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Mechanistic Basis for Reduced Viral and Enzymatic Fitness of HIV-1 Reverse Transcriptase Containing Both K65R and M184V Mutations

2003; Elsevier BV; Volume: 279; Issue: 1 Linguagem: Inglês

10.1074/jbc.m308806200

1083-351X

Jérôme Deval, Kirsten L. White, Michael D. Miller, Neil Parkin, Jérôme Courcambeck, Philippe Halfon, Boulbaba Selmi, Joëlle Boretto, Bruno Canard,

HIV/AIDS Research and Interventions

HIV-1 drug resistance mutations are often inversely correlated with viral fitness, which remains poorly described at the molecular level. Some resistance mutations can also suppress resistance caused by other resistance mutations. We report the molecular mechanisms by which a virus resistant to lamivudine with the M184V reverse transcriptase mutation shows increased susceptibility to tenofovir and can suppress the effects of the tenofovir resistance mutation K65R. Additionally, we report how the decreased viral replication capacity of resistant viruses is directly linked to their decreased ability to use natural nucleotide substrates and that combination of the K65R and M184V resistance mutations leads to greater decreases in viral replication capacity. All together, these results define at the molecular level how nucleoside-resistant viruses can be driven to reduced viral fitness. HIV-1 drug resistance mutations are often inversely correlated with viral fitness, which remains poorly described at the molecular level. Some resistance mutations can also suppress resistance caused by other resistance mutations. We report the molecular mechanisms by which a virus resistant to lamivudine with the M184V reverse transcriptase mutation shows increased susceptibility to tenofovir and can suppress the effects of the tenofovir resistance mutation K65R. Additionally, we report how the decreased viral replication capacity of resistant viruses is directly linked to their decreased ability to use natural nucleotide substrates and that combination of the K65R and M184V resistance mutations leads to greater decreases in viral replication capacity. All together, these results define at the molecular level how nucleoside-resistant viruses can be driven to reduced viral fitness. Nucleoside analogue drugs are core constituents of antiretroviral regimens to control HIV-1 1The abbreviations used are: HIV-1, human immunodeficiency virus, type I; NRTI, nucleoside reverse transcriptase inhibitor; RT, reverse transcriptase; 3TC, 2′,3′-dideoxy-3′thiacythidine; 3TC-TP, 2′,3′-dideoxy-3′thiacythidine 5′-triphosphate; tenofovir-PP, tenofovir diphosphate; dNTP, 2′-deoxynucleoside 5′-triphosphate; ddNTP, 2′,3′-dideoxynucleoside 5′-triphosphate; Inorganic pyrophosphate, PPi; kpol, burst rate. infection (1.De Clercq E. J. Clin. Virol. 2001; 22: 73-89Crossref PubMed Scopus (241) Google Scholar). Once activated to the 5′-triphosphate state in the infected cell, they target the viral reverse transcriptase (RT), a specialized DNA polymerase encoded by the viral pol gene. In the clinic, however, treatment failures can occur that result in the development of drug-resistant virus. Both the high adaptive capacity of HIV-1 and the incomplete suppression of viral replication are responsible for the selection of drug-resistant viruses (2.Menendez-Arias L. Trends Pharmacol. Sci. 2002; 23: 381-388Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 3.Miller V. Larder B.A. Antivir. Ther. 2001; 6: 25-44PubMed Google Scholar). Controlling the appearance and spread of drug-resistant viruses requires novel drugs of increased potency that do not readily elicit resistance, and that are active against resistant viruses. The potency of a nucleoside RT inhibitor (NRTI) as an inhibitor of viral replication depends on several