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• W4234935877 abstract "Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) variants with the K65R or L74V substitution display resistance to several nucleoside analogs. An in vitro dNTP exclusion assay revealed an increased fidelity for K65R RT compared with wild-type RT, but little change for L74V RT. When the forward mutation rates were measured via a gap-filling assay, the K65R variant displayed an 8-fold decrease in the overall mutation rate (1.0 × 10−3 versus 8.6 × 10−3 for wild-type HIV-1 RT), whereas the rate for the L74V variant was closer to that for wild-type RT (5.0 × 10−3). The increase in overall fidelity observed for K65R RT is the largest reported for any drug-resistant HIV-1 RT variant. Nucleotide sequence analysis oflacZα mutants generated by variant RTs indicated that K65R RT displays uniform reduction in most types of errors, whereas L74V RT does not. Modeling the substitutions into the x-ray structure of the ternary complex revealed that the major influence of Leu74 in stabilizing the templating base is unaffected by Val substitution, whereas the K65R substitution appears to increase the stringency of dNTP binding. It is speculated that the increased fidelity of K65R RT is due to an altered interaction with the dNTP substrate. Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) variants with the K65R or L74V substitution display resistance to several nucleoside analogs. An in vitro dNTP exclusion assay revealed an increased fidelity for K65R RT compared with wild-type RT, but little change for L74V RT. When the forward mutation rates were measured via a gap-filling assay, the K65R variant displayed an 8-fold decrease in the overall mutation rate (1.0 × 10−3 versus 8.6 × 10−3 for wild-type HIV-1 RT), whereas the rate for the L74V variant was closer to that for wild-type RT (5.0 × 10−3). The increase in overall fidelity observed for K65R RT is the largest reported for any drug-resistant HIV-1 RT variant. Nucleotide sequence analysis oflacZα mutants generated by variant RTs indicated that K65R RT displays uniform reduction in most types of errors, whereas L74V RT does not. Modeling the substitutions into the x-ray structure of the ternary complex revealed that the major influence of Leu74 in stabilizing the templating base is unaffected by Val substitution, whereas the K65R substitution appears to increase the stringency of dNTP binding. It is speculated that the increased fidelity of K65R RT is due to an altered interaction with the dNTP substrate. human immunodeficiency virus human immunodeficiency virus type 1 reverse transcriptase In the absence of an effective vaccine against human immunodeficiency virus infections, long-term therapy with combinations of anti-retrovirals continues to be the only option for infected individuals. Despite the use of multiple drugs in therapy regimens, the rapid replication of HIV1 (1Wei X. Ghosh S.K. Taylor M.E. Johnson V.A. Emini E.A. Deutsch P. Lifson J.D. Bonhoeffer S. Nowak M.A. Hahn B.H. Saag M.S. Shaw G.M. Nature. 1995; 373: 117-122Crossref PubMed Scopus (2914) Google Scholar,2Ho D.D. Neumann A.U. Perelson A.S. Chen W. Leonard J.M. Markowitz M. Nature. 1995; 373: 123-126Crossref PubMed Scopus (3789) Google Scholar) and its inherently high genetic variability (3Hahn B.H. Shaw G.M. Taylor M.E. Redfield R.R. Markham P.D. Salahuddin S.Z. Wong-Staal F. Gallo R.C. Parks E.S. Parks W.P. Science. 1986; 232: 1548-1553Crossref PubMed Scopus (388) Google Scholar, 4Saag M.S. Hahn B.H. Gibbons J. Li Y. Parks E.S. Parks W.P. Shaw G.M. Nature. 1988; 334: 440-444Crossref PubMed Scopus (292) Google Scholar) lead to the emergence of drug-resistant variants and ultimately to clinical failure (5Larder B.A. Skalka A.M. Goff S.P. Reverse Transcriptase. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993: 205-222Google Scholar, 6Richman D.D. AIDS Res. Hum. Retroviruses. 1992; 8: 1065-1071Crossref PubMed Scopus (54) Google Scholar). Owing to the long-term nature of both the disease and the treatment, drug-resistant HIV variants are associated with infected individuals for extended periods of time. Therefore, it is important to understand the influence of such mutations on viral properties such as replicative fitness, fidelity, and mutation rates. We previously reported that certain nucleoside analog-resistance mutations confer an increased dNTP insertion fidelity on the HIV-1 RTin vitro. These include the multi-2′,3′-dideoxynucleoside analog-resistant E89G RT variant (7Drosopoulos W.C. Prasad V.R. J. Virol. 