From the Department of Biological Chemistry and
Molecular Pharmacology, Harvard Medical School, Boston,
Massachusetts 02115 and the ¶ Institut Pasteur, 28 rue du Dr Roux,
75724 Paris, France
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ABSTRACT |
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Human immunodeficiency virus type 1 is resistant
to 3'-azido-3'-deoxythymidine (AZT) when four amino acid substitutions
(D67N, K70R, T215F, and K219Q) are present simultaneously in its
reverse transcriptase. Wild-type and AZT-resistant reverse
transcriptases show identical binding to a 3'-azido-3'-deoxythymidine
5'-monophosphate (AZTMP)-terminated primer/RNA template. On DNA
templates, the equilibrium dissociation constant
(KD) for primer/template and AZT-resistant reverse
transcriptase (RT) (KD = 4.1 nM) is
similar to that of the wild-type enzyme (KD = 6.2 nM). However, koff is 4-25-fold
lower for the AZT-resistant enzyme than for the wild-type enzyme,
depending on the nucleotide and the template. The kinetic decay of a
wild-type RT/primer/AZTMP-terminated DNA template complex is biphasic.
Seventy percent of the initial complex decays with a rate constant
greater than 0.05 s1, and 30% with a rate constant of
0.0017 s
1. Decay of an AZT-resistant RT/AZTMP-terminated
primer/DNA template complex is monophasic, with a rate constant of
0.0018 s
1. The last two nucleotides at the 3' end of the
AZTMP-terminated DNA primer in complex with AZT-resistant RT, but not
wild-type RT, and a DNA template are protected from exonuclease
digestion, suggesting that enhanced binding of the 3' end of the
AZTMP-terminated DNA primer to reverse transcriptase is involved in the
mechanism of AZT resistance by human immunodeficiency virus type 1.
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INTRODUCTION |
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Reverse transcriptase (RT)1 of the human immunodeficiency virus type 1 (HIV-1) is a DNA- and RNA-dependent DNA polymerase that synthesizes a double-stranded DNA from the viral (+) RNA genome (1). Most of the replicative steps of the HIV-1 genome are dependent on the RT encoded by the viral pol gene. This dependence of the replication cycle on RT has directed much of the anti-HIV-1 chemotherapy strategy toward this enzyme (2). Nucleoside analogues were among the first RT inhibitors described (3, 4). Once taken up by the infected eukaryotic cell, most nucleoside analogues require phosphorylation of their 5'-hydroxyl group by cellular kinases to convert them to the 5'-triphosphate, substrates for RT. Most of these analogues do not possess a free 3'-hydroxyl group, and chain termination occurs once they are added to the nascent viral DNA. Termination is believed to account for the observed inhibition of viral growth. It is not known whether incorporation of the analogue in vivo occurs during DNA- or RNA-dependent DNA synthesis, or both.
3'-Azido-3'-deoxythymidine (AZT) is a prototype of the chain-terminating analogues (4). 3'-Azido-3'-deoxythymidine 5'-triphosphate (AZTTP) is over a thousandfold more efficient as a substrate for RT than for human polymerases (5-7), and is as good a substrate as the natural substrate dTTP for RT. Since AZTTP and dTTP pools are comparable in cultured cells (8), one would expect that replication and hence viral growth would be inhibited completely (9). However, decreased sensitivity to AZT appears gradually over time together with the rapid emergence of mutations in the viral pol gene (2, 10). This decreased sensitivity of the virus to AZT is called AZT resistance, and correlates with a specific pattern of mutations in the pol gene (11, 12). One particular set of amino acid changes, D67N, K70R, T215F/Y, and K219Q, gives rise to a 120-fold AZT-resistant virus; the corresponding RT will be referred to as AZT-resistant RT throughout this paper.
