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Mechanistic Studies to Understand the Progressive Development of Resistance in Human Immunodeficiency Virus Type 1 Reverse Transcriptase to Abacavir

2002; Elsevier BV; Volume: 277; Issue: 43 Linguagem: Inglês

10.1074/jbc.m205303200

1083-351X

Adrian S. Ray, Aravind Basavapathruni, Karen S. Anderson,

HIV-related health complications and treatments

Abacavir has been shown to select for multiple resistant mutations in the human immunodeficiency type 1 (HIV-1) pol gene. In an attempt to understand the molecular mechanism of resistance in response to abacavir, and nucleoside analogs in general, a set of reverse transcriptase mutants were studied to evaluate their kinetics of nucleotide incorporation and removal. It was found that, similar to the multidrug-resistant mutant reverse transcriptase (RT)Q151M, the mutations L74V, M184V, and a triple mutant containing L74V/Y115F/M184V all caused increased selectivity for dGTP over the active metabolite of abacavir (carbovir triphosphate). However, the magnitude of resistance observed in cell culture to abacavir in previous studies was less than that observed to other compounds. Our mechanistic studies suggest that this may be due to carbovir triphosphate decreasing the overall effect on its efficiency of incorporation by forming strong hydrophobic interactions in the RT active site. Unlike RTAZTR, no increase in the rate of ATP- or PPi-mediated chain terminator removal relative to RTWT could be detected for any of the mutants. However, marked decreases in the steady-state rate may serve as a mechanism for increased removal of a chain-terminating carbovir monophosphate by increasing the time spent at the primer terminus for some of the mutants studied. The triple mutant showed no advantage in selectivity over RTM184V and was severely impaired in its ability to remove a chain terminator, giving no kinetic basis for its increased resistance in a cellular system. Biochemical properties including percentage of active sites, fidelity, and processivity may suggest that the triple mutant's increased resistance to abacavir in cell culture is perhaps due to a fitness advantage, although further cellular studies are needed to verify this hypothesis. These data serve to further the understanding of how mutations in RT confer resistance to nucleoside analogs. Abacavir has been shown to select for multiple resistant mutations in the human immunodeficiency type 1 (HIV-1) pol gene. In an attempt to understand the molecular mechanism of resistance in response to abacavir, and nucleoside analogs in general, a set of reverse transcriptase mutants were studied to evaluate their kinetics of nucleotide incorporation and removal. It was found that, similar to the multidrug-resistant mutant reverse transcriptase (RT)Q151M, the mutations L74V, M184V, and a triple mutant containing L74V/Y115F/M184V all caused increased selectivity for dGTP over the active metabolite of abacavir (carbovir triphosphate). However, the magnitude of resistance observed in cell culture to abacavir in previous studies was less than that observed to other compounds. Our mechanistic studies suggest that this may be due to carbovir triphosphate decreasing the overall effect on its efficiency of incorporation by forming strong hydrophobic interactions in the RT active site. Unlike RTAZTR, no increase in the rate of ATP- or PPi-mediated chain terminator removal relative to RTWT could be detected for any of the mutants. However, marked decreases in the steady-state rate may serve as a mechanism for increased removal of a chain-terminating carbovir monophosphate by increasing the time spent at the primer terminus for some of the mutants studied. The triple mutant showed no advantage in selectivity over RTM184V and was severely impaired in its ability to remove a chain terminator, giving no kinetic basis for its increased resistance in a cellular system. Biochemical properties including percentage of active sites, fidelity, and processivity may suggest that the triple mutant's increased resistance to abacavir in cell culture is perhaps due to a fitness advantage, although further cellular studies are needed to verify this hypothesis. These data serve to further the understanding of