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• W2023942953 abstract "Nucleoside analogues are currently used to treat human immunodeficiency virus infections. The appearance of up to five substitutions (A62V, V75I, F77L, F116Y, and Q151M) in the viral reverse transcriptase promotes resistance to these drugs, and reduces efficiency of the antiretroviral chemotherapy. Using pre-steady state kinetics, we show that Q151M and A62V/V75I/F77L/F116Y/Q151M substitutions confer to reverse transcriptase (RT) the ability to discriminate an analogue relative to its natural counterpart, and have no effect on repair of the analogue-terminated DNA primer. Discrimination results from a selective decrease of the catalytic rate constant k pol: 18-fold (from 7 to 0.3 s−1), 13-fold (from 1.9 to 0.14 s−1), and 12-fold (from 13 to 1 s−1) in the case of ddATP, ddCTP, and 3′-azido-3′-deoxythymidine 5′-triphosphate (AZTTP), respectively. The binding affinities of the triphosphate analogues for RT remain unchanged. Molecular modeling explains drug resistance by a selective loss of electrostatic interactions between the analogue and RT. Resistance was overcome using α-boranophosphate nucleotide analogues. Using A62V/V75I/F77L/F116Y/Q151M RT, k polincreases up to 70- and 13-fold using α-boranophosphate-ddATP and α-boranophosphate AZTTP, respectively. These results highlight the general capacity of such analogues to circumvent multidrug resistance when RT-mediated nucleotide resistance originates from the selective decrease of the catalytic rate constantk pol. Nucleoside analogues are currently used to treat human immunodeficiency virus infections. The appearance of up to five substitutions (A62V, V75I, F77L, F116Y, and Q151M) in the viral reverse transcriptase promotes resistance to these drugs, and reduces efficiency of the antiretroviral chemotherapy. Using pre-steady state kinetics, we show that Q151M and A62V/V75I/F77L/F116Y/Q151M substitutions confer to reverse transcriptase (RT) the ability to discriminate an analogue relative to its natural counterpart, and have no effect on repair of the analogue-terminated DNA primer. Discrimination results from a selective decrease of the catalytic rate constant k pol: 18-fold (from 7 to 0.3 s−1), 13-fold (from 1.9 to 0.14 s−1), and 12-fold (from 13 to 1 s−1) in the case of ddATP, ddCTP, and 3′-azido-3′-deoxythymidine 5′-triphosphate (AZTTP), respectively. The binding affinities of the triphosphate analogues for RT remain unchanged. Molecular modeling explains drug resistance by a selective loss of electrostatic interactions between the analogue and RT. Resistance was overcome using α-boranophosphate nucleotide analogues. Using A62V/V75I/F77L/F116Y/Q151M RT, k polincreases up to 70- and 13-fold using α-boranophosphate-ddATP and α-boranophosphate AZTTP, respectively. These results highlight the general capacity of such analogues to circumvent multidrug resistance when RT-mediated nucleotide resistance originates from the selective decrease of the catalytic rate constantk pol. human immunodeficiency virus reverse transcriptase 3′-azido-3′-deoxythymidine 2′,3′-didehydro-2′,3′-dideoxythymidine (−)-β-l-2′,3′-dideoxy-3′-l-thiacytidine 3′-azido-3′-deoxythymidine 5′-monophosphate 3′-azido-3′-deoxythymidine 5′-triphosphate 2′,3′-dideoxyadenine 5′-triphosphate 2′,3′-dideoxycytidine 5′-triphosphate 2′-deoxynucleoside 5′-triphosphate 2′,3′-dideoxynucleoside 5′-triphosphate inorganic pyrophosphate borano The human immunodeficiency virus (HIV)1 infects more than 40 million individuals in the world. 3′-Azido-3′-deoxythymidine (AZT, zidovudine) was the first antiretroviral drug to receive approval from the FDA in 1987 to treat HIV-1-infected patients. AZT is a nucleoside analogue acting on viral replication. It is metabolically activated by cellular kinases of the host cell to its corresponding triphosphate form AZTTP before reaching its target, reverse transcriptase (RT). RT is an essential viral DNA polymerase responsible for viral DNA synthesis. AZTTP is a poor substrate for cellular DNA polymerases, but is incorporated into the nascent viral DNA strand by RT with the same efficiency as its natural nucleotide counterpart dTTP. Because AZT lacks a 3′-hydroxyl group (3′-OH) on its ribose moiety, AZTMP is incorporated into DNA and viral DNA synthesis is terminated. The prolonged use of AZT as the sole drug in the clinic has resulted in the emergence of AZT-resistant viruses (1Larder B.A. Darby G. Richman D.D. Science. 