Amino acid residues in HIV-2 reverse transcriptase that restrict the development of nucleoside analogue resistance through the excision pathway

Abstract

Nucleoside reverse transcriptase (RT) inhibitors (NRTIs) are the backbone of current antiretroviral treatments. However, the emergence of viral resistance against NRTIs is a major threat to their therapeutic effectiveness. In HIV-1, NRTI resistance-associated mutations either reduce RT-mediated incorporation of NRTI triphosphates (discrimination mechanism) or confer an ATP-mediated nucleotide excision activity that removes the inhibitor from the 3' terminus of DNA primers, enabling further primer elongation (excision mechanism). In HIV-2, resistance to zidovudine (3'-azido-3'-deoxythymidine (AZT)) and other NRTIs is conferred by mutations affecting nucleotide discrimination. Mutations of the excision pathway such as M41L, D67N, K70R, or S215Y (known as thymidine-analogue resistance mutations (TAMs)) are rare in the virus from HIV-2-infected individuals. Here, we demonstrate that mutant M41L/D67N/K70R/S215Y HIV-2 RT lacks ATP-dependent excision activity, and recombinant virus containing this RT remains susceptible to AZT inhibition. Mutant HIV-2 RTs were tested for their ability to unblock and extend DNA primers terminated with AZT and other NRTIs, when complexed with RNA or DNA templates. Our results show that Met⁷³ and, to a lesser extent, Ile⁷⁵ suppress excision activity when TAMs are present in the HIV-2 RT. Interestingly, recombinant HIV-2 carrying a mutant D67N/K70R/M73K RT showed 10-fold decreased AZT susceptibility and increased rescue efficiency on AZT- or tenofovir-terminated primers, as compared with the double-mutant D67N/K70R. Molecular dynamics simulations reveal that Met⁷³influences β3-β4 hairpin loop conformation, whereas its substitution affects hydrogen bond interactions at position 70, required for NRTI excision. Our work highlights critical HIV-2 RT residues impeding the development of excision-mediated NRTI resistance.

Keywords: ATP-dependent excision; DNA replication; antiretroviral drugs; drug resistance; human immunodeficiency virus (HIV); nucleoside/nucleotide analogue; reverse transcription.

Figure 1.

Amino acid sequence differences within residues 1–250 of HIV-1_BH10 and HIV-2_ROD RTs and mutant HIV-2_ROD RTs analyzed in this study. a, sequence alignments showing differences between HIV-1 and HIV-2 RTs at positions associated with the excision pathway of resistance to thymidine analogues. Differences between both RTs are highlighted in red. Amino acids shown in blue were introduced in one or more HIV-2_ROD mutant RTs generated in this study. b, HIV-2_ROD RTs analyzed in this study and their abbreviated designations.

Figure 2.

ATP-mediated excision of AZTMP from RNA/DNA template–primers by WT and mutant RTs. Assays were carried out with 31/21-mer RNA–DNA complexes (sequences shown below the upper left panel). The inhibitor was first incorporated at position +1 (indicated with an asterisk) of the 21-nucleotide primer (lane P) to generate a 22-nucleotide product (lane B). Excision of the inhibitor and further primer extension in the presence of 3.2 mm ATP and a mixture of dNTPs led to the formation of a fully extended 31-nucleotide product. Aliquots were removed 2, 4, 6, 8, 10, 12, 15, and 20 min after the addition of ATP. Time courses of the excision reactions are shown in three panels, each one containing data from different sets of related RTs. Time courses obtained with WT HIV-2 RT and mutant 5M are included in all panels for comparison. All dNTPs in the assays were supplied at 200 μm, except for dATP whose concentration was 2 μm. Template–primer and active RT concentrations in these assays were 30 and 24 nm, respectively. Represented values (means ± S.D., error bars) were obtained from at least three independent experiments.

Figure 3.

