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. 2015 May 14;59(6):3189–3196. doi: 10.1128/AAC.00315-15

Subtype-Specific Analysis of the K65R Substitution in HIV-1 That Confers Hypersusceptibility to a Novel Nucleotide-Competing Reverse Transcriptase Inhibitor

Hong-Tao Xu a, Susan P Colby-Germinario a, Peter K Quashie a, Richard Bethell b,*, Mark A Wainberg a,c,d,✉
PMCID: PMC4432161  PMID: 25779585

Abstract

Compound A is a novel nucleotide-competing HIV-1 reverse transcriptase (RT) inhibitor (NcRTI) that selects for a unique W153L substitution that confers hypersusceptibility to tenofovir, while the K65R substitution in RT confers resistance against tenofovir and enhances susceptibility to NcRTIs. Although the K65R substitution is more common in subtype C viruses, the impact of subtype variability on NcRTI susceptibility has not been studied. In the present study, we performed experiments with compound A by using purified recombinant RT enzymes and viruses of subtypes B and C and circulating recombinant form CRF_A/G. We confirmed the hypersusceptibility of K65R substitution-containing RTs to compound A for subtype C, CRF_A/G, and subtype B. Steady-state kinetic analysis showed that K65R RTs enhanced the susceptibility to compound A by increasing binding of the inhibitor to the nucleotide binding site of RT in a subtype-independent manner, without significantly discriminating against the natural nucleotide substrate. These data highlight the potential utility of NcRTIs, such as compound A, for treatment of infections with K65R substitution-containing viruses, regardless of HIV-1 subtype.

INTRODUCTION

Currently, over 35 million people are living with HIV, according to the World Health Organization (http://www.who.int/hiv/en/). Genetic diversity is characteristic of HIV-1 due to the error-prone nature of its reverse transcriptase (RT) enzyme and its high viral replication rate. The HIV-1 epidemic has rapidly evolved to include 9 major known circulating subtypes (A to D, F to H, J, and K) and over 60 known circulating recombinant forms (CRFs) that show 25 to 35% overall genetic variation; this includes 10 to 15% diversity in RT (1–4). HIV-1 subtype C accounts for 50 to 55% of all HIV infections worldwide, while HIV-1 subtype B is the most prevalent subtype in developed countries and accounts for ∼12% of global HIV infections. CRF01_AE and CRF02_AG are two globally predominant CRFs that are found in Southeast Asia and West/Central Africa, respectively.

Current standard anti-HIV-1 therapy, termed highly active antiretroviral therapy (HAART), consists of three or more antiretroviral compounds from six distinct classes, including nucleoside or nucleotide reverse transcriptase inhibitors (NRTIs), nonnucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), fusion inhibitors, entry inhibitors, and integrase inhibitors (INIs) (for reviews, see references 5 and 6). Most currently available antiretrovirals (ARVs) were developed based on the ability to block replication of subtype B viruses, but the development of resistance to all ARVs is a major obstacle in the face of long-term treatment success (7, 8). The high genetic diversity of HIV-1 subtypes may lead to distinct pathways to drug resistance (9–12), necessitating the development of novel effective ARVs that possess distinct mechanisms and superior resistance profiles for all HIV-1 subtypes.

Inhibitors that target RT constitute the largest class of ARVs and are key components of HAART. HIV-1 RT is a multifunctional heterodimeric enzyme that possesses both RNA- and DNA-dependent DNA polymerase (RDDP and DDDP) activities as well as an RNase H activity (13). Two categories of RT inhibitors include nucleoside analogues (NRTIs) and nonnucleoside RT inhibitors (NNRTIs). The former are analogues of natural nucleosides and act as competitive chain terminators by halting DNA synthesis due to lack of a 3′-OH group. NNRTIs are noncompetitive allosteric inhibitors which block DNA synthesis by binding to a hydrophobic binding pocket (8, 14, 15). Two major mechanisms account for resistance to NRTIs: discrimination and excision (2, 16). Discrimination is based on the decreased incorporation of NRTIs by a mutated RT, whereas excision is based on the enhanced ability of a mutated RT to excise an incorporated chain termination inhibitor from the viral DNA terminus.

More recently, a novel category of RT inhibitors, termed nucleotide-competing HIV-1 RT inhibitors (NcRTIs), was identified (17–21). The compounds in this class, typified by the indolopyridone INDOPY-1 (17–19), act as nucleotide-competing inhibitors of RT but not as chain terminators (17, 19, 22). Although structurally unrelated to nucleotides, the NcRTIs compete with incoming nucleotide substrates and reversibly inhibit their binding to the RT active site to form dead-end complexes. Note that the NRTI resistance substitutions M184V and Y115F emerged under the selective pressure of INDOPY-1, resulting in diminished susceptibility to this agent (17, 19), indicating that INDOPY-1 displays overlapping resistance with certain NRTIs, such as lamivudine (3TC), emtricitabine (FTC), and abacavir (ABC).

A novel NcRTI, termed compound A, was recently identified that retains potency against HIV-1 M184V variants (21). Compound A exhibits a unique resistance profile and selects for a novel W153L substitution in RT in cell culture, and its structure was disclosed previously (21). Interestingly, W153L substitution-containing viruses are hypersusceptible to tenofovir (TFV), and the W153L substitution was able to reverse the effects of the K65R substitution in regard to resistance to TFV (21, 23). The mechanism of hypersusceptibility to TFV conferred by the W153L substitution is an increased efficiency of incorporation of TFV-diphosphate (23).

In addition, the K65R RT substitution confers hypersusceptibility to compound A (21, 23) and is a signature substitution for TFV (24–26). Subtype C HIV-1 develops the K65R substitution more rapidly than subtype B does (11), because of differential template usage (27). However, it is not known whether variations among RTs of different HIV-1 subtypes can affect NcRTI inhibition of either wild-type (WT) viruses or viruses containing the K65R substitution. Therefore, we generated HIV-1 RT enzymes and viruses of subtypes B and C and CRF_A/G. Our data showed that all of the K65R substitution-containing viruses tested had impaired viral replication and were hypersusceptible to compound A; the latter finding was confirmed in studies performed with recombinant RT enzymes. Compound A exhibited the highest affinity for RT/primer template complexes at sites following thymidine incorporation. This is the first report on the mechanisms responsible for the hypersusceptibility of viruses containing the K65R resistance substitution to compound A and other NcRTIs. Steady-state kinetic analysis showed that K65R RTs enhanced the susceptibility to compound A by increasing binding of the inhibitor to the nucleotide binding site of RT.

MATERIALS AND METHODS

Chemicals, cells, and nucleic acids.

The NcRTI compound A was obtained from Boehringer Ingelheim Canada Ltd. (Laval, QC, Canada) (21). The HEK293T cell line was obtained from the American Type Culture Collection (ATCC). The following reagents and cells were obtained through the NIH AIDS Research and Reference Reagent Program: the infectious molecular clone pNL4-3, from Malcolm Martin; and TZM-bl (JC53-bl) cells, from John C. Kappes, Xiaoyun Wu, and Tranzyme Inc.