1989; 243: 1731-1734Crossref PubMed Scopus (1350) Google Scholar). A set of six specific substitutions on RT (M41L, D67N, K70R, T215Y or F, L210W, and K219E or Q) gives rise to high level AZT resistance (2Larder B.A. Kemp S.D. Science. 1989; 246: 1155-1158Crossref PubMed Scopus (1016) Google Scholar), the appearance of T215F or Y being the most important substitution. A long awaited mechanism of AZT resistance because of these mutations has been proposed, based on biochemical studies using purified reverse transcriptase: AZT-resistant RT is able to catalyze a primer-unblocking reaction related to pyrophosphorolysis (3Arion D. Kaushik N. McCormick S. Borkow G. Parniak M.A. Biochemistry. 1998; 37: 15908-15917Crossref PubMed Scopus (313) Google Scholar, 4Meyer 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) to remove the chain-terminating AZTMP. This “repair” reaction allows the RT to resume elongation of the primer DNA. To circumvent limitations because of resistance and to increase the efficacy of antiretroviral regimens, several other nucleoside analogues such as 2′,3′-dideoxynucleosides (ddNs) have been developed and used in the clinic. Unfortunately, combination therapies using a mixture of AZT and ddNs often give rise to multiple dideoxynucleoside-resistant viruses that are no longer sensitive to either AZT or dideoxynucleosides (5Shirasaka T. Kavlick M.F. Ueno T. Gao W.Y. Kojima E. Alcaide M.L. Chokekijchai S. Roy B.M. Arnold E. Yarchoan R. Mitsuya H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2398-2402Crossref PubMed Scopus (363) Google Scholar). Multidrug-resistant RTs isolated from these viruses carry another set of five mutations (A62V, V75I, F77L, F116Y, and Q151M) that were shown to significantly reduce sensitivity to AZT, ddI, ddC, and d4T in vivo (6Maeda Y. Venzon D.J. Mitsuya H. J. Infect. Dis. 1998; 177: 1207-1213Crossref PubMed Scopus (119) Google Scholar, 7Kosalaraksa P. Kavlick M.F. Maroun V., Le, R. Mitsuya H. J. Virol. 1999; 73: 5356-5363Crossref PubMed Google Scholar). Q151M is a key mutation in this type of multiple dideoxynucleoside resistance (8Iversen A.K. Shafer R.W. Wehrly K. Winters M.A. Mullins J.I. Chesebro B. Merigan T.C. J. Virol. 1996; 70: 1086-1090Crossref PubMed Google Scholar, 9Garcia-Lerma J.G. Gerrish P.J. Wright A.C. Qari S.H. Heneine W. J. Virol. 2000; 74: 9339-9346Crossref PubMed Scopus (53) Google Scholar). Q151M appears first during combination therapy with a concomitant increase in the virus load of infected patients. Multidrug resistance and viral fitness are increased further with the sequential appearance of up to four additional amino acid substitutions mentioned above. Q151M-based reverse transcriptase variants have been studied at the biochemical level (10Lennerstrand J. Hertogs K. Stammers D.K. Larder B.A. J. Virol. 2001; 75: 7202-7205Crossref PubMed Scopus (52) Google Scholar, 11Ueno T. Mitsuya H. Biochemistry. 1997; 36: 1092-1099Crossref PubMed Scopus (63) Google Scholar). Steady-state kinetics of single nucleotide incorporation showed that resistance levels of A62V/V75I/F77L/F116Y/Q151M RT (here referred to as Q151Mcomplex RT) had risen up to 10-fold for ddATP and 14-fold for AZTTP. These results suggested that multidrug resistance is because of an altered recognition of the incoming nucleotide analogue, as opposed to the resistance induced by the repair reaction described previously in the case of AZT. Discrimination of the 5′-triphosphate analogue at the RT active site has been described as a mechanism of nucleoside drug resistance. The incorporation efficiency of an analogue into DNA, and hence its DNA termination potency, depends on two factors. First, the nucleotide analogue has to compete for binding to the RT active site with