Mechanistic Understanding of an Altered Fidelity Simian Immunodeficiency Virus Reverse Transcriptase Mutation, V148I, Identified in a Pig-tailed Macaque
2003; Elsevier BV; Volume: 278; Issue: 32 Linguagem: Inglês
10.1074/jbc.m211754200
ISSN1083-351X
AutoresTracy L. Diamond, George P. Souroullas, Kellie K. Weiss, Kwi Y. Lee, Robert A. Bambara, Stephen Dewhurst, Baek Kim,
Tópico(s)HIV/AIDS Research and Interventions
ResumoWe have recently reported that the reverse transcriptase (RT) of SIVMNE 170 (170), which is a representative viral clone of the late symptomatic phase of infection with the parental strain, SIVMNE CL8 (CL8), has a largely increased fidelity, compared with the CL8 RT. In the present study, we analyzed the mechanistic alterations of the high fidelity 170 RT variant. First, we found that among several 170 RT mutations, only one, V148I, is solely responsible for the fidelity increase over the CL8 RT. This V148I mutation lies near the Gln-151 residue that we recently found is important to the low fidelity of RT and the binding of incoming dNTPs. Second, we compared dNTP binding affinity (K d) and catalysis (k pol) of the CL8 RT and the CL8-V148I RT using pre-steady state kinetic analysis. In this experiment, the high fidelity CL8-V148I RT has largely decreased binding to both correct and incorrect dNTP without altering k pol. The fidelity increase imparted by the V148I mutation is likely because of the major reduction seen in RT binding to dNTPs. This parallels our findings with the Q151N mutant. Third, site-directed mutagenesis targeting amino acid residue 148 has revealed that a valine amino acid at this position is essential to RT infidelity. Based on these findings, we discuss possible structural impacts of residue 148 (and mutations at this site) on the interaction of RT with incoming dNTPs and infer how alterations in these properties may relate to viral replication and fitness. We have recently reported that the reverse transcriptase (RT) of SIVMNE 170 (170), which is a representative viral clone of the late symptomatic phase of infection with the parental strain, SIVMNE CL8 (CL8), has a largely increased fidelity, compared with the CL8 RT. In the present study, we analyzed the mechanistic alterations of the high fidelity 170 RT variant. First, we found that among several 170 RT mutations, only one, V148I, is solely responsible for the fidelity increase over the CL8 RT. This V148I mutation lies near the Gln-151 residue that we recently found is important to the low fidelity of RT and the binding of incoming dNTPs. Second, we compared dNTP binding affinity (K d) and catalysis (k pol) of the CL8 RT and the CL8-V148I RT using pre-steady state kinetic analysis. In this experiment, the high fidelity CL8-V148I RT has largely decreased binding to both correct and incorrect dNTP without altering k pol. The fidelity increase imparted by the V148I mutation is likely because of the major reduction seen in RT binding to dNTPs. This parallels our findings with the Q151N mutant. Third, site-directed mutagenesis targeting amino acid residue 148 has revealed that a valine amino acid at this position is essential to RT infidelity. Based on these findings, we discuss possible structural impacts of residue 148 (and mutations at this site) on the interaction of RT with incoming dNTPs and infer how alterations in these properties may relate to viral replication and fitness. One of the most unique enzymatic properties of lentiviral DNA polymerases (i.e. human and simian immunodeficiency virus (HIV-1 1The abbreviations used are: HIV-1, human immunodeficiency virus, type 1; SIV, simian immunodeficiency virus; RT, reverse transcriptase; T/P, template-primer complex; AZTTP, 3′-Azido-3′-deoxythymidine 5′-triphosphate; DTT, dithiothreitol.1The abbreviations used are: HIV-1, human immunodeficiency virus, type 1; SIV, simian immunodeficiency virus; RT, reverse transcriptase; T/P, template-primer complex; AZTTP, 3′-Azido-3′-deoxythymidine 5′-triphosphate; DTT, dithiothreitol. and SIV) reverse transcriptases (RT)) is their highly error-prone mode of DNA synthesis (1Preston B.D. Poiesz B.J. Loeb L.A. Science. 