p38 MAPK activation is required for HIV-1-related apoptosis. (A) p38 MAPK is activated by HIV-1 infection. Immunoblot analysis of protein extracted from PBMCs or Jurkat T cells infected with mock or NL4-3 virus. Total cellular protein extract (50 μg) was extracted 24 hours after infection and analyzed by 12% SDS-PAGE. p38 and phospho-p38 MAPK activation was detected by using specific antibodies that recognize phospho-p38 MAPK (top) only when phosphorylated or the p38 MAPK (middle). HIV infection strongly activates phosphorylation in both cell types. The same lysates were blotted with anti-p24^gag(bottom) to monitor viral infection. (B-C) HIV-1-induced apoptosis is blocked by p38 MAPK inhibitor. Human PBMCs were infected with (B) NL4-3 Wt virus or (C) different HIV-clade specific primary viral isolates. Cells were uninfected (mock) or infected with HIV-1 virus with or without addition of 1 μM p38 inhibitor-I or -II (SB203580 or RWJ67657, respectively). Cells were collected 2 days after infection and stained with annexin-V-FITC; p24^gag-PE and HIV-induced apoptosis analysis was performed. The values indicated frequencies of positive cells of each quadrangle. Data were representative of 3 (B) or 2 (C) independent experiments. Both p38 inhibitors block HIV-driven apoptosis. (D) p38 MAP kinase inhibitors block HIV-induced caspase-3 activity. Cell lysates were prepared from the groups described in panel B. Total protein from each cell lysate (100 μg) was used for the colorimetric caspase assay as described in “Materials and methods.” Each column represents the mean ± standard deviation from triplicate samples derived from 1 of 3 independent experiments. All these experiments gave similar results.