its natural nucleotide counterpart. This binding competition is reflected in the comparison of the analogue dissociation constant (K d) with that of its natural nucleotide competitor. Second, once the nucleotide analogue is bound to the RT active site, it has to be incorporated into DNA with a favorable catalytic rate constant k pol. The overall efficiency of incorporation of a given analogue is thus the ratiok pol/K d. This ratio offers a precise comparison between the chain termination properties of the analogues. Knowledge of the intracellular nucleotide analogue concentrations in the target cell allows one to predict the average number of chain termination events during viral DNA replication (12Reardon J.E. Biochemistry. 1992; 31: 4473-4479Crossref PubMed Scopus (137) Google Scholar). The resistance of Q151M and Q151Mcomplex RT toward various analogues has been studied using purified RT, but steady-state kinetics do not give access to the dissociation and catalytic rate constantsK d and k pol, respectively. Therefore, the precise mechanism by which Q151M and related substitutions confer nucleotide resistance to RT remains to be detailed. We have previously described nucleotide analogues that are modified on their α-phosphate by a borano (BH 3−) group (13Meyer P. Schneider B. Sarfati S. Deville-Bonne D. Guerreiro C. Boretto J. Janin J. Veron M. Canard B. EMBO J. 2000; 19: 3520-3529Crossref PubMed Scopus (72) Google Scholar). The R p diastereoisomer of these α-boranophosphate (BH3-dNTPs) analogues acts as a substrate for the RT, and as a chain terminator when it is devoid of a 3′-OH group. Recently, we have determined the precise basis of dideoxynucleotide analogue resistance promoted by the K65R substitution in RT (14Selmi 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). K65R RT is responsible for a decrease in the rate of incorporation of the nucleotide analogues specifically. We have also shown that this resistance can be suppressed by the presence of the BH3 group at the α-position of ddNTPs. Indeed, the presence of the BH3 group does not influence the binding of the analogue to the RT active site, but greatly enhances the catalytic rate constant, k pol, of incorporation of the dideoxy analogue specifically. This suppression of drug resistance provides mechanistic proof for the involvement of the catalytic step, but is also an elegant way to overcome resistance in vitro. These results allowed us to propose that these analogues could be similarly useful with other RTs showing nucleotide analogue resistance by a related mechanism. In addition to an ill defined resistance mechanism, the incorporation properties of BH3-dNTP analogues are not known for these clinically important Q151M variants. In this study, we show that a decrease in the rate constant of the phosphodiester bond formation is critical in the mechanism of multidrug resistance by Q151M and Q151Mcomplex RT. Based on this result, we make use of BH3-nucleotide analogues to overcome resistance and recover sensitivity to these nucleotide analogue inhibitors. The wild-type RT gene construct p66RTB served as a template for directed mutagenesis using the QuikChange kit from Stratagene, to obtain both Q151M and the Q151Mcomplex RT. All constructs were verified by DNA sequencing. The recombinant RTs were co-expressed with HIV-1 protease in Escherichia coli to get p66/p51 heterodimers, which were later purified using affinity chromatography as described (15Boretto J. Longhi S. Navarro J.M. Selmi B. Sire J. Canard B. Anal. Biochem. 