1996; 70: 4834-4838Crossref PubMed Google Scholar) and the (−)-2′,3′-dideoxy-3′-thiacytidine-resistant M184V variant (8Wainberg M.A. Drosopoulos W.C. Salomon H. Hsu M. Borkow G. Parniak M. Gu Z. Song Q. Manne J. Islam S. Castriota G. Prasad V.R. Science. 1996; 271: 1282-1285Crossref PubMed Scopus (293) Google Scholar). These results have been confirmed by other groups, who showed increases in dNTP insertion fidelity as well as mispair extension fidelity of these RT variants (9Pandey V.N. Kaushik N. Rege N. Sarafianos S.G. Yadav P.N. Modak M.J. Biochemistry. 1996; 35: 2168-2179Crossref PubMed Scopus (141) Google Scholar, 10Rubinek T. Bakhanashvili M. Taube R. Avidan O. Hizi A. Eur. J. Biochem. 1997; 247: 238-247Crossref PubMed Scopus (37) Google Scholar, 11Oude Essink B.B. Back N.K.T. Berkhout B. Nucleic Acids Res. 1997; 25: 3212-3217Crossref PubMed Scopus (75) Google Scholar, 12Hsu M. Inouye P. Rezende L. Richard N. Li Z. Prasad V.R. Wainberg M.A. Nucleic Acids Res. 1997; 25: 4532-4536Crossref PubMed Scopus (61) Google Scholar). The hypothesis that such nucleoside analog-resistance mutations may reduce the overall mutation rates was tested in vitro by measuring the forward mutation rate of recombinant wild-type and mutant HIV-1 RTs using a gapped duplex assay with lacZα as a reporter. Interestingly, such assays revealed no changes in the overall mutation rate by the nucleoside analog-resistance mutations E89G and M184V (13Drosopoulos W.C. Prasad V.R. J. Virol. 1998; 72: 4224-4230Crossref PubMed Google Scholar). That this might be due to an increase in other types of errors was suggested by the finding that the E89G RT variant displays a 10–29-fold increased efficiency of mispair extension with some of the mispaired template-primer termini (14Hamburgh M.E. Drosopoulos W.C. Prasad V.R. Nucleic Acids Res. 1998; 26: 4389-4394Crossref PubMed Scopus (16) Google Scholar). These results are in agreement with the report that, under selection pressure in culture with anti-protease drugs, the M184V HIV variant leads to the emergence of resistant variants at a frequency comparable to wild-type RT (15Balzarini J. Pelemans H. Karlsson A. De Clercq E. Kleim J.-P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13152-13157Crossref PubMed Scopus (43) Google Scholar). In contrast to the M184V and E89G mutants, another 3TC-resistant variant, M184I RT, revealed a 4-fold decrease in the forward mutation rate compared with the wild-type enzyme (16Rezende L.F. Drosopoulos W.C. Prasad V.R. Nucleic Acids Res. 1998; 26: 3066-3072Crossref PubMed Scopus (69) Google Scholar). This suggested that by examining the forward mutation rates of additional mutants, one may be able to identify HIV-1 RT variants with larger changes in mutation rate. Insight into residues that constitute the dNTP-binding pocket as well as mechanisms by which mutations at different sites may confer drug resistance or increased fidelity was provided by the recent crystal structure of a covalently trapped catalytic complex of HIV-1 RT with template-primer and dTTP (17Huang H. Chopra R. Verdine G.L. Harrison S.C. Science. 1998; 282: 1669-1675Crossref PubMed Google Scholar). In this structure, the presence of dNTP substrate in the binding pocket led to a conformational change involving an inward movement of the finger β3–β4 hairpin loop, bringing it closer to the active site. In that conformation, the residues in the β3–β4 hairpin loop, many of which are implicated in nucleoside analog resistance, directly contact the incoming dNTP as well as indirectly influence its binding via interactions with the templating base. For example, the ammonium group of Lys65contacts the γ-phosphate of the dNTP, whereas its aliphatic portion interacts with Arg72. Similarly, Leu74 locks the templating base tightly in place and contacts the side chain of Arg72 and Gln151, which in turn stack on the base of the dNTP. All of these residues except Arg72 are altered in variant viruses displaying resistance to nucleoside analogs. A Lys-to-Arg substitution at codon 65 associated with resistance to ddC and cross-resistance to 3TC and ddI was first reported by Gu et al. (18Gu Z. Gao Q. Fang H. Salomon H. Parniak M.A. Goldberg E. Cameron J. Wainberg M.A. Antimicrob. Agents Chemother. 