Hypersusceptibility mechanism of Tenofovir-resistant HIV to EFdA
© Michailidis et al.; licensee BioMed Central Ltd. 2013
Received: 22 April 2013
Accepted: 13 June 2013
Published: 24 June 2013
The K65R substitution in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is the major resistance mutation selected in patients treated with first-line antiretroviral tenofovir disoproxil fumarate (TDF). 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA), is the most potent nucleoside analog RT inhibitor (NRTI) that unlike all approved NRTIs retains a 3'-hydroxyl group and has remarkable potency against wild-type (WT) and drug-resistant HIVs. EFdA acts primarily as a chain terminator by blocking translocation following its incorporation into the nascent DNA chain. EFdA is in preclinical development and its effect on clinically relevant drug resistant HIV strains is critically important for the design of optimal regimens prior to initiation of clinical trials.
Here we report that the K65R RT mutation causes hypersusceptibility to EFdA. Specifically, in single replication cycle experiments we found that EFdA blocks WT HIV ten times more efficiently than TDF. Under the same conditions K65R HIV was inhibited over 70 times more efficiently by EFdA than TDF. We determined the molecular mechanism of this hypersensitivity using enzymatic studies with WT and K65R RT. This substitution causes minor changes in the efficiency of EFdA incorporation with respect to the natural dATP substrate and also in the efficiency of RT translocation following incorporation of the inhibitor into the nascent DNA. However, a significant decrease in the excision efficiency of EFdA-MP from the 3’ primer terminus appears to be the primary cause of increased susceptibility to the inhibitor. Notably, the effects of the mutation are DNA-sequence dependent.
We have elucidated the mechanism of K65R HIV hypersusceptibility to EFdA. Our findings highlight the potential of EFdA to improve combination strategies against TDF-resistant HIV-1 strains.
KeywordsHIV-1 RT EFdA K65R
Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is the major target of antiretroviral drug treatments. RT inhibitors constitute the largest class of HIV-1 drugs and are grouped in two separate categories. The first category consists of the nucleos(t)ide RT inhibitors (NRTIs), which are analogs of the natural nucleosides. Most NRTIs lack a 3’-OH and act as chain terminators by blocking DNA polymerization [1–8]. The other group includes the nonnucleoside RT inhibitors (NNRTIs), which are non-competitive RT inhibitors with respect to either dNTP or nucleic acid substrates and block DNA synthesis by binding to a hydrophobic pocket of RT [9–15]. Highly Active Antiretroviral Therapies (HAART) are based on combinations of antiretrovirals and have helped extend the lives of HIV-1 patients. However, the efficacy of combination therapies is being challenged by the selection of drug-resistant variants of HIV-1.
There are two major mechanisms of NRTI resistance [16, 17]. The first is the discrimination mechanism, which is based on decreased incorporation of the nucleotide analog into the elongating DNA over the canonical dNTP substrate [16, 18–21]. An example of this type of resistance is conferred by the M184V mutation, which decreases HIV susceptibility to lamivudine (3TC) and emtricitabine (FTC) [20–24]. The second mechanism is the excision mechanism, which is based on the enhanced ability of the mutant RT to remove the chain-terminating inhibitor from the DNA terminus [25–28] through a phosphorolytic reaction that uses primarily adenosine triphosphate (ATP) as a substrate. Upon removal of the inhibitor DNA synthesis resumes. The excision reaction is facilitated by Excision Enhancement Mutations (EEMs), typically M41L, D67N, K70R, T215Y/F, L210W, and K219E/Q, which are also known as Thymidine Associated Mutations (TAMs) because they were historically linked to resistance to thymidine analogs AZT and d4T [29, 30].
Tenofovir disoproxil fumarate (TDF) is one of the most prescribed anti-HIV drugs, and is described as a key component of all first-line regimens in the DHHS HIV guidelines (http://aidsinfo.nih.gov/contentfiles/lvguidelines/adultandadolescentgl.pdf). The K65R mutation in HIV-1 RT is the signature mutation selected during tenofovir-based therapy. Viruses carrying K65R have reduced susceptibility to tenofovir and other NRTIs, but remain susceptible to zidovudine (AZT) [31–36]. This mutation has also been associated with a reduction in viral replication capacity, NRTI excision, NRTI incorporation, and dNTP incorporation [37–43]. Recent crystallographic data suggest that the K65R mutation disrupts the interaction between the side chains of 65R and 72R resulting in structural changes that lead to NRTI resistance .
We have previously shown that a series of NRTIs with 4’-substitutions and a 3’-OH group are very potent inhibitors of WT and multi-drug resistant HIV-1. The most effective of these compounds is the adenosine analog 4’-ethynyl-2-fluoro-2’-deoxyadenosine (EFdA) [45, 46]. We have demonstrated that EFdA acts in a DNA-sequence specific manner, primarily inhibiting DNA synthesis as an immediate chain terminator, but less often, at some DNA sequences can also act as a delayed chain terminator . Compounds that exhibit this novel mechanism of inhibition have been dubbed Translocation Defective Reverse Transcriptase Inhibitors (TDRTIs) .
In an effort to investigate the effect of EFdA against drug-resistant strains of HIV-1 we found that RT mutation K65R confers hypersusceptibility to EFdA. We carried out a series of biochemical experiments to elucidate the mechanism of this phenomenon and we propose here that K65R increases the susceptibility to EFdA mainly by suppressing the ATP- or PPi-dependent repair of EFdA-MP-terminated DNA. Understanding the molecular basis of K65R hypersusceptibility to EFdA may lead to new and more effective combination therapies.