how mutations in RT confer resistance to nucleoside analogs. Human immunodeficiency virus (HIV), 1The abbreviations used are: HIV, human immunodeficiency virus; RT, reverse transcriptase; NRTI, nucleoside reverse transcriptase inhibitor; CBV, carbovir; AZT, β-d-(+)-3′-azido-3′-deoxythymidine; 3TC, β-l-(-)-2′,3′-dideoxy-3′-thiacytidine; D4T, β-d-(+)-2′,3′-didehydro-3′-deoxythymidine; D4G, β-d-(+)-2′,3′-didehydro-2′,3′-dideoxyguanosine (the suffix -MP or -TP is added to the drug abbreviations to indicate their monophosphate or triphosphate form, respectively); WT, wild type; CBVR, carbovir-resistant triple mutant containing L74V/Y115F/M184V; AZTR, AZT-resistant quadruple mutant containing D67N/K70R/T215Y/K219Q. the causative agent of AIDS, requires reverse transcriptase (RT) to copy its single-stranded RNA genome into a double-stranded DNA copy for integration into the host cell genome. Although almost all aspects of the HIV-1 life cycle have been targeted (1De Clercq E. J. Med. Chem. 1995; 38: 2491-2517Google Scholar, 2De Clercq E. Clin. Microbiol. Rev. 1997; 10: 674-693Google Scholar, 3Mitsuya H. Yarchoan R. Kageyama S. Broder S. FASEB J. 1991; 5: 2369-2381Google Scholar), a majority of the drugs that have been effective in clinical trials are nucleoside reverse transcriptase inhibitors (NRTIs). However, treatment with NRTIs is limited by their toxicity to the host (often through their interaction with mitochondrial DNA polymerase γ (4Parker W.B. Cheng Y.-C. J. NIH Res. 1994; 6: 57-61Google Scholar, 5Martin J.L. Brown C.E. Matthews-Davis N. Reardon J.E. Antimicrob. Agents Chemother. 1994; 38: 2743-2749Google Scholar, 6Johnson A.A. Ray A.S. Hanes J.W. Suo Z. Colacino J.M. Anderson K.S. Johnson K.A. J. Biol. Chem. 2001; 276: 40847-40857Google Scholar, 7Feng J. Johnson A. Johnson K. Anderson K. J. Biol. Chem. 2001; 276: 23832-23837Google Scholar)) and the ability of the virus to mutate and gain resistance (8Larder B.A. J. Gen. Virol. 1994; 75: 951-957Google Scholar). Other factors that affect the ability of these inhibitors to reduce viral replication are uptake, transport, metabolism, and incorporation of the drug. All clinically used nucleoside analogs lack 3′-hydroxyl groups and are metabolically activated by host cellular kinases to their triphosphate forms. Compounds currently approved by the Food and Drug Administration are β-d-(+)-3′-azido-3′-deoxythymidine (AZT or zidovudine), β-d-(+)-2′,3′-didehydro-3′-deoxythymidine (D4T or stavudine), β-l-(−)-2′,3′-dideoxy-3′-thiacytidine (3TC or lamivudine), β-d-(+)-2′,3′-dideoxycytidine, β-d-(+)-2′,3′-dideoxyinosine, (1S,4R)-4-[2-amino-6-(cyclopropyl-amino)-9H-purin-9-yl]-2-cyclopentene-1-methanol succinate (abacavir or ziagen), and (R)-9-(2-phosphonylmethoxy-propyl)adenine (PMPA). HIV-1's high rate of replication and the lack of proofreading by RT during viral replication leads to frequent mutations (9Preston B.D. Poiez B. Loeb L.A. Science. 1988; 242: 1168-1171Google Scholar, 10Roberts J.D. Bebenek K. Kunkel T.A. Science. 1988; 242: 1171-1173Google Scholar, 11Ji J. Loeb L.A. Biochemistry. 1992; 31: 954-958Google Scholar, 12Coffin J.M. Science. 1995; 267: 483-489Google Scholar). Distinct mutations occur in the presence of different NRTIs, and their temporal occurrence is often predictable (13Shirasaka 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: 562-566Google Scholar, 14Larder B.A. Kemp S.D. Science. 1989; 246: 1155-1158Google Scholar, 15Kellam P. Boucher C.A. Larder B.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1934-1938Google Scholar, 16Shafer 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. et al.J. Infect. Dis. 1994; 169: 722-729Google Scholar, 17Tisdale M. Alnadaf T. Cousens D. Antimicrob. Agents Chemother. 1997; 41: 1094-1098Google Scholar, 18Moyle G.J. J. Antimicrob. Chemother. 