FIG 4.

PR mutations abolish or significantly decrease Env-mediated cell-cell fusion. HEK293T cells were separately transfected with pRK empty vector, WT, or mutant Env (gp160) expression construct together with pCMV-Rev. HEK293T cells transfected without plasmid DNA were used as a mock control. (A) At 24 h posttransfection, HEK293T cells were collected and lysed for immunoblotting analysis with antibodies to gp160/gp120, gp41, or GAPDH. (B and C) At 24 h posttransfection, the cells were stained with gp120 monoclonal antibody (2G12) at 4°C to measure the cell surface gp120 expression using flow cytometry. Average percentages (B) and the mean fluorescence intensity (C) of gp120-positive cells from four independent experiments were determined. (D) Transfected HEK293T cells were cocultured with TZM-bl cells for 24 h and then lysed for firefly luciferase activity measurement of Env-mediated cell-cell fusion. Average percentages of HIV-1 Env-mediated cell-cell fusion from three independent experiments are shown, with the value for the WT set as 100%. All experiments were performed with triplicate samples and repeated at least three times, and means ± standard errors of the means are shown. Dunnett's multiple-comparison test was used for statistical analysis. *, P < 0.05; **, P < 0.01; ***, P < 0.0001, for the comparison of the result with an individual mutant Env to that with WT Env.