The K65R RT mutation enhances susceptibility of HIV to EFdA
EC 50 determination of EFdA and TDF in single cycle cell-based assays
EC50 ± SD (nM) (Fold change)
3.2 ± 0.7
32 ± 6
1.3 ± 0.4
96 ± 3
The K65R mutation enhances susceptibility of RT to EFdA-TP
Enhancement of hypersusceptibility to EFdA-TP under ATP-based excision conditions
IC50(nM) of EFdA-TP ± SD (Fold change)
Hypersusceptibility enhancement in the presence of ATP
With 3.5 mM ATP
186 ± 40a (1)b
318 ± 99a (1)b
125 ± 28a (0.7)b
131 ± 28a (0.4)b
Sequences of oligonucleotides used in this study
5’CCA TAG ATA GCA TTG GTG CTC GAA CAG TGA C
5’Cy3 GTC ACT GTT CGA GCA CCA
5’CCA TAG ATA GCA TTG GTG CTC GAA CA
5’Cy3 TGT TCG AGC ACC AAT GCT
5’AAA AAA AAA TGG ATA CAT ATG GTT AAA GTA T
5’Cy3 ATA CTT TAA CCA TAT GTA TCC
5’Cy3 CCA TAG ATA GCA T TG GTG CTC GAA CAG TGA CAA TCA GTG TAG A
5’TCT ACA CTG ATT GTC ACT GTT CGA GCA CCA
The K65R RT mutation does not enhance susceptibility to EFdA by significantly affecting incorporation of the inhibitor
Steady state kinetic parameters for EFdA-TP and dATP incorporation by WT and K65R HIV-1 RTs
K m (μM)
kcat/K m (min-1 · μM-1)
T d26 /P d18-P5
2.38 ± 0.27
1.58 ± 0.12
0.66 ± 0.04
3.26 ± 0.42
5.99 ± 0.81
0.92 ± 0.14
1.83 ± 0.34
1.74 ± 0.42
T d31 /P d18-P0
0.33 ± 0.07
6.32 ± 0.14
0.23 ± 0.01
4.73 ± 0.33
0.42 ± 0.01
3.62 ± 0.71
0.31 ± 0.02
3.07 ± 0.15
T d31A /P d21
0.37 ± 0.08
3.64 ± 0.57
0.19 ± 0.06
3.54 ± 0.44
1.06 ± 0.11
3.68 ± 0.28
0.53 ± 0.05
3.38 ± 0.29
The K65R RT mutation does not enhance susceptibility to EFdA by significantly affecting enzyme translocation on EFdA-MP-terminated template/primers
ATP- and PPi-dependent Excision/Rescue of EFdA-MP
Tenofovir is a major component of current antiviral therapies (http://aidsinfo.nih.gov/contentfiles/lvguidelines/adultandadolescentgl.pdf) and new HIV drugs are likely to be used in patients that have failed tenofovir-based treatment. Hence, the ability of novel HIV inhibitors to efficiently block tenofovir-resistant viruses is critical for their potential utility as HIV therapeutics. The clinical cut-off for tenofovir resistance is defined as a 2.1-fold reduction in virological response. It is associated with the presence of the tenofovir-resistance signature mutation K65R in the reverse transcriptase gene . We report here that EFdA is highly potent against tenofovir-resistant K65R HIV, and inhibits this mutant 2.5-fold more efficiently than WT HIV. Given the fact that clinical resistance to tenofovir is considered a 2.1-fold decrease in susceptibility, we consider a 2-fold increase in susceptibility as significant hypersusceptibility. Understanding the mechanism by which HIV becomes resistant or more susceptible to EFdA could allow us to overcome drug resistance challenges and improve the current combination therapies. We have previously demonstrated that EFdA is highly efficient in suppressing viral replication of clinical isolates harboring signature mutations to other NRTIs and NNRTIs, including isolates containing 3TC/FTC resistance mutation M184V; TAMs or Q151M complex mutations that confer resistance to AZT, d4T, and abacavir; and nevirapine and efavirenz resistance mutations K103N and Y181C . In addition, we have recently shown that EFdA is 3 logs more potent in SIV inhibition than tenofovir, AZT, and 3TC, and EFdA treatment decreases viral load in SIV-infected macaques by 3–4 logs within 1 week of SIV therapy and eventually to non-detectable levels . The present study demonstrates that the K65R tenofovir-resistance RT mutation confers HIV hypersensitivity to EFdA compared to WT HIV. Other studies have shown that NRTI resistance mutations can confer enhanced susceptibility to other NRTIs. Specifically, the K65R and to a lesser extent the L74V RT mutations have been reported to suppress AZT resistance [43, 52–55]. In addition, we have previously reported that K65R and L74V HIVs can be hypersusceptible to NRTIs with 4’-ethynyl substitutions [45, 56]. The NNRTI-resistance mutation Y181C also increases susceptibility to AZT [57, 58]. Moreover, the 3TC/FTC-resistance mutation M184V also increases HIV sensitivity to AZT by decreasing the excision efficiency of AZT-MP [22, 53, 59–61]. Finally, we have recently shown that the 172K polymorphism can enhance susceptibility to both NRTIs and NNRTIs .
We have provided virological and biochemical data demonstrating that the K65R RT mutation confers enhanced sensitivity to EFdA. We reported here that the mechanism of hypersensitivity is mainly through reduced excision of the chain terminating EFdA-MP. Our findings demonstrated that EFdA is a very potent NRTI and it could be used not only against WT HIV but also against tenofovir-resistant HIVs. The primary resistance mutation for EFdA is M184V and combination with tenofovir could be similar to the pair of mutations for 3TC/AZT combination. Unlike AZT and 3TC which are analogs of different deoxynucleosides, EFdA and tenofovir are both deoxyadenosine analogs and would theoretically compete to each other. However, they are activated/phosphorylated by different pathways . Therefore, combination of EFdA with tenofovir could help suppress K65R resistance. This conclusion has significant potential therapeutic implications. Moreover, EFdA would be a good candidate in salvage therapies for patients that fail tenofovir-treatment due to K65R resistance.
