Mechanistic Insights into the Suppression of Drug Resistance by Human Immunodeficiency Virus Type 1 Reverse Transcriptase Using α-Boranophosphate Nucleoside Analogs

Jérôme Deval,Karine Alvarez,Boulbaba Selmi,Marielle Bermond,Joëlle Boretto,Catherine Guerreiro,Laurence Mulard,Bruno Canard

doi:10.1074/jbc.m411559200

Abstract

A class of amino acid substitutions in drug-resistant HIV-1 reverse transcriptase (RT) is responsible for the selectively impaired incorporation of the nucleotide analog inhibitor into DNA. We have shown previously that alpha-boranophosphate nucleoside analogs suppress RT-mediated resistance when the catalytic rate is responsible for drug resistance such as in the case of K65R and dideoxy (dd)NTPs, and Q151M toward AZTTP and ddNTPs. Here, we extend this property to BH3-d4TTP and BH3-3TCTP toward their clinically relevant mutants Q151M and M184V, respectively. Pre-steady-state kinetics on mutants of the Q151M RT family reveal a 3-5-fold resistance to d4TTP. This resistance is suppressed using BH3-d4TTP. Likewise, resistance to 3TCTP by M184V RT (30-fold) and K65R/M184V RT (180-fold) is suppressed using BH3-3TCTP because of a 160-fold acceleration of the catalytic constant kpol. Mechanistic insights into the rate enhancement were obtained using various alpha-boranophosphate nucleotides. The presence of the BH3 group renders kpol independent of amino acid substitutions present in RT. Indeed, the approximately 100-fold decrease in polymerase activity caused by the R72A substitution is restored to wild-type levels using BH3-dTTP. Metal ion titration studies show that alpha-boranophosphate nucleoside analogs enhance 3-8-fold the binding of Mg2+ ions to the active site of the RT.DNA.dNTP complex and alleviate the requirement of critical amino acids involved in phosphodiester bond formation. To our knowledge, this is the first example of rescue of polymerase activity by means of a nucleotide analog.

Highlights

Reverse transcriptase (RT)1 is one of the major retroviral targets in the fight against Agence Nationale de Recherche sur le SIDA (AIDS) because it plays an essential role in human immunodeficiency virus type-1 (HIV-1) replica
We have shown previously that ␣-boranophosphate nucleoside analogs suppress RT-mediated resistance when the catalytic rate is responsible for drug resistance such as in the case of K65R and dideoxyNTPs, and Q151M toward AZTTP and ddNTPs
We extend this property of drug resistance suppression to two other ␣-boranophosphate nucleotide analogs, BH3-d4TTP and BH3-3TCTP toward corresponding clinically relevant drug resistance mutations Q151M and M184V, respectively

Summary

EXPERIMENTAL PROCEDURES

HIV-RT Plasmid Constructions, Enzyme Preparations, and Reagents—The wild-type RT gene construct p66RTB was used to obtain mutant RT genes as described previously [22]. The reaction was performed by mixing a solution containing 50 nM (active sites) HIV-1 RT bound to 100 nM primer-template in RT buffer (50 mM Tris-HCl, pH 8.0, 50 mM KCl, 0.05% Triton X-100) and a variable concentration of dNTP in 6 mM MgCl2. To determine Kd(MgCl2)C, one should vary the total magnesium concentration and measure [Mg2ϩ]free in the reaction mix simultaneously with the titration of active enzyme [RT1⁄7DNA1⁄7dNTP1⁄7Mg2ϩ]. Together with the use of a magnesium chelator buffer, one could solve all equations involving magnesium and individual dissociation constants relative to RT, nucleotides, and DNA, such as described in Refs. For the sake of simplicity of comparison between boronated and nonboronated derivatives, all magnesium-binding species (i.e. RT, DNA, and nucleotide) were kept constant in the reactions, and only [Mg2ϩ]total was varied in nucleotide incorporation assays. Kd,app(Mg2ϩ) and kapp,max were determined from hyperbolic curve fitting

RESULTS

DISCUSSION

TABLE IV