Decreased recognition of AZTTP in favor of dTTP (AZTTP discrimination) by AZT-resistant RT occurs only to a modest level (4-fold), if at all (13-16). When pools of dTTP and AZTTP are comparable, AZTTP discrimination can be reflected by the ratio of dTTP over AZTTP incorporation efficiencies (kcat/Km)dTTP/(kcat/Km)AZTTP during a single nucleotide incorporation event. Kerr and Anderson (15) found similar kcat values for wild-type and AZT-resistant RT. Comparison of Km values for substrates and inhibitors can be misleading because Km is composed of several kinetic constants (17, 18). However, if AZT resistance arises through a more stringent selection of dTTP relative to AZTTP, RT should be able to discriminate several hundredfold against AZTTP during the more than 4000 events of incorporation of T opposite A along the HIV-1 genome (9). It is unlikely that this high degree of discrimination would not be reflected in KmAZTTP.
A possible alternative mechanism to achieve resistance to AZT would be to remove AZTMP once it has been incorporated at the 3' end of the primer. Such a model of AZT resistance involving repair of the AZTMP-terminated DNA chain does not imply a high degree of discrimination during the incorporation of the inhibitor AZTTP relative to the natural substrate dTTP. If repair is extremely efficient without any nucleotide discrimination, the synthesis of viral DNA would be simply delayed. Thus, the combined effect of a modest discrimination of the inhibitor in conjunction with repair of the inhibitor-terminated DNA chain is an attractive possibility to account for the 120-fold increase in IC50 provided by the set of amino acid changes described above. Since RT does not have a proofreading 3' to 5' exonuclease activity, one must invoke another enzyme to remove the chain-terminating AZTMP. Although eukaryotic cells have a variety of 3' to 5' exonucleases, one obvious candidate for repair of prematurely terminated DNA chains is by RT itself, through pyrophosphorolysis, the reversal of nucleotide polymerization. In this reaction, RT in complex with an AZTMP-terminated primer/template binds pyrophosphate (PPi) and catalyzes the formation of AZTTP at the expense of primer length. Intracellular PPi concentrations in human lymphocytes are around 130 µM (19), but the Kd (PPi) of RT is several mM, making pyrophosphorolysis an unfavored reaction compared with nucleotide polymerization (18, 20). However, once the chain-terminator has been added at the 3' end of the primer, nucleotide polymerization can no longer occur and hence does not compete with pyrophosphorolysis. Therefore, the removal of the chain-terminating AZTMP through pyrophosphorolysis depends on the presence of RT in complex with AZTMP-terminated primer/template and PPi concentration. In this case, the dNTP concentration is relevant only if dNTPs compete with PPi for the same binding site as true competitive inhibitors. Thus, any factor increasing the lifetime of RT in complex with AZTMP-terminated primer/template should favor pyrophosphorolysis. In case repair is not provided by RT itself, a stable complex made of RT bound to the AZTMP-terminated primer/template would impede access of auxiliary proteins to the terminated primer.
With these observations in mind, we compared binding of wild-type and AZT-resistant RT to various primer/templates having either a hydroxyl or an azido group at the 3' end of the primer. We show that AZT-resistant RT has a slower off-rate than wild-type RT from its primer/template, and that the presence of AZTMP at the primer 3' end is responsible for additional specific nucleic acid/protein contacts.
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EXPERIMENTAL PROCEDURES |
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Proteins and Reagents-- The gene encoding bacteriophage T4 polynucleotide kinase was mutated so as to eliminate phosphatase activity. The phosphatase-free T4 polynucleotide kinase was purified and provided by S. Tabor, Harvard Medical School. Escherichia coli exonuclease III (Exo III) was purchased from New England Biolabs. 3'-Deoxynucleoside 5'-triphosphates (dNTPs) and all 32P-labeled nucleotides were from Amersham Pharmacia Biotech. Oligonucleotides were 5' 32P-labeled using phosphatase-free bacteriophage T4 polynucleotide kinase purified and provided by S. Tabor. p-Azidophenacyl bromide was from Sigma. The RNA template and DNA oligonucleotide containing a unique phosphorothioate linkage at nucleotide position 15 were obtained from Oligos, Etc. DNA primers were synthesized by the Biopolymers Laboratory, Harvard Medical School. RNA and DNA primers were purified by electrophoresis through an 18% polyacrylamide gel, and oligonucleotides were extracted from the gel using standard methods.