1994; 38: 275-280Crossref PubMed Scopus (203) Google Scholar). This mutation was subsequently shown to confer cross-resistance to (9R)-2-phosphonylmethoxyethyladenine (19Gu Z. Salomon H. Cherrington J.M. Mulato A.S. Chen M.S. Yarchoan R. Foli A. Sogocio K.M. Wainberg M.A. Antimicrob. Agents Chemother. 1995; 39: 1888-1891Crossref PubMed Scopus (70) Google Scholar) and to bisisopropyloxymethylcarbonyl (20Srinivas R.V. Fridland A. Antimicrob. Agents Chemother. 1998; 42: 1484-1487Crossref PubMed Google Scholar). A Leu-to-Val substitution at codon 74 is the most frequently described mutation in patients receiving ddI (21St. Clair M.H. Martin J.L. Tudor-Williams G. Bach M.C. Vavro C.L. King D.M. Kellam P. Kemp S.D. Larder B.A. Science. 1991; 253: 1557-1559Crossref PubMed Scopus (645) Google Scholar, 22Winters M. Shafer R. Jellinger R. Mamtora G. Gingeras T. Merigan T. Antimicrob. Agents Chemother. 1997; 41: 757-762Crossref PubMed Google Scholar) and confers a 5–10-fold decrease in sensitivity to ddI. The L74V substitution has also been shown to confer cross-resistance to ddC and 3TC. Interestingly, although both mutations can be found in a single patient, they usually exist as separate viral quasi-species (22Winters M. Shafer R. Jellinger R. Mamtora G. Gingeras T. Merigan T. Antimicrob. Agents Chemother. 1997; 41: 757-762Crossref PubMed Google Scholar). The functionally critical positions of these two residues suggest that the K65R and L74V substitutions may profoundly influence the polymerase fidelity of HIV-1 RT. In this study, we report that whereas one nucleoside analog-resistance mutation (K65R) enhances in vitro fidelity and considerably decreases the forward mutation rate of HIV-1 RT, the other (L74V) does not affect either. Results derived from modeling these substitutions into the structure of the ternary complex and a possible mechanism for their influence on polymerase fidelity are presented. The bacteriophage M13mp2 was used to prepare the gapped duplex DNA substrate. M13 phage was grown inEscherichia coli strain NR9099 (Δ(pro-lac),thi, ara, recA56/F′ (proAB,lacIqZΔM15)) to prepare single-stranded and replicative form DNAs. E. coli strain MC1061 (hsdR,hsdM+, araD, Δ(ara,leu), Δ(lacIPOZY), galU,galK, strA) was used to generate mutant phage. The α-complementation strain of E. coli, CSH50 (Δ(pro-lac), thi, ara,strA/F′ (proAB, lacIqZΔM15,traD36)), was used to visualize the phenotype of the mutant phage. Recombinant heterodimeric K65R mutant RT was generated as described (23Gu Z. Fletcher R.S. Arts E.J. Wainberg M.A. Parniak M.A. J. Biol. Chem. 1994; 269: 28118-28122Abstract Full Text PDF PubMed Google Scholar, 24Fletcher R.S. Hollenschak G. Nagy E. Arion D. Borkow G. Gu Z. Wainberg M.A. Parniak M.A. Protein Expression Purif. 1996; 7: 27-32Crossref PubMed Scopus (59) Google Scholar). L74V RT employed in this study was a homodimeric preparation purified as described by Ueno and Mitsuya (25Ueno T. Mitsuya H. Biochemistry. 1997; 36: 1092-1099Crossref PubMed Scopus (63) Google Scholar). K65R RT used in the single dNTP exclusion assay was a homodimeric preparation. All enzymes were shown to be free of nuclease contamination. Primer extension reactions were performed using a 5′-32P-labeled DNA 28-mer primer annealed to a 55-mer DNA template using wild-type, K65R, or L74V RT in the absence of one dNTP: 5′-CGCTTTCAGGTCCCTGTTCGGGCGCCAC-3′ and 5′-TTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACCTGAAAGCG-3′, respectively. Reactions were initiated by combining equal volumes (10 μl) of enzyme/template-primer solution and dNTP/salt solution in which all four dNTPs were present or in which one of the dNTPs was omitted. The enzyme/template-primer solution contained 100 nm template-primer and increasing concentrations of the wild-type (0.64, 0.85, and 1.28 nm) or K65R (1.13, 1.50, and 1.88 nm) enzyme and the wild-type (0.64, 0.85, and 1.05 nm) or L74V (22.5, 30.0, and 37.5 