factors. First, the NRTI must be activated to the 5′-triphosphate state by cellular kinases. Second, the analogue 5′-triphosphate must be an efficient substrate for RT that competes significantly at its cellular concentration with its natural dNTP counterpart to terminate viral DNA chain extension. Third, it is increasingly apparent that stability of the analogue-terminated DNA and inefficient excision by RT are key factors in sustained inhibition. Viral drug resistance has been shown to involve the second and third steps listed above (4.Selmi B. Deval J. Boretto J. Canard B. Antiviral Therapy. 2003; 8: 143-154PubMed Google Scholar). There is intense ongoing research aimed at elucidating resistance mechanisms at the molecular level. In most cases, the corresponding substituted RT (referred to as mutant, mutated, or resistant therein) has altered biochemical properties accounting for viral resistance. At present, two classes of resistance mechanisms for NRTIs have been characterized. The first class involves an increased discrimination of the nucleotide analogue 5′-triphosphate at the RT active site relative to the natural substrate dNTP. Discrimination can be achieved either through selective decreased binding of the 5′-triphosphate analogue (reflected by an increase in the binding equilibrium constant Kd), or at the catalytic step of incorporation of the analogue 5′-monophosphate into viral DNA (reflected by a decrease of kpol value, the catalytic constant of incorporation of the nucleotide analogue into DNA). Lamivudine (3TC) and 2′-3′ dideoxynucleosides (ddNs, e.g. ddI and ddC) typically select resistance mutations conferring increased discrimination properties to the resistant RT (5.Krebs R. Immendorfer U. Thrall S.H. Wohrl B.M. Goody R.S. Biochemistry. 1997; 36: 10292-10300Crossref PubMed Scopus (135) Google Scholar, 6.Selmi B. Boretto J. Sarfati S.R. Guerreiro C. Canard B. J. Biol. Chem. 2001; 276: 48466-48472Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 7.Feng J.Y. Anderson K.S. Biochemistry. 1999; 38: 9440-9448Crossref PubMed Scopus (121) Google Scholar). The other class of resistance mechanisms involves repair of the analogue-terminated DNA chain. In this mechanism, the resistant RT exhibits enhanced repair (also named excision or unblocking) of the analogue monophosphate-terminated DNA chain. The repair reaction utilizes pyrophosphate (PPi) or a nucleoside 5′-triphosphate (NTP) as a co-substrate to unblock the analogue 5′-monophosphate terminated DNA chain in a reaction chemically similar to pyrophosphorolysis, the reversal of the polymerization reaction (8.Meyer P.R. Matsuura S.E. Mian A.M. So A.G. Scott W.A. Mol. Cell. 1999; 4: 35-43Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 9.Arion D. Kaushik N. McCormick S. Borkow G. Parniak M.A. Biochemistry. 1998; 37: 15908-15917Crossref PubMed Scopus (313) Google Scholar). Zidovudine (AZT) or stavudine (d4T) can select for RT mutations such as M41L, D67N, K70R, L210W, T215Y/F, or K219(Q/E/N/R) that act by this repair mechanism. As a group, these mutations are referred to as thymidine analogue-associated mutations or TAMs (10.Larder B. Skalka A.M. Goff S.P. Reverse Transcriptase. CSHL Press, 1992: 205-222Google Scholar, 11.Schinazi R.F. Larder B.A. Mellors J.W. Int. Antiviral News. 2000; 8: 65-91Google Scholar). As a result of acquiring drug resistance, drug-resistant viruses often have a replicative disadvantage relative to wild-type viruses in the absence of drug (12.Quinones-Mateu M.E. Arts E.J. Drug. Resist. Updat. 