1988; 242: 1168-1171Crossref PubMed Scopus (676) Google Scholar, 2Williams K.J. Loeb L.A. Curr. Top. Microbiol. Immunol. 1992; 176: 165-180PubMed Google Scholar). The anti-viral immune selection drives the viral diversity that may result from the fast replication of lentiviruses (3Coffin J.M. Science. 1995; 267: 483-489Crossref PubMed Scopus (1681) Google Scholar, 4Coffin J.M. AIDS. 1996; 10: 75-84PubMed Google Scholar, 5Rouzine I.M. Rodrigo A. Coffin J.M. Microbiol. Mol. Biol. Rev. 2001; 65: 151-185Crossref PubMed Scopus (129) Google Scholar) and the efficient mutation synthesis catalyzed by lentiviral RTs (2Williams K.J. Loeb L.A. Curr. Top. Microbiol. Immunol. 1992; 176: 165-180PubMed Google Scholar). As recently reported, it is increasingly apparent that infidelity of lentiviral RTs provides the raw material for the viral genomic hypermutation that allows lentiviruses to efficiently evolve and escape from various anti-viral selective pressures (6O'Neil P.K. Sun G. Yu H. Ron Y. Dougherty J.P. Preston B.D. J. Biol. Chem. 2002; 277: 38053-38061Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Unlike DNA polymerases involved in the cellular genomic replication process, lentiviral RTs lack the 3′ to 5′ proofreading nuclease activity, which would normally help prevent mutagenic DNA synthesis. However, this is not the only factor contributing to the low fidelity of lentiviral RTs because RTs of onco-retroviruses lacking the proofreading exonuclease activity, such as murine leukemia virus and avian myeloblastosis virus, have 10–18-fold higher fidelity than lentiviral RTs (7Roberts J.D. Preston B.D. Johnston L.A. Soni A. Loeb L.A. Kunkel T.A. Mol. Cell. Biol. 1989; 9: 469-476Crossref PubMed Scopus (142) Google Scholar). This suggests that lentiviral RTs harbor DNA polymerase active sites with unique molecular characteristics responsible for their unfaithful DNA synthesis.Kinetic studies with DNA polymerases suggest that mutation synthesis, which generates a mismatch at the 3′ end of newly synthesized DNA, interrupts processive DNA polymerization (8Johnson K.A. Annu. Rev. Biochem. 1993; 62: 685-713Crossref PubMed Scopus (504) Google Scholar). HIV-1 RT is highly efficient in the incorporation of incorrect dNTPs (misinsertion). In addition, compared with other known DNA polymerases involved in genomic replication, HIV-1 RT more efficiently extends the mismatched primer created by a misinsertion (9Yu H. Goodman M.F. J. Biol. Chem. 1992; 267: 10888-10896Abstract Full Text PDF PubMed Google Scholar). Possibly, the high capability of HIV-1 RT to extend a mismatched primer may compensate for the disruption of processive DNA synthesis caused by frequent misinsertion events during viral genomic replication by HIV-1 RT.The interaction of DNA polymerases with incoming dNTPs is one of the key elements that determines DNA polymerase fidelity. Mutations in the dNTP binding domain, O helix, of pol I DNA polymerase family members, such as the Klenow fragment of Escherichia coli DNA polymerase (10Kuchta R.D. Mizrahi V. Benkovic P.A. Johnson K.A. Benkovic S.J. Kuchta M. Biochemistry. 1987; 26: 8410-8417Crossref PubMed Scopus (338) Google Scholar, 11Carroll S.S. Cowart M. Benkovic S.J. Biochemistry. 