The HIV-1 RT gene cloned into a M13 filamentous bacteriophage vector to give mpRT4 has been described (21). The RT gene was excised from the mpRT4 construct and subcloned into a plasmid expression vector as follows. mpRT4 DNA was digested with EcoRI restriction enzyme, and the resulting 5'-overhangs were eliminated using dNTPs and the Klenow fragment of E. coli DNA polymerase I. The DNA was then digested with HindIII and subcloned into the plasmid vector pTrc99a (Amersham Pharmacia Biotech) at its dephosphorylated NcoI and HindIII restriction sites to give p66-WT. E. coli XL1-Blue (Stratagene) harboring p66-WT was used to express the RT gene. mpRT4 was also used as the starting material to construct a RT gene carrying four mutated codons producing the amino acid changes D67N, K70R, T215F, and K219Q, using the single-stranded template method. The resulting RT gene was subcloned into the same expression vector as p66-WT to produce p66-Res. To construct a gene encoding the p51 subunit counterpart of either p66-WT or p66-Res, p66-WT or p66-Res DNA was digested with KpnI and HindIII, dephosphorylated, and ligated with a double-stranded, phosphorylated synthetic oligonucleotide. The latter oligonucleotide was designed so as to maintain the correct reading frame of the RT gene, contain KpnI and HindIII restriction site sequences to be ligated at each end, and introduce a stop codon at the desired position in order to produce a p51-WT or p51-Res protein containing 440 amino acids. All plasmid constructs containing the RT genes were verified by restriction enzyme analysis and by DNA sequencing.Expression and Purification of Enzymes--
Ten-liter cultures
of bacteria harboring p66-WT or p66-Res plasmids were grown up at
37 °C in 2YT medium (16 g/liter Tryptone, 10 g/liter yeast extract,
5 g/liter NaCl containing 100 µg/ml ampicillin. Bacteria were grown
to an A600 = 1, and
isopropyl-1-thio--D-galactopyranoside was added to a
final concentration of 1 mM. After 3 h of incubation, the cells were harvested by centrifugation. All subsequent steps were
at 4 °C. The pellet (about 100 g of wet cells) was resuspended in 100 ml of 10% sucrose, 40 mM Tris-HCl, pH 8.0, 25 mM EDTA. Lysozyme was added to 1 mg/ml together with 0.5 mM phenylmethylsulfonyl fluoride and 1 mM
benzamidine. After a 15-min incubation, cells were lysed with an
equal volume of solution containing 0.4% Nonidet P-40, 10 mM dithiothreitol (DTT), 0.5 mM
phenylmethylsulfonyl fluoride, and 1 mM benzamidine.
Following another 15-min incubation with gentle agitation, the mixture
was brought to 0.5 M NaCl and sonicated six times for
20 s at maximum output using a Branson 450 Sonifier. The solution
was sedimented at 100,000 × g for 1 h in a
Beckman SW27 swinging bucket rotor, and the supernatant fluid was
collected to yield fraction I (200 ml, about 20 mg/ml protein).