The plasmid pRT6H-PROT was a generous gift from Stuart F. J. Le Grice, National Institutes of Health, Bethesda, MD (28). The subtype C HIV-1 infectious clone pINDIE-C1 and the CRF_A/G HIV-1 infectious clone p97GH-AG2 were kindly provided by Michiyuki Matsuda, International Medical Center of Japan, Tokyo, Japan.

An HIV-1 RNA template of ∼500 nucleotides (nt), spanning the region from the 5′-untranslated region (UTR) to the primer binding site, was transcribed in vitro from AccI-linearized pHIV-PBS DNA (29) by using an Ambion T7-MEGAshortscript kit (Invitrogen, Burlington, ON, Canada) as described previously (30). The oligonucleotides used in this study were synthesized by Integrated DNA Technologies Inc. (Coralville, IA) and purified by polyacrylamide-urea gel electrophoresis. For 5′-end labeling of oligonucleotides with [γ-32P]ATP, an Ambion KinaseMax kit was used, followed by purification through Ambion NucAway spin columns according to protocols provided by the supplier (Invitrogen, Burlington, ON, Canada).

Recombinant reverse transcriptase expression and purification.

The plasmid pRT6H-PROT (28), in which the RT coding region is from HIV-1 HXB2, was kindly provided by S. F. J. Le Grice. For construction of subtype C, subtype B, and CRF_A/G HIV-1 RT heterodimers, the expression plasmids cRT6H-PROT, bRT6H-PROT, and a/gRT6H-PROT were employed together with the RT coding regions of subtype C HIV-1 molecular clone pINDIE-C1 (GenBank accession number AB023804) (31), subtype B HIV-1 molecular clone pNL4-3 (GenBank accession number AF324493), and CRF_A/G HIV-1 molecular clone p97GH-AG2 (GenBank accession number AB052867), respectively, which were subcloned into pRT6H-PROT by a standard PCR cloning procedure to replace the original HXB2 RT coding region. Mutant DNA constructs were generated by using a Stratagene QuikChange II XL site-directed mutagenesis kit (Agilent Technologies Canada Inc., Mississauga, ON, Canada). The presence of mutations and the accuracies of the RT coding sequences were verified by DNA sequencing.

Recombinant RTs were expressed in heterodimeric form and purified as described previously (28, 32). In brief, RT expression in Escherichia coli M15(pREP4) (Qiagen, Mississauga, ON, Canada) was induced with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) at room temperature. Purification was accomplished by a batch method using Ni-nitrilotriacetic acid (NTA) metal affinity chromatography (QIAexpressionist; Qiagen, Mississauga, ON, Canada). Hexahistidine-tagged RT was eluted using an imidazole gradient. RT-containing fractions were pooled, passed through DEAE-Sepharose (GE Healthcare, Mississauga, ON, Canada), and further purified using SP-Sepharose (GE Healthcare, Mississauga, ON, Canada). Fractions containing purified RT were pooled, dialyzed against storage buffer (50 mM Tris-HCl [pH 7.8], 50 mM NaCl, and 50% glycerol), and concentrated to 4 to 8 mg/ml with Centricon Plus-20 MWCO30 filter units (Millipore, Etobicoke, ON, Canada).

The RNA-dependent DNA polymerase activity of each recombinant RT preparation was assayed using a poly(rA)/oligo(dT)12–18 template/primer pair (Midland Certified Reagent Company, Midland, TX) essentially as described previously (33, 34). Each 50-μl reaction mixture contained 25 μg/ml poly(rA)/oligo(dT)12–18, 50 mM Tris (pH 7.8), 2 mM ATP, 5 mM MgCl2, 60 mM KCl, 10 mM dithiothreitol (DTT), 5 μM dTTP with 2.5 μCi of [3H]dTTP, and various amounts of wild-type or mutated RT. Reactions were performed at 37°C, and aliquots of 15 μl were removed at 3 min, 9 min, and 15 min and quenched with 0.2 ml of 10% cold trichloroacetic acid (TCA) and 20 mM sodium pyrophosphate. The radioactivity of incorporated products was analyzed by liquid scintillation spectrometry. Specific activities were calculated as described previously (30, 35).

Filter-based DNA synthesis inhibition assay.

Filter-based DNA synthesis inhibition assays were performed essentially as reported previously (21), with some modifications. Each reaction mixture (50 μl) contained 50 mM Tris-HCl (pH 7.8), 5 mM MgC12, 2 mM ATP, 60 mM KCl, 5 mM DTT, 1 μM [3H]dTTP, 5 μg/ml of the template/primer poly(rA)/oligo(dT)12–18 (Midland Certified Reagent Company, Midland, TX), a recombinant RT normalized to the others by activity, and various amounts of the NcRTI compound A. The radioactivities of incorporated products were analyzed by use of a model 1450 MicroBeta TriLux microplate scintillation and luminescence counter (PerkinElmer, Waltham, MA).

Determination of steady-state kinetic parameters for inhibition by the NcRTI compound A.

The Km for dTTP and the Ki for the NcRTI compound A were determined by filter-binding assays using the homopolymeric RNA/DNA poly(rA)/oligo(dT)12–18 substrate. The reaction mixture (50 μl) contained 50 mM Tris-HCl (pH 7.8), 5 mM MgC12, 2 mM ATP, 60 mM KCl, 5 mM DTT, 5 μg/ml of the template/primer poly(rA)/oligo(dT)12–18 (Midland Certified Reagent Company, Midland, TX), recombinant RT enzyme, different concentrations of [3H]dTTP (0.2 μM to 40 μM), and various amounts of the NcRTI compound A. After incubation for 30 min at 37°C, the reaction mixtures were terminated by adding 0.2 ml of 10% cold TCA and 20 mM sodium pyrophosphate and incubating them for at least 30 min on ice. The precipitated products were filtered onto a 96-well MultiScreen HTS FC filter plate (Millipore, Etobicoke, ON, Canada) that was prewet with 150 μl assay buffer prior to use and sequentially washed with 200 μl of 10% TCA and 150 μl of 95% ethanol. The radioactivities of incorporated products were analyzed using a model 1450 MicroBeta TriLux microplate scintillation and luminescence counter (PerkinElmer, Waltham, MA). The experiments were done in triplicate, and at least three independent experiments were performed in each instance. Data were fitted to a competitive inhibition equation by using GraphPad Prism5.0 software (GraphPad Software, San Diego, CA).

Primer extension assay.

The same RNA template and 5′-end-32P-labeled D25 primer (T/P) as those described previously (36) were used to assess the inhibitory effects of the NcRTI compound A on DNA synthesis by use of purified recombinant RT enzymes in time course experiments. Final reaction mixtures contained 40 nM T/P, equal amounts of RT enzymes in terms of activity, 50 mM Tris-HCl (pH 7.8), 2 mM ATP, and 50 mM NaCl. Reactions were initiated by adding 6 mM MgCl2 and a 50 μM concentration of each deoxynucleoside triphosphate (dNTP) in the presence or absence of a fixed concentration of the NcRTI compound A or 20 μM ddTTP or ddCTP. The reactions were allowed to proceed for 10 s, 30 s, 60 s, 5 min, 10 min, 30 min, and 60 min, and then 2 volumes of stop solution (90% formamide, 10 mM EDTA, and 0.1% each of xylene cyanol and bromophenol blue) was added to stop each reaction. Reaction products were separated by 6% denaturing polyacrylamide gel electrophoresis and analyzed using a Molecular Dynamics Typhoon phosphorimager system (GE Healthcare, Mississauga, ON, Canada).