nm) enzyme. The dNTP/salt solution contained 250 μm dNTPs in 50 mm Tris-Cl (pH 8), 60 mm NaCl, 20 mm dithiothreitol, 0.05% IGEPAL, and 10 mmMgCl2. The reactions were terminated after 6 min by the addition of 20 μl of stop solution (95% formamide and 20 mm EDTA). Ten microliters of boiled reaction mixture were loaded onto a 10% urea-polyacrylamide gel and electrophoresed for 2.5 h at 100 watts. A gapped duplex DNA substrate (M13mp2) was used as template-primer for DNA synthesis reactions using purified wild-type or mutant RT. DNA synthesis was performed in a 25-μl reaction volume containing 75 mm Tris-Cl (pH 8); 80 mm KCl; 6 mm MgCl2; 10 mm dithiothreitol; 500 μm each dATP, dCTP, dGTP, and dTTP (Roche Molecular Biochemicals); 59 ng of gapped duplex DNA; and 500 ng of purified RT. The reactions were incubated at 37 °C for 1 h and then stopped by adjusting the EDTA concentration to 20 mm. Complete synthesis across the gapped region was confirmed by agarose gel electrophoresis. Two independent DNA synthesis reactions were performed for each enzyme examined in this study. Successfully filled-in gapped duplex DNA was electroporated into E. coli strain MC1061. Electroporations were performed in two to four batches on separate days for each DNA synthesis reaction. Cells were allowed to recover for 10 min and then mixed with a log-phase culture of E. coli strain CSH50. Cell mixtures were overlaid onto M9 plates containing 0.2 mmisopropyl-β-d-thiogalactopyranoside (Sigma) and 0.195 mm 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside (X-gal; Labscientific, Inc., Livingston NJ). Plates were incubated at 37 °C for ∼15 h and then screened for β-galactosidase activity by looking for plaques that did not display the dark-blue wild-type phenotype. Mutant plaques were picked from the plates and stored in 1 ml of 0.9% saline at 4 °C. Mutants were confirmed by mixing with an equal number of wild-type phage, plating on M9 plates, and comparing the phenotype of the mutant against that of the wild type. Mutation frequencies were determined by dividing the number of confirmed mutants by the total number of plaques screened. Background mutation frequency was determined by electroporating unfilled gapped duplex DNA and scoring for mutant plaques as described above. Single-stranded DNA was prepared from mutant plaques as described previously (26Bebenek K. Kunkel T.A. Methods Enzymol. 1995; 262: 217-232Crossref PubMed Scopus (192) Google Scholar), and DNA sequence was determined using the Sequenase 2.0 DNA sequencing kit (Amersham Pharmacia Biotech). Sequenced templates were resolved on 6% sequencing gels using the GENOMYX LR DNA sequencer. Error rates were calculated as described previously (26Bebenek K. Kunkel T.A. Methods Enzymol. 1995; 262: 217-232Crossref PubMed Scopus (192) Google Scholar). Briefly, the mutation frequency for the RTs was corrected by subtracting the background mutation frequency. The corrected mutation frequency was multiplied by the percentage of all mutations represented by the class being examined (e.g.percentage of frameshifts). This number was divided by 0.6 (the probability of a mutation being expressed by E. coli) and then divided by the total number of sites where the mutation can be detected. Statistical differences between the proportion of mutations at specific sites and the proportion of mutations of a particular class were assessed using Fisher's exact test and the χ2 test. Models of the two mutants K65R and L74V were built into the structure of HIV-1 RT complexed with template-primer and dTTP using the computer graphics program O (27Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13009) Google Scholar). The most frequent rotamer of the valine as placed automatically by the program was retained. The arginine residue was adjusted manually to avoid steric clashes and to optimize hydrogen-bonding distances. In our attempt to measure the influence of the K65R and L74V mutations on the polymerase fidelity and mutation rates of HIV-1 RT, we first used the single dNTP exclusion assay (popularly termed the minus dNTP assay). This assay allows a polymerase to copy a heteropolymeric template using a 5′-end-labeled primer in the absence of a single dNTP (in the presence of the other three dNTPs) (28Kim B. Hathaway T.R. Loeb L.A. Biochemistry. 