FIG 4.

HIV-1 replicates in quiescent CD4⁺ T lymphocytes treated with exosomes from Nef-expressing cells. (A) HIV-1 replication in resting CD4⁺ T lymphocytes treated with exosomes from cells expressing either wt Nef or Nef_4EA. A total of 10⁵ quiescent CD4⁺ T lymphocytes were challenged with 120 μU of exosomes from 293T cells transfected with wt Nef- or Nef_4EA-expressing vector, or the empty vector (Ctrl), and then infected with 50 ng of a T-tropic HIV-1 strain. As a control, quiescent cells were challenged with HIV-1 alone (Nil). Three days later, the cells were analyzed for HIV-1 expression. Shown are the mean percentages of HIV-1 positive cells as calculated from duplicate conditions using resting CD4⁺ T lymphocytes from seven healthy donors. The interdonor mean percentages + SD are also shown. *, P < 0.05. (B) Detection of Nef in cells transfected with either wt Nef or Nef_4EA and in exosomes purified from the respective supernatants. Shown is the anti-Nef Western blot analysis of both cells and exosomes from 293T cells transiently transfected with vectors expressing wt Nef, Nef_4EA, or the empty vector (Ctrl). Signals from cellular Nef were normalized with β-actin detection, while anti-ICAM-1 analysis served to normalize exosome signals. The molecular markers are given in kDa. Results are representative of results from two independent experiments.