Preparation of site-directed mutant subtype B, subtype C, and CRF_A/G HIV-1 stocks.

Site-directed mutagenesis reactions were carried out using a Stratagene QuikChange II XL site-directed mutagenesis kit to construct subtype B, subtype C, and CRF_A/G HIV-1 infectious clones that harbored the desired substitutions in the RT gene. For this purpose, the molecular clones pNL4-3, pINDIE-C1, and p97GH-AG2 were employed. DNA sequencing of the complete RT region was performed to verify the absence of spurious substitutions and the presence of the desired substitutions in the RT coding sequences. Recombinant HIV-1 WT and mutant viruses were generated by transfection of the corresponding proviral plasmid DNAs into HEK293T cells by use of Lipofectamine 2000 (Invitrogen, Burlington, ON, Canada) according to the manufacturer's instructions. Viral supernatants were harvested at 48 h posttransfection, centrifuged for 5 min at 800 × g to remove cellular debris, filtered through a 0.45-μm-pore-size filter, aliquoted, and stored at −80°C. Levels of p24 antigen in viral supernatants were measured by use of a Perkin-Elmer HIV-1 p24 antigen enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's instructions (Perkin-Elmer Life Sciences, Boston, MA). Virion-associated RT activity was assessed as described previously (23).

Measurements of HIV-1 RC in TZM-bl cells.

The relative replication capacities (RCs) of the recombinant WT HIV-1 clones from subtype B, subtype C, and CRF_A/G and their K65R substitution-containing variants were evaluated by means of a noncompetitive infectivity assay using TZM-bl cells, as previously described (37, 38). Twenty thousand cells per well were added in triplicate to a 96-well culture plate in 100 μl Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Burlington, ON, Canada) supplemented with 10% fetal bovine serum (Invitrogen, Burlington, ON, Canada), 1% penicillin-streptomycin, and 1% l-glutamine (Invitrogen, Burlington, ON, Canada). Viral stocks of both wild-type and mutant viruses were normalized on the basis of p24 antigen levels, and recombinant viruses were serially diluted from viral stock suspensions. After 4 h, 50 μl of DMEM was removed from each well and replaced with 50 μl of virus dilution; a control well did not contain virus. Viruses and cells were cocultured for 48 h, after which 100 μl of Promega luciferase assay Bright-Glo reagent (Fisher Scientific, Ottawa, ON, Canada) was added and luciferase activity was measured in a model 1450 MicroBeta TriLux microplate scintillation and luminescence counter (PerkinElmer, Waltham, MA). The viral replication level of each virus was expressed as the percentage of relative light units (RLU) with reference to the subtype B WT virus.

Analysis of phenotypic drug susceptibility in TZM-bl cells.

Phenotypic susceptibility analyses of RT inhibitors were performed with subtype B, subtype C, and CRF_A/G HIV-1 clones in a TZM-bl cell-based assay as described previously (37, 39, 40). Briefly, RT inhibitors at various concentrations were added to TZM-bl cells (104 cells/well) in 96-well plates grown in 100 μl supplemented medium. Immediately after drug addition, cells were infected with WT or mutant viruses. At 48 h postinfection, cells were rinsed with 100 μl phosphate-buffered saline and lysed with 50 μl/well Promega luciferase assay cell lysis reagent (Fisher Scientific, Ottawa, ON, Canada). Cell lysates were then transferred to a white, opaque 96-well plate (Corning, Tewksbury, MA). Promega luciferase assay reagent (Fisher Scientific, Ottawa, ON, Canada) was added to each well, and the number of RLU/well was measured by use of a PerkinElmer 1450 MicroBeta TriLux microplate scintillation and luminescence counter (PerkinElmer, Waltham, MA). The 50% effective concentration (EC50) was calculated using GraphPad Prism5.0 software (GraphPad Software, San Diego, CA).

RESULTS

Purification of recombinant HIV-1 RT and specific activity analysis.

Recombinant heterodimeric (p66/p51) RTs of subtypes B and C and CRF_A/G were purified to >95% homogeneity at equivalent p66/p51 molar ratios (Fig. 1A). To determine the specific activities of the recombinant enzyme preparations, DNA polymerase activity was measured using the synthetic poly(rA)/oligo(dT)12–18 template/primer in a 15-min initial rate reaction mixture, and the calculated initial velocities were divided by the concentration of enzyme used (Fig. 1B). Wild-type RTs from each subtype shared similar activities. All mutant enzymes containing the K65R substitution had impaired specific activity compared with that of the WT enzyme, i.e., ∼60% of wild-type activity, in agreement with previous results obtained with subtype B K65R RT (33, 35).

FIG 1.

FIG 1

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Expression, purification, and determination of the specific activity and TFV susceptibility of recombinant subtype C and B HIV-1 RTs. (A) Coomassie brilliant blue staining of purified heterodimer RTs after 8% SDS-PAGE. MW, molecular size standards, in kilodaltons; BWT, HIV-1 subtype B wild-type RT; CWT, HIV-1 subtype C wild-type RT; BK65R, HIV-1 subtype B RT harboring the K65R substitution; CK65R, HIV-1 subtype C RT harboring the K65R substitution; A/GWT and A/GK65R, HIV-1 CRF_A/G WT RT and that harboring the K65R substitution, respectively. The positions of purified recombinant RT heterodimers are indicated on the right. (B) Specific activities of recombinant RT enzymes as assessed using the poly(rA)/oligo(dT) template/primer as described in Materials and Methods. All specific activities are expressed as percentages of the subtype B WT RT specific activity, with the percent standard deviation for duplicate samples also indicated. These data are representative of at least two independent experiments from two independent preparations of recombinant RTs, with similar results being obtained each time.

K65R substitutions in the RTs of HIV-1 subtypes B and C and CRF_A/G enhance NcRTI susceptibility in both biochemical and cell-based assays.

Cell culture assays showed that HIV-1 subtype B harboring the K65R substitution exhibited an enhanced susceptibility to the NcRTI compound A (21, 23). We compared the impacts of the K65R substitution on susceptibility to compound A among HIV-1 subtypes B and C and CRF_A/G by a cell-free recombinant RT assay and cell-based assays. We generated recombinant WT and K65R RT enzymes from subtypes B and C and CRF_A/G and determined the 50% inhibitory concentrations (IC50s) of compound A by measuring inhibition using the homopolymeric poly(rA)/oligo(dT)12–18 template/primer substrate. All three K65R mutant enzymes displayed 0.3- to 0.4-fold changes in IC50 compared to the WT enzymes (Table 1). We also generated recombinant viruses containing the K65R substitution in the three HIV-1 molecular clones and determined the EC50s of compound A by measuring the inhibition of viral replication in TZM-bl cultures. All three K65R mutant viruses showed 0.2- to 0.3-fold changes in EC50 compared to the WT (Table 2), demonstrating that the K65R substitution enhanced the inhibitory effects of the NcRTI compound A, regardless of subtype, and conferred hypersusceptibility to compound A. These data are in agreement with observations that the K65R substitution in subtype B enhances NcRTI susceptibility (17, 19, 21, 23).

