3) performed in triplicates is shown. (B) Cells (3 × 104/0.1 ml) were infected with a VSV-G–pseudotyped HIV (20 ng p24/ml), and p24 production was evaluated in cell supernatants at day 5 p.i. Means ± SD of two independent experiments expressed as percentages of p24 production of control cells are shown. (C) Immunoblot of Rab27a protein levels. Actin was used as a loading control. (D) Cell viability was determined at day 7 after puromycin selection by annexin V staining and propidium iodide exclusion followed by FACS analysis. Dot plots from a representative experiment (n = 4) are shown. Percentage of death cells (annexin V+/propidium iodide+) is indicated. (E–G) Control (closed circles) and Rab27a-silenced Jurkat cells (open circles; 3 × 104/0.1 ml) were infected with HIV (IIIb strain; 50 ng p24/ml). The production of p24 (E) and the percentages of p24-positive cells (F and G) were determined by ELISA and FACS analysis, respectively. Representative dot plots (F) and the percentages of infected cells (G) from a representative experiment (n = 6) are shown. (H) Virus release assay. Cells were spinoculated with a high MOI of VSV-G–pseudotyped HIV strain. 48 h later, the amounts of cell-associated and released virus were analyzed by immunoblotting. (I and J) Control and Rab27a-silenced PBMCs (I) or PBMCs from a GS patient and an age-matched control (J) were infected with a VSV-G–pseudotyped HIV-1 (200 and 50 ng/ml p24, respectively). The production of p24 was evaluated at different days p.i. Results from a representative experiment (n = 3 and n = 2) performed in triplicates are expressed as the means ± SD. (K) Quantification of p24 antigen in cell supernatants from control (closed triangles) or Rab27a-silenced (open triangles) MDMs (5 × 104/0.1 ml) infected with HIV-1(BaL strain; 50 ng p24/ml). Kinetics of p24 production from a representative experiment are shown in the left graph. p24 production by cells from seven different blood donors at day 5 p.i. is shown in the right graph. Asterisks indicate statistically significant differences from the control: **, P < 0.01. KD, knockdown.

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