FIG. 3.

Effect of prostratin on resting CD4⁺ T cells infected with HIV-1. PHA-activated PBMC depleted of monocytes, of CD8⁺ cells, and of CD25⁺/CD69⁺/HLA-DR⁺ cells were inoculated with HIV-1 or HDV on day 10 postactivation. After their return to resting phase (20 days postinfection), residual CD25⁺/CD69⁺/HLA-DR⁺ cells were removed and the remaining cells were treated with 10 μM prostratin, TPA (100 nM), or soluble anti-CD3/CD28 monoclonal antibodies (1 μg/ml) in the presence of 10 μM AZT. Cells were analyzed by means of fluorescence-activated cell sorter (FACS) analysis or Alu PCR on day 3 after application of prostratin. (A) Time schedule of the experiment. (B) Alu PCR of 10-fold serial dilutions of genomic DNA extracted from mock-treated CD4⁺ T cells (lanes a and b) 20 (lane a) or 24 (lane b) days after PHA stimulation or from prostratin-treated cells at day 24 (lane c). Nested PCR amplifications were conducted with the following primer pairs: a first round with primer pair Alu and L1 (L1 is an antisense primer localized in LTR) (+Alu) and a control round from which the forward Alu primer was omitted (−Alu) were followed by a second round of PCR with HIV-1-specific primers from the U3 part of the HIV-1 LTR. Amplification products were run on agarose gels and visualized by means of ethidium bromide staining. (C) Percentages of noninfected CD25⁺ cells, HDV-inoculated GFP⁺ cells, and HIV-1-infected p24gag⁺ cells that were left nonreactivated (control) (open bars) or were activated with prostratin (black bars), TPA (dark-gray-shaded bars), or CD3/CD28 (light-gray-shaded bars). The means of at least 4 experiments (14 experiments for prostratin-activated cells) are shown.