1998; 37: 5831-5839Crossref PubMed Scopus (42) Google Scholar, 29Preston B.D. Poiesz B.J. Loeb L.A. Science. 1988; 242: 1168-1171Crossref PubMed Scopus (685) Google Scholar). Analysis of the resulting products yields a gross estimate of the degree of dNTP insertion and mispair extension fidelity for polymerases lacking exonuclease function. Thus, when the polymerase reaches a template base for which the complementary dNTP is missing, further polymerization will depend on the degree of error-proneness of the polymerase requiring both dNTP misinsertion and mispair extension activities. Wild-type HIV-1 RT displayed many products beyond this “barrier” (Fig. 1, Wild type lanes), indicating that both misinsertion and mispair extension occur at a high efficiency. When wild-type RT is compared with K65R RT, it is clear that K65R RT tended to generate very few products beyond the template site that served as a barrier (Fig. 1). In contrast, however, L74V RT behaved similarly to wild-type RT in displaying multiple additional products (Fig. 1). Thus, our results suggest that the substitutions at two critical residues, Leu74 and Lys65, influence the polymerase fidelity of HIV-1 RT differentially. In view of its increased fidelity in the gel assay, we wished to test the influence of the K65R substitution in HIV-1 RT on its mutation rate using an M13-based forward mutation assay with lacZα as a reporter. Screening 32,614 plaques led to 113 mutants, corresponding to a background-corrected mutation frequency of 1.06 × 10−3 (Table I). This represents an 8-fold decrease over that of wild-type HIV-1 RT, which displayed a mutation rate of 8.6 × 10−3 (Table I). To date, K65R is the first nucleoside analog-resistance mutation in HIV-1 RT with such a large decrease in mutation rate.Table IOverall mutation frequencies of the three RTsRTNo. of plaques screened1-aNumbers represent pooled totals from two independent fill-in reactions.No. of mutants1-aNumbers represent pooled totals from two independent fill-in reactions.Mutation frequency (×10−3)1-bAll numbers were corrected for a background mutation frequency of 2.4 × 10−3.Wild-type13,6621388.6K65R32,6141131.06L74V20,5101535.051-a Numbers represent pooled totals from two independent fill-in reactions.1-b All numbers were corrected for a background mutation frequency of 2.4 × 10−3. Open table in a new tab The gapped duplex substrate was then filled-in with L74V RT, and the mutation frequency was determined as described above. However, the mutation frequency of L74V RT was 5 × 10−3, which represents a <2-fold decrease over that of wild-type HIV-1 RT (8.6 × 10−3) (Table I). The wild-type residues at positions 65 and 74 make key contacts with the incoming dNTP and stabilize the templating base, respectively. Thus, the differential effect on mutation rate by substitutions at both residues that confer resistance to nucleoside analogs is intriguing. It appears that the alteration at Lys65 leads to an ability to discriminate against several 2′-deoxy-NTP analogs as well as against non-Watson-Crick base-paired incoming dNTPs. The effects of the L74V substitution, on the other hand, appear to be limited to discrimination against 2′-deoxy analogs. We sought to investigate the basis for this disparity between the two variant RTs tested. The M13 single-stranded circular DNAs from 105 plaques representing mutations by K65R RT were randomly selected for sequence determination. A comparison of the mutation spectra of wild-type and K65R RTs (Fig. 2,upper panel) revealed some interesting features. First, K65R RT appeared to display an increased frameshift fidelity over wild-type RT. Frameshift mutations are a prominent feature of HIV-1 RT, and such mutations are formed at high efficiency even when forward mutation rates are estimated using an HIV-1 envelope sequence in vitro (30Ji J. Loeb L.A. Virology. 1994; 199: 323-330Crossref PubMed Scopus (71) Google Scholar) or in a single cycle infection assay. 