1991; 30: 804-813Crossref PubMed Scopus (116) Google Scholar) and the thermostable Taq polymerase (12Li Y. Korolev S. Waksman G. EMBO J. 1998; 17: 7514-7525Crossref PubMed Scopus (653) Google Scholar, 13Li Y. Mitaxov V. Waksman G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9491-9496Crossref PubMed Scopus (103) Google Scholar, 14Suzuki M. Avicola A.K. Hood L. Loeb L.A. J. Biol. Chem. 1997; 272: 11228-11235Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 15Suzuki M. Yoshida S. Adman E.T. Blank A. Loeb L.A. J. Biol. Chem. 2000; 275: 32728-32735Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), alter their fidelity. Mutations in several dNTP-binding residues of HIV-1 RT were also found to affect RT fidelity. For example, alterations in the Arg-72 residue of HIV-1 RT, which interacts with the triphosphate moiety of incoming dNTPs, increase RT fidelity; however, these alterations can dramatically decrease RT activity (16Lewis D.A. Bebenek K. Beard W.A. Wilson S.H. Kunkel T.A. J. Biol. Chem. 1999; 274: 32924-32930Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). In contrast, mutations in the Tyr-115 residue that interacts with base-sugar moieties of the incoming dNTP reduce RT fidelity (17Martin-Hernandez A.M. Gutierrez-Rivas M. Domingo E. Menendez-Arias L. Nucleic Acids Res. 1997; 25: 1383-1389Crossref PubMed Scopus (52) Google Scholar, 18Cases-Gonzalez C.E. Gutierrez-Rivas M. Menendez-Arias L. J. Biol. Chem. 2000; 275: 19759-19767Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar).Kinetic parameters indicating the dNTP binding affinity and chemical catalysis of DNA polymerases have been determined under reaction conditions where DNA polymerase molecules prebound to the template-primer complex (T/P), incorporate a single nucleotide; this is referred to as the pre-steady state condition (8Johnson K.A. Annu. Rev. Biochem. 1993; 62: 685-713Crossref PubMed Scopus (504) Google Scholar). We recently reported a pre-steady state kinetic study with another high fidelity HIV-1 RT mutant, Q151N. The Gln-151 residue, encoded in the β8–αE loop of RT, directly interacts with the 3′ OH of the incoming dNTP. Molecular modeling suggested that the Q151N mutation disconnects the interaction of the RT with the 3′ OH of the incoming dNTP. Our pre-steady state kinetic study demonstrated that the Q151N mutant specifically slows down the binding step (K d) for all incoming dNTPs (correct and incorrect) but not the chemistry step (k pol). In fact, the binding of the Q151N mutant to incorrect dNTP was reduced to such an extent that we were not able to determine an accurate K d value (19Weiss K.K. Bambara R.A. Kim B. J. Biol. Chem. 2002; 277: 22662-22669Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Therefore, this kinetic study suggests that the Gln-151 RT residue interacts with the 3′ OHs of both correct and incorrect incoming dNTPs, thereby providing support for the existence of a common mechanism involved in the binding of both correct and incorrect dNTPs.Met-184 RT mutations of HIV-1 RT, M184V and M184I, are the only in vivo lentiviral RT mutations previously found to have moderately increased fidelity. These mutations were initially isolated from virus strains that exhibited resistance to a nucleoside RT inhibitor, 3TC. HIV-1 RTs containing the M184V and M184I mutations showed 1.8- and 3.5-fold increased fidelities, respectively, compared with wild type HIV-1 RT, as determined by the M13 lacZα forward mutation assay (20Rezende L.F. Drosopoulos W.C. Prasad V.R. Nucleic Acids Res. 1998; 26: 3066-3072Crossref PubMed Scopus (69) Google Scholar). A pre-steady state kinetic study with M184V RT showed reduced binding affinity to both correct and incorrect dNTPs (21Feng J.Y. Anderson K.S. Biochemistry. 