FIG 1.

HIV-1 replicates in quiescent CD4⁺ T lymphocytes cocultured in transwell plates with activated CD4⁺ T lymphocytes infected by HIV-1. (A) Inhibition of exosome release from HIV-1-infected cells treated with GW4869 and spiroepoxide. A total of 3 ×10⁶ CD4⁺ T lymphocytes were activated with 2 μg/ml of PHA and, 2 days later, infected with VSV-G wt HIV-1. After an additional 2 days, the cells were treated with GW4869 and spiroepoxide for 16 h. Finally, exosomes in the supernatants were isolated and quantified in terms of mU of AchE. Values are the means + SD of results for duplicates from three independent experiments. *, P < 0.05. (B) FACS analysis for the HIV-1 CAp24 expression in activated CD4⁺ T lymphocytes from cocultures with resting lymphocytes. CD4⁺ T lymphocytes were activated and infected as described for panel A. Two days after infection, the cells were put in the upper chambers of transwell plates, and quiescent CD4⁺ T lymphocytes were seeded in the bottom chambers. The cocultures were run in the presence or not of either AZT or inhibitors of exosome release. Three days later, supernatants and cells from both the upper and bottom chambers were analyzed. The FACS analysis has been carried out in activated CD4⁺ T lymphocytes uninfected or infected with VSV-G wt HIV-1 in the presence or not of either AZT or the inhibitors of exosome release, GW4869 and spiroepoxide. The results are representative of data from three independent experiments. (C) HIV-1 CAp24 levels in supernatants of cocultures. nd, not detectable. Values are the means + SD of results for triplicates from one experiment representative of three independent experiments. (D) Infectivity of HIV-1 released by activated CD4⁺ T lymphocytes in the presence of GW4869 and spiroepoxide as measured on Rev-CEM indicator cells. Values are the means + SD of results for triplicates from one experiment representative of two independent experiments. (E) HIV-1 replication in resting CD4⁺ T lymphocytes. The cells were washed, treated with trypsin, and then scored for the expression of HIV-1 by means of intracellular FACS analysis. Shown are the mean percentages of HIV-1-positive cells as calculated from duplicate conditions using resting CD4⁺ T lymphocytes from three healthy donors. The interdonor mean percentages + SD are also shown. *, P < 0.05.