Figure 9.

HIV-1–expressing dimerization-defective Nef mutants show attenuated replication and endogenous Itk activation in donor PBMCs. PBMCs were isolated from normal donors and stimulated with PHA and IL-2 for 3 days. The cells were then infected with HIV-1–expressing WT Nef, dimerization-defective mutants of Nef or virus that does not express Nef (ΔNef; viral input in each case 40 pg/ml p24). Infected PBMCs were primed with anti-CD3/anti-CD28 antibodies on day 5, and on day 7 they were fixed and stained for HIV-1 p24 Gag and active Itk (anti-pTyr⁵¹¹ antibody) followed by flow cytometry. A, representative flow cytometry plots for unstained, uninfected cells (Control), uninfected stained cells (Uninfected), and cells infected with WT, ΔNef, and dimerization-defective Nef mutant viruses. The number in the gating box indicates the percentage of p24⁺ infected cells present in each culture. B, viral replication was quantified as p24 Gag release into the culture supernatant. The results from three independent determinations are shown with the horizontal bar representing the mean value ± S.E. Student's t tests show that p24 values for ΔNef and each Nef mutant virus are significantly different from WT (p < 0.05 in each case). This experiment was repeated three times with comparable results; a representative example is shown. C, percentage of Itk pTyr⁵¹¹⁺ cells present in each infected (p24⁺) cell population. This experiment was performed three times, and the results are plotted as percentages of pTyr⁵¹¹⁺ cells relative to control cells infected with WT HIV-1 ± S.E. The values for ΔNef and each Nef mutant virus are significantly different from WT (p < 0.01 in each case).