DNA synthesis profile of HIV-1 RT at the range of the dNTP concentrations found in HIV-1 target cell types. A 5′-end ³²P-labled 19-mer DNA primer (P) annealed to a 184-nt RNA template (T) encoding a portion of the HIV-1 NL4-3 glycosaminoglycan gene was extended by HIV-1 RT (40 nm; green circle) with the dNTP concentration found in human primary activated CD4 T cells (1x, dATP, 23 nm; dGTP, 20 nm; dCTP, 30 nm; TTP, 32 nm) (22) or its three serial dilutions (1:4 (1/4x), 1:8 (1/8x), and 1:16 (1/16x)) for 0. 5, 1, 2, 4, and 8 min under the reaction conditions described under “Experimental Procedures.” These reactions were analyzed by 10% polyacrylamide-urea denaturing gels. The pause sites were marked by an asterisk. F, fully extended product; P, unextended primer. C, no RT control.

Comparison of dNTP and rNTP incorporation rates of HIV-1 RT at the nucleotide concentrations found in human macrophages. Incorporation kinetics of HIV-1 RT (green circle) for individual dNTPs and rNTPs at their concentrations found in macrophages (see Table 1) were compared by single nucleotide extension reactions described under “Experimental Procedures.” A, a representative time course reaction set for dATP alone, ATP alone, or both dATP and ATP at their macrophage concentrations. P, unextended 17-mer primer; E, extended 18-mer product. C, no RT control. B, the percentages of the primer extension at early time points were plotted to compare the incorporation rate for each of dNTPs or rNTPs alone as well as both dNTPs and rNTPs at their macrophage concentrations. C, identical reactions were conducted with SIV_agm Sab-1 RT (SIV). D, steady state K_m values of HIV-1 RT to rNTPs determined by single nucleotide incorporation assay. E, comparison of rNTP concentrations (black bars) of macrophages and PBMC and the K_m values (red zone) of HIV-1 RT to rNTPs. Gray bars, average concentrations of dNTPs of the two target cell types obtained from Table 1.

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.

Comparison of dNTP concentrations of human primary macrophages and activated PBMCs with steady state K_m and pre-steady state K_d values of HIV-1 RT to dNTP substrates. The dNTP concentrations of macrophages and activated PBMC, which were determined by LC-MS/MS and summarized in Table 1, are plotted with black bars. The gray bars represent dNTP concentrations of human primary macrophages and activated CD4⁺ T cells previously determined by the enzyme-based dNTP assay (22). The red and blue zones represent the ranges of the previously determined HIV-1 K_m (22, 37–39) and K_d (34, 40, 41) values of HIV-1 RT to dNTP substrates, respectively.

HIV-1 RT mediated incorporation of rNTPs at the intracellular concentrations found in primary human HIV-1 target cell types. A, the ratios of the rNTP and dNTP concentrations in human primary macrophages and activated PBMCs (see Table 1) are compared. The calculated -fold differences of the dNTP and rNTP concentration disparity in macrophages and activated PBMCs are marked for each of four rNTP and dNTP pairs. B, a 23-mer DNA primer (P) annealed to an RNA 40-mer template (T) (10 nm template-primer complex) was extended by HIV-1 RT (200 nm) at macrophage (M) and PBMC (PB) dNTP and rNTP concentrations (see Table 1) in the presence of [α-³²P[UTP with a 1:690 ratio to the nonradioactive UTP. The reactions were terminated by 10 mm EDTA and inactivation at 95 °C for 3 min, purified with a Qiagen nucleotide removal column, and mixed with an equal amount of the 23-mer 5′-end ³²P-labeled DNA primer as a loading control (LC) before application to 15% polyacrylamide denaturing gels (left). The fully extended product density (F) in the macrophage and T cell nucleotide reactions was quantified and compared after normalization by the loading control for calculating the -fold difference of the rNTP incorporation between macrophages and T cell nucleotide pools (right). C, a time course DNA synthesis by HIV-1 RT (20 nm) with the 5′-end ³²P-labeled 17-mer primer annealed to the RNA 40-mer template (2 nm template-primer) at the macrophage dNTP concentration (see Table 1) for 2, 4, 8, 16, and 20 min in the presence and absence of macrophage rNTP pools measured in Table 1. C, no RT control; F, fully extended product; P, unextended primer.

Extension of DNA primers containing 3′-end dCMP and rCMP by HIV-1 RT in macrophage dNTP concentrations. A, diagram of the template (T) and primers (P) used in this experiment. Two 5′-end ³²P-labeled DNA primers containing dCMP or CMP at their 3′ ends were individually annealed to the RNA 40-mer template. These two template-primers were extended by HIV-1 RT (40 nm; green circle), and single nucleotide (dATP) extension time courses were preformed with dTTP for the first nucleotide incorporation. B, the two template-primers were also extended by HIV-1 RT with both dATP and dGTP for the first and second dNTP incorporations at their macrophage concentrations (see Table 1), respectively, from the 3′-end dCMP or rCMP primers. The reactions were conducted for 30, 60, 90, 120, 240, 480, and 720 s. P, unextended primer; E1, one nucleotide extended product; E2, two nucleotide extended products; C, no RT control.