Abstract
Human immunodeficiency virus type 1 reverse transcriptase (RT) is an error-prone DNA polymerase. Structural determinants of its fidelity are incompletely understood. RT/template primer contacts have been shown to influence its fidelity and sensitivity to nucleoside analog inhibitors. The Phe(61) residue, located within the beta 3 sheet of the finger subdomain, is highly conserved among retroviral RTs. The crystal structure of a ternary complex revealed that Phe(61) contacts the first and second bases of the 5'-template overhang. To determine whether such contacts influence the dNTP-binding pocket, we performed a limited vertical scanning mutagenesis (Phe --> Ala, Leu, Trp, or Tyr) at Phe(61). The F61A mutant displayed the highest increase in fidelity, followed by the F61L and F61W variants, which had intermediate phenotypes. F61Y RT had a minimal effect. The increase in fidelity of the F61A mutant was corroborated by a 12-fold decrease in its forward mutation rate. The Phe(61) mutant RTs also displayed large reductions in sensitivity to 2',3'-dideoxythymidine triphosphate and 2',3'-dideoxy,2'3'-didehydrothymidine triphosphate. Mutants displaying the largest increase in fidelity (F61A and F61L) were also the most resistant. These results suggest that contacts between the finger subdomain of human immunodeficiency virus type 1 RT and the template 5'-overhang are important determinants of the geometry of the dNTP-binding pocket.
Highlights
To date, the lack of success in the development of an effective vaccine against human immunodeficiency virus (HIV)1 infections has led to the practice of long term administration of cocktails of antiretroviral drugs for the efficient suppression of viremia
Mutants displaying the largest increase in fidelity (F61A and F61L) were the most resistant. These results suggest that contacts between the finger subdomain of human immunodeficiency virus type 1 reverse transcriptase (RT) and the template 5-overhang are important determinants of the geometry of the dNTP-binding pocket
We carried out a structure-based mutagenesis of Phe61 in HIV-1 RT to determine whether proposed contacts between this residue and the extended single-stranded template play a functional role in polymerase fidelity and sensitivity to nucleoside analog drugs
Summary
Materials—Poly(rA) and oligo(dT) were purchased from Amersham Biosciences. 16 S rRNA, dNTPs, and ddTTP were purchased from Roche Molecular Biochemicals. Similar reactions were carried out with a 16 S rRNA template annealed to a 22-nt DNA primer, VP200, and contained 0.84 nM (25 ng) RT and 50 M each of dATP, dCTP, and dGTP For both sets of reactions the dTTP substrate concentration was varied from 0.1 to 50 M, and the amount of DNA synthesis was determined by measuring [␣-32P]dTMP incorporation. M13mp DNA containing a singlestranded gap of 361 nucleotides was prepared as specified (27) and used as a template-primer for fill-in DNA synthesis by wild type and F61A mutant RTs. Fill-in reactions (25 l) contained 80 mM KCl, 75 mM Tris-Cl (pH 8.0), 10 mM dithiothreitol, 6 mM MgCl2, 0.5 mM each dATP, dCTP, dGTP, and dTTP, 75 ng of gapped duplex DNA, and 170 nM (500 ng) of purified RT. IC50 values of each NRTI for a given RT variant were determined by fitting results from at least three independent experiments to a dose-response curve using nonlinear regression (GraphPad Software Inc.)
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