1999; 38: 9440-9448Crossref PubMed Scopus (121) Google Scholar). Structural studies have proposed that the β-branched side chain of the valine (or isoleucine) at position 184 in HIV-1 RT blocks the entry of incoming dNTP, including 3TCTP and incorrect dNTP, into the active site (22Sarafianos 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: 10027-10032Crossref PubMed Scopus (282) Google Scholar).We found recently that an in vivo SIV RT variant, called SIVMNE 170 (170) RT, has an enhanced replication fidelity. SIVMNE 170 virus is a molecular clone isolated at the late stage of infection of a pig-tailed macaque (M87004) initially infected with a molecular clone of SIVMNE CL8 (CL8) (23Overbaugh J. Rudensey L.M. Papenhausen M.D. Benveniste R.E. Morton W.R. J. Virol. 1991; 65: 7025-7031Crossref PubMed Google Scholar, 24Rudensey L.M. Kimata J.T. Benveniste R.E. Overbaugh J. Virology. 1995; 207: 528-542Crossref PubMed Scopus (61) Google Scholar). The 170 RT has ∼11-fold higher fidelity than the initial low fidelity CL8 RT. Sequence analysis revealed that the 170 high fidelity RT has six mutations in the DNA polymerase domain of RT, compared with the low fidelity CL8 RT. Identification of the 170 high fidelity variant suggests that RT fidelity may change during the course of viral infection. Indeed, the 170 RT is the first in vivo RT variant isolated during the course of natural infection (no antiretroviral drug treatment) with such an increase in fidelity (25Diamond T.L. Kimata J. Kim B. J. Biol. Chem. 2001; 276: 23624-23631Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar).In the present study, we investigated kinetic and structural properties of the high fidelity 170 RT protein. First, we identified a 170 RT mutation solely responsible for the fidelity increase of the 170 RT. Second, we determined the reaction step affected by the 170 high fidelity mutation by employing the pre-steady state kinetic assay. Third, we examined the effect of other types of mutations at the 148 residue of CL8 RT on RT fidelity. Finally, based on these kinetic and mechanistic analyses, we proposed a model for the structural impact made by the in vivo high fidelity 170 RT mutation.MATERIALS AND METHODSPlasmids, Strains, and Chemicals—E. coli DH5α (Invitrogen) was used for construction of plasmids and BL21 (Novagen, WI) for overexpression of SIVMNE RT proteins. SIVMNE CL8 and 170 RT proteins were previously purified from pBK90 and 125 (25Diamond T.L. Kimata J. Kim B. J. Biol. Chem. 2001; 276: 23624-23631Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), respectively, which are pHis/NdeI plasmids (26Kim B. Methods (Orlando). 1997; 12: 318-324Google Scholar) encoding full-length RT genes, fused at the N terminus to six histidine residues. pBK33 (26Kim B. Methods (Orlando). 1997; 12: 318-324Google Scholar) was used for the bacterial expression of HIV-1 RT proteins. SIVMNE and HIV-1 RTs (p66/p66 homodimers) were purified from the pHis/NdeI plasmids cloned as noted below. All restriction endonucleases were obtained from New England Biolabs.Construction of SIVMNE CL8-V148I, 170-I148V RT Derivatives, and the V148I HIV-1 RT Mutant—In order to make a pHis/NdeI CL8-V148I RT clone, pBK90 expressing SIVMNE CL8 RT clone was amplified with 170V148I R primer (5′-TCCACCCCTGAGGCAGAATCTTATAAATGTATCG-3′) and 5′ HSG primer (5′-TATGTTGTGTGGAATTGT-3′) using Taq polymerase (New England Biolabs). The Bsu36I site is shown in boldface and the complement to the Ile-148 codon is underlined. NdeI and Bsu36I were used to insert the V148I fragment into the SIVMNE CL8 RT plasmid. Clones were sequenced using 5′ HSG (see above) and the ABI system (PerkinElmer Life Sciences). The pHis/NdeI 170-I148V RT clone was made similarly using CL8V148I R primer (5′-TCCACCCCTGAGGCAGAACCTTATAAATGTATCG-3′) and 5′ HSG (see above) to amplify from pBK125 expressing SIVMNE 170 RT and inserting the mutated fragment into pBK125 at the