TABLE 1.

Susceptibilities of WT and site-directed mutant K65R HIV-1 RT enzymes to the NcRTI compound A as assessed in a cell-free RT assay

RT enzyme Mean IC50 ± SD (nM) (fold change in resistance)a
Subtype B Subtype C CRF_A/G
WT 48.1 ± 7.2 44.3 ± 9.3 50.1 ± 9.1
K65R variant 19.3 ± 6.4 (0.4) 13.2 ± 3.1 (0.3) 14.2 ± 4.2 (0.3)
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a

IC50 values were determined in RT assays. Data represent the averages for two or three representative experiments, with standard deviations (SD) also shown. All IC50s for K65R enzymes were significantly decreased compared to those of the relevant wild-type RT counterparts (P < 0.05 by the Wilcoxon rank sum test). Fold changes represent the changes in IC50 for mutated compared to WT RT enzymes.

TABLE 2.

Susceptibilities of WT and site-directed K65R mutant HIV-1 strains to the NcRTI compound A as assessed in TZM-bl cultures

Virus EC50 ± SD (nM) (fold change in resistance)a
Subtype B Subtype C CRF_A/G
WT 12.5 ± 3.5 9.5 ± 3.2 10.1 ± 3.0
K65R mutant 3.0 ± 1.2 (0.2) 2.9 ± 0.9 (0.3) 3.1 ± 1.1 (0.3)
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a

EC50 values were determined in TZM-bl cell cultures. Data represent the averages and SD for two representative experiments. All EC50s for K65R viruses were significantly decreased compared to those of the relevant wild-type viruses (P < 0.05 by the Wilcoxon rank sum test). Fold changes represent the changes in EC50 for mutated compared to WT viruses.

Effect of the K65R substitution on inhibition of DNA synthesis by compound A in gel-based assays.

Inhibition of DNA synthesis by compound A was also monitored in time course experiments using a heteropolymeric RNA/DNA template/primer substrate and a 5′-end-labeled DNA primer (D25) annealed to a 471-nt RNA template derived from the 5′ end of the HIV-1 genome to mimic physiologically relevant conditions. Levels of RT were adjusted based on production of the full-length product in the absence of inhibitor (Fig. 2B). Figure 2B shows that subtype B K65R RT in the presence of compound A showed a pausing profile similar to that of WT RT but that an enhanced band intensity and earlier pausing of DNA synthesis were present, resulting in lower yields of full-length product (Fig. 2B). Hence, the K65R substitution is responsible for hypersusceptibility to compound A, in agreement with the results of the filter-binding assay with homopolymeric T/P and with the results of the cell-based assay. Similar results were obtained with RT enzymes of subtype C and CRF_A/G (not shown).

FIG 2.

FIG 2

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Sequence dependence and effect of the K65R substitution on compound A inhibition of DNA synthesis. (A) Graphic representation of the RNA template (T) and DNA primer (P) strands synthesized by purified subtype B wild-type (WT) and K65R recombinant RT enzymes. The arrows indicate hot spots for inhibition by the NcRTI compound A, while the numbers are the positions of nucleotides incorporated ahead of the 3′ terminus of the primer. (B) Inhibition of DNA synthesis was monitored in time course experiments in the absence (−) or presence (+) of the NcRTI compound A, using a 5′-end-labeled DNA primer (D25) annealed to a 471-nt RNA template as the substrate; the resulting full-length DNA (FL DNA) was 471 nt long. The sizes of some fragments of the 32P-labeled 25-bp DNA ladder (Invitrogen) are indicated to the left, in nucleotides (nt). The reactions were stopped at 10 s (10″), 30 s (30″), 60 s (60″), 5 min (5′), 10 min (10′), 30 min (30′), and 60 min (60′). All reaction products were resolved by denaturing 6% polyacrylamide gel electrophoresis and visualized by phosphorimaging. Positions of the 32P-labeled D25 primer (32P-D25) and the 471-nt FL DNA product are indicated on the right. Lanes 1 and 2 show control reactions in the presence of ddTTP at 10 s and 60 min, respectively; lanes 3 and 4 show control reactions in the presence of ddCTP at 10 s and 60 min, respectively. A representative image from one of three independent experiments from which similar results were obtained is shown. (C) Enlarged view of the boxed region in panel B to show the hot spots for inhibition by the NcRTI compound A. “T” or “C” on the right represents the incorporated dTTP or dCTP substrate, respectively, and the numbers indicate the positions of incorporated nucleotides ahead of the 3′ terminus of the primer. Inhibition by the NcRTI compound A predominantly followed a thymidine incorporation pattern.

Inhibition by another NcRTI, INDOPY-1, predominantly follows incorporation of thymidine (17), and compound A is a competitive RT inhibitor with respect to the incoming nucleotide substrate (21). However, it is not clear whether inhibition by compound A is sequence dependent. To address this question, we employed a heteropolymeric RNA/DNA substrate derived from HIV-1 and used a gel-based time course primer extension assay. Control reactions in the presence of ddTTP or ddCTP were also performed to determine hot spots of inhibition of DNA synthesis (Fig. 2B). In the absence of an inhibitor, shorter extension products indicate enzyme pausing. The results showed that additional distinct bands were observed in the presence of the NcRTI compound A following incorporation of pyrimidines, predominantly thymidine (Fig. 2B and C), while the incoming base in the template did not seem to affect inhibition by compound A. These distinct bands in the presence of inhibitor vanished over time, indicating that compound A binding is reversible and that its mechanism of action does not involve either base pairing or chain termination. These findings provide the first direct evidence that the binding of an NcRTI is not dependent on base-like complementarities with the template and that the interaction of compound A with RT is favored by pyrimidines, mainly thymidine, at the primer terminus.

Steady-state kinetic analysis of inhibition of RT by compound A.

Subtype B K65R HIV-1 and a recombinant RT enzyme were previously shown to be hypersusceptible to compound A (21). It was hypothesized that diminished incorporation of the natural nucleotide substrates by the K65R variant may favor competition with nucleotide binding by compound A, resulting in the hypersusceptibility phenotype. To determine the effects of K65R substitutions in subtype B and C and CRF_A/G RTs on inhibition by compound A, we measured the steady-state kinetic constant Km for the natural nucleotide substrate dTTP and the inhibition constant Ki for compound A, using a homopolymeric RNA/DNA substrate (Table 3). The Ki/Km ratios were calculated to determine the ability of each RT enzyme to selectively bind compound A relative to the natural nucleotide substrate. The steady-state Km values of the K65R RTs for dTTP were not significantly changed compared to those of the WT RTs, suggesting that K65R RTs of all three subtypes bind/incorporate the natural dTTP substrate with an efficiency similar to that of WT RT. However, the Ki values of the K65R RTs for compound A were significantly decreased compared to those of their WT counterparts (P ≤ 0.01). The K65R RTs showed a decrease in the Ki/Km ratio of 0.2-fold compared with the corresponding WT RTs for compound A, suggesting that there was increased binding of this inhibitor. These findings indicate that the K65R substitution in HIV-1 RT, regardless of subtype, enhances susceptibility to the NcRTI compound A through increased binding, without significantly decreasing binding and/or incorporation of the natural dTTP nucleotide.