2001; 292: 139-147Crossref PubMed Scopus (48) Google Scholar). All enzymes were quantified 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, Beverly, MA). γ-32P-Labeled adenosine 5′-triphosphate was purchased from Amersham Biosciences. The synthesis and purification of α-boranophosphate nucleotide analogues (R pdiastereoisomer) has been described (Ref. 14Selmi 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 and references therein). Pre-steady state kinetics were performed using dATP, dTTP, dCTP, ddATP, AZTTP, ddCTP, BH3-ddATP, and BH3-AZTTP in conjunction with wild-type, Q151M, and Q151Mcomplex RT. Rapid quench experiments were performed with a Kintek Instruments 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′-ATACTTTAACCATATGTATCC-3′) annealed to a 31-mer template 31T-RT (5′-TTTTTTTTTAGGATACATATGGTTAAAGTAT-3′) for the incorporation of dTTP/AZTTP, a 31A-RT (5′-AAAAAAAAATGGATACATATGGTTAAAGTAT-3′) for the incorporation of dATP/ddATP, or a 31C-RT primer (5′-TTTTTTTTTGGGATACATATGGTTAAAGTAT-3′) for the incorporation of dCTP/ddCTP. For the incorporation of natural nucleotides, the reaction was performed by mixing a solution containing 50 nm (active sites) of HIV-1 RT bound to 100 nm 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 nucleotide analogue inhibitors were conducted with excess concentrations of enzyme (200 nm) over the primer/template duplex (100 nm). These conditions were chosen to eliminate the influence of the enzyme turnover rate (k ss), which interferes in the measurements of low incorporation rates (16Brandis J.W. Edwards S.G. Johnson K.A. Biochemistry. 1996; 35: 2189-2200Crossref PubMed Scopus (43) Google Scholar). The products of reactions were analyzed using sequencing gel electrophoresis (14% acrylamide, 8 m urea in TBE buffer), and quantified using photostimulated plates and FujiImager. The formation of product (P) over time was fitted with a burst equation, (P)=A×(1­exp(−kapp×t))+kss×tEquation 1 where A is the amplitude of the burst,k app is the apparent kinetic constant of formation of the phosphodiester bond, and k ss is the enzyme turnover rate, i.e. the kinetic constant of the steady-state linear phase. The dependence ofk app on dNTP concentration is described by the hyperbolic equation, kapp=kpol×[dNTP]/(Kd+[dNTP])Equation 2 where K d and k pol are the equilibrium constant and the catalytic rate constant of the dNTP for RT, respectively. K d andk pol were determined from curve-fitting using Kaleidagraph (Synergy Software, Reading, PA). For both pyrophosphorolysis and ATP-mediated unblocking assays, the 5′-32P-labeled 21-mer DNA primer was annealed to a 35-mer (5′-GGTCCGTTGCATGCGGATACATATGGTTAAAGTAT-3′) DNA template at a 50 nm DNA concentration. DNA polymerization was initiated by the addition of wild-type, Q151M, or Q151Mcomplex RT (100 nm) and nucleotides (5 μm AZTTP, and 25 μm each of dATP, dCTP, and dGTP) for 15 min at 37 °C in RT buffer supplemented with 6 mm MgCl2. The unblocking reaction was started by adding dTTP to reach a final concentration of 25 μm in the presence of either pyrophosphate (PPi) or ATP. In this manner, unblocking is performed in the presence of the next correct nucleotide binding on top of the terminated primer, under conditions approximating those found in the infected cell. Aliquots were withdrawn during the time course of the reaction, and products were analyzed using denaturing gel electrophoresis. The % of unblocked primer is the ratio of extension products larger than 25 nucleotides over those larger than 24 nucleotides, multiplied by 100. Modeling was based on the 3.2-Å crystal structure of the complex of HIV-1 reverse transcriptase with a DNA template/primer and a deoxynucleoside triphosphate (17Huang H. Chopra R. Verdine G.L. Harrison S.C. Science. 1998; 282: 1669-1675Crossref PubMed Scopus (1358) Google Scholar) (Protein Data Bank code 1RTD). Structures were displayed, modified, and analyzed using the graphic program TURBO (18Roussel A. Cambillau C. Graphics S. Silicon Graphics Directory. Silico Graphics, Mountain View, CA1991: 97Google Scholar). Mutations were manually built and the whole structure was subsequently submitted to several rounds of minimization of the energy function, in which the crystallographic contribution has been removed, using the program crystallography NMR software (19Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D. 1998; 54: 905-921Crossref PubMed Scopus (16957) Google Scholar). Preliminary validation of the method was performed on the deposited coordinates of the complex using an identical protocol for minimization. The substitutions A62V/V75I/F77L/F116Y/Q151M have been described to confer resistance to various nucleoside