NdeI and Bsu36I sites. The Bsu36I site is shown in boldface, and the complement to the Val148 codon is underlined in the CL8V148I R primer. Clones were sequenced using the fmol DNA cycle sequencing system (Promega, WI) and SIV 111/112F primer (5′-TTTTTTCTTAAGGACGCATATTTCTCCATACC-3′) in order to identify the V148I mutation and with SIVseq280 primer (5′-AATACCACACCCTGCAGGAC-3′) in order to verify that the backbone is SIVMNE 170.For the construction of the HIV-1 RT V148I mutant, the V148I mutation was also created by PCR-based site-directed mutagenesis using a primer containing the V148I mutation (5′-TTTTAGATATCAGTACAATATCCTTCCACAGGGATGGAAAGG-3′; the V148I codon is shown in boldface, and the EcoRV restriction site is underlined) and the BamHI primer (5′-AAAGGATCCCCAGCAATATTCCAAAG-3′; the BamHI site is underlined). The wild type sequence of pBK33 (26Kim B. Methods (Orlando). 1997; 12: 318-324Google Scholar) between the unique EcoRV (near codon 144) and BamHI (near codon 155) sites was replaced with the EcoRV- and BamHI-digested PCR product made with these two primers.Random Site-directed Mutagenesis of Val-148 Residue—In order to generate CL8 RT with a variety of amino acids present at residue 148, pBK90 expressing SIVMNE CL8 RT clone was amplified with CL8 V148X R primer (5′-TCCACCCCTGAGGCAGSXXCTTATAAATGTATCG-3′; S refers to C or G, and X refers to G, A, T, or C) and 5′ HSG primer (5′-TATGTTGTGTGGAATTGT-3′) using Taq polymerase (New England Biolabs). The Bsu36I site is shown in boldface, and the complement to the random 148 codon is underlined. NdeI and Bsu36I were used to insert the V148X fragment into the SIVMNE CL8 RT plasmid. Clones were sequenced using the fmol DNA cycle sequencing system (Promega, WI) and SIV 111/112F primer (5′-TTTTTTCTTAAGGACGCATATTTCTCCATACC-3′) in order to identify the codon present at position 148 and verify the presence of the CL8 codon at position 173.Purification of SIVMNE and HIV-1 RT Proteins—Homodimeric (p66/p66) RT proteins were purified using a modification of our purification protocol for HIV-1 RT as described previously (26Kim B. Methods (Orlando). 1997; 12: 318-324Google Scholar). Expression of SIVMNE and HIV-1 RTs was induced by addition of 1 mm isopropyl-1-thio-β-d-galactopyranoside to log phase E. coli BL21 with the RT expression plasmids described above, grown in 2× YT to an A 600 of 0.5. Cells were then harvested by centrifugation, and the pellets were resuspended and frozen (–70 °C) with 10 ml of 1× binding buffer and lysozyme (200 μg/ml). All buffers and binding resin used in this work were purchased from Novagen (WI). Frozen cells were thawed and lysed on ice for ∼2 h. The lysed cells were centrifuged (27,000 × g), and after addition of 10 ml of fresh 1× binding buffer the supernatant was applied to a charged 5-ml His Bind column (1 × 5 cm). The resin was prepared by successive washes with deionized water (15 ml), 1× charge buffer (15 ml), and 1× binding buffer (15 ml). All chromatographic steps were carried out at 4 °C at a flow rate of 20 ml/h. Following application of the crude supernatant solution, the column was washed with 1× binding buffer (15 ml) and a mixture of 1× binding buffer and 1× wash buffer (7:3, 10 ml). RT proteins were eluted with 1× elute buffer (20 ml); 90% of the recovered RT protein was released from the resin in the first 8 ml. Fractions containing purified RT proteins were analyzed by electrophoresis in a 12% SDS-polyacrylamide stacking gel. Fractions containing RT were dialyzed against 1× dialysis buffer (50 mm Tris-Cl, pH 7.5, 1 mm EDTA, 200 mm NaCl, 10% glycerol) for 16 h and 1× dialysis buffer with 1 mm DTT for 3 h. In this protocol the purity of the RT proteins was typically greater than 95%, as estimated by visual inspection of Coomassie Blue-stained gels.M13mp2 lacZα Forward Mutation Assay—The mutant frequencies for our SIVMNE RT proteins were measured essentially as described previously (27Bebenek K. Kunkel T.A. Methods Enzymol. 