1997; 40: 767-777Google Scholar). Initial mutations are often responsible for resistance to the compound, whereas later mutations increase the fitness of the mutant virus (19Maeda Y. Venzon D.J. Mitsuya H. J. Infect. Dis. 1998; 177: 1207-1213Google Scholar). The mutations that are responsible for conferring nucleoside drug resistance are primarily clustered in three regions of the protein as illustrated in Fig. 1. These include the dNTP binding site (region II), the site near the n + 1 templating base (region III), and the putative ATP binding site (region I). These mutations have been shown to cause resistance by two distinct mechanisms. The first mechanism of resistance appears to be related to a change in the incorporation of the activated drug into the replicating viral genome. These mutations are often found to be in direct contact with the incoming dNTP in the active site of RT and impede nucleotide analog incorporation and thereby show altered reaction kinetics (Fig. 1, region II) (20Feng J.Y. Anderson K.S. Biochemistry. 1999; 38: 9440-9448Google Scholar, 21Ray A.S. Anderson K.S. Nucleosides Nucleotides Nucleic Acids. 2001; 20: 1247-1250Google Scholar, 22Ray A.S. Yang Z. Shi J. Hobbs A. Schinazi R.F. Chu C.K. Anderson K.S. Biochemistry. 2002; 41: 5150-5162Google Scholar, 23Sluis-Cremer N. Arion D. Kaushik N. Lim H. Parniak M.A. Biochem. J. 2000; 348: 77-82Google Scholar, 24Krebs R. Immendörfer U. Thrall S.H. Wöhrl B.M. Goody R.S. Biochemistry. 1997; 36: 10292-10300Google Scholar, 25Vaccaro J.A. Anderson K.S. Biochemistry. 1998; 37: 14189-14194Google Scholar, 42Gu Z. Gao Q. Fang H. Parniak M.A. Brenner B.G. Wainberg M.A. Leukemia. 1994; 8 Suppl. 1: 166-169Google Scholar). This has been best illustrated by steric hindrance interfering with 3TCTP binding in the active site of HIV-1 RT containing a Met184 to Val mutation (20Feng J.Y. Anderson K.S. Biochemistry. 1999; 38: 9440-9448Google Scholar, 26Gao H.Q. Boyer P.L. Sarafianos S.G. Arnold E. Hughes S.H. J. Mol. Biol. 2000; 300: 403-418Google Scholar, 27Sarafianos S.G. Das K. Clark Jr., A.D. Ding J. Boyer P.L. Hughes S.H. Arnold E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10027-10032Google Scholar). Additionally, other mutations have also been noted that contact then + 1 templating base and appear to cause resistance at the level of incorporation by repositioning the active site in an unfavorable orientation for analog incorporation (Fig. 1, region III) (28Jacobo-Molina A. Ding J. Nanni R.G. Clark A.D., Jr., Lu, X. Tantillo C. Williams R.L. Kamer G. Ferris A.L. Clark P. Hizi A. Hughes S.H. Arnold E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6320-6324Google Scholar, 29Tantillo C. Ding J. Jacobo-Molina A. Nanni R.G. Boyer P.L. Hughes S.H. Pauwels R. Andries K. Janssen P.A. Arnold E. J. Mol. Biol. 1994; 243: 369-387Google Scholar, 30Boyer P.L. Tantillo C. Jacobo-Molina A. Nanni R.G. Ding J. Arnold E. Hughes S.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4882-4886Google Scholar, 31Ueno T. Shirasaka T. Mitsuya H. J. Biol. Chem. 1995; 270: 23605-23611Google Scholar, 32Selmi B. Boretto J. Navarro J.M. Sire J. Longhi S. Guerreiro C. Mulard L. Sarfati S. Canard B. J. Biol. Chem. 2001; 276: 13965-13974Google Scholar). The second mechanism of resistance appears to involve removal of the chain-terminator. RT does not contain 3′ to 5′ exonuclease activity in the same sense as traditional polymerases, but the reverse of the forward reaction catalyzed by the polymerase active site can serve to remove a 3′ chain-terminating compound. The largest amount of work done to understand this mechanism of resistance is in the case of AZT resistance. AZT-resistant mutations occur in a pocket that is connected to the triphosphate binding region of the active site (Fig.1, region I) (33Huang H. Verdine G.L. Chopra R. Harrison S.C. Science. 1998; 282: 1669-1675Google Scholar, 34Boyer P.L. Sarafianos S.G. Arnold E. Hughes S.H. J. Virol. 2001; 75: 4832-4842Google Scholar, 43Fitzgibbon J.E. Howell R.M. Haberzettl C.A. Sperber S.J. Gocke D.J. Dubin D.T. Antimicrob. Agents Chemother. 1992; 36: 153-157Google Scholar). Reports have shown that AZT-resistant mutations, in the absence of causing large changes in incorporation (25Vaccaro J.A. Anderson K.S. Biochemistry. 1998; 37: 14189-14194Google Scholar, 35Kerr S.G. Anderson K.S. Biochemistry. 1997; 36: 14064-14070Google Scholar), may cause an increase in the rate of pyrophosphorolysis (36Arion D. Kaushik N. McCormick S. Borkow G. Parniak M.A. Biochemistry. 1998; 37: 15908-15917Google Scholar) and ATP-mediated removal (34Boyer P.L. Sarafianos S.G. Arnold E. Hughes S.H. J. Virol. 