2002; 5: 224-233Crossref PubMed Scopus (77) Google Scholar). There is a growing body of evidence showing that some of these replication-impaired (or unfit) viruses might be less pathogenic and better controlled than wild-type viruses (13.Sufka S.A. Ferrari G. Gryszowka V.E. Wrin T. Fiscus S.A. Tomaras G.D. Staats H.F. Patel D.D. Sempowski G.D. Hellmann N.S. Weinhold K.J. Hicks C.B. J. Infect. Dis. 2003; 187: 1027-1037Crossref PubMed Scopus (64) Google Scholar). In addition, some NRTIs such as 3TC and AZT (14.Gotte M. Arion D. Parniak M.A. Wainberg M.A. J. Virol. 2000; 74: 3579-3585Crossref PubMed Scopus (162) Google Scholar, 15.Boyer P.L. Sarafianos S.G. Arnold E. Hughes S.H. J. Virol. 2002; 76: 3248-3256Crossref PubMed Scopus (90) Google Scholar) select mutant RTs that have antagonistic resistance mutations where resistance to 3TC decreases resistance to AZT. Treatment combining AZT with 3TC leads to a more sustained efficacy than treatment with either alone, although virus resistant to both drugs will eventually evolve without adequate viral load suppression. Consequently, it is possible that certain combinations of drugs might deliberately lead to a genetically defined virus with altered fitness and a resistance profile such that the virus remains fully or partially susceptible to drugs in the current or next regimen. Tenofovir disoproxil fumarate is an orally bioavailable form of tenofovir that has shown potent and durable efficacy in phase III clinical trials (16.Schooley R.T. Ruane P. Myers R.A. Beall G. Lampiris H. Berger D. Chen S.S. Miller M.D. Isaacson E. Cheng A.K. Aids. 2002; 16: 1257-1263Crossref PubMed Scopus (267) Google Scholar). Tenofovir is an acyclic nucleotide phosphonate analogue of AMP that is active on RT in its diphosphate form (tenofovir-PP). The RT mutation K65R appears to be the only mutation selected for by tenofovir in vitro and in vivo, and results in low level resistance to tenofovir (17.Wainberg M.A. Miller M.D. Quan Y. Salomon H. Mulato A.S. Lamy P.D. Margot N.A. Anton K.E. Cherrington J.M. Antivir. Ther. 1999; 4: 87-94PubMed Google Scholar, 18.Margot N.A. Isaacson E. McGowan I. Cheng A.K. Schooley R.T. Miller M.D. Aids. 2002; 16: 1227-1235Crossref PubMed Scopus (139) Google Scholar). This resistance is partially to fully suppressed when present in combination with the 3TC/emtricitabine (FTC)-resistance mutation M184V (17.Wainberg M.A. Miller M.D. Quan Y. Salomon H. Mulato A.S. Lamy P.D. Margot N.A. Anton K.E. Cherrington J.M. Antivir. Ther. 1999; 4: 87-94PubMed Google Scholar, 18.Margot N.A. Isaacson E. McGowan I. Cheng A.K. Schooley R.T. Miller M.D. Aids. 2002; 16: 1227-1235Crossref PubMed Scopus (139) Google Scholar, 19.White K.L. Margot N.A. Wrin T. Petropoulos C.J. Miller M.D. Naeger L.K. Antimicrob. Agents Chemother. 2002; 46: 3437-3446Crossref PubMed Scopus (174) Google Scholar). In this study, we focused on RT resistance mutations acting through discrimination of the nucleotide analogue relative to its natural counterpart, specifically K65R, M184V, and the multidrug-resistant Q151M complex mutations, and their effects on drug susceptibility, RT enzymatic function, and viral replication capacity. We give a mechanistic basis for the reduced resistance, as well as the impaired viral and enzymatic fitness of HIV-1 reverse transcriptase containing both K65R and M184V mutations. Phenotypic Assays of Drug Susceptibility—Phenotypic data for tenofovir, zidovudine (AZT), lamivudine (3TC), didanosine (ddI), stavudine (d4T), and abacavir were obtained for patient-derived recombinant viruses present in the Virco library using the Antivirogram assay (Virco Central Virological Laboratory, Mechelen, Belgium). The antiretroviral histories of patients from whom these viruses were obtained are unknown. Viruses were chosen based on their RT genotype of M184V alone (n = 10), K65R alone (n = 5), K65R in combination with M184V (n = 5), and the combination of Q151M, V75I, F77L, and F116Y (n = 5). Assay of Viral Replication Capacity—Replication capacity was measured using a single cycle virus growth assay as previously described with patient-derived recombinant viruses present in the ViroLogic library (ViroLogic, S. San Francisco, CA) (19.White K.L. Margot N.A. Wrin T. Petropoulos C.J. Miller M.D. Naeger L.K. Antimicrob. Agents Chemother. 