1995; 262: 217-232Crossref PubMed Scopus (192) Google Scholar). M13mp2 DNA containing a 361-nucleotide single-stranded gap was prepared as specified. Gapped M13mp2 DNA (1 μg) was incubated with RT (250 nm) at 37 °C for 20 min under the conditions described above for the misincorporation assays. The extended gapped DNAs were analyzed by 0.7% agarose gel for verifying the production of double-stranded M13 DNA with filled gaps. The extended gapped DNAs were transformed to MC1016 cells, and the transformed cells were plated to M9 plates containing 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal) and isopropyl-1-thio-β-d-galactopyranoside with CSH50 lawn cells. The mutant frequency was determined as the ratio of mutant (pale blue and clear) plaques to mutant plus wild type (dark blue) plaques as described.Misincorporation Assay with DNA or RNA Templates—Procedures were modified from those of Preston et al. (1Preston B.D. Poiesz B.J. Loeb L.A. Science. 1988; 242: 1168-1171Crossref PubMed Scopus (676) Google Scholar). The RNA T/P was prepared by annealing a 40-mer RNA (5′-AAGCUUGGCUGCAGAAUAUUGCUAGCGGGAAUUCGGCGCG-3′, Dharmacon Research) to the 17-mer A primer (5′-CGCGCCGAATTCCCGCT-3′; template-primer, 2.5:1, Invitrogen) 32P-labeled at the 5′ end by T4 polynucleotide kinase (New England Biolabs). The DNA T/P was prepared by annealing a 40-mer DNA (5′-AAGCTTGGCTGCAGAATATTGCTAGCGGGAATTCGGCGCG-3′), which encodes the same sequence as the 40-mer RNA template, to the 17-mer used for DNA template-primer preparation. Assay mixtures (20 μl) contained 10 nm T/P, RT proteins as specified in figure legends, or exo– T7 DNA polymerase (Sequenase version 2.0 DNA polymerase, U. S. Biochemical Corp.), 3 or 4 dNTPs (250 μm each), 25 mm Tris-HCl, pH 8.0, 100 mm KCl, 2 mm DTT, 5 mm MgCl2, 10 μm (dT)20, and 0.1 mg/ml bovine serum albumin (New England Biolabs). Reactions were incubated at 37 °C for 5 min and terminated by 10 μlof40mm EDTA, 99% formamide. Reaction products were immediately denatured by incubating at 95 °C for 5 min and analyzed by electrophoresis in 14% polyacrylamide-urea gels.Extension of Mismatched Primers—To measure the capability of RT to extend mismatched primer, we used two different mismatched primers annealed to either the RNA or DNA template used in misincorporation assay. 32P-Labeled 16-mer G/T mismatched primer (5′-CGCGCCGAATTCCCGT-3′, mismatch underlined) or 19-mer C/A primer (5′-CGCGCCGAATTCCCGCTAA-3′, mismatch underlined) was annealed to the 40-mer RNA or DNA templates (see above), respectively. For control matched primer, we used 32P-labeled C primer (5′-CGCGCCGAATTCCCG-3′) annealed to the 40-mer RNA or DNA templates (see above). Extension conditions for this extension assay were the same as those used for the misincorporation assay above, except for the different T/Ps. The reactions were also analyzed by electrophoresis on a 14% denaturing sequencing gel.Pre-steady State Kinetic Assay—Pre-steady state burst and single-turnover experiments were employed to examine the transient kinetics associated with a single nucleotide incorporating onto the 3′ end of a 32P-labeled 17-mer A primer (see above) annealed to a 40-mer RNA template (see above). We used 20 μl of T/P pre-incubated with purified SIV RT protein and reaction buffer (25 mm Tris-HCl, pH 8.0, 40 mm KCl, 2 mm DTT, 5 mm MgCl2, 0.1 mg/ml bovine serum albumin). This mixture was injected into one sample tube of the rapid quench machine (Kintek). An equal volume of dNTP pre-incubated with Mg2+ (10 mm) was injected into the other sample tube. The polymerization reaction was initiated by rapidly mixing the two reactants and was terminated by adding 0.25 m EDTA at different time points.In the pre-steady state burst experiments, T/P (150 nm) was present in excess over active RT (∼50 or 150 nm purified protein), and the reaction was initiated by addition of 800 μm dATP (correct). These experiments were used to determine the active site concentrations of the RT proteins (see data analysis Refs. 8Johnson K.A. Annu. Rev. Biochem. 