Cells and viruses
TZM-bl cells (CCR5 transduced HeLa-CD4/LTR-β-gal and luciferase cells) were obtained from the AIDS Research and Reference Reagent Program, the National Institutes of Health (NIH). 293T and TZM-bl cells were maintained in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100 μg/ml streptomycin, and used for transfection and antiviral assays, respectively.
K65R RT mutation was introduced by site-directed mutagenesis as described previously [63, 64]. Briefly, the desired mutations were introduced into the Xma I - NheI region (759 bp) of pTZNX1, which encodes nucleotides Gly-15 to Ala-267 of HIV-1 RT. After mutagenesis, the XmaI - NheI cassettes were inserted back into pNL101 and confirmed by sequencing. Viral stocks were obtained by transfection of each molecular clone into 293T cells using Fugene 6 (Roche, Mannheim, Germany), harvested and stored at −80°C until use.
Cell-based drug susceptibility assays
Single-replication-cycle drug susceptibility assays were performed in triplicates using TZM-bl cells. TZM-bl cells were infected with diluted virus stock at 400,000 relative light units (RLU) in the presence of increasing concentrations of RTIs and cultured for 48 h. The luciferase marker gene expressions were measured using the Bright-Glo (Promega, WI). Susceptibility to NRTIs was calculated as the concentration that reduces RLU (infection) by 50% (50% effective concentration [EC50]). The data were obtained from the results of at least three independent experiments and the P values were determined using t-test statistical analysis.
Enzymes and nucleic acids
HIV-1 RTs were expressed in JM-109 (Invitrogen) bacteria and purified by nickel affinity chromatography and monoQ anion exchange chromatography as previously described [46, 65–69]. Oligonucleotides used in this study were chemically synthesized and purchased from Integrated DNA Technologies (Coralville, IA). Sequences of the DNA substrates are shown in Table 3. Deoxynucleotide triphosphates and dideoxynucleotide triphosphates were purchased from Fermentas (Glen Burnie, MD). EFdA was synthesized by Yamasa Corporation (Chiba, Japan) as described before . Using EFdA as starting material the triphosphate form EFdA-TP was synthesized by TriLink BioTechnologies (San Diego, CA). Concentrations of nucleotides and EFdA-TP were calculated spectrophotometrically on the basis of absorption at 260 nm and their extinction coefficients. All nucleotides were treated with inorganic pyrophosphatase (Roche Diagnostics) as described previously  to remove traces of PPi contamination that might interfere with the rescue assay.
Enzymatic drug susceptibility assays
Inhibition of HIV-1 RT-catalyzed DNA Synthesis by EFdA-TP
DNA template was annealed to 5’-Cy3 labeled DNA primer (3:1 molar ratio) (Td31/Pd18-P0). To monitor primer extension, the DNA/DNA hybrid (20 nM) was incubated at 37°C with WT or K65R HIV-1 RT (20 nM) in a buffer containing 50 mM Tris (pH 7.8) and 50 mM NaCl (RT buffer). Subsequently, varying amounts of EFdA-TP were added and the reactions were initiated by the addition of 6 or 10 mM MgCl2 in a final volume of 20 μl. All dNTPs were present at a final concentration of 1 μM in the presence or absence of 3.5 mM ATP. The reactions were terminated after 50 minutes by adding equal volume of 100% formamide containing traces of bromophenol blue. The products were resolved on 15% polyacrylamide 7 M urea gels. In this and in subsequent assays the gels were scanned with a Typhoon FLA 9000 PhosphorImager (GE Healthcare, NJ). The bands corresponding to fully-extended product were quantified using Multi Gauge software. The results of at least four independent experiments were plotted as percent full extension using one site-competition nonlinear regression in GraphPad Prism 4 to determine the mean and standard deviation of the IC50 for EFdA-TP.
Single nucleotide incorporation of dATP and EFdA-TP by WT and K65R RTs
Steady-state kinetic parameters K m and k cat for incorporation of EFdA-TP or dATP were determined using single nucleotide incorporation in gel-based assays under saturating concentrations of T/P (10-fold excess over RT). Reactions were carried out in RT buffer, 6 mM MgCl2, 100 nM Td26/Pd18-P5 or Td31/Pd18-P0 or Td31A/Pd21 (Table 3) and 10 nM WT or K65R HIV-1 RT in a final volume of 20 μl and stopped at indicated reaction times. The products were resolved and quantified as described above. K m and k cat were determined graphically using the Michaelis-Menten equation. Reactions were carried out in two to four independent experiments to determine the mean and standard deviation values.
Site-specific Fe2+footprinting assay
Site-specific Fe2+ footprints were monitored on 5'-Cy3-labeled DNA templates. 100 nM of 5’-Cy3-Td43/Pd30 was incubated with 600 nM WT or K65R HIV-1 RT in a buffer containing 120 mM sodium cacodylate (pH 7), 20 mM NaCl, 6 mM MgCl2, and 1 μM EFdA-TP, to allow quantitative chain-termination. Prior to the treatment with Fe2+, complexes were pre-incubated for 7 min with increasing concentrations of the next incoming nucleotide (dTTP). The complexes were treated with ammonium iron sulfate (1 mM) as previously described [46, 47]. This reaction relies on autoxidation of Fe2+ to create a local concentration of hydroxyl radicals, which cleave the DNA at the nucleotide closest to the Fe2+ specifically bound to the RNase H active site. These experiments were performed at least twice.
ATP- and PPi-dependent excision and rescue of T/PEFdA-MP
ATP-dependent rescue of T/PEFdA-MP
Template/primer with EFdA-MP at the 3’ primer terminus (T/PEFdA-MP) was prepared by incubating 500 nM Td31/Pd18-P0 with 1 μM HIV-1 RT in RT buffer and 6 mM MgCl2. EFdA-TP was added into the reaction and the mixture was incubated at 37°C for 1 h. After incorporation of EFdA-TP, the T/PEFdA-MP was purified using the QIAquick nucleotide removal kit (Qiagen, Valencia, CA). Under these conditions, the extension of T/P to T/PEFdA-MP was complete. 20 nM of purified Td31/Pd18-P0-EFdA-MP was incubated with 60 nM WT or K65R HIV-1 RT in the presence of 3.5 mM ATP, 100 μM dATP, 0.5 μM dTTP, and 10 μM ddGTP in RT buffer and 10 mM MgCl2. Aliquots of the reaction were stopped at different time points (0–90 min). The data from at least four independent experiments were analyzed using GraphPad Prism 4.