2002; 46: 3437-3446Crossref PubMed Scopus (174) Google Scholar, 20.Deeks S.G. Wrin T. Liegler T. Hoh R. Hayden M. Barbour J.D. Hellmann N.S. Petropoulos C.J. McCune J.M. Hellerstein M.K. Grant R.M. N. Engl. J. Med. 2001; 344: 472-480Crossref PubMed Scopus (604) Google Scholar). The antiretroviral histories of patients from whom these viruses were obtained are unknown. Viruses were chosen that contained no NRTI-associated or protease inhibitor (PI)-associated resistance mutations (wild-type, n = 1307), K65R alone (n = 17), M184V alone (n = 291), K65R plus M184V (n = 12), or Q151M complex plus M184V (n = 8), all in the absence of thymidine analogue-associated mutations (TAMs) or PI-associated resistance mutations. None of the samples contained mixtures at positions 65, 151, or 184. Briefly, a retroviral vector was constructed using the NL4-3 infectious molecular clone of HIV-1. The vector contains a luciferase expression cassette replacing the HIV-1 envelope (21.Petropoulos C.J. Parkin N.T. Limoli K.L. Lie Y.S. Wrin T. Huang W. Tian H. Smith D. Winslow G.A. Capon D.J. Whitcomb J.M. Antimicrob. Agents Chemother. 2000; 44: 920-928Crossref PubMed Scopus (576) Google Scholar). NL4-3 protease and RT sequences were replaced with corresponding patient-derived sequences. Recombinant virus stocks were generated by co-transfecting 293 cells with retroviral vector DNA pools and an expression vector that produces the amphotropic murine leukemia virus envelope protein. After normalization, the amount of luciferase activity in cells infected with recombinant pseudotyped virus was used as a direct measure of replication capacity, i.e. the ability of the virus to complete a single cycle of viral replication. Relative replication capacity was assessed by comparing the amount of luciferase activity produced by mutant recombinant viruses to the amount of luciferase activity produced by the control NL4-3 virus and adjusted to reflect the difference between NL4-3 and the average replication capacity of wild-type (drug-sensitive) clinical isolates. HIV-RT Plasmid Constructions, Enzyme Preparations, and Re-agents—The wild-type RT bacterial expression gene construct p66RTB was used to obtain K65R, M184V, K65R/M184V, and Q151M RT as described (22.Boretto J. Longhi S. Navarro J.M. Selmi B. Sire J. Canard B. Anal. Biochem. 2001; 292: 139-147Crossref PubMed Scopus (48) Google Scholar). All constructs were verified by DNA sequencing. The recombinant RTs were co-expressed with HIV-1 protease in Escherichia coli in order to obtain p66/p51 heterodimers, which were later purified using affinity chromatography. All enzymes were quantitated by active-site titration before biochemical studies. DNA oligonucleotides were obtained from Invitrogen. Oligonucleotides were 5′-32P-labeled using T4 polynucleotide kinase (New England Biolabs, MA). γ-32P-labeled adenosine 5′-triphosphate was purchased from Amersham Biosciences. Pre-steady State Kinetics of Single Nucleotide Incorporation Into DNA—Pre-steady state kinetics were performed using dATP, dTTP, dCTP, dGTP, tenofovir-PP and 3TC-TP in conjunction with wild-type, K65R, M184V, K65R/M184V, and Q151M RT. Rapid quench experiments were performed with a Kintek instrument Model RQF-3 using reaction times ranging from 10 ms to 30 s. All indicated concentrations are final. The primer DNA/DNA oligonucleotides used for the rapid reaction were a 5′-labeled 21-mer primer (5′-ATA CTT TAA CCA TAT GTA TCC-3′) annealed to a 31-mer