1993; 62: 685-713Crossref PubMed Scopus (504) Google Scholar and 28Johnson K.A. Methods Enzymol. 1995; 249: 38-61Crossref PubMed Scopus (193) Google Scholar). The pre-steady state single-turnover experiments were used to determine the dNTP concentration dependence of the purified SIV RT proteins. In the presence of varying dATP concentrations (in the range of 600 nm to2mm), active RT (100 nm) was used in slight excess of T/P (90 nm). In reactions involving incorrect dNTPs, the experiments were performed manually at longer time points and used a higher concentration of RT (700 nm) (10Kuchta R.D. Mizrahi V. Benkovic P.A. Johnson K.A. Benkovic S.J. Kuchta M. Biochemistry. 1987; 26: 8410-8417Crossref PubMed Scopus (338) Google Scholar, 28Johnson K.A. Methods Enzymol. 1995; 249: 38-61Crossref PubMed Scopus (193) Google Scholar).Product Analysis—The reactions were analyzed by 14% denaturing sequencing gel electrophoresis. The extended product in each reaction was quantified by the Cyclone PhosphorImager (PerkinElmer Life Sciences).Data Analysis—Pre-steady state kinetic data was analyzed using nonlinear regression. Equations were generated with the program Kaleidagraph version 3.51 (Synergy Software, Reading, PA). Data points obtained during the burst experiment were fit to the burst Equation 1, [product]=A(1-exp(-kobst)+ksst)(Eq. 1) The value A is the amplitude of the burst, which reflects the actual concentration of enzyme that is in active form. k obs is the observed first-order rate constant for dNTP incorporation, whereas k ss is the observed steady state rate constant (8Johnson K.A. Annu. Rev. Biochem. 1993; 62: 685-713Crossref PubMed Scopus (504) Google Scholar, 10Kuchta R.D. Mizrahi V. Benkovic P.A. Johnson K.A. Benkovic S.J. Kuchta M. Biochemistry. 1987; 26: 8410-8417Crossref PubMed Scopus (338) Google Scholar, 28Johnson K.A. Methods Enzymol. 1995; 249: 38-61Crossref PubMed Scopus (193) Google Scholar). Data from single turnover experiments were fit to a single exponential equation that measures the rate of dNTP incorporation (k obs) per given dNTP concentration ([dNTP]). These results can then be used to determine K d, the dissociation constant for dNTP binding to the RT-T/P binary complex. This was done by fitting the data to the following hyperbolic Equation 2, kobs=kpol[dNTP]/(Kd+[dNTP])(Eq. 2) From this equation, we could then identify the following kinetic constants for each RT during pre-steady state kinetics: k pol, the maximum rate of dNTP incorporation, and K d, equilibrium dissociation constant for the interaction of dNTP with the E·DNA complex (8Johnson K.A. Annu. Rev. Biochem. 1993; 62: 685-713Crossref PubMed Scopus (504) Google Scholar).[dNTP] Dependence Assay for Primer Extension Capability—To measure the ability of RT to perform multiple rounds of DNA synthesis in the presence of low concentrations of dNTP, the RNA T/P (A primer/RNA; see above) was extended by identical activities of either CL8 or CL8-V148I RT with dNTP concentrations ranging from 25 nm to 100 μm. Other than dNTP concentrations, reactions were performed using the same conditions as described for the misincorporation assay above. Reactions were applied to a 14% polyacrylamide-urea gel for analysis.AZTTP Susceptibility Assay—The drug susceptibility assay performed