PPi-dependent rescue of T/PEFdA-MP
20 nM of purified Td31/Pd18-P0-EFdA-MP was incubated at 37°C with 60 nM WT or K65R HIV-1 RT in the presence of 150 μM PPi, 100 μM dATP, 0.5 μM dTTP, and 10 μM ddGTP in RT buffer and 6 mM MgCl2. Aliquots of the reaction were stopped at different times (0–40 min). The data from at least two independent experiments were plotted using GraphPad Prism 4.
Molecular models of dATP and EFdA-TP in the active site of WT HIV RT were made using PDB ID 1 T05  as a starting model (WT HIV RT in complex with tenofovir diphosphate). A molecular model of EFdA-TP in the active site of K65R HIV RT was made using PDB ID 3JYT  as a starting model (K65R HIV RT in complex with dATP). The sketch module of SYBYL (Version 7.3.5, Tripos International, St. Louis, MO) was used to make dATP and EFdA-TP molecules. dATP and EFdA-TP were each superposed to tenofovir diphosphate in the WT complex, after which the tenofovir diphosphate was removed. Gasteiger-Huckel charges were calculated and molecular minimization of the WT-dATP and WT-EFdA-TP were performed in SYBYL using the Powell method. SYBYL was also used to add the 2-fluoro and 4’-ethynyl groups to dATP in the K65R complex. Gasteiger-Huckel charges were then calculated and molecular minimization was performed as for the WT complexes.
Human immunodeficiency virus
Nucleoside reverse transcriptase inhibitor
Translocation-defective RT inhibitor
Tenofovir disoproxil fumarate
Template/primer possessing EFdA-MP at the 3'-primer terminus (or T/P chain terminated by EFdA-MP).
Dr. Tatiana Ilina’s contribution in the preparation of the RT plasmids is acknowledged. This work was supported, in whole or in part, by National Institutes of Health grants AI076119, AI074389, AI076119-S1, AI076119-02S1, AI100890, AI099284, and GM103368 (S. G. S.) and AI079801 (M. A. P.).
- Parniak MA, Sluis-Cremer N: Inhibitors of HIV-1 reverse transcriptase. Advances in pharmacology (San Diego, Calif). 2000, 49: 67-109.View ArticleGoogle Scholar
- Sharma PL, Nurpeisov V, Hernandez-Santiago B, Beltran T, Schinazi RF: Nucleoside inhibitors of human immunodeficiency virus type 1 reverse transcriptase. Curr Top Med Chem. 2004, 4: 895-919.View ArticlePubMedGoogle Scholar
- Hammer SM, Saag MS, Schechter M, Montaner JS, Schooley RT, Jacobsen DM, Thompson MA, Carpenter CC, Fischl MA, Gazzard BG, et al: Treatment for adult HIV infection: 2006 recommendations of the International AIDS Society–USA panel. Top HIV Med. 2006, 14: 827-843.PubMedGoogle Scholar
- Schinazi RF, Hernandez-Santiago BI, Hurwitz SJ: Pharmacology of current and promising nucleosides for the treatment of human immunodeficiency viruses. Antiviral Res. 2006, 71: 322-334. 10.1016/j.antiviral.2006.03.012.View ArticlePubMedGoogle Scholar
- De Clercq E: Anti-HIV drugs. Verh K Acad Geneeskd Belg. 2007, 69: 81-104.PubMedGoogle Scholar
- Sluis-Cremer N, Tachedjian G: Mechanisms of inhibition of HIV replication by non-nucleoside reverse transcriptase inhibitors. Virus Res. 2008, 134: 147-156. 10.1016/j.virusres.2008.01.002.PubMed CentralView ArticlePubMedGoogle Scholar
- Deval J: Antimicrobial strategies: inhibition of viral polymerases by 3'-hydroxyl nucleosides. Drugs. 2009, 69: 151-166. 10.2165/00003495-200969020-00002.View ArticlePubMedGoogle Scholar
- Sarafianos SG, Marchand B, Das K, Himmel DM, Parniak MA, Hughes SH, Arnold E: Structure and Function of HIV-1 Reverse Transcriptase: Molecular Mechanisms of Polymerization and Inhibition. J Mol Biol. 2009, 385: 693-713. 10.1016/j.jmb.2008.10.071.PubMed CentralView ArticlePubMedGoogle Scholar
- Ren J, Esnouf R, Garman E, Somers D, Ross C, Kirby I, Keeling J, Darby G, Jones Y, Stuart D, et al: High resolution structures of HIV-1 RT from four RT-inhibitor complexes. Nat Struct Biol. 1995, 2: 293-302. 10.1038/nsb0495-293.View ArticlePubMedGoogle Scholar
- Ding J, Das K, Hsiou Y, Sarafianos SG, Clark AD, Jacobo-Molina A, Tantillo C, Hughes SH, Arnold E: Structure and functional implications of the polymerase active site region in a complex of HIV-1 RT with a double-stranded DNA template-primer and an antibody Fab fragment at 2.8 A resolution. J Mol Biol. 1998, 284: 1095-1111. 10.1006/jmbi.1998.2208.View ArticlePubMedGoogle Scholar
- Kohlstaedt LA, Wang J, Friedman JM, Rice PA, Steitz TA: Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor. Science. 1992, 256: 1783-1790. 10.1126/science.1377403.View ArticlePubMedGoogle Scholar
- Rodgers DW, Gamblin SJ, Harris BA, Ray S, Culp JS, Hellmig B, Woolf DJ, Debouck C, Harrison SC: The structure of unliganded reverse transcriptase from the human immunodeficiency virus type 1. Proc Natl Acad Sci USA. 1995, 92: 1222-1226. 10.1073/pnas.92.4.1222.PubMed CentralView ArticlePubMedGoogle Scholar
