Nef is required for HIV-1-mediated FasL induction and efficient p38 activation. (A) Expression and analysis of the proviral expression constructs. A frameshift mutation (5′ Nef) was introduced in the nef gene in the proviral constructs as described in “Materials and methods.” Cell lysates derived from 293T cells transfected with pNef, pNL4-3 Wt, or pNL4-3 ΔNef HIV-1 proviral constructs were separated by 12% SDS-PAGE and then transferred to nitrocellulose membranes. Samples were probed with an HIV-1 Nef-specific antiserum. Nef was not expressed in the pNL4-3 ΔNef constructs. WB indicates Western blot. (B) HIV-1 infection was monitored by measuring p24 levels in culture supernatants derived from PBMCs infected with NL4-3 Wt or NL4-3 ΔNef virus. Data were obtained 96 hours after infection. These experiments were repeated 3 times and similar results were obtained. Error bars represent the mean ± standard deviation from triplicate samples derived from 1 of 3 independent experiments. (C) Induction of FasL expression by NL4-3 Wt or NL4-3 ΔNef viruses in human PBMCs. Cells (1 × 10⁶) were infected with NL4-3 Wt or NL4-3 ΔNef virions and then treated with or without 1 μM p38 inhibitor RWJ67657 as indicated. Two days after infection, an equal number of cells (analysis was performed on a gated low forward scatter and CD24[HAS]-positive cells) were assayed for intracellular p24^gag and surface FasL expression by flow cytometry as described in “Materials and methods.” The values indicated frequencies of positive cells of each quadrangle. Data were representative of 3 independent experiments and observations of similar suppression of FasL were obtained. (D) Nef-deleted viruses failed to induce phosphorylation of p38. Western blot analysis of protein extracted from Jurkat cells infected with mock, NL4-3 Wt, or NL4-3 ΔNef virus. Samples were prepared 24 hours after infection as described in “Materials and methods” and immunoblotted with p38MAPK and phospho-p38MAPK-specific antibodies as indicated.