analogues such as AZT, ddI, ddC, and d4T (5Shirasaka T. Kavlick M.F. Ueno T. Gao W.Y. Kojima E. Alcaide M.L. Chokekijchai S. Roy B.M. Arnold E. Yarchoan R. Mitsuya H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2398-2402Crossref PubMed Scopus (363) Google Scholar, 20Shirasaka T. Yarchoan R. O'Brien M.C. Husson R.N. Anderson B.D. Kojima E. Shimada T. Broder S. Mitsuya H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 562-566Crossref PubMed Scopus (130) Google Scholar, 21Shafer R.W. Iversen A.K. Winters M.A. Aguiniga E. Katzenstein D.A. Merigan T.C. J. Infect. Dis. 1995; 172: 70-78Crossref PubMed Scopus (96) Google Scholar, 22Shafer R.W. Kozal M.J. Winters M.A. Iversen A.K. Katzenstein D.A. Ragni M.V. Meyer III, W.A. Gupta P. Rasheed S. Coombs R. J. Infect. Dis. 1994; 169: 722-729Crossref PubMed Scopus (198) Google Scholar). In the clinic, this resistance pattern is selected in 3 to 16% of viruses isolated from patients treated with AZT and ddI or ddC. Enhanced nucleotide selectivity has been implicated as the mechanism of Q151M-based resistance (23Ueno T. Shirasaka T. Mitsuya H. J. Biol. Chem. 1995; 270: 23605-23611Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), as in the case of the single amino acid substitutions M184V (resistance to 3TC (24Feng J.Y. Anderson K.S. Biochemistry. 1999; 38: 9440-9448Crossref PubMed Scopus (121) Google Scholar)) and K65R (resistance to ddI and ddC (14Selmi 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)). However, it is not known how discrimination is achieved at the Q151Mcomplex RT active site. In other words, it is not known whether nucleotide analogues are poorly bound to the resistant RT nucleotide binding site relative to their natural counterparts, or are bound to resistant RT with the same affinity as their natural counterparts but poorly incorporated into DNA at the catalytic step, as it is the case for K65R RT and ddNTPs (14Selmi 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). On the other hand, it has been shown that AZT-resistant RT (bearing also multiple substitutions such as D67N/K70R/T215F or Y or K219E or Q) is acting through a primer rescue (or unblocking) mechanism preferentially using NTP as pyrophosphate donors (4Meyer 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). It was thus of interest to determine whether the substitutions A62V/V75I/F77L/F116Y/Q151M would increase the natural ability of wild-type RT to unblock the analogue-terminated primer. We tested the efficiency of pyrophosphorolytic- and ATP-mediated repair of an AZTMP-terminated DNA primer by RT. A 5′-32P-labeled 21-mer primer annealed to a specific 35-mer template is extended using RT and a mixture of nucleotides in which dTTP is replaced by AZTTP (Fig.1 A, step ❶). For the rescue reaction, the AZTMP-terminated 25-mer is incubated at various times in the presence of dTTP and ATP or PPi. ATP or PPiunblocks the primer, allowing RT to use dTTP now present in the reaction mixture and resume elongation up to a 35-mer (Fig.1 A, step ❷). In the case of ATP as the pyrophosphate donor, the unblocking activity exhibited by wild-type and Q151M RT are comparable (Fig. 1, B and C). The Q151Mcomplex RT shows a slightly impaired ATP-mediated ability to unblock the primer, relative to wild-type. Similar results are obtained for pyrophosphorolysis using PPi as the unblocking agent (Fig. 1, B and C). None of the substituted RT seems to have any improved primer unblocking ability that could account for resistance to AZTTP under various experimental conditions (not shown). We conclude that primer rescue is not involved in multinucleoside drug resistance exhibited by RT bearing Q151M-type substitutions. This finding is consistent with the results detailed below showing that discrimination of nucleotide analogues at the active site can account for drug resistance. Using pre-steady state kinetics with a single nucleotide incorporation, we tested the possibility that either K d or k pol are different between wild-type RT and Q151M-based mutants. Fig.2 shows a typical data set of rapid incorporation of a saturating concentration of