- Hsiou Y, Ding J, Das K, Clark AD, Hughes SH, Arnold E: Structure of unliganded HIV-1 reverse transcriptase at 2.7 A resolution: implications of conformational changes for polymerization and inhibition mechanisms. Structure. 1996, 4: 853-860. 10.1016/S0969-2126(96)00091-3.View ArticlePubMedGoogle Scholar
- Spence RA, Kati WM, Anderson KS, Johnson KA: Mechanism of inhibition of HIV-1 reverse transcriptase by nonnucleoside inhibitors. Science. 1995, 267: 988-993. 10.1126/science.7532321.View ArticlePubMedGoogle Scholar
- Rittinger K, Divita G, Goody RS: Human immunodeficiency virus reverse transcriptase substrate-induced conformational changes and the mechanism of inhibition by nonnucleoside inhibitors. Proc Natl Acad Sci USA. 1995, 92: 8046-8049. 10.1073/pnas.92.17.8046.PubMed CentralView ArticlePubMedGoogle Scholar
- Sluis-Cremer N, Arion D, Parniak MA: Molecular mechanisms of HIV-1 resistance to nucleoside reverse transcriptase inhibitors (NRTIs). Cell Mol Life Sci. 2000, 57: 1408-1422. 10.1007/PL00000626.View ArticlePubMedGoogle Scholar
- Menendez-Arias L: Mechanisms of resistance to nucleoside analogue inhibitors of HIV-1 reverse transcriptase. Virus Res. 2008, 134: 124-146. 10.1016/j.virusres.2007.12.015.View ArticlePubMedGoogle Scholar
- Martin-Hernandez AM, Domingo E, Menendez-Arias L: Human immunodeficiency virus type 1 reverse transcriptase: role of Tyr115 in deoxynucleotide binding and misinsertion fidelity of DNA synthesis. EMBO J. 1996, 15: 4434-4442.PubMed CentralPubMedGoogle Scholar
- Gao G, Orlova M, Georgiadis MM, Hendrickson WA, Goff SP: Conferring RNA polymerase activity to a DNA polymerase: a single residue in reverse transcriptase controls substrate selection. Proc Natl Acad Sci USA. 1997, 94: 407-411. 10.1073/pnas.94.2.407.PubMed CentralView ArticlePubMedGoogle Scholar
- Gao HQ, Boyer PL, Sarafianos SG, Arnold E, Hughes SH: The role of steric hindrance in 3TC resistance of human immunodeficiency virus type-1 reverse transcriptase. J Mol Biol. 2000, 300: 403-418. 10.1006/jmbi.2000.3823.View ArticlePubMedGoogle Scholar
- Sarafianos SG, Das K, Clark AD, Ding J, Boyer PL, Hughes SH, Arnold E: Lamivudine (3TC) resistance in HIV-1 reverse transcriptase involves steric hindrance with beta-branched amino acids. Proc Natl Acad Sci USA. 1999, 96: 10027-10032. 10.1073/pnas.96.18.10027.PubMed CentralView ArticlePubMedGoogle Scholar
- Tisdale M, Kemp SD, Parry NR, Larder BA: Rapid in vitro selection of human immunodeficiency virus type 1 resistant to 3'-thiacytidine inhibitors due to a mutation in the YMDD region of reverse transcriptase. Proc Natl Acad Sci USA. 1993, 90: 5653-5656. 10.1073/pnas.90.12.5653.PubMed CentralView ArticlePubMedGoogle Scholar
- Feng JY, Anderson KS: Mechanistic studies examining the efficiency and fidelity of DNA synthesis by the 3TC-resistant mutant (184V) of HIV-1 reverse transcriptase. Biochemistry. 1999, 38: 9440-9448. 10.1021/bi990709m.View ArticlePubMedGoogle Scholar
- Back NK, Nijhuis M, Keulen W, Boucher CA, Oude Essink BO, van Kuilenburg AB, van Gennip AH, Berkhout B: Reduced replication of 3TC-resistant HIV-1 variants in primary cells due to a processivity defect of the reverse transcriptase enzyme. EMBO J. 1996, 15: 4040-4049.PubMed CentralPubMedGoogle Scholar
- Arion D, Kaushik N, McCormick S, Borkow G, Parniak MA: Phenotypic mechanism of HIV-1 resistance to 3'-azido-3'-deoxythymidine (AZT): increased polymerization processivity and enhanced sensitivity to pyrophosphate of the mutant viral reverse transcriptase. Biochemistry. 1998, 37: 15908-15917. 10.1021/bi981200e.View ArticlePubMedGoogle Scholar
- Meyer PR, Matsuura SE, So AG, Scott WA: Unblocking of chain-terminated primer by HIV-1 reverse transcriptase through a nucleotide-dependent mechanism. Proc Natl Acad Sci USA. 1998, 95: 13471-13476. 10.1073/pnas.95.23.13471.PubMed CentralView ArticlePubMedGoogle Scholar
- Meyer PR, Matsuura SE, Mian AM, So AG, Scott WA: A mechanism of AZT resistance: an increase in nucleotide-dependent primer unblocking by mutant HIV-1 reverse transcriptase. Mol Cell. 1999, 4: 35-43. 10.1016/S1097-2765(00)80185-9.View ArticlePubMedGoogle Scholar
- Meyer PR, Matsuura SE, Tolun AA, Pfeifer I, So AG, Mellors JW, Scott WA: Effects of specific zidovudine resistance mutations and substrate structure on nucleotide-dependent primer unblocking by human immunodeficiency virus type 1 reverse transcriptase. Antimicrob Agents Chemother. 2002, 46: 1540-1545. 10.1128/AAC.46.5.1540-1545.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Boyer PL, Sarafianos SG, Arnold E, Hughes SH: Selective excision of AZTMP by drug-resistant human immunodeficiency virus reverse transcriptase. J Virol. 2001, 75: 4832-4842. 10.1128/JVI.75.10.4832-4842.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Larder BA, Kemp SD: Multiple mutations in HIV-1 reverse transcriptase confer high-level resistance to zidovudine (AZT). Science. 1989, 246: 1155-1158. 10.1126/science.2479983.View ArticlePubMedGoogle Scholar