2J. Dougherty, personal communication. A comparison of the frameshift mutation rates of K65R and wild-type RTs showed a 16-fold decrease for K65R (1/40,650 for wild-type RTversus 1/666,666 for K65R RT) (TableII). Second, a large substitution hot spot (a hot spot is defined as five or more occurrences of a particular type of error at the same site) at position −36 in the regulatory sequence of the lacZα gene observed for wild-type HIV-1 RT (26 out of 138 mutations) was significantly reduced in the case of K65R RT (10 out of 105 mutations; p = 0.043) (Fig. 2,upper panel; and Table II). In fact, 50% of both frameshift and substitution mutations by wild-type RT were represented in hot spots, whereas only 28% of those by K65R RT were in hot spots. Except for hot spots at positions −36 and 145 (which together accounted for 61% of mutations in hot spots), most other mutations by K65R barely exceeded our criterion to be termed hot spots (see above) (Fig. 2, upper panel).Table IISummary of error rates for various classes of mutationsMutation typeWild-type RTK65R RTL74V RTNo. of errorsError rateNo. of errorsError rateNo. of errorsError rateAll classes1381 /17,651051 /144,9271071 /33,333Frameshifts351 /40,650141 /666,6662-ap < 0.025 when compared with errors made by the wild-type enzyme.421 /45,6622-ap < 0.025 when compared with errors made by the wild-type enzyme. At runs321 /23,753121 /4,166,6662-ap < 0.025 when compared with errors made by the wild-type enzyme.381 /27,0272-ap < 0.025 when compared with errors made by the wild-type enzyme. At non-runs131 /222,22221 /2,500,00041 /227,272Base substitutions1031 /11,876911 /84,0332-ap < 0.025 when compared with errors made by the wild-type enzyme.651 /25,6412-ap < 0.025 when compared with errors made by the wild-type enzyme.2-a p < 0.025 when compared with errors made by the wild-type enzyme. Open table in a new tab Furthermore, the frameshift hot spot at positions 106–108 observed with wild-type RT was absent in the spectra of K65R RT (p = 0.03) (Table III). The hot spot at position 145 observed for K65R RT was reduced in the case of wild-type RT (p = 0.023) (Table III). Approximately 2% of the templates generated by the K65R enzyme had two mutations, as opposed to 6% of those generated by wild-type RT.Table IIIComparison of error rates at specific hot spotsMutation type and positionWild-type RTK65R RTL74V RTNo. of errorsError rateNo. of errorsError rateNo. of errorsError rateBase substitutions −6621 /483151 /11,9040CD3-aCD, cannot be determined. −36261 /370101 /59883-bp < 0.05 when compared with errors made by the wild-type enzyme.141 /917 −3581 /120331 /20,00051 /2570 8451 /192611 /59,88011 /14,285 8951 /192621 /29,8500CD3-cp < 0.09 when compared with errors made by the wild-type enzyme. 9041 /241571 /86200CD 11251 /192621 /29,85021 /66,660 13711 /100011 /59,88051 /26313-cp < 0.09 when compared with errors made by the wild-type enzyme. 14531 /320991 /66313-bp < 0.05 when compared with errors made by the wild-type enzyme.181 /7193-bp < 0.05 when compared with errors made by the wild-type enzyme.Frameshifts 70–7351 /770211 /25,64061 /8620 88–9051 /57770CD41 /9708 106–10861 /48140CD3-bp < 0.05 when compared with errors made by the wild-type enzyme.71 /5555 132–13611 /487831 /83,33371 /76923-bp < 0.05 when compared with errors made by the wild-type enzyme. 137–13991 /320941 /45,454111 /3571All1381 /17651051 /144,9271071 /33,3333-a CD, cannot be determined.3-b p < 0.05 when compared with errors made by the wild-type enzyme.3-c p < 0.09 when compared with errors made by the wild-type enzyme. Open table in a new tab K65R RT displayed a uniformly reduced rate for all kinds of errors. Although the reduction in frameshift mutation rate for K65R RT was more striking (16-fold) than that for base substitution errors (7-fold) (Table II), a remarkable feature of this RT variant is that it displayed a very large reduction (175-fold decrease), compared with the wild type, in the rate of frameshift mutations at homopolymeric runs (stretches of sequence containing the same nucleotide). Most nucleoside analog-resistance mutations tested in this assay so far (E89G, M184V, M184I, Q151M, and VILYM) have showed a <3-fold reduction in this type of error. An exception to this, M184I, showed an 8-fold reduction in frameshifts at runs of nucleotides. Sequence determination of the single-stranded phage DNAs derived from 107 mutant plaques generated by L74V RT led to the spectrum of mutations (Fig. 