2001; 75: 4832-4842Google Scholar, 37Meyer P.R. Matsuura S.E. Mian A.M., So, A.G. Scott W.A. Mol. Cell. 1999; 4: 35-43Google Scholar). There are some conflicting reports on whether the kinetic rate of pyrophosphorolysis is in fact increased by AZT-resistant mutations, and mounting evidence suggests that there is no increase in the rate of removal by PPi (34Boyer P.L. Sarafianos S.G. Arnold E. Hughes S.H. J. Virol. 2001; 75: 4832-4842Google Scholar). It may be argued, however, that this does not mean that PPi does not play a role in AZT resistance. Studies have shown that there is a slower rate of primer-template release by the AZT-resistant mutant (38Canard B. Sarfati S.R. Richardson C.C. J. Biol. Chem. 1998; 273: 14596-14604Google Scholar) and a tendency to form a stable complex, less sensitive to inhibition by the presence of the next correct nucleotide, at the incorporated AZTMP nucleotide (39Meyer P.R. Matsuura S.E. Schinazi R.F., So, A.G. Scott W.A. Antimicrob. Agents Chemother. 2000; 44: 3465-3472Google Scholar). Accordingly, this may increase the overall amount of removal by prolonging the time RT spends at the 3′ terminus no matter what the removing agent (PPi or ATP) and in the absence of any marked change in the kinetic rate of removal. These results taken together suggest that both modes of removal may be active in vivo (40Lennerstrand J. Hertogs K. Stammers D.K. Larder B.A. J. Virol. 2001; 75: 7202-7205Google Scholar), although further studies are needed to clarify inconsistencies in the literature. To better understand resistance to the Food and Drug Administration-approved compound abacavir and the mechanism of resistance to NRTIs in general, transient kinetic methodology was employed with abacavir's active metabolite (CBVTP, Fig. 2) (41Faletto M.B. Miller W.H. Garvey E.P., St. Clair M.H. Daluge S.M. Good S.S. Antimicrob. Agents Chemother. 1997; 41: 1099-1107Google Scholar) and a subset of mutants found in an in vitro drug selection study (17Tisdale M. Alnadaf T. Cousens D. Antimicrob. Agents Chemother. 1997; 41: 1094-1098Google Scholar). Conclusions were further tested using a clinically relevant multidrug-resistant mutant (Q151M (16Shafer 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. et al.J. Infect. Dis. 1994; 169: 722-729Google Scholar)) and an AZT-resistant mutant (RTAZTR, D67N/K70R/T215Y/K219Q (14Larder B.A. Kemp S.D. Science. 1989; 246: 1155-1158Google Scholar, 15Kellam P. Boucher C.A. Larder B.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1934-1938Google Scholar)). Our study looked at both incorporation and removal (by PPi and ATP) along with other biochemical properties of these mutants. Results showed that the resistance of mutants seen in cell culture could not always be explained by incorporation alone. None of the abacavir-selected mutants showed increases in PPi- or ATP-mediated removal rates, but some had significantly slower primer-template release rates, which is discussed as a possible mode of facilitating nucleotide removal. Abacavir's resistance profile, in light of the large number of mutations coupled with the relatively small changes in biological and kinetic behavior, may best be described as "minimization of resistance rather than avoidance." This, in part, may be the result of CBVTP's inability to effectively mimic dGTP and a dampening of the overall reaction kinetics by strong hydrophobic interactions in the RT active site. RTWT, RTY115F, RTAZTR, and RTM184V clones were generously provided by Stephen Hughes, Paul Boyer, and Andrea Ferris (NCI-Frederick). RTL74V and RTQ151Mclones were created from the Hughes clone and kindly provided by Phillip Furman, Joy Feng, and Jerry Jeffrey (Triangle Pharmaceuticals). The triple mutant L74V/Y115F/M184V (RTCBVR) was made by sequentially adding mutations to the Hughes RTM184V clone using the Stratagene QuikChange kit. All RT clones were sequenced to verify correct sequence. The N-terminal histidine-tagged heterodimeric p66/p51 enzymes were purified as previously described (47Kerr S.G. Anderson K.S. Biochemistry. 1997; 36: 14056-14063Google Scholar, 48Feng J.Y. Anderson K.S. Biochemistry. 