- Winters MA, Shafer RW, Jellinger RA, Mamtora G, Gingeras T, Merigan TC: Human immunodeficiency virus type 1 reverse transcriptase genotype and drug susceptibility changes in infected individuals receiving dideoxyinosine monotherapy for 1 to 2 years. Antimicrob Agents Chemother. 1997, 41: 757-762.PubMed CentralPubMedGoogle Scholar
- Harrigan PR, Stone C, Griffin P, Najera I, Bloor S, Kemp S, Tisdale M, Larder B: Resistance profile of the human immunodeficiency virus type 1 reverse transcriptase inhibitor abacavir (1592U89) after monotherapy and combination therapy. CNA2001 Investigative Group. J Infect Dis. 2000, 181: 912-920. 10.1086/315317.View ArticlePubMedGoogle Scholar
- Margot NA, Lu B, Cheng A, Miller MD, Study T: Resistance development over 144 weeks in treatment-naive patients receiving tenofovir disoproxil fumarate or stavudine with lamivudine and efavirenz in Study 903. HIV Med. 2006, 7: 442-450. 10.1111/j.1468-1293.2006.00404.x.View ArticlePubMedGoogle Scholar
- Wainberg MA, Miller MD, Quan Y, Salomon H, Mulato AS, Lamy PD, Margot NA, Anton KE, Cherrington JM: In vitro selection and characterization of HIV-1 with reduced susceptibility to PMPA. Antivir Ther. 1999, 4: 87-94.PubMedGoogle Scholar
- Margot NA, Isaacson E, McGowan I, Cheng AK, Schooley RT, Miller MD: Genotypic and phenotypic analyses of HIV-1 in antiretroviral-experienced patients treated with tenofovir DF. AIDS. 2002, 16: 1227-1235. 10.1097/00002030-200206140-00004.View ArticlePubMedGoogle Scholar
- Feng JY, Myrick FT, Margot NA, Mulamba GB, Rimsky L, Borroto-Esoda K, Selmi B, Canard B: Virologic and enzymatic studies revealing the mechanism of K65R- and Q151M-associated HIV-1 drug resistance towards emtricitabine and lamivudine. Nucleosides Nucleotides Nucleic Acids. 2006, 25: 89-107. 10.1080/15257770500379157.View ArticlePubMedGoogle Scholar
- Shah FS, Curr KA, Hamburgh ME, Parniak M, Mitsuya H, Arnez JG, Prasad VR: Differential influence of nucleoside analog-resistance mutations K65R and L74V on the overall mutation rate and error specificity of human immunodeficiency virus type 1 reverse transcriptase. J Biol Chem. 2000, 275: 27037-27044.PubMedGoogle Scholar
- Frankel FA, Invernizzi CF, Oliveira M, Wainberg MA: Diminished efficiency of HIV-1 reverse transcriptase containing the K65R and M184V drug resistance mutations. AIDS. 2007, 21: 665-675. 10.1097/QAD.0b013e3280187505.View ArticlePubMedGoogle Scholar
- Parikh UM, Zelina S, Sluis-Cremer N, Mellors JW: Molecular mechanisms of bidirectional antagonism between K65R and thymidine analog mutations in HIV-1 reverse transcriptase. AIDS. 2007, 21: 1405-1414. 10.1097/QAD.0b013e3281ac229b.View ArticlePubMedGoogle Scholar
- White KL, Margot NA, Ly JK, Chen JM, Ray AS, Pavelko M, Wang R, McDermott M, Swaminathan S, Miller MD: A combination of decreased NRTI incorporation and decreased excision determines the resistance profile of HIV-1 K65R RT. AIDS. 2005, 19: 1751-1760. 10.1097/01.aids.0000189851.21441.f1.View ArticlePubMedGoogle Scholar
- Deval J, Navarro JM, Selmi B, Courcambeck J, Boretto J, Halfon P, Garrido-Urbani S, Sire J, Canard B: 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. 2004, 279: 25489-25496. 10.1074/jbc.M313534200.View ArticlePubMedGoogle Scholar
- Deval J, White KL, Miller MD, Parkin NT, Courcambeck J, Halfon P, Selmi B, Boretto J, Canard B: Mechanistic basis for reduced viral and enzymatic fitness of HIV-1 reverse transcriptase containing both K65R and M184V mutations. J Biol Chem. 2004, 279: 509-516.View ArticlePubMedGoogle Scholar
- White KL, Chen JM, Feng JY, Margot NA, Ly JK, Ray AS, Macarthur HL, McDermott MJ, Swaminathan S, Miller MD: The K65R reverse transcriptase mutation in HIV-1 reverses the excision phenotype of zidovudine resistance mutations. Antivir Ther. 2006, 11: 155-163.PubMedGoogle Scholar
- Das K, Bandwar RP, White KL, Feng JY, Sarafianos SG, Tuske S, Tu X, Clark AD, Boyer PL, Hou X, et al: Structural basis for the role of the K65R mutation in HIV-1 reverse transcriptase polymerization, excision antagonism, and tenofovir resistance. J Biol Chem. 2009, 284: 35092-35100. 10.1074/jbc.M109.022525.PubMed CentralView ArticlePubMedGoogle Scholar