2, lower panel) that reveals two key features. Although the mutations by the wild-type and K65R RTs were fairly well distributed throughout the target sequence, those induced by L74V RT were generally clustered at hot spots, with ∼70% of mutations localized to hot spots. In addition, the frameshift mutations produced by L74V RT constitute a sizable proportion of mutations, comprising 40% of the mutations identified, most of which were of the −1 type (loss of 1 base). Most of the errors made by L74V RT were at homopolymeric runs. The base substitution hot spot at position 89 seen for wild-type RT was absent in the spectrum for L74V RT (p = 0.07) (TableIII). Position 145 represents the strongest hot spot for L74V RT mutations (18 out of 107, 17% of total), whereas wild-type RT generated only 2% of its errors at this site (p < 0.0001) (Table III). Also, L74V RT generated frameshift hot spots at positions 132–136, whereas wild-type HIV-1 RT did not (p < 0.03) (Table III). All three RTs share certain hot spots, such as a base substitution hot spot at position −36 and a frameshift hot spot at positions 137–139. The results reported in this work show that certain mutations in the β3–β4 hairpin loop, e.g. K65R, cause an increased polymerase fidelity as well as a global reduction in the mutation frequency of HIV-1 RT. In contrast, another substitution in the same hairpin loop, L74V, despite its critical role in the fidelity of dNTP insertion, neither increases polymerase fidelity nor affects the overall mutation frequency, suggesting that substitution with valine does not perturb the normal interactions of the wild-type residue at position 74. The K65R RT variant displayed a general decrease in the frequency of most types of errors, the most significant feature being a very large reduction in the rate of frameshift mutations (175-fold lower than that of wild-type HIV-1 RT) at homopolymeric runs. It is known that −1 frameshift mutations at runs are the most frequent of all errors that HIV-1 RT makes. In fact, for wild-type HIV-1 RT, the rate of −1 frameshift mutations is >90-fold higher at runs than non-runs (31Bebenek K. Abbotts J. Roberts J.D. Wilson S.H. Kunkel T.A. J. Biol. Chem. 1989; 264: 16948-16956Abstract Full Text PDF PubMed Google Scholar). Although not as striking as the reduction in the rate of frameshifts at homopolymeric runs, K65R RT also displayed a general reduction in the rates of base substitution hot spots at homopolymeric runs (Table II). Klarmann et al. (32Klarmann G.J. Schauber C.A. Preston B.D. J. Biol. Chem. 1993; 268: 9793-9802Abstract Full Text PDF PubMed Google Scholar) previously reported that wild-type HIV-1 RT displays a tendency to pause at homopolymeric runs. Interestingly, as shown by Arion et al. (33Arion D. Borokov G. Gu Z. Wainberg M.A. Parniak M.A. J. Biol. Chem. 1996; 271: 19860-19864Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), K65R RT also displays a significantly enhanced polymerase processivity compared with the wild-type enzyme. An elegant series of studies have demonstrated a direct link between termination probability and frameshift fidelity of HIV-1 RT (34Bebenek K. Beard W.A. Darden T.A. Li L. Prasad R. Luton B.A. Gorenstein D.G. Wilson S.H. Kunkel T.A. Nat. Struct. Biol. 1997; 4: 194-197Crossref PubMed Scopus (114) Google Scholar, 35Bebenek K. Beard W.A. Casas-Finet J.R. Kim H.R. Darden T.A. Wilson S.H. Kunkel T.A. J. Biol. Chem. 1995; 270: 19516-19523Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). The increased processivity is thought to be due to a decreased rate of template-primer dissociation. Taken together, these results suggest that a decreased pausing by K65R RT may be responsible for a decreased rate of mutations at homopolymeric runs of nucleotides, especially those involving single base deletions. As this form of error constitutes a key feature of mutagenesis by HIV-1 RT, the residue Lys65 appears to be a key determinant of the normal error-prone synthesis by this enzyme. Our results suggest that residue 65 in wild-type RT plays a critical role in polymerase fidelity. In the structure of RT complexed with template and primer DNA and dTTP (ternary complex), th" @default.
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