TABLE 3.

Steady-state kinetic analysis of relative inhibitory capacities of compound A with WT and K65R RT enzymes

HIV-1 RT enzyme Km (dTTP) (μM)a Ki (NcRTI) (nM)b Ki/Km (fold change)c
Subtype B WT 4.1 ± 0.1 23.1 ± 3.2 5.6 (1.0)
Subtype B K65R variant 5.4 ± 0.2 7.1 ± 1.4 1.3 (0.2)
Subtype C WT 3.5 ± 0.2 25.2 ± 2.9 7.2 (1.0)
Subtype C K65R variant 4.2 ± 0.3 5.8 ± 1.2 1.4 (0.2)
CRF_A/G WT 3.8 ± 0.2 25.3 ± 3.8 6.7(1.0)
CRF_A/G K65R variant 4.9 ± 0.3 6.2 ± 2.1 1.3 (0.2)
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a

Km values are means ± standard deviations obtained for three independent experiments.

b

Ki values are means ± standard deviations obtained for three independent experiments. All Ki values for K65R mutants were significantly decreased compared to those for the relevant wild-type RTs (P ≤ 0.01 by two-tailed Student's t test).

c

Fold change in Ki/Km relative to the relevant HIV-1 subtype WT strain.

The K65R substitution in RT diminishes the viral replication capacity of HIV subtypes B and C and CRF_A/G.

The K65R substitution in RT of subtype B HIV-1 has been shown to impair the viral replication capacity (11, 26, 41). We next wished to investigate the impact of the K65R substitution on viral replication in subtype C and CRF_A/G, as well as subtype B, in parallel. For this purpose, we used a well-established noncompetitive high-throughput infectivity assay with TZM-bl cells infected with recombinant HIV-1 strains that were normalized in terms of inoculum on the basis of the p24 antigen level (37, 38). The levels of infectivity of the various WT and mutant viruses were determined by measuring the luciferase activity at 48 h postinfection. The results showed that the replication of all mutant viruses containing the K65R substitution was diminished compared to that of each of the relevant WT viruses (Fig. 3). At a p24 input of 1.5 × 105 pg/ml, the relative replication capacity (RC) of a virus containing the K65R substitution was decreased to 36 to 48% that of the WT, consistent with previous observations for subtype B (11, 26, 41).

FIG 3.

FIG 3

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Comparative analysis of the effect of the K65R substitution on viral replication capacity among different HIV-1 subtypes. WT and mutated viruses were normalized by the p24 level and used to infect TZM-bl cells. Luciferase activity was measured at 48 h postinfection to monitor viral replication. The relative infectivity of each virus compared to the subtype B WT virus is shown on the y axis, whereas the x axis denotes the input of p24. Values are means for three independent experiments, and error bars represent standard deviations.

DISCUSSION

Biochemical, virological, and clinical studies of HIV-1 subtype B and non-B subtypes have shown that genetic diversity can affect drug resistance pathways and the outcome of HAART (9, 10, 12). For example, the K65R substitution is known to occur at a higher frequency among individuals infected with HIV-1 subtype C (42–44), which may limit the success of some TFV-based regimens (45, 46). Our lab has shown that this may be due to template usage in subtype C under TFV pressure (11). Others have shown that the K65R substitution may be less frequent in subtype A than in other subtypes (47).

It is important to understand the basis for these differences, since this information may help to guide the development of novel ARVs that possess unique resistance profiles. The NcRTIs INDOPY-1 and compound A are two such molecules that retain potency against subtype B HIV-1 and hypersusceptibility of viruses containing the K65R substitution (17, 21). In this study, we have shown that compound A is active against WT HIV-1 RTs of subtype C and CRF_A/G as well as subtype B. Moreover, the K65R RTs of all three subtype variants are hypersusceptible to compound A. Cell culture phenotyping assays confirmed these results.

The K65R substitution is a major resistance substitution for TFV, and it also decreases susceptibility to most N(t)RTIs, i.e., abacavir (ABC), didanosine (ddI), emtricitabine (FTC), lamivudine (3TC), and stavudine (d4T), with zidovudine (AZT; also known as ZDV) being a notable exception (48). Our experiments have revealed that the overall enzyme activity and susceptibility to compound A of HIV-1 subtype C and CRF_A/G RTs are comparable to those of subtype B RT and that the K65R substitution, when present in subtype C and CRF_A/G RTs, diminishes enzyme activity and viral replicative capacity in a manner similar to that seen with subtype B. Thus, all K65R HIV-1 strains may have lower fitness levels than those of WT viruses. The K65R substitution confers resistance to NRTIs by means of discrimination, meaning that the K65R substitution favors the natural dNTP substrate over NRTIs by decreasing the binding affinity and/or incorporation rate of NRTIs (49–52). Previously, it was shown that the K65R substitution confers hypersusceptibility to the prototype NcRTI INDOPY-1 by discriminating against the dNTP substrate through enhanced binding of the inhibitor and diminished binding of the natural dNTP substrate (19). Our biochemical data now show that K65R variants in all three subtypes tested bind/incorporate natural dNTP substrates with efficiencies similar to that of WT RT. These data are in agreement with previous observations showing that the K65R substitution has hardly any impact on the Km values for natural dNTP substrates (52, 53). However, the Ki values of K65R RTs for compound A were significantly decreased compared to that of each WT enzyme. K65R RTs from all three subtype variants showed a decrease in Ki/Km of 0.2-fold compared to each relevant WT RT for compound A. This suggests that the K65R substitution results in increased binding of this inhibitor.

Our primer extension experiments using an HIV heterodimeric RNA template showed that the hot spots of inhibition of DNA synthesis by compound A predominantly follow a thymidine incorporation pattern. This is in agreement with observations that INDOPY-1 binds preferentially following incorporation of a pyrimidine (T > C) (17). This further indicates that compound A, which belongs to the benzo[4,5]furo[3,2,d]pyrimidin-2-one (BFPY) series of NcRTIs, shares an RT binding mode similar to that of INDOPY-1, although different resistance mutational profiles can distinguish the two series of compounds.