1999; 38: 55-63Google Scholar). dGTP was purchased from AmershamBiosciences. The (−)-CBVTP was generously provided by Dr. William B. Parker (Southern Research Institute, Birmingham, AL). The compound was further purified by high pressure liquid chromatography utilizing a gradient from 20 to 60% 1 mEt3NH+(HCO3)− in water and an Amersham Biosciences ion exchange column (mono Q HR 5/5). Its identity was verified using liquid chromatography/electrospray ionization mass spectrometry. The concentration of purified CBVTP was determined using the extinction coefficient ε253 = 13,260m−1 cm−1 (49White E.L. Parker W.B. Macy L.J. Shaddix S.C. McCaleb G. Secrist III, J.A. Vince R. Shannon W.M. Biochem. Biophys. Res. Commun. 1989; 161: 393-398Google Scholar). Primers and templates used for incorporation and removal studies are shown in Table I, and all of the DNA oligonucleotides were synthesized on an Applied Biosystems 380A DNA synthesizer (Keck DNA synthesis facility, Yale University) and purified using 20% polyacrylamide denaturing gel electrophoresis. The R45-mer was synthesized and purified by New England Biolabs. D30-CBVMP was made using RTWT as previously described (6Johnson A.A. Ray A.S. Hanes J.W. Suo Z. Colacino J.M. Anderson K.S. Johnson K.A. J. Biol. Chem. 2001; 276: 40847-40857Google Scholar).Table ISequence of primers and templates used to study CBVMP incorporation and removalNameSequenceDNA 23-mer5′ -GCC TCG CAG CCG TCC AAC CAA CTDNA 30-mer5′ -GCC TCG CAG CCG TCC AAC CAA CTC AAC CTCDNA 31-mer5′ -GCC TCG CAG CCG TCC AAC CAA CTC AAC CTC GDNA 30-CBVMP5′ -GCC TCG CAG CCG TCC AAC CAA CTC AAC CTC VaV, CBVMP.DNA 45-mer3′ -CGG AGC GTC GGC AGG TTG GTT GAG TTG GAG CTA GGT TAC GGC AGGRNA 45-mer3′ -CGG AGC GUC GGC AGG UUG GUU GAG UUG GAGCUA GGU UAC GGC AGGDNA 45-mer (removal)3′ -CGG AGC GTC GGC AGG TTG GTT GAG TTG GAG CGA GGT TAC GGC AGGTemplating bases for single nucleotide incorporation and removal studies are shown in boldface italic type, and sites of expected RNase H cleavage during incorporation into D30/R45 at 18 and 21 base pairs from the site of incorporation are underlined.a V, CBVMP. Open table in a new tab Templating bases for single nucleotide incorporation and removal studies are shown in boldface italic type, and sites of expected RNase H cleavage during incorporation into D30/R45 at 18 and 21 base pairs from the site of incorporation are underlined. The 23-, 30-, and 31-mer DNAs and 45-mer RNA were 5′-32P-labeled with T4 polynucleotide kinase (New England Biolabs) as previously described (50Kati W.M. Johnson K.A. Jerva L.F. Anderson K.S. J. Biol. Chem. 1992; 267: 25988-25997Google Scholar). [γ-32P]ATP was purchased from Amersham Biosciences. Biospin columns for the removal of excess [γ-32P]ATP were purchased from Bio-Rad. Annealing of the DNA primers (D23, D30, D31, or D30-CBVMP) and 45-mer DNA and RNA templates were carried out by adding a 1:1.4 molar ratio of purified primer to 45-mer at 90 °C for 5 min, 50 °C for 10 min, and ice for 10 min. The annealed primer and template were then analyzed using 15% nondenaturing polyacrylamide gel electrophoresis to ensure complete annealing. Concentrations of the oligonucleotides were estimated by UV absorbance at 260 nm using calculated extinction coefficients. Rapid chemical quench experiments were performed as previously described with a KinTek Instruments model RQF-3 rapid quench-flow apparatus (47Kerr S.G. Anderson K.S. Biochemistry. 1997; 36: 14056-14063Google Scholar, 50Kati W.M. Johnson K.A. Jerva L.F. Anderson K.S. J. Biol. Chem. 1992; 267: 25988-25997Google Scholar). A pre-steady-state kinetic analysis was used to examine the incorporation and removal of dGMP or CBVMP into a DNA/DNA or DNA/RNA duplex. To analyze rates of incorporation of less than 2 s−1, single-turnover experiments were used. The reactions were carried out by rapid mixing of a solution containing the preincubated complex of 250 nm HIV-1 RT (active site concentration) and 50 nm 5′-labeled DNA/DNA duplex with a solution of 10 mm MgCl2 and varying concentrations of the next correct dNTP in the presence of 50 mm Tris-Cl, 50 mm NaCl, at pH 7.8 and 37 °C (all concentrations represent the final concentration after mixing). Single-turnover experiments were also used to study removal, misincorporation, and processivity. Misincorporation was studied in the same manner, except the DNA 31-mer primer was used to allow for the observation of incorporation of dGMP opposite a templating dTMP. In processivity experiments, elongation of the 23-mer DNA with the 45-mer DNA