- Kawamoto A, Kodama E, Sarafianos SG, Sakagami Y, Kohgo S, Kitano K, Ashida N, Iwai Y, Hayakawa H, Nakata H, et al: 2'-deoxy-4'-C-ethynyl-2-halo-adenosines active against drug-resistant human immunodeficiency virus type 1 variants. Int J Biochem Cell Biol. 2008, 40: 2410-2420. 10.1016/j.biocel.2008.04.007.View ArticlePubMedGoogle Scholar
- Michailidis E, Marchand B, Kodama EN, Singh K, Matsuoka M, Kirby KA, Ryan EM, Sawani AM, Nagy E, Ashida N, et al: Mechanism of inhibition of HIV-1 reverse transcriptase by 4'-Ethynyl-2-fluoro-2'-deoxyadenosine triphosphate, a translocation-defective reverse transcriptase inhibitor. J Biol Chem. 2009, 284: 35681-35691. 10.1074/jbc.M109.036616.PubMed CentralView ArticlePubMedGoogle Scholar
- Marchand B, Gotte M: Site-specific footprinting reveals differences in the translocation status of HIV-1 reverse transcriptase. Implications for polymerase translocation and drug resistance. J Biol Chem. 2003, 278: 35362-35372. 10.1074/jbc.M304262200.View ArticlePubMedGoogle Scholar
- Ray AS, Murakami E, Basavapathruni A, Vaccaro JA, Ulrich D, Chu CK, Schinazi RF, Anderson KS: Probing the molecular mechanisms of AZT drug resistance mediated by HIV-1 reverse transcriptase using a transient kinetic analysis. Biochemistry. 2003, 42: 8831-8841. 10.1021/bi034435l.View ArticlePubMedGoogle Scholar
- Tu X, Das K, Han Q, Bauman JD, Clark AD, Hou X, Frenkel YV, Gaffney BL, Jones RA, Boyer PL, et al: Structural basis of HIV-1 resistance to AZT by excision. Nat Struct Mol Biol. 2010, 17: 1202-1209. 10.1038/nsmb.1908.PubMed CentralView ArticlePubMedGoogle Scholar
- Winters B, Montaner J, Harrigan PR, Gazzard B, Pozniak A, Miller MD, Emery S, van Leth F, Robinson P, Baxter JD, et al: Determination of clinically relevant cutoffs for HIV-1 phenotypic resistance estimates through a combined analysis of clinical trial and cohort data. J Acquir Immune Defic Syndr. 2008, 48: 26-34. 10.1097/QAI.0b013e31816d9bf4.View ArticlePubMedGoogle Scholar
- Murphey-Corb M, Rajakumar P, Michael H, Nyaundi J, Didier PJ, Reeve AB, Mitsuya H, Sarafianos SG, Parniak MA: Response of simian immunodeficiency virus to the novel nucleoside reverse transcriptase inhibitor 4'-ethynyl-2-fluoro-2'-deoxyadenosine in vitro and in vivo. Antimicrob Agents Chemother. 2012, 56: 4707-4712. 10.1128/AAC.00723-12.PubMed CentralView ArticlePubMedGoogle Scholar
- St Clair MH, Martin JL, Tudor-Williams G, Bach MC, Vavro CL, King DM, Kellam P, Kemp SD, Larder BA: Resistance to ddI and sensitivity to AZT induced by a mutation in HIV-1 reverse transcriptase. Science. 1991, 253: 1557-1559. 10.1126/science.1716788.View ArticlePubMedGoogle Scholar
- Quan Y, Gu Z, Li X, Liang C, Parniak MA, Wainberg MA: Endogenous reverse transcriptase assays reveal synergy between combinations of the M184V and other drug resistance-conferring mutations in interactions with nucleoside analog triphosphates. J Mol Biol. 1998, 277: 237-247. 10.1006/jmbi.1997.1592.View ArticlePubMedGoogle Scholar
- Bazmi HZ, Hammond JL, Cavalcanti SC, Chu CK, Schinazi RF, Mellors JW: In vitro selection of mutations in the human immunodeficiency virus type 1 reverse transcriptase that decrease susceptibility to (−)-beta-D-dioxolane-guanosine and suppress resistance to 3'-azido-3'-deoxythymidine. Antimicrob Agents Chemother. 2000, 44: 1783-1788. 10.1128/AAC.44.7.1783-1788.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Parikh UM, Bacheler L, Koontz D, Mellors JW: The K65R mutation in human immunodeficiency virus type 1 reverse transcriptase exhibits bidirectional phenotypic antagonism with thymidine analog mutations. J Virol. 2006, 80: 4971-4977. 10.1128/JVI.80.10.4971-4977.2006.PubMed CentralView ArticlePubMedGoogle Scholar
- Kodama EI, Kohgo S, Kitano K, Machida H, Gatanaga H, Shigeta S, Matsuoka M, Ohrui H, Mitsuya H: 4'-Ethynyl nucleoside analogs: potent inhibitors of multidrug-resistant human immunodeficiency virus variants in vitro. Antimicrob Agents Chemother. 2001, 45: 1539-1546. 10.1128/AAC.45.5.1539-1546.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Larder BA: 3'-Azido-3'-deoxythymidine resistance suppressed by a mutation conferring human immunodeficiency virus type 1 resistance to nonnucleoside reverse transcriptase inhibitors. Antimicrob Agents Chemother. 1992, 36: 2664-2669. 10.1128/AAC.36.12.2664.PubMed CentralView ArticlePubMedGoogle Scholar
- Selmi B, Deval J, Alvarez K, Boretto J, Sarfati S, Guerreiro C, Canard B: The Y181C substitution in 3'-azido-3'-deoxythymidine-resistant human immunodeficiency virus, type 1, reverse transcriptase suppresses the ATP-mediated repair of the 3'-azido-3'-deoxythymidine 5'-monophosphate-terminated primer. J Biol Chem. 2003, 278: 40464-40472. 10.1074/jbc.M302928200.View ArticlePubMedGoogle Scholar