was a modification of the drug susceptibility assay as described by Klarmann et al. (29Klarmann G.J. Smith R.A. Schinazi R.F. North T.W. Preston B.D. J. Biol. Chem. 2000; 275: 359-366Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Briefly, the RNA T/P was prepared by annealing the 40-mer RNA template (see above) to the 17-mer primer A (see above) 32P-labeled at the 5′ end by T4 polynucleotide kinase (New England Biolabs). Assay mixtures (20 μl) contained 10 nm T/P, SIV RT proteins as specified in the figure legends, 4 dNTPs (see figure legend for concentrations), 0–200 μm AZTTP (see figure legend for specific concentrations), 25 mm Tris-HCl, pH 8.0, 100 mm KCl, 2 mm DTT, 5 mm MgCl2,2 μm (dT)20, and 0.1 mg/ml bovine serum albumin (New England Biolabs). Reactions were incubated at 37 °C for 10 min and terminated by addition of 10 μlof40mm EDTA, 99% formamide. Reaction products were immediately denatured by incubating at 95 °C for 5 min and analyzed by electrophoresis in 14% polyacrylamide-urea gels.RESULTSIdentification of the 170 RT Mutation Responsible for the Fidelity Increase—Our previous study (25Diamond T.L. Kimata J. Kim B. J. Biol. Chem. 2001; 276: 23624-23631Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar) showed that the high fidelity 170 RT has six mutations at the DNA polymerase domain, compared with the sequence of the CL8 RT. We tested which of the six SIVMNE 170 RT mutations is (are) responsible for the fidelity increase of the SIVMNE 170 RT. Among the six SIVMNE 170 RT mutations, an SIVMNE CL8 RT derivative containing V148I (CL8-V148I) was initially constructed because this Val-148 residue lies near the active site of RT. After purification of the CL8-V148I RT protein, we measured the fidelity of the CL8 and CL8-V148I RTs by the M13 lacZα forward mutation assay. As shown in Table I, the CL8-V148I RT has about 8 times higher fidelity, compared with the SIVMNE CL8 RT, indicating that the V148I mutation increases the fidelity of SIVMNE CL8 RT. In addition, the CL8-V148I RT has similar high fidelity to the 170 RT containing all six mutations (Table I). These data suggest that the V148I mutation contributes to the fidelity increase observed in the 170 RT. In addition, the other five SIVMNE 170 RT mutations (I73M, K173R, S211G, Y303F, and N332S) are unlikely to be involved in the fidelity increase. The CL8 K173R mutant RT, for example, had identical fidelity to CL8 RT by misincorporation assay (data not shown).Table IFidelity of SIVMNE RT proteins determined by M13 lacZα forward mutation assaySIVMNE RT proteinsTotal plaquesaNumbers represent pooled totals from at least two independent fill-in reactions.Mutant plaquesaNumbers represent pooled totals from at least two independent fill-in reactions.Mutant frequencyaNumbers represent pooled totals from at least two independent fill-in reactions. (×10-3)Fold decreaseCL8 RT7,92014117.8 ± 1.3CL8-V148I RT7,580172.2 ± 0.07×8.2170 RT9,240222.4 ± 0.2×7.4170-I148V RT6,75013019.3 ± 1.6×0.92a Numbers represent pooled totals from at least two independent fill-in reactions. Open table in a new tab Next, we confirmed the effect of the V148I mutation on RT fidelity by testing whether reversion of the V148I mutation in the high fidelity SIVMNE 170 RT reduces its fidelity. We constructed and purified an SIVMNE 170 RT derivative containing five of the six SIVMNE 170 RT mutations except for the V148I mutation, called 170-I148V RT. As shown in Table I, the 170-I148V RT showed an ∼10-fold reduced fidelity, compared with the high fidelity 170 RT. In addition, similar low mutation frequencies were seen between the 170-I148V and CL8 RTs (Table I). Therefore, these results suggest that the V148I mutation is resp
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