Inhibition of HIV replication in primary T lymphocytes by IFN-α. CD4⁺ T lymphocytes were cultured in the presence of increasing concentrations of IFN-α (0 to 10,000 IU/ml) for 24 h and then exposed to HIV (1, 10, or 100 ng of p24/0.5 ml/10⁶ cells). IFN was maintained in the cultures. The accumulation of Gag p24 in the culture supernatant over time was measured by ELISA, and the percentage of Gag⁺ cells in the culture was determined by flow cytometry (A). Data are representative of four independent experiments. (B) Means and SDs of Gag p24 levels in the supernatants, measured in four independent experiments. The value 100% corresponds to the amount of Gag p24 obtained in the absence of IFN the day of peak virus replication (day 4 or day 6 postinfection). (C) Means and SD of the percentage of Gag⁺ cells at early time points (peak virus replication in the absence of IFN) and late time points (2 days postpeak), measured in four independent experiments using in each experiment 1, 10, and 100 ng of p24/0.5 ml/10⁶ cells (corresponding to low, intermediate, and high MOIs, respectively). The asterisks indicate significant pairwise differences compared to infections of untreated cells (P < 0.01). p.i., postinfection.

Inhibition of a Vpu-defective HIV strain by IFN-α. CD4⁺ T lymphocytes were cultured in the absence or in the presence of IFN-α (1,000 IU/ml) for 24 h and then exposed to WT or ΔVpu HIV. The accumulation of Gag p24 in the culture supernatants over time was measured by ELISA in untreated cells (A) and in IFN-treated cultures (B). The percentage of Gag⁺ cells in untreated lymphocyte cultures (D) or in IFN-treated cells (E) was determined by flow cytometry. The depicted experiment corresponds to infection using 50 ng of p24, whereas means and SDs for p24 (C) and Gag⁺ cells (F) were calculated in three independent experiments using 10, 50, and 100 ng of p24. The value 100% corresponds to cultures infected by WT virus. The asterisks indicate significant pairwise differences compared to infections of untreated cells by the WT virus (for panel C, P < 0.01; for panel F, P < 0.05). p.i., postinfection.

HIV emerging in treated cultures remains susceptible to IFN. Comparison of the susceptibility to IFN of viruses issued from IFN-treated or untreated cultures. Primary T lymphocytes or MT4R5 cells were cultured in the presence of 1,000 IU/ml IFN-α and infected by HIV (not shown). Viruses produced in the supernatants were used to infect primary T lymphocytes (A) or MT4R5 cells (B) that had been pretreated with increasing concentrations if IFN-α. By comparison, IFN-naive viruses, collected from T lymphocytes and MT4R5-infected cultures, were used in parallel to infect untreated or IFN-treated cells (right panels). In each cell type, IFN-naive and IFN-treated viruses displayed comparable levels of inhibition by IFN. p.i., postinfection.

Inhibition of cell-to-cell HIV transfer by IFN. Cell-to-cell virus transfer among primary T lymphocytes was measured by a flow cytometry-based assay. Cocultures were established between HIV-infected donor cells and target cells. In the experiment shown, approximately 25% of the donor cell population was productively infected. We used a low donor-to-target (D:T) cell ratio (C; 1 donor to 8 targets) and a high ratio (C; 1 donor to 2 targets). Before the coculture, target cells were treated with different concentrations of IFN-α. The increase in the percentage of Gag⁺ cells among target T lymphocytes was followed over time. The mean effect and SD measured at 48 h in five independent experiments using low (B; 1:4 to 1:9) or high (O; 1:1 to 1:2) ratios of donor to target cells are reported. Asterisks indicate significant pairwise differences compared to coculture with untreated cells (P < 0.05).

Effects of IFN and of HIV infection on primary T lymphocytes survival. (A) The percentage of nonviable cells (primary CD4⁺ T lymphocytes) was followed over time in cultures treated with the indicated concentrations of IFN-α, by staining with the nucleic acid dye 7-AAD. The lower panel shows cell cultures exposed to HIV (low MOI of 1 ng of p24/0.5 ml/10⁶ cells). (B) The percentage of productively infected cells in HIV-exposed cultures was followed over time by intracellular Gag staining. Overall, IFN did not affect the proportion of nonviable cells in uninfected cultures while it reduced the extent of the HIV-induced cell death in a dose-dependent manner. p.i., postinfection.

Inhibition of primary HIV isolates by IFN-α. Replication of four primary HIV isolates (BX08, 132W, BON, and KAS) in CD4⁺ T lymphocytes was measured in the absence and presence of 1,000 IU/ml of IFN-α. The accumulation of Gag p24 in the culture supernatant (left panels) and the percentage of Gag⁺ cells in the cultures (right panels) were followed over time. Data shown were obtained using the following amounts of p24/0.5 ml/10⁶ cells for infection: BX08, 0.5 ng; 132W, 5 ng; BON, 5 ng; KAS, 10 ng. Data are representative of three independent experiments. p.i., postinfection.

Effect of the time of addition of IFN on the inhibition of virus spread. Primary CD4⁺ T lymphocytes (A) or MT4R5 cells (B) were treated with IFN-α (1,000 IU/ml) 24 h before exposure to HIV (pretreatment) or 48 h postinfection (postinfection treatment). Virus spread was followed over time by determining the percentage of Gag⁺ cells. Postinfection treatment induced only a marginal delay in virus spread. Pretreatment was performed also using a potent combination of reverse transcriptase inhibitors (AZT/NVP; green line), which successfully inhibited the appearance of Gag⁺ cells, while postinfection treatment with reverse transcriptase inhibitors prevented further virus spread. Data are representative of four independent experiments for which means and SDs at the time of peak virus replication are shown. Asterisks indicate significant pairwise differences compared to virus replication in untreated cells (P < 0.05). p.i., postinfection; PBL, peripheral blood lymphocytes.