The K65R substitution also confers hypersusceptibility to an experimental 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) compound (54). EFdA is a potent NRTI that is unique in that it possesses a 3′-OH group. EFdA acts mainly as a delayed chain terminator by blocking translocation following its incorporation into a nascent DNA strand (54). The primary resistance substitution for EFdA is M184V, which also confers resistance to lamivudine (3TC) and emtricitabine (FTC). Thus, both EFdA and the NcRTI INDOPY-1 display overlapping resistance profiles with certain NRTIs, which may limit their ultimate use in combination anti-HIV-1 therapy. The improved antiviral potency and unique resistance profile of compound A make it or successor molecules promising candidates for use with TFV in combination therapy to circumvent the emergence of the K65R substitution in various HIV-1 subtypes.

The primary resistance substitution for compound A is W153L, which confers hypersusceptibility to TFV via an increased efficiency of incorporation (23). Conversely, the primary substitution for TFV is K65R, which results in hypersusceptibility to compound A via increased binding. Although the pharmacological profile of compound A makes it unsuitable for clinical development, the synthesis and evaluation of derivatives of compound A will be worthwhile if such compounds possess good tolerability and pharmacokinetics (55) while sharing the resistance profile of compound A.

The novelty of this study lies in the subtype-specific analysis of the K65R substitution in HIV-1 RT that confers hypersusceptibility to compound A. Although HIV-1 subtypes B and C and CRF_ A/G are the most important in terms of the worldwide epidemic, future work should also include other viral subtypes and circulating recombinant forms.

ACKNOWLEDGMENTS

This work was supported by research grants from the Canadian Institutes of Health Research (CIHR).

Richard Bethell was an employee of Boehringer Ingelheim (Canada) Ltd., Laval, Quebec, Canada, when this study was initiated. We have no other conflicts of interest to declare.

