Figure 3 : Inhibition of HIV-1 propagation by CASP3*.

From: Therapeutic potential of HIV protease-activable CASP3

Figure 3

(a) Schematic representation of the experimental approach. SUP-T1 cells were infected with MLV carrying the CASP3* to produce cells constitutively expressing CASP3*. These cells were infected with HIV-1, and the replication efficiency of HIV-1 was measured using viral p24CA antigen in the cell culture medium. (b) Inhibition of HIV-1NL4-3 replication in SUP-T1/CASP3* cells. Cells were exposed to high- or low-dose viral preparation yielding 25 ng or 1.3 ng of p24CA per 1 × 105 cells, respectively. The viral p24CA concentration in the culture supernatant was measured at 6–9 d post-viral infection. Results shown were obtained from two independent isolates of SUP-T1/CASP3* and control cells. (c) Analysis of SUP-T1/CASP3* cell fate after HIV-1 entry. Luciferase activity of cells infected with either an HIV-1-based lentiviral vector or an MLV vector was measured at 4 d post-infection. RLU, relative light units. (d) Flow cytometric detection of apoptotic SUP-T1/Cont and SUP-T1/CASP3* cells by HIV-1NL4-3 infection. The percentage of Annexin V Alexa Fluor 488 positive cells was measured at 6 h post-nelfinavir (NFV) exposure, post-HIV-1 infection, and post-HIV-1 infection in the presence of 2 µM NFV, and subtracted from the percentage of Annexin V positive cells in untreated controls. The live cell fraction was gated according to scatter to analyze the early phase of apoptotic cell death. Data from two independent experiments are shown. For this experiment, cells from isolate #2 were used. (e) Analysis of SUP-T1/CASP3* cell fate upon HIV-1 production. The SUP-T1/CASP3* cells were transduced with HIV-1 Gag-pol or Gag by either MLV vector (left panel) or transfection (right panel), and the expression of transduced proteins was detected at 7 d post-gene transduction by Western blot. For the transfection experiment, cells were maintained in the presence of 0.5 µM NFV to detect the effect of Pol on cell survical.