template 31T-RT (5′-TTT TTT TTT AGG ATA CAT ATG GTT AAA GTA T-3′) for the incorporation of dTTP, a 31A-RT (5′-AAA AAA AAA TGG ATA CAT ATG GTT AAA GTA T-3′) for the incorporation of dATP/tenofovir-PP, a 31C-RT (5′-TTT TTT TTT GGG ATA CAT ATG GTT AAA GTA T-3′) for the incorporation of dCTP/3TC-TP and 31G-RT (5′-TTT TTT TTT CGG ATA CAT ATG GTT AAA GTA T-3′) for the incorporation of dGTP. For natural nucleotides, the reaction was performed by mixing a solution containing 50 nm (active sites) of HIV-1 RT bound to 100 nm of primer/template in RT buffer (50 mm Tris-HCl, pH 8.0, 50 mm KCl, 0.05% Triton X-100), and a variable concentration of dNTP in 6 mm MgCl2. Reactions involving 3TC-TP and tenofovir-PP were conducted with excess concentrations of enzyme (200 nm) over primer/template duplex (100 nm). These conditions were chosen to eliminate the influence of the enzyme turnover rate (kss) that interferes in the measurements of low incorporation rates. The products of reactions were analyzed using sequencing gel electrophoresis (14% acrylamide, 8 m urea in Tris borate/EDTA buffer), and quantified using photo-stimulated plates and FujiImager. The formation of product (P) over time was fitted with a burst equation shown in Equation 1, (P)=A·(1-exp(-(kapp·t))+kss·t(Eq. 1) where A is the amplitude of the burst, kapp is the apparent kinetic constant of formation of the phosphodiester bond, and kss is the enzyme turnover rate, i.e. the kinetic constant of the steady-state linear phase. The dependence of kapp on dNTP concentration is described by the hyperbolic Equation 2, kapp=kpol·[dNTP](Kd+[dNTP])(Eq. 2) where Kd and kpol are the equilibrium constant and the catalytic rate constant of the dNTP for RT, respectively. Kd and kpol were determined from curve-fitting using Kaleidagraph (Synergy Software, PA). Molecular Modeling of Tenofovir-PP in the RT Active Site—The molecular modeling was performed with GenMol (23.Pepe G. Guiliani G. Loustalet S. Halfon P. Eur. J. Med. Chem. 2002; 37: 865-872Crossref PubMed Scopus (14) Google Scholar, 24.Pepe G. Siri D. Stud. Phys. Theor. Chem. 1990; 71: 93-101Google Scholar, 25.Pepe G. Seres B. Laporte D. Del Re C. J. Theor. Biol. 1985; 115: 571-593Crossref Scopus (29) Google Scholar) with its all-atom force field, from the x-ray crystal structure of wild type ternary complex RT-DNA-dTTP (26.Huang H. Chopra R. Verdine G.L. Harrison S.C. Science. 1998; 282: 1669-1675Crossref PubMed Scopus (1360) Google Scholar). We extracted amino acids surrounding the catalytic DNA polymerase domain within a radius of 25 Å. This model of HIV-RT included the residues of both p66 and p51 domain, DNA template/primer, two catalytic magnesium ions, and dTTP. Close attention was given for both magnesium ions, because they have a key role in positioning the incoming dNTP or NRTI and in catalysis. In order to prepare the closed RT-DNA-dTTP complex (26.Huang H. Chopra R. Verdine G.L. Harrison S.C. Science. 1998; 282: 1669-1675Crossref PubMed Scopus (1360) Google Scholar), the authors used a ddGTP primer terminus positioned at the so-called "Priming site" (P site). We replaced ddGTP by dGTP and interactions of its 3′-OH with Mg2+ were optimized. In the crystal structure, both magnesium ions were complexed only with residues Asp-185, Asp-110, and Val-111. By a conformational analysis, we optimized the complexation process of both Mg2+. The complexation process of Mg2+, with the incoming nucleotide via its triphosphate chain and residues Asp-110, Val-111, and Asp-185, was found to be the same as in the crystal structure of the ternary complex made of RT-DNA-dTTP. The complexation of the second Mg2+ was optimized. Positions of Asp-186 and Asp-110 side chains were optimized in order to obtain a better complexation process. This second Mg2+ was complexed with Asp-186 (Oδ1 carboxylate side chain), Asp-185 (Oδ2 carboxylate side chain), and 3′-OH dGTP primer terminus. All minimizations were conducted with Linux RedHat 7.3 workstations, on a 2.0 GHz bi-Xeon Pentium° 4 processor. Hydrogen atoms were added to the enzyme, DNA duplex and dTTP within the Biopolymer module of GenMol. Hydrogen positions were then optimized. The construction of the nucleotide triphosphate was based on x-ray conformation of dTTP present in the ternary complex RT-DNA-dTTP. All-Atom charges were computed with GenMol for RT-DNA, both Mg2+, dATP and tenofovir-PP (24.Pepe G. Siri D. Stud. Phys. Theor. Chem. 1990; 71: 93-101Google Scholar, 25.Pepe G. Seres B. Laporte D. Del Re C. J. Theor. Biol. 1985; 115: 571-593Crossref Scopus (29) Google Scholar). The substituted RT structures were obtained by changing selected amino acid (M184V). The adenine at the position 5 of the template was mutated to the counterpart of the incoming nucleotide dATP and tenofovir-PP (A to T). The conformation of 5th template nucleotide and the side chain of selected amino acid were minimized within the Biopolymer module of GenMol, using a conformational analysis method. Finally, the incoming dATP or tenofovir-PP were docked into the DNA-polymerase active site from the positioning of dTTP present in the x-ray structure ternary complex (nucleoside binding site, N site). The resulting ternary complexes were optimized using GenMol All-Atom force field. The interplay between suppressive mutations and viral fitness bears potential to increase the efficacy of antiretroviral drug regimens. The aim of our study was to determine the potential for tenofovir in such a strategy. We investigated the mechanism of drug resistance to tenofovir by its most relevant mutation K65R in order to identify which drug would be best combined with tenofovir in terms of resistance suppression, as well as resistance mutations that would maximize the alteration of viral replication capacity. HIV-1 Genotypes Associated with Reduced Susceptibility to Tenofovir—Table I shows antiviral susceptibilities determined using the Virco Antivirogram for patient-derived samples. Patient samples were selected that contained M184V alone, K65R alone, K65R plus M184V, or the Q151M substitution with additional resistance mutations including V75I, F77L, and F116Y in all cases (Q151M complex). When compared with the wild-type, viruses containing M184V demonstrated high level susceptibility changes to 3TC (>50-fold) and no significant changes for all other nucleosides and tenofovir. Viruses containing K65R showed decreased susceptibility for 3TC, tenofovir, ddI, and abacavir (2.0-7.7-fold), but were not significantly changed for AZT and d4T (1.1- and 1.6-fold, respectively). The K65R and M184V double mutants showed high-level susceptibility changes for lamivudine (>50-fold) and further decreased susceptibility for ddI and abacavir (2.5- and 7.6-fold, respectively). For tenofovir, AZT, and d4T, however, the susceptibility values for the K65R + M184V double mutant were improved relative to K65R alone (1.7-, 0.7- and 1.0-fold relative to wild-type, respectively). The Q151M complex viruses showed high-level resistance to all NRTIs (>11-fold), except to lamivudine and tenofovir, where the susceptibility changes were minimal compared with wild-type (2.1- and 1.8-fold, respectively). In summary for tenofovir, full in vitro activity against M184V was maintained. Low level phenotypic changes were demonstrated for the K65R mutant viruses, and this was partially reversed when M184V was also present. These results with patient-derived HIV isolates parallel two published studies with site-directed recombinant viruses that showed significantly increased tenofovir susceptibility for