REFERENCES

  • 1.Johnson VA, Brun-Vezinet F, Clotet B, Gunthard HF, Kuritzkes DR, Pillay D, Schapiro JM, Richman DD. 2008. Update of the drug resistance mutations in HIV-1: Spring 2008. Top HIV Med 16:62–68. [DOI] [PubMed] [Google Scholar]
  • 2.Menendez-Arias L. 2008. Mechanisms of resistance to nucleoside analogue inhibitors of HIV-1 reverse transcriptase. Virus Res 134:124–146. doi: 10.1016/j.virusres.2007.12.015. [DOI] [PubMed] [Google Scholar]
  • 3.Taylor BS, Hammer SM. 2008. The challenge of HIV-1 subtype diversity. N Engl J Med 359:1965–1966. doi: 10.1056/NEJMc086373. [DOI] [PubMed] [Google Scholar]
  • 4.Robertson DL, Anderson JP, Bradac JA, Carr JK, Foley B, Funkhouser RK, Gao F, Hahn BH, Kalish ML, Kuiken C, Learn GH, Leitner T, McCutchan F, Osmanov S, Peeters M, Pieniazek D, Salminen M, Sharp PM, Wolinsky S, Korber B. 2000. HIV-1 nomenclature proposal. Science 288:55–56. doi: 10.1126/science.288.5463.55d. [DOI] [PubMed] [Google Scholar]
  • 5.Arts EJ, Hazuda DJ. 2012. HIV-1 antiretroviral drug therapy. Cold Spring Harb Perspect Med 2:a007161. doi: 10.1101/cshperspect.a007161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Arribas JR, Eron J. 2013. Advances in antiretroviral therapy. Curr Opin HIV AIDS 8:341–349. doi: 10.1097/COH.0b013e328361fabd. [DOI] [PubMed] [Google Scholar]
  • 7.Menendez-Arias L. 2010. Molecular basis of human immunodeficiency virus drug resistance: an update. Antiviral Res 85:210–231. doi: 10.1016/j.antiviral.2009.07.006. [DOI] [PubMed] [Google Scholar]
  • 8.Sarafianos SG, Marchand B, Das K, Himmel DM, Parniak MA, Hughes SH, Arnold E. 2009. Structure and function of HIV-1 reverse transcriptase: molecular mechanisms of polymerization and inhibition. J Mol Biol 385:693–713. doi: 10.1016/j.jmb.2008.10.071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Singh K, Flores JA, Kirby KA, Neogi U, Sonnerborg A, Hachiya A, Das K, Arnold E, McArthur C, Parniak M, Sarafianos SG. 2014. Drug resistance in non-B subtype HIV-1: impact of HIV-1 reverse transcriptase inhibitors. Viruses 6:3535–3562. doi: 10.3390/v6093535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Brenner BG, Lowe M, Moisi D, Hardy I, Gagnon S, Charest H, Baril JG, Wainberg MA, Roger M. 2011. Subtype diversity associated with the development of HIV-1 resistance to integrase inhibitors. J Med Virol 83:751–759. doi: 10.1002/jmv.22047. [DOI] [PubMed] [Google Scholar]
  • 11.Brenner BG, Oliveira M, Doualla-Bell F, Moisi DD, Ntemgwa M, Frankel F, Essex M, Wainberg MA. 2006. HIV-1 subtype C viruses rapidly develop K65R resistance to tenofovir in cell culture. AIDS 20:F9–F13. doi: 10.1097/01.aids.0000232228.88511.0b. [DOI] [PubMed] [Google Scholar]
  • 12.Martinez-Cajas JL, Pant-Pai N, Klein MB, Wainberg MA. 2008. Role of genetic diversity amongst HIV-1 non-B subtypes in drug resistance: a systematic review of virologic and biochemical evidence. AIDS Rev 10:212–223. [PubMed] [Google Scholar]
  • 13.Goff SP. 1990. Retroviral reverse transcriptase: synthesis, structure, and function. J Acquir Immune Defic Syndr 3:817–831. [PubMed] [Google Scholar]
  • 14.Singh K, Marchand B, Kirby KA, Michailidis E, Sarafianos SG. 2010. Structural aspects of drug resistance and inhibition of HIV-1 reverse transcriptase. Viruses 2:606–638. doi: 10.3390/v2020606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Ren J, Stammers DK. 2008. Structural basis for drug resistance mechanisms for non-nucleoside inhibitors of HIV reverse transcriptase. Virus Res 134:157–170. doi: 10.1016/j.virusres.2007.12.018. [DOI] [PubMed] [Google Scholar]
  • 16.Menendez-Arias L. 2013. Molecular basis of human immunodeficiency virus type 1 drug resistance: overview and recent developments. Antiviral Res 98:93–120. doi: 10.1016/j.antiviral.2013.01.007. [DOI] [PubMed] [Google Scholar]
  • 17.Jochmans D, Deval J, Kesteleyn B, Van Marck H, Bettens E, De Baere I, Dehertogh P, Ivens T, Van Ginderen M, Van Schoubroeck B, Ehteshami M, Wigerinck P, Gotte M, Hertogs K. 2006. Indolopyridones inhibit human immunodeficiency virus reverse transcriptase with a novel mechanism of action. J Virol 80:12283–12292. doi: 10.1128/JVI.00889-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Zhang Z, Walker M, Xu W, Shim JH, Girardet JL, Hamatake RK, Hong Z. 2006. Novel nonnucleoside inhibitors that select nucleoside inhibitor resistance mutations in human immunodeficiency virus type 1 reverse transcriptase. Antimicrob Agents Chemother 50:2772–2781. doi: 10.1128/AAC.00127-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Ehteshami M, Scarth BJ, Tchesnokov EP, Dash C, Le Grice SF, Hallenberger S, Jochmans D, Gotte M. 2008. Mutations M184V and Y115F in HIV-1 reverse transcriptase discriminate against “nucleotide-competing reverse transcriptase inhibitors.” J Biol Chem 283:29904–29911. doi: 10.1074/jbc.M804882200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Jegede O, Khodyakova A, Chernov M, Weber J, Menendez-Arias L, Gudkov A, Quinones-Mateu ME. 2011. Identification of low-molecular weight inhibitors of HIV-1 reverse transcriptase using a cell-based high-throughput screening system. Antiviral Res 91:94–98. doi: 10.1016/j.antiviral.2011.05.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Rajotte D, Tremblay S, Pelletier A, Salois P, Bourgon L, Coulombe R, Mason S, Lamorte L, Sturino CF, Bethell R. 2013. Identification and characterization of a novel HIV-1 nucleotide-competing reverse transcriptase inhibitor series. Antimicrob Agents Chemother 57:2712–2718. doi: 10.1128/AAC.00113-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Ehteshami M, Nijhuis M, Bernatchez JA, Ablenas CJ, McCormick S, de Jong D, Jochmans D, Gotte M. 2013. Formation of a quaternary complex of HIV-1 reverse transcriptase with a nucleotide-competing inhibitor and its ATP enhancer. J Biol Chem 288:17336–17346. doi: 10.1074/jbc.M112.433441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Xu HT, Colby-Germinario SP, Oliveira M, Rajotte D, Bethell R, Wainberg MA. 2014. Effects of the W153L substitution in HIV reverse transcriptase on viral replication and drug resistance to multiple categories of reverse transcriptase inhibitors. Antimicrob Agents Chemother 58:4515–4526. doi: 10.1128/AAC.02729-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Gu Z, Gao Q, Fang H, Salomon H, Parniak MA, Goldberg E, Cameron J, Wainberg MA. 1994. Identification of a mutation at codon 65 in the IKKK motif of reverse transcriptase that encodes human immunodeficiency virus resistance to 2′,3′-dideoxycytidine and 2′,3′-dideoxy-3′-thiacytidine. Antimicrob Agents Chemother 38:275–281. doi: 10.1128/AAC.38.2.275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Wainberg MA, Miller MD, Quan Y, Salomon H, Mulato AS, Lamy PD, Margot NA, Anton KE, Cherrington JM. 1999. In vitro selection and characterization of HIV-1 with reduced susceptibility to PMPA. Antivir Ther 4:87–94. [DOI] [PubMed] [Google Scholar]
  • 26.White KL, Margot NA, Wrin T, Petropoulos CJ, Miller MD, Naeger LK. 2002. Molecular mechanisms of resistance to human immunodeficiency virus type 1 with reverse transcriptase mutations K65R and K65R+M184V and their effects on enzyme function and viral replication capacity. Antimicrob Agents Chemother 46:3437–3446. doi: 10.1128/AAC.46.11.3437-3446.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Coutsinos D, Invernizzi CF, Xu H, Moisi D, Oliveira M, Brenner BG, Wainberg MA. 2009. Template usage is responsible for the preferential acquisition of the K65R reverse transcriptase mutation in subtype C variants of human immunodeficiency virus type 1. J Virol 83:2029–2033. doi: 10.1128/JVI.01349-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Le Grice SF, Gruninger-Leitch F. 1990. Rapid purification of homodimer and heterodimer HIV-1 reverse transcriptase by metal chelate affinity chromatography. Eur J Biochem 187:307–314. doi: 10.1111/j.1432-1033.1990.tb15306.x. [DOI] [PubMed] [Google Scholar]
  • 29.Arts EJ, Li X, Gu Z, Kleiman L, Parniak MA, Wainberg MA. 1994. Comparison of deoxyoligonucleotide and tRNA(Lys-3) as primers in an endogenous human immunodeficiency virus-1 in vitro reverse transcription/template-switching reaction. J Biol Chem 269:14672–14680. [PubMed] [Google Scholar]
