FIGURE 6.

Inhibition of HIV-1 reverse transcription by 3′-deoxyadenosine chain terminator (rACT) in human primary macrophage and its cytotoxicity. A, cytotoxicity of rACT in human primary CD4⁺ T cell, U937, and CHME5. These three types of cells were cultured and treated with different concentrations of rACT up to 1 mm for 2 days, and the percentages of live and dead cells were determined by FACS and/or trypan blue staining. The percentage of the live cells in the absence of rACT was used for normalization (100%), and the base line cell death of these cell types in the absence of rACT was less than 5%. The cytotoxicity of rACT is shown in the FACS described in B by the propidium iodide staining. B, human primary macrophages were preincubated with different concentrations of rACT (0, 10, 100, and 100 μm) for 24 h and then transduced with HIV-GFP vector (equal p24 amounts). The percentages of GFP-positive macrophages and propidium iodide-positive macrophages in a representative donor, which were determined by FACS at 7 days post-transduction, are shown. C, the percentages of the GFP-positive macrophages in three donors are summarized. D, the genomic DNAs were extracted from the transduced macrophages in B and were applied for the quantitative 2-LTR circle PCR as described under “Experimental Procedures.” The viral production was determined by p24 ELISA with the collected media. These experiments were performed in triplicate on cells from three different blood donors. E, the percentages of the GFP-positive activated CD4⁺ T cells from two donors. The transduction and drug treatment were conducted identically as in D except the infection duration was 2 days. Error bars, S.D.