Kinetics of the ATP-dependent excision of AZTMP and d4TMP from DNA/DNA template–primer (D38/25PGA). Time courses of the excision reactions of AZTMP- and d4TMP-terminated primers (26-mer) annealed to their corresponding 38-nucleotide DNA templates (30 nm) were determined in the presence of 3.2 mm ATP (a and b, respectively). The excision reactions were catalyzed by WT and mutant RTs (40 nm active-site concentration). Aliquots were removed 2, 5, 10, 15, 20, 30, 45, 60, and 80 min after the addition of ATP. Represented values were obtained from at least three independent experiments.

Figure 4.

ATP-mediated excision of nucleoside analogues from RNA/DNA and DNA/DNA template–primers by WT HIV-2 RT and mutants M73K, D67N/K70R, and D67N/K70R/M73K. Assays were carried out with 31/21-mer RNA–DNA complexes (a) or 38/25-mer DNA–DNA complexes (b and c) (sequences shown above excision reaction time courses). The inhibitors (AZT in a and b, and tenofovir in c) were first incorporated at position +1 (indicated by asterisks) to generate 22- or 26-nucleotide products. Excision of the inhibitor and further primer extension in the presence of 3.2 mm ATP and a mixture of dNTPs led to the formation of fully extended primers of 31 or 38 nucleotides. Aliquots were removed at the appropriate times after ATP addition. Time courses of the excision reactions are shown below. The dNTPs in the assays carried out with RNA–DNA complexes were supplied at 200 μm, except for dATP whose concentration was 2 μm. In assays using DNA–DNA complexes, the dNTP concentration was kept at 100 μm, except for dATP (AZT-terminated primers) or dTTP (tenofovir-terminated primers) which was supplied at 1 μm. Template–primer and active RT concentrations in these assays were 30 and 24 nm, respectively. Represented values (means ± S.D. error bars) were obtained from at least three independent experiments.

Figure 5.

Comparison of the structural models of WT and mutant HIV-1 RTs D67N/K70R/K73M and D67N/K70R complexed with double-stranded DNA and an incoming dNTP. a, ribbon representation of the polypeptide backbone around the nucleotide-binding site of the RT. The side chains of Val⁶⁰, Arg⁷⁰, and Met⁷³/Lys⁷³ and the incoming dNTP are shown with stick and sphere representations. In the double-mutant, molecular dynamics simulations predict the formation of a hydrogen bond between amido and hydroxyl groups of the RT (Arg⁷⁰) and the incoming dNTP, respectively (dotted line). The distance between the relevant atoms involved is 2.4 Å in this structure, but in the D67N/K70R/K73M RT model, this value increases up to 6.2 Å. b, view of the β3–β4 hairpin loop, the incoming dNTP, and the two Mg²⁺ ions, based on the structural alignment of the three modeled RTs. The catalytically competent WT HIV-1 RT is shown in orange, the D67N/K70R RT in green, and the D67N/K70R/K73M RT in red. Side chains at positions 60 (Val in all cases), 70 (Lys/Arg), and 73 (Met/Lys) are represented with sticks. Spheres are used to indicate the location of Val⁶⁰ and Met⁷³ in the structure of the triple mutant. The hydrogen bond between Arg⁷⁰ and the incoming dNTP, predicted for the D67N/K70R RT, is shown with a dotted line.

Figure 6.

ATP-mediated excision of AZTMP from DNA/DNA template–primers by HIV-1 RT mutants D67N/K70R and D67N/K70R/K73M. Assays were carried out with 38/25-mer DNA–DNA complexes (sequences shown below the upper panel). The inhibitor was first incorporated at position +1 (indicated with an asterisk) of the 25-nucleotide primer (lane P) to generate a 26-nucleotide product (lane B). Excision of the inhibitor and further primer extension in the presence of 3.2 mm ATP and a mixture of dNTPs led to the formation of a fully extended 38-nucleotide product. Aliquots were removed 2, 4, 6, 8, 10, 12, 15, and 20 min after the addition of ATP. Time courses of the excision reactions are shown below. All dNTPs in the assays were supplied at 100 μm, except for dATP whose concentration was 1 μm. Template–primer and active RT concentrations in these assays were 30 and 24 nm, respectively. Represented values (means ± S.D. error bars) were obtained from at least three independent experiments.