  • 30.Xu HT, Quan Y, Asahchop E, Oliveira M, Moisi D, Wainberg MA. 2010. Comparative biochemical analysis of recombinant reverse transcriptase enzymes of HIV-1 subtype B and subtype C. Retrovirology 7:80. doi: 10.1186/1742-4690-7-80. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Mochizuki N, Otsuka N, Matsuo K, Shiino T, Kojima A, Kurata T, Sakai K, Yamamoto N, Isomura S, Dhole TN, Takebe Y, Matsuda M, Tatsumi M. 1999. An infectious DNA clone of HIV type 1 subtype C. AIDS Res Hum Retroviruses 15:1321–1324. doi: 10.1089/088922299310223. [DOI] [PubMed] [Google Scholar]
  • 32.Le Grice SF, Cameron CE, Benkovic SJ. 1995. Purification and characterization of human immunodeficiency virus type 1 reverse transcriptase. Methods Enzymol 262:130–144. doi: 10.1016/0076-6879(95)62015-X. [DOI] [PubMed] [Google Scholar]
  • 33.Miller MD, Lamy PD, Fuller MD, Mulato AS, Margot NA, Cihlar T, Cherrington JM. 1998. Human immunodeficiency virus type 1 reverse transcriptase expressing the K70E mutation exhibits a decrease in specific activity and processivity. Mol Pharmacol 54:291–297. [DOI] [PubMed] [Google Scholar]
  • 34.Quan Y, Brenner BG, Marlink RG, Essex M, Kurimura T, Wainberg MA. 2003. Drug resistance profiles of recombinant reverse transcriptases from human immunodeficiency virus type 1 subtypes A/E, B, and C. AIDS Res Hum Retroviruses 19:743–753. doi: 10.1089/088922203769232548. [DOI] [PubMed] [Google Scholar]
  • 35.Xu HT, Martinez-Cajas JL, Ntemgwa ML, Coutsinos D, Frankel FA, Brenner BG, Wainberg MA. 2009. Effects of the K65R and K65R/M184V reverse transcriptase mutations in subtype C HIV on enzyme function and drug resistance. Retrovirology 6:14. doi: 10.1186/1742-4690-6-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Xu HT, Asahchop EL, Oliveira M, Quashie PK, Quan Y, Brenner BG, Wainberg MA. 2011. Compensation by the E138K mutation in HIV-1 reverse transcriptase for deficits in viral replication capacity and enzyme processivity associated with the M184I/V mutations. J Virol 85:11300–11308. doi: 10.1128/JVI.05584-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Asahchop EL, Oliveira M, Wainberg MA, Brenner BG, Moisi D, Toni T, Tremblay CL. 2011. Characterization of the E138K resistance mutation in HIV-1 reverse transcriptase conferring susceptibility to etravirine in B and non-B HIV-1 subtypes. Antimicrob Agents Chemother 55:600–607. doi: 10.1128/AAC.01192-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Xu HT, Quan Y, Schader SM, Oliveira M, Bar-Magen T, Wainberg MA. 2010. The M230L nonnucleoside reverse transcriptase inhibitor resistance mutation in HIV-1 reverse transcriptase impairs enzymatic function and viral replicative capacity. Antimicrob Agents Chemother 54:2401–2408. doi: 10.1128/AAC.01795-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Schader SM, Oliveira M, Ibanescu RI, Moisi D, Colby-Germinario SP, Wainberg MA. 2012. In vitro resistance profile of the candidate HIV-1 microbicide drug dapivirine. Antimicrob Agents Chemother 56:751–756. doi: 10.1128/AAC.05821-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Asahchop EL, Wainberg MA, Oliveira M, Xu H, Brenner BG, Moisi D, Ibanescu IR, Tremblay C. 2013. Distinct resistance patterns to etravirine and rilpivirine in viruses containing nonnucleoside reverse transcriptase inhibitor mutations at baseline. AIDS 27:879–887. doi: 10.1097/QAD.0b013e32835d9f6d. [DOI] [PubMed] [Google Scholar]
  • 41.Frankel FA, Invernizzi CF, Oliveira M, Wainberg MA. 2007. Diminished efficiency of HIV-1 reverse transcriptase containing the K65R and M184V drug resistance mutations. AIDS 21:665–675. doi: 10.1097/QAD.0b013e3280187505. [DOI] [PubMed] [Google Scholar]
  • 42.Doualla-Bell F, Avalos A, Brenner B, Gaolathe T, Mine M, Gaseitsiwe S, Oliveira M, Moisi D, Ndwapi N, Moffat H, Essex M, Wainberg MA. 2006. High prevalence of the K65R mutation in human immunodeficiency virus type 1 subtype C isolates from infected patients in Botswana treated with didanosine-based regimens. Antimicrob Agents Chemother 50:4182–4185. doi: 10.1128/AAC.00714-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Doualla-Bell F, Avalos A, Gaolathe T, Mine M, Gaseitsiwe S, Ndwapi N, Novitsky VA, Brenner B, Oliveira M, Moisi D, Moffat H, Thior I, Essex M, Wainberg MA. 2006. Impact of human immunodeficiency virus type 1 subtype C on drug resistance mutations in patients from Botswana failing a nelfinavir-containing regimen. Antimicrob Agents Chemother 50:2210–2213. doi: 10.1128/AAC.01447-05. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Doualla-Bell F, Gaolathe T, Avalos A, Cloutier S, Ndwapi N, Holcroft C, Moffat H, Dickinson D, Essex M, Wainberg MA, Mine M. 2009. Five-year follow up of genotypic resistance patterns in HIV-1 subtype C infected patients in Botswana after failure of thymidine analogue-based regimens. J Int AIDS Soc 12:25. doi: 10.1186/1758-2652-12-25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Wainberg MA, Brenner BG. 2012. The impact of HIV genetic polymorphisms and subtype differences on the occurrence of resistance to antiretroviral drugs. Mol Biol Int 2012:256982. doi: 10.1155/2012/256982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Wainberg MA, Brenner BG. 2010. Role of HIV subtype diversity in the development of resistance to antiviral drugs. Viruses 2:2493–2508. doi: 10.3390/v2112493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Gupta RK, Chrystie IL, O'Shea S, Mullen JE, Kulasegaram R, Tong CY. 2005. K65R and Y181C are less prevalent in HAART-experienced HIV-1 subtype A patients. AIDS 19:1916–1919. doi: 10.1097/01.aids.0000189860.36688.e5. [DOI] [PubMed] [Google Scholar]
  • 48.Wensing AM, Calvez V, Gunthard HF, Johnson VA, Paredes R, Pillay D, Shafer RW, Richman DD. 2014. 2014 update of the drug resistance mutations in HIV-1. Top Antivir Med 22:642–650. [PMC free article] [PubMed] [Google Scholar]
  • 49.Deval J, Navarro JM, Selmi B, Courcambeck J, Boretto J, Halfon P, Garrido-Urbani S, Sire J, Canard B. 2004. A loss of viral replicative capacity correlates with altered DNA polymerization kinetics by the human immunodeficiency virus reverse transcriptase bearing the K65R and L74V dideoxynucleoside resistance substitutions. J Biol Chem 279:25489–25496. doi: 10.1074/jbc.M313534200. [DOI] [PubMed] [Google Scholar]
  • 50.Selmi B, Boretto J, Sarfati SR, Guerreiro C, Canard B. 2001. Mechanism-based suppression of dideoxynucleotide resistance by K65R human immunodeficiency virus reverse transcriptase using an alpha-boranophosphate nucleoside analogue. J Biol Chem 276:48466–48472. doi: 10.1074/jbc.M107003200. [DOI] [PubMed] [Google Scholar]
  • 51.White KL, Chen JM, Margot NA, Wrin T, Petropoulos CJ, Naeger LK, Swaminathan S, Miller MD. 2004. Molecular mechanisms of tenofovir resistance conferred by human immunodeficiency virus type 1 reverse transcriptase containing a diserine insertion after residue 69 and multiple thymidine analog-associated mutations. Antimicrob Agents Chemother 48:992–1003. doi: 10.1128/AAC.48.3.992-1003.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.White KL, Margot NA, Ly JK, Chen JM, Ray AS, Pavelko M, Wang R, McDermott M, Swaminathan S, Miller MD. 2005. A combination of decreased NRTI incorporation and decreased excision determines the resistance profile of HIV-1 K65R RT. AIDS 19:1751–1760. doi: 10.1097/01.aids.0000189851.21441.f1. [DOI] [PubMed] [Google Scholar]
  • 53.Ly JK, Margot NA, MacArthur HL, Hung M, Miller MD, White KL. 2007. The balance between NRTI discrimination and excision drives the susceptibility of HIV-1 RT mutants K65R, M184V and K65R+M184V. Antivir Chem Chemother 18:307–316. doi: 10.1177/095632020701800603. [DOI] [PubMed] [Google Scholar]
  • 54.Michailidis E, Ryan EM, Hachiya A, Kirby KA, Marchand B, Leslie MD, Huber AD, Ong YT, Jackson JC, Singh K, Kodama EN, Mitsuya H, Parniak MA, Sarafianos SG. 2013. Hypersusceptibility mechanism of tenofovir-resistant HIV to EFdA. Retrovirology 10:65. doi: 10.1186/1742-4690-10-65. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Sturino CF, Bousquet Y, James CA, DeRoy P, Duplessis M, Edwards PJ, Halmos T, Minville J, Morency L, Morin S, Thavonekham B, Tremblay M, Duan J, Ribadeneira M, Garneau M, Pelletier A, Tremblay S, Lamorte L, Bethell R, Cordingley MG, Rajotte D, Simoneau B. 2013. Identification of potent and orally bioavailable nucleotide competing reverse transcriptase inhibitors: in vitro and in vivo optimization of a series of benzofurano[3,2-d]pyrimidin-2-one derived inhibitors. Bioorg Med Chem Lett 23:3967–3975. doi: 10.1016/j.bmcl.2013.04.043. [DOI] [PubMed] [Google Scholar]

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