dTTP or AZTTP by Q151M RT, with constant values reported in TableI. K d, the nucleotide affinity, is calculated as the nucleotide concentration that gives half of the maximum incorporation rate k pol (Fig. 2). The incorporation efficiency (k pol/K d) is used to calculate the selectivity factor: (k pol/K d)dNTP/(k pol/K d)analogue. A selectivity factor greater than 1 means that the enzyme discriminates the analogue over the natural nucleotide. Finally, the resistance of the RT to the inhibitor is the ratio between the selectivity of the mutant and the selectivity of the wild-type enzyme. The enzyme affinity for dTTP (K d = 14 μm) is very similar to that for AZTTP (K d = 18 μm). However, the k pol (AZTTP) value drops from 17 s−1 for dTTP to 4.3 s−1. This means that the rate of a step before or at the creation of the phosphodiester bond decreases 4-fold when AZTMP is incorporated in comparison with its natural counterpart dTTP.Table IPre-steady state kinetic constants of WT RT, Q151M RT, and Q151Mcomplex RT mutants for DNA templatesAdTTP/AZTTPNucleotideWT RTQ151M RTQ151Mcomplex RTdTTPAZTTPdTTPAZTTPdTTPAZTTPK d(μm)aKd andk pol were determined as described under “Experimental Procedures.” Standard deviations were <20%.17bValue from Selmi et al.(24).7.114189.79.9k pol(s−1)aKd andk pol were determined as described under “Experimental Procedures.” Standard deviations were <20%.13bValue from Selmi et al.(24).13174.37.61.1k pol/K d (s−1μm−1)0.751.81.20.240.790.11SelectivitycSelectivity and resistance were determined as described under “Results.” The selectivity is the ratio of [k pol/K d (nucleotide analogue)]/k pol/K d(nucleotide)].0.4X5X7.2XResistancecSelectivity and resistance were determined as described under “Results.” The selectivity is the ratio of [k pol/K d (nucleotide analogue)]/k pol/K d(nucleotide)].12X17XBdATP/ddATPNucleotideWT RTQ151M RTQ151Mcomplex RTdATPddATPdATPddATPdATPddATPK d(μm)aKd andk pol were determined as described under “Experimental Procedures.” Standard deviations were <20%.7.5bValue from Selmi et al.(24).8.0153.94111k pol(s−1)aKd andk pol were determined as described under “Experimental Procedures.” Standard deviations were <20%.50bValue from Selmi et al.(24).7.2500.69990.38k pol/K d (s−1μm−1)6.70.913.20.172.30.036SelectivitycSelectivity and resistance were determined as described under “Results.” The selectivity is the ratio of [k pol/K d (nucleotide analogue)]/k pol/K d(nucleotide)].7.4 X18 X66 XResistancecSelectivity and resistance were determined as described under “Results.” The selectivity is the ratio of [k pol/K d (nucleotide analogue)]/k pol/K d(nucleotide)].2.4X9XCdCTP/ddCTPNucleotideWT RTQ151M RTQ151Mcomplex RTdCTPddCTPdCTPddCTPdCTPddCTPK d(μm)aKd andk pol were determined as described under “Experimental Procedures.” Standard deviations were <20%.7.9bValue from Selmi et al.(24).5.4124.614.22.1k pol(s−1)aKd andk pol were determined as described under “Experimental Procedures.” Standard deviations were <20%.7.3bValue from Selmi et al.(24).1.98.60.516.00.14k pol/K d (s−1μm−1)0.930.340.690.110.420.066SelectivitycSelectivity and resistance were determined as described under “Results.” The selectivity is the ratio of [k pol/K d (nucleotide analogue)]/k pol/K d(nucleotide)].2.7X6.3X6.4XResistancecSelectivity and resistance were determined as described under “Results.” The selectivity is the ratio of [k pol/K d (nucleotide analogue)]/k pol/K d(nucleotide)].2.3X2.4XThe resistance is determined by the ratio of selectivityWT RT/selectivitymutant.a Kd andk pol were determined as described under “Experimental Procedures.” Standard deviations were <20%.b Value from Selmi et al.(24Feng J.Y. Anderson K.S. Biochemistry. 