Abstract
The 2',3'-dideoxy-3'-thiacytidine drug-resistant M184I HIV-1 reverse transcriptase (RT) has been shown to synthesize DNA with decreased processivity compared with the wild-type RT. M184A displays an even more severe processivity defect. However, the basis of this decreased processivity has been unclear, and both primer-template binding and dNTP interaction defects have been proposed to account for it. In this study, we show that the altered properties of the M184I and M184A RT mutants that we have measured, including decreased processivity, a slower rate of primer extension, and increased strand transfer activity, can all be explained by a defect in dNTP utilization. These alterations are observed only at low dNTP concentration and vanish as the dNTP concentration is raised. The mutant RTs exhibit a normal dissociation rate from a DNA primer-RNA template while paused during synthesis. Slower than normal synthesis at physiological dNTP concentration, coupled with normal dissociation from the primer-template, results in the lowered processivity. The mutant RTs exhibit normal DNA 3'-end-directed and RNA 5'-end-directed ribonuclease H activity. The reduced rate of DNA synthesis causes an increase in the ratio of ribonuclease H to polymerase activity thereby promoting increased strand transfer. These latter results are consistent with an observed higher rate of recombination by HIV-1 strains with Met-184 mutations.
Highlights
The 2,3-dideoxy-3-thiacytidine drug-resistant M184I HIV-1 reverse transcriptase (RT) has been shown to synthesize DNA with decreased processivity compared with the wild-type RT
We show that the altered properties of the M184I and M184A RT mutants that we have measured, including decreased processivity, a slower rate of primer extension, and increased strand transfer activity, can all be explained by a defect in dNTP utilization
The drug-resistant mutant of HIV-1 RT M184I, which is usually the first resistance mutation to appear in patients treated with 3TC, displays a moderate decrease in processivity compared with the WT RT [15,16,17]
Summary
Materials—HIV-1 nucleocapsid protein NCp7-(1–72) was chemically synthesized as described [27]. The final 12-l reaction mixture contained 4 nM primer, 2 nM donor RNA, 2 units of RT (25 nM WT, 28 nM M184I or 30 nM M184A), 5 g (ϳ1000-fold excess over the substrates) oligo(dT)-poly(rA), 50 mM Tris-HCl (pH 8.0), 6 mM MgCl2, 50 mM KCl, 1 mM dithiothreitol, 1 mM EDTA and dNTP concentrations as described. In all cases the donor template and primer were annealed as described above and preincubated with RT at room temperature for 5 min prior to the initiation of the reactions by addition of MgCl2 and dNTPs. The final reaction mixtures contained 2 nM substrates, 50 mM TrisHCl (pH 8.0), 6 mM MgCl2, 50 mM KCl, 1 mM dithiothreitol, 1 mM EDTA, 2 units of RT (25 nM wild-type, 28 nM M184I, or 30 nM M184A), and varying dNTPs concentrations as described. Reaction products were separated on a 10% polyacrylamide/urea gel and visualized and quantitated as described above
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