viruses with K65R plus M184V relative to K65R alone (17.Wainberg M.A. Miller M.D. Quan Y. Salomon H. Mulato A.S. Lamy P.D. Margot N.A. Anton K.E. Cherrington J.M. Antivir. Ther. 1999; 4: 87-94PubMed Google Scholar, 18.Margot N.A. Isaacson E. McGowan I. Cheng A.K. Schooley R.T. Miller M.D. Aids. 2002; 16: 1227-1235Crossref PubMed Scopus (139) Google Scholar). In order to understand the molecular mechanisms involved in resistance and suppression of resistance, the incorporation of the active metabolite of tenofovir, tenofovir-PP, by RTs carrying these mutations was investigated using pre-steady state kinetics.Table ISummary of tenofovir and nucleoside analogue susceptibilitiesMean fold decrease in susceptibility from wild-type (range)aFold change versus wild-type control using the Virco Antivirogram assay with tenofovir, zidovudine (AZT), lamivudine (3TC), didanosine (ddI), stavudine (d4T), and abacavirResistance groupNTenofovirAZT3TCddId4TAbacavirK65R52.7bStatistically significant decrease in susceptibility as compared to a panel of 10 viruses with wild-type HIV sequence (p < 0.01, Student's t test) (1.8-3.8)1.1 (0.7-1.9)7.7bStatistically significant decrease in susceptibility as compared to a panel of 10 viruses with wild-type HIV sequence (p < 0.01, Student's t test) (1.1-15.6)2.0bStatistically significant decrease in susceptibility as compared to a panel of 10 viruses with wild-type HIV sequence (p < 0.01, Student's t test) (0.9-3.2)1.6 (1.0-2.0)2.9bStatistically significant decrease in susceptibility as compared to a panel of 10 viruses with wild-type HIV sequence (p < 0.01, Student's t test) (2.3-3.5)M184V100.7 (0.3-1.3)0.9 (0.2-1.5)>50bStatistically significant decrease in susceptibility as compared to a panel of 10 viruses with wild-type HIV sequence (p < 0.01, Student's t test) (>50)1.0 (0.3-2.4)1.4 (0.4-2.8)1.3 (0.9-2.4)K65R + M184V51.7 (0.4-3.5)0.7 (0.4-1.1)>50bStatistically significant decrease in susceptibility as compared to a panel of 10 viruses with wild-type HIV sequence (p < 0.01, Student's t test) (>50)2.5bStatistically significant decrease in susceptibility as compared to a panel of 10 viruses with wild-type HIV sequence (p < 0.01, Student's t test) (1.8-2.9)1.0 (0.4-1.7)7.6bStatistically significant decrease in susceptibility as compared to a panel of 10 viruses with wild-type HIV sequence (p < 0.01, Student's t test) (2.3-14.3)Q151M complex51.8bStatistically significant decrease in susceptibility as compared to a panel of 10 viruses with wild-type HIV sequence (p < 0.01, Student's t test) (1.1-3.0)43bStatistically significant decrease in susceptibility as compared to a panel of 10 viruses with wild-type HIV sequence (p < 0.01, Student's t test) (9.6-85)2.1bStatistically significant decrease in susceptibility as compared to a panel of 10 viruses with wild-type HIV sequence (p < 0.01, Student's t test) (1.3-2.6)13bStatistically significant decrease in susceptibility as compared to a panel of 10 viruses with wild-type HIV sequence (p < 0.01, Student's t test) (6.4-31)20bStatistically significant decrease in susceptibility as compared to a panel of 10 viruses with wild-type HIV sequence (p < 0.01, Student's t test) (6.1-57)11bStatistically significant decrease in susceptibility as compared to a panel of 10 viruses with wild-type HIV sequence (p < 0.01, Student's t test) (3.0-24)a Fold change versus wild-type control using the Virco Antivirogram assay with tenofovir, zidovudine (AZT), lamivudine (3TC), didanosine (ddI), stavudine (d4T), and abacavirb Statistically significant decrease in susceptibility as compared to a panel of 10 viruses with wild-type HIV sequence (p < 0.01, Student's t test) Open table in a new tab Mechanisms of RT-mediated Tenofovir-PP Resistance—A nucleotide analogue 5′-triphosphate is