FIGURE 3.

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.