- Boucher CA, Cammack N, Schipper P, Schuurman R, Rouse P, Wainberg MA, Cameron JM: High-level resistance to (−) enantiomeric 2'-deoxy-3'-thiacytidine in vitro is due to one amino acid substitution in the catalytic site of human immunodeficiency virus type 1 reverse transcriptase. Antimicrob Agents Chemother. 1993, 37: 2231-2234. 10.1128/AAC.37.10.2231.PubMed CentralView ArticlePubMedGoogle Scholar
- Boyer PL, Sarafianos SG, Arnold E, Hughes SH: The M184V mutation reduces the selective excision of zidovudine 5'-monophosphate (AZTMP) by the reverse transcriptase of human immunodeficiency virus type 1. J Virol. 2002, 76: 3248-3256. 10.1128/JVI.76.7.3248-3256.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Gotte M, Arion D, Parniak MA, Wainberg MA: The M184V mutation in the reverse transcriptase of human immunodeficiency virus type 1 impairs rescue of chain-terminated DNA synthesis. J Virol. 2000, 74: 3579-3585. 10.1128/JVI.74.8.3579-3585.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Hachiya A, Marchand B, Kirby KA, Michailidis E, Tu X, Palczewski K, Ong YT, Li Z, Griffin DT, Schuckmann MM, et al: HIV-1 reverse transcriptase (RT) polymorphism 172K suppresses the effect of clinically relevant drug resistance mutations to both nucleoside and non-nucleoside RT inhibitors. J Biol Chem. 2012, 287: 29988-29999. 10.1074/jbc.M112.351551.PubMed CentralView ArticlePubMedGoogle Scholar
- Hachiya A, Gatanaga H, Kodama E, Ikeuchi M, Matsuoka M, Harada S, Mitsuya H, Kimura S, Oka S: Novel patterns of nevirapine resistance-associated mutations of human immunodeficiency virus type 1 in treatment-naive patients. Virology. 2004, 327: 215-224. 10.1016/j.virol.2004.07.001.View ArticlePubMedGoogle Scholar
- Hachiya A, Kodama EN, Sarafianos SG, Schuckmann MM, Sakagami Y, Matsuoka M, Takiguchi M, Gatanaga H, Oka S: Amino acid mutation N348I in the connection subdomain of human immunodeficiency virus type 1 reverse transcriptase confers multiclass resistance to nucleoside and nonnucleoside reverse transcriptase inhibitors. J Virol. 2008, 82: 3261-3270. 10.1128/JVI.01154-07.PubMed CentralView ArticlePubMedGoogle Scholar
- Bauman JD, Das K, Ho WC, Baweja M, Himmel DM, Clark AD, Oren DA, Boyer PL, Hughes SH, Shatkin AJ, Arnold E: Crystal engineering of HIV-1 reverse transcriptase for structure-based drug design. Nucleic Acids Res. 2008, 36: 5083-5092. 10.1093/nar/gkn464.PubMed CentralView ArticlePubMedGoogle Scholar
- Schuckmann MM, Marchand B, Hachiya A, Kodama EN, Kirby KA, Singh K, Sarafianos SG: The N348I mutation at the connection subdomain of HIV-1 reverse transcriptase decreases binding to nevirapine. J Biol Chem. 2010, 285: 38700-38709. 10.1074/jbc.M110.153783.PubMed CentralView ArticlePubMedGoogle Scholar
- Ndongwe TP, Adedeji AO, Michailidis E, Ong YT, Hachiya A, Marchand B, Ryan EM, Rai DK, Kirby KA, Whatley AS, et al: Biochemical, inhibition and inhibitor resistance studies of xenotropic murine leukemia virus-related virus reverse transcriptase. Nucleic Acids Res. 2012, 40: 345-359. 10.1093/nar/gkr694.PubMed CentralView ArticlePubMedGoogle Scholar
- Kirby KA, Marchand B, Ong YT, Ndongwe TP, Hachiya A, Michailidis E, Leslie MD, Sietsema DV, Fetterly TL, Dorst CA, et al: Structural and Inhibition Studies of the RNase H Function of Xenotropic Murine Leukemia Virus-Related Virus Reverse Transcriptase. Antimicrob Agents Chemother. 2012, 56 (4): 2048-61. 10.1128/AAC.06000-11.PubMed CentralView ArticlePubMedGoogle Scholar
- Sarafianos SG, Clark AD, Tuske S, Squire CJ, Das K, Sheng D, Ilankumaran P, Ramesha AR, Kroth H, Sayer JM, et al: Trapping HIV-1 reverse transcriptase before and after translocation on DNA. J Biol Chem. 2003, 278: 16280-16288. 10.1074/jbc.M212911200.View ArticlePubMedGoogle Scholar
- Ohrui H, Kohgo S, Hayakawa H, Kodama E, Matsuoka M, Nakata T, Mitsuya H: 2'-deoxy-4'-C-ethynyl-2-fluoroadenosine: a nucleoside reverse transcriptase inhibitor with highly potent activity against wide spectrum of HIV-1 strains, favorable toxic profiles, and stability in plasma. Nucleosides Nucleotides Nucleic Acids. 2007, 26 (10-12): 1543-6. 10.1080/15257770701545218.View ArticlePubMedGoogle Scholar
- Biaglow JE, Kachur AV: The generation of hydroxyl radicals in the reaction of molecular oxygen with polyphosphate complexes of ferrous ion. Radiat Res. 1997, 148: 181-187. 10.2307/3579576.View ArticlePubMedGoogle Scholar
- Tuske S, Sarafianos SG, Clark AD, Ding J, Naeger LK, White KL, Miller MD, Gibbs CS, Boyer PL, Clark P, et al: Structures of HIV-1 RT-DNA complexes before and after incorporation of the anti-AIDS drug tenofovir. Nat Struct Mol Biol. 2004, 11: 469-474. 10.1038/nsmb760.View ArticlePubMedGoogle Scholar
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