characterized by its efficiency of incorporation (kpol/Kd) into DNA as compared with that of its natural counterpart. For a given RT, comparing kpol/Kd values between a natural dNTP and its corresponding analogue is a convenient manner to evaluate selectivity for (or discrimination of) the analogue. Comparing selectivity between RTs defines in vitro resistance (or susceptibility) at the enzymatic level. The results are presented in Table II and Fig. 1. K65R was found to be the most tenofovir-PP resistant RT, in accordance with the fact that this is the only tenofovir-selected mutation that has been shown to develop in vitro and in tenofovir-treated patients to date (17.Wainberg M.A. Miller M.D. Quan Y. Salomon H. Mulato A.S. Lamy P.D. Margot N.A. Anton K.E. Cherrington J.M. Antivir. Ther. 1999; 4: 87-94PubMed Google Scholar, 18.Margot N.A. Isaacson E. McGowan I. Cheng A.K. Schooley R.T. Miller M.D. Aids. 2002; 16: 1227-1235Crossref PubMed Scopus (139) Google Scholar). This mutation is known to occur at low frequency during regimens making use of dideoxynucleosides (ddC and ddI) as well as for abacavir. The molecular mechanism of resistance to ddNs by K65R is known in great detail: ddNTPs bind to K65R RT with the same affinity as dNTPs, but their incorporation rate is decreased selectively, accounting for resistance (6.Selmi B. Boretto J. Sarfati S.R. Guerreiro C. Canard B. J. Biol. Chem. 2001; 276: 48466-48472Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar).Table IIPre-steady state kinetic constants of dATP/tenofovir-PP and dCTP/3TC-TP incorporation by HIV-1 RTsNucleotide HIV-1 RTdATPTenofovir-PPSelectivitybSelectivity and resistance were determined as described under "Results." The selectivity is the ratio of [kpol/Kd (nucleotide analogue)]/[kpol/Kd (nucleotide)]. The resistance is determined by the ratio of selectivityWTRT/selectivitymutantResistance foldbSelectivity and resistance were determined as described under "Results." The selectivity is the ratio of [kpol/Kd (nucleotide analogue)]/[kpol/Kd (nucleotide)]. The resistance is determined by the ratio of selectivityWTRT/selectivitymutantk polK dkpol/Kdk polK dkpol/Kds-1 aKd and kpol were determined as described under "Experimental Procedure." S.D. were <20%μmaKd and kpol were determined as described under "Experimental Procedure." S.D. were <20%s-1 μm-1s-1 aKd and kpol were determined as described under "Experimental Procedure." S.D. were <20%μmaKd and kpol were determined as described under "Experimental Procedure." S.D. were <20%s-1 μm-1WT50cValue from Selmi et al. (6)7.5cValue from Selmi et al. (6)6.77.0230.3221K65R126.91.70.32180.017974.4M184V33181.86.9300.2380.4K65R/M184V20370.561660.015371.7Nucleotide HIV-1 RTdCTP3TC-TPSelectivitybSelectivity and resistance were determined as described under "Results." The selectivity is the ratio of [kpol/Kd (nucleotide analogue)]/[kpol/Kd (nucleotide)]. The resistance is determined by the ratio of selectivityWTRT/selectivitymutantResistance foldbSelectivity and resistance were determined as described under "Results." The selectivity is the ratio of [kpol/Kd (nucleotide analogue)]/[kpol/Kd (nucleotide)]. The resistance is determined by the ratio of selectivityWTRT/selectivitymutantk polK dkpol/Kdk polK dkpol/Kds-1 aKd and kpol were determined as described under "Experimental Procedure." S.D. were <20%μmaKd and kpol were determined as described under "Experimental Procedure." S.D. were <20%s-1 μm-1s-1 aKd and kpol were determined as described under "Experimental Procedure." S.D. were <20%μmaKd and kpol were determined as described under "Experimental Procedure." S.D. were <20%s-1 μm-1WT7.3bSelectivity and resistance were determined as described under "Results." The selectivity is the ratio of [kpol/Kd (nucleotide analogue)