Polymerase Assays-- RT activity was determined either by monitoring the formation of radioactively labeled nucleic acid product using ion-exchange paper discs (21) (assay 1) or by measuring the incorporation of a single nucleotide using the gel assay described below (assay 2). Polymerase activities during enzyme purification and re-association were determined using assay 1. Reactions were carried out in RT buffer (50 mM Tris-HCl, pH 8.0, 50 mM KCl, 10 mM MgCl2, 0.05% Triton X-100) in the presence of 50 µg/ml poly(rA)/oligo(dT), 30 µM each of dATP, dCTP, dGTP, and [3H]dTTP (36 µCi/µmol). Control experiments showed that the presence of dGTP, dATP, and dCTP in assays using poly(rA)/oligo(dT) did not affect the reaction. Hence, for convenience all reaction mixtures for RT activity using assay 1 contained the four dNTPs. After addition of RT, aliquots were withdrawn at several time points and spotted onto DE-81 paper discs that had been previously soaked in 50 mM EDTA. Paper discs were washed three times for 10 min in 0.3 M ammonium formate, pH 8.0, 2 times in ethanol, and dried. The radioactivity bound to the filters was determined by liquid scintillation counting. One unit of enzyme catalyzes the incorporation of 1 nmol of DE-81-absorbable dTMP in 10 min at 37 °C.
For assay 2, the incorporation of a single nucleotide was monitored using a primer extension reaction followed by polyacrylamide gel electrophoresis. Except when indicated, only one nucleoside triphosphate was present in the reaction. A 21-mer DNA primer (5'-ATACTTTAACCATATGTACC-3') was 5' end-labeled with [Determination of Equilibrium Dissociation (KD) and On-rate (kon) Constants for DNA Template and RT Dimers-- RT was titrated with the same 5'-labeled primer/template as above for polymerase assay 2 to determine the equilibrium dissociation constant (KD) (17, 20, 22). RT (40-50 nM final concentration) was allowed to bind to different concentrations of primer/template in RT buffer at 0 °C. After incubation for 1-2 min, the reaction was divided into three portions. Each of the three reactions was incubated at 23 °C for 2 min before addition of dNTP-Mg2+ to yield the final desired concentration in 5 mM MgCl2. For each reaction, after rapid manual mixing of the dNTP with the RT/primer/template, two aliquots were withdrawn and quenched at times 5 and 15 s, 8 and 20 s, or 10 and 30 s for the three reactions, respectively, using formamide gel loading buffer. Products were analyzed by 18% polyacrylamide gel electrophoresis as described above and quantitated using photostimulable plates on a Fuji Imaging apparatus.
The time course of product formation was biphasic, with an initial burst of product formation too fast for manual sampling, followed by a slower, linear phase. The burst amplitudes were determined by linear extrapolation of the slow phase on the ordinate axis. Comparisons with experiments conducted using a rapid quench apparatus (Kintech) gave essentially the same burst amplitude values. It was found that the manual initial mixing of reagents was the limiting step to obtain comparable pre-steady-state values. However, after 3-4 s, the linear time course was indistinguishable from time points obtained with the rapid quench apparatus, yielding an accurate extrapolation of burst amplitude values. Burst amplitudes were plotted against primer/template concentration. The equilibrium constant KD for binding of the enzyme to the primer template, and total active enzyme E0 were determined from the fit of the data to the quadratic equation shown below, where D0 is total primer/template concentration.
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(Eq. 1) |
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(Eq. 2) |
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(Eq. 3) |
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(Eq. 4) |
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(Eq. 5) |
Synthesis and Purification of Primers Containing a p-Azidophenacyl Photo-activable Probe-- Upon UV irradiation at 365 nm, aromatic azido groups react with a variety of protein side chains, such as carboxylic acids and aromatic rings, to form a covalent bond. Attachment of a photoreactive p-azidophenacyl moiety to DNA primers was as described (24, 25). Phosphorothioate-containing oligonucleotides (2 mg) were dissolved in 0.3 ml of 150 mM potassium phosphate buffer, pH 7.0, mixed with 0.75 ml of p-azidophenacyl bromide (5 mg/ml in methanol), and incubated for 3 h at room temperature. Reaction products were precipitated with excess ethanol, redissolved in water, and lyophilized. The modified oligonucleotides were purified from unreacted reagents using C18 reverse-phase HPLC. Annealing of the various primer/templates was as described (22). The enzymatic phosphorylation of the 5' end of the oligonucleotide primer was not affected by the photoprobe (data not shