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, 6 (1), e16242

K70Q Adds High-Level Tenofovir Resistance to "Q151M Complex" HIV Reverse Transcriptase Through the Enhanced Discrimination Mechanism

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K70Q Adds High-Level Tenofovir Resistance to "Q151M Complex" HIV Reverse Transcriptase Through the Enhanced Discrimination Mechanism

Atsuko Hachiya et al. PLoS One.

Abstract

HIV-1 carrying the "Q151M complex" reverse transcriptase (RT) mutations (A62V/V75I/F77L/F116Y/Q151M, or Q151Mc) is resistant to many FDA-approved nucleoside RT inhibitors (NRTIs), but has been considered susceptible to tenofovir disoproxil fumarate (TFV-DF or TDF). We have isolated from a TFV-DF-treated HIV patient a Q151Mc-containing clinical isolate with high phenotypic resistance to TFV-DF. Analysis of the genotypic and phenotypic testing over the course of this patient's therapy lead us to hypothesize that TFV-DF resistance emerged upon appearance of the previously unreported K70Q mutation in the Q151Mc background. Virological analysis showed that HIV with only K70Q was not significantly resistant to TFV-DF. However, addition of K70Q to the Q151Mc background significantly enhanced resistance to several approved NRTIs, and also resulted in high-level (10-fold) resistance to TFV-DF. Biochemical experiments established that the increased resistance to tenofovir is not the result of enhanced excision, as K70Q/Q151Mc RT exhibited diminished, rather than enhanced ATP-based primer unblocking activity. Pre-steady state kinetic analysis of the recombinant enzymes demonstrated that addition of the K70Q mutation selectively decreases the binding of tenofovir-diphosphate (TFV-DP), resulting in reduced incorporation of TFV into the nascent DNA chain. Molecular dynamics simulations suggest that changes in the hydrogen bonding pattern in the polymerase active site of K70Q/Q151Mc RT may contribute to the observed changes in binding and incorporation of TFV-DP. The novel pattern of TFV-resistance may help adjust therapeutic strategies for NRTI-experienced patients with multi-drug resistant (MDR) mutations.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Clinical course of patient and drug resistance profile.
(A) The two clinical isolates were collected from the patient at the time points indicated by triangles. Both isolates had no known resistant mutations in the protease region. During the period indicated by asterisk, LPV was administrated but the patient demonstrated poor adherence due to undesirable side effects. After instruction on the use of antiretroviral drugs, the viral loads successfully decreased below the detection limit (<50 copies/ml). (B) Phenotypic drug susceptibility assays of clinical isolates in at least three independent experiments are shown as a relative increase in EC50 compared to HIV-1 NL4-3 strain which served as WT (see also Table S1). (C) Mutations observed in the isolates that are defined as the NRTI and NNRTI resistance associated mutations deposited in the HIV Drug Resistance Database maintained by International AIDS Society 2009 and the Stanford University (http://hivdb.stanford.edu/) were shown. Abbreviations of drugs used: d4T, stavudine; ddI, didenosine; EFV, efavirenz; TFV-DF, tenofovir disoproxil fumarate; LPV, lopinovir; AZT, zidovudine; 3TC, lamivudine; ABC, abacavir; FTC, emtricitabine; NVP, nevirapine.
Figure 2
Figure 2. NRTI resistance of HIVs with mutations at RT residue 70 in the background of WT or Q151Mc.
Antiviral activities of HIV-1s carrying mutations at residue 70 (K70R, K70G, K70E, K70T, K70N, or K70Q) in the WT (A) or Q151Mc (B) background were determined by the MAGIC5 assay. The data for each clone were compared to WT (A) and Q151Mc (B) HIV-1 and are shown as fold increase; AZT (red), ddI (green), d4T (cyan), 3TC (orange), ABC (blue), and TFV-DF (purple). Error bars represent standard deviations from at least three independent experiments (see also Table S2 and Table S3). The asterisk indicates statistically significant in EC50 values (P<0.0001 by t-test).
Figure 3
Figure 3. Effects of RT mutations K70Q, Q151Mc, or K70Q/Q151Mc on DNA primer extension activity and on ATP-based excision activities.
(A) Effect of varying concentrations of TFV-DP on the primer extension activities of HIV-1 WT and mutant RTs. The experiments were carried out in the presence (▪) or absence (▴) of 3.5 mM ATP (B) Time dependence of ATP-based rescue of TFV-terminated primers. TFV-terminated T31/P18 oligos (20 nM) were incubated with 60 nM RT and 3.5 mM ATP. The reaction mixture also included excess of competing dATP (100 µM) that prevented reincorporation of TFV-DP and 0.5 µM dTTP, and 10 µM ddGTP that allowed extension of the rescued primer by two nucleotides and chain termination. Rescue products (WT [▪], K70Q [▴], Q151Mc [▾] and K70Q/Q151Mc [♦]) were analyzed at indicated time points. (C) ATP-based rescue was dependent on concentration of ATP. Reactions were as in (B), but for 30 minutes and at varying concentrations of ATP. Rescue products at 7 mM ATP are defined as 100% product formed.
Figure 4
Figure 4. Pre-steady state kinetics of incorporation of dATP or TFV-DP by WT and K70Q/Q151Mc HIV-1 RTs.
Single-nucleotide incorporation of dATP (panels A, B, and E) or TFV-DP (panels C, D, and F) by WT (panels A, C, E, and F) and K70Q/Q151Mc (panels B, D, E, and F). Formation of extended primer products in the reactions with WT RT and K70Q/Q151Mc RT were measured at 5 ms to 5 s time points, using the following dATP concentrations: 0.5 (▪), 1 (□), 2.5 (▴), 5 (*), 10 (♦), 20 (◊), 50 (▾) and 75 µM (+). Incorporation of TFV was measured at 0.1–10 s reactions and at the following TFV-DP concentrations: 0.75 (▪), 1.5 (□), 3.75 (▴), 7.5 (*), 15 (♦), 37.5 (◊) and 75 µM (+) for reactions with WT RT (panel C), and 1.5 (□), 7.5 (*), 15 (♦), 37.5 (◊), 55 (▾), 75 (+), 112.5 (•), 150 (○) and 200 µM (x) for reactions with K70Q/Q151Mc RT (panel D). (E) The amplitudes of the burst phases from the dATP reactions shown in panels A (WT, [♦]) and B (K70Q/Q151Mc, [▪]) were plotted as a function of dATP concentrations. (F) The amplitudes of the burst phases from the TFV-DP reactions shown in panels C (WT, [♦]) and D (K70Q/Q151Mc, [▪]) were plotted as a function of TFV-DP concentrations. The solid lines in panels A, B, C, and D represent the best fit of data to the burst equation. Each point represents the average values of three experiments.
Figure 5
Figure 5. Stereo view of TFV-DP in the polymerase active site of WT RT and K70Q/Q151Mc RT.
WT RT residues are shown as cyan sticks, K70Q/Q151Mc RT residues are shown as purple sticks. The primer strand is shown as dark gray sticks, template strand as light gray sticks. The fingers and palm subdomains are shown as blue and red cartoons, respectively.

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