FIGURE 4.

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.