or RNA template was followed in the presence of 125 μm (final) of all four dNTPs. Polymerization was quenched at various time points by the addition of 0.3 m (final) EDTA. Single-turnover conditions were also used to study the removal of chain-terminating nucleotides. Either 2 mm PPi(J.T. Baker catalogue no. 3850-1) or 3.2 mm ATP (Sigma catalogue no. A2383) were mixed with a solution containing 250 nm HIV-1 RT prebound to 50 nm 5′-labeled chain-terminated DNA/DNA duplex with 10 mmMgCl2 in the presence of 50 mm Tris, 50 mm NaCl, at pH 7.8 and 37 °C (all concentrations represent the final concentration after mixing). Removal studies using the original D45/D31 template-primer were unsuccessful in that no removal of a chain-terminating CBVMP and the D31-mer (containing dGMP at the 3′ terminus) was only extended under these conditions. The observed incorporation may have been due to ribonucleotide incorporation or a small amount of deoxynucleotide contamination. The inefficient nucleotide-dependent removal when the next correct incorporation is complementary to the removing nucleotide has been previously noted (51Meyer P.R. Matsuura S.E., So, A.G. Scott W.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13471-13476Google Scholar). In order to alleviate this problem, the templating base was changed from dTMP to dGMP in the D45-mer template for removal studies. To obtain observed rates of incorporation for faster reactions and to measure the steady-state rate of incorporation, pre-steady-state burst experiments were used. Pre-steady-state bursts were done under the same conditions as those described for a single-turnover experiment, except the amount of primer-template (300 nm final) was in 3-fold excess of enzyme (100 nm active sites final). Products were analyzed by 20% polyacrylamide gel electrophoresis and quantified using a Bio-Rad GS525 molecular imager. Data were fit by nonlinear regression using the program KaleidaGraph version 3.09 (Synergy Software, Reading, PA). Results from pre-steady-state burst experiments were fit to a burst equation: [Product] = A(1 − exp(−k obsd t) +k ss t), where A represents the amplitude of the burst that correlates with the concentration of active enzyme, k obsd is the observed first-order rate constant for dNTP or analog incorporation, andk ss is the observed steady-state rate constant. Data from single-turnover incorporation experiments were fit to a single exponential equation: [Product] = A(1 − exp(−k obsd t)). L74V incorporation of CBVMP into a DNA/DNA primer-template showed two exponential phases of incorporation, and data were fit to a double exponential equation [Product] = A 1(1 − exp(−k obsd1 t)) +A 2(1 − exp(−k obsd2 t)). Although double exponential behavior under single-turnover conditions has been previously noted (22Ray A.S. Yang Z. Shi J. Hobbs A. Schinazi R.F. Chu C.K. Anderson K.S. Biochemistry. 2002; 41: 5150-5162Google Scholar), L74V incorporation of CBVMP was unique in that it could be observed under pre-steady-state burst conditions. Single-turnover removal experiments were fit to a single exponential decay: [Product] =A(exp(−k obsd t)). The dissociation constant (K d) of dNTP binding to the complex of RT and primer-template during dNMP incorporation was calculated by fitting the observed rate constants at different concentrations of dNTP to the following hyperbolic equation:k obsd = (k pol[dNTP])/(K d + [dNTP]), where k pol is the maximum first-order rate constant for dNMP incorporation and K d is the equilibrium dissociation constant for the interaction of dNTP with theE·DNA complex. Errors reported represent the deviation of points from the curve fit generated by KaleidaGraph or were calculated by standard statistical analysis (52Skoog D.A. Leary J.J. Principles of Instrumental Analysis. 4th Ed. Saunders College Publishing, New York1992Google Scholar). In the current study, we compare the activities of RTWT and a number of drug-resistant mutants in terms of incorporation and removal of dGMP and CBVMP (CBVTP shown in Fig.2) into model oligonucleotide substrates (Table I). Experiments were also done to further characterize the effects of some of the mutations on biochemical properties such as misincorporation and processivity. A series of pre-steady-state bursts and single-turnover experiments were conducted in order to determine the kinetic parameters for correct and incorrect single nucleotide incorporation directed by a DNA or RNA template. Kinetic constants determined include the maximum rate of incorporation (k pol) and the equilibrium dissociation constant (K d). From these values, the incorporation efficiency was calculated (k pol/K d) and used as a means for comparing different substrates and enzymes. The observed rate of dNMP removal from the end of a terminated chain was also determined at a single concentration of PPi(k pyro) and ATP (k ATP). This information was used as a quantitative basis for understanding these RT mutants and how their presence may confer resistance to abacavir. When primer-template is in slight excess over RT, the kinetics of nucleotide incorporation during the first enzyme turnover as well as multiple turnovers can be examined. During the incorporation of all natural dNMPs by RT, this results in the observation of a burst of product formation, as prebound substrate is turned over, followed by a linear phase reflecting the overall rate-limiting step of product release (50Kati W.M. Johnson K.A. Jerva L.F. Anderson K.S. J. Biol. Chem. 1992; 267: 25988-25997Google Scholar). All of the RT mutants showed a burst of product formation during incorporation of dGMP (an example of a burst of CBVMP incorporation is shown in Fig.4 A). The amplitude of the fast phase of product formation can be directly correlated to the amount of active enzyme, and when compared with the total protein concentration (determined by absorbance at 280 nm), the percentage of active protein can be calculated. These analyses showed that none of the mutants had a significantly lower active site concentration than RTWT (25%) and that many had different activity depending on the primer-template (Fig.3). During DNA-directed polymerization, RTL74V and RTCBVR(L74V/Y115F/M184V) showed the highest percentage of active sites (around 40%). During RNA-directed polymerization, RTM184Vand RTCBVR showed the highest level of activity (around 50%). RTCBVR was the only protein with at least a 10% higher percentage of active sites during both DNA- and RNA-directed polymerization than RTWT.Figure 3Percentage of active sites for DNA- and RNA-directed DNA synthesis by HIV-1 RT mutants. Values obtained by comparing the concentration of protein obtained by absorbance at 280 nm and the amplitude of pre-steady-state burst experiments looking at either DNA- or RNA-directed dGMP incorporation. Values represent the mean ± S.D. of at least four independent experiments.View Large Image Figure ViewerDownload (PPT) The presence of a pre-steady-state burst during the incorporation of an analog suggests that it is being incorporated by a similar kinetic mechanism as natural nucleotides. A pre-steady-state burst of product formation was observed for the incorporation of CBVMP by RTWT and all mutants studied (CBVMP incorporation by RTCBVR shown in Fig.4 A). To better understand the effect of mutations at positions 74, 115, and 184 both alone and in combination and a multidrug-resistant mutation at position 151 (locations of mutations shown in Fig. 1), pre-steady-state incorporation studies were carried out. By fitting the observed rates of nucleotide incorporation (k obsd) for DNA- and RNA-directed polymerization at different concentrations of dGTP for each of the RT mutants to hyperbolic curves, the maximum rates of dGMP incorporation (k pol) and the binding constants for dGTP (K d) were obtained. Fig. 4 shows the typical kinetic data obtained and how it is quantitated during transient kinetic studies (in this case the DNA-directed incorporation of CBVMP by RTCBVR). First, a pre-steady-state burst is done to determine whether, like a natural nucleotide, the rate-limiting step follows chemistry (Fig. 4 A). A set of single-turnover experiments were then done at varying nucleotide concentrations to determine the observed rate (Fig. 4 B), and the observed rates were then plotted against nucleotide concentration and fit to a hyperbolic curve to determine the K d andk pol (Fig. 4 C). In general, thek pol values for different mutants were all equal to or faster than those obtained for RTWT incorporation of dGMP; however, the