1999; 38: 9440-9448Crossref PubMed Scopus (121) Google Scholar).c Selectivity and resistance were determined as described under “Results.” The selectivity is the ratio of [k pol/K d (nucleotide analogue)]/k pol/K d(nucleotide)]. Open table in a new tab The resistance is determined by the ratio of selectivityWT RT/selectivitymutant. Wild-type RT does not discriminate AZTTP relative to dTTP (Table I, part A). Catalytic rate constants for both substrates are the same (13 s−1). However, the nucleotide affinity is better for AZTTP than dTTP. Wild-type RT has a slightly higher overall incorporation efficiency (k pol/K d) for AZTTP (1.8 s−1 μm−1) than for dTTP (0.7 s−1 μm−1). Q151M RT, as shown above (Fig. 2), does not alter nucleotide affinity significantly but induces a drop in k pol from 17 s−1for dTTP to 4.3 s−1 for AZTTP, decreasing the incorporation efficiency 5-fold. Comparison of incorporation efficiencies of wild-type and Q151M RT leads to a 12-fold resistance mainly provided by a k pol effect. When the four other mutations (A62V, V75I, F77L, and F116Y) are added to the Q151M background, the k pol constant further decreases 7-fold from 7.7 s−1 for dTTP to 1.1 s−1 for AZTTP. On the other hand, the affinity for the Q151Mcomplex RT is unchanged (K d = 10 μm). Taken together, these constants bring the AZTTP incorporation efficiency down to 0.11 s−1μm−1, which is responsible for a 7.2-fold discrimination, and a 17-fold resistance. We conclude that resistance to AZTTP is observed with the Q151M mutation on reverse transcriptase at the catalytic step, and this resistance is further increased in the case of the Q151Mcomplex RT. Same single nucleotide incorporation assays were performed to study resistance to ddATP (TableI, part B). As observed previously, wild-type RT discriminates ddATP against dATP. Unlike dTTP/AZTTP incorporation data, the affinity of wild-type RT for dATP (K d = 7.5 μm) is very similar to that of ddATP (K d = 8 μm). However, the catalytic rate constantk pol(ddATP) is 7-fold lower thank pol(dATP). This decrease brings the catalytic efficiency k pol/K d (ddATP) down to 0.91 s−1 μm−1, and is responsible for a 7.4-fold discrimination. Pre-steady state constants of dATP incorporation by Q151M RT are very similar to those measured using wild-type RT. However, k pol decreases 73-fold from 50 s−1 for dATP to 0.69 s−1 for ddATP, resulting in an incorporation efficiency of 0.17 s−1 μm−1. A 18-fold discrimination of ddATP by Q151M RT is obtained, resulting in an overall 2.4-fold resistance relative to wild-type RT. In the case of the Q151Mcomplex RT, the incorporation rate is even lower (k pol(ddATP) = 0.38 s−1), inducing a 66-fold discrimination and a 9-fold resistance. Resistance to ddCTP was analyzed in a similar fashion (Table I, part C). We noticed that like ddATP, wild-type RT originally discriminates ddCTP from dCTP, with a 3-fold lower catalytic efficiency (0.93 s−1 μm−1 for dCTP to 0.34 s−1 μm−1 for ddCTP) originating from a decrease in thek pol value. The Q151M substitution increases this discrimination further: k poldecreases from 1.9 s−1 to 0.5 s−1 yielding in a 2.3-fold resistance to ddCTP. The affinity for the nucleotide analogue remains unchanged. The Q151Mcomplex RT displays a very low incorporation rate down to 0.14 s−1, a 43-fold decrease relative to k pol(dATP). However, because of a higher affinity to ddCTP, the overall resistance of Q151Mcomplex RT to ddCTP remains in the vicinity to that of Q151M RT. We conclude that, as it is the case for AZTTP, resistance to ddATP and ddCTP is observed with Q151M RT at the catalytic step, and this resistance is further increased with Q151McomplexRT. Fig. 3 summarizes the evolution of RT-mediated resistance by discrimination of AZTTP and ddNTPs. Resistance is mostly acquired by the Q151M substitution in the case of AZTTP (12-fold), and ddCTP (2.3-fold). On the other hand, the addition of the other four mutations is required to reach high ddATP resistance levels (9-fold), whereas Q151M alone is responsible for only a 2.5-fold increase. The affinity constant K d does not play any significant role in the discrimination process. Table Ishows that there is no drop in affinity for ddATP, ddCTP, nor AZTTP upon the appearance of resistance substitutions. Instead, there is a direct dependence of Q151M-based resistance to AZTTP and ddNTPs upon the catalytic constant k pol (Fig. 3,A and B). 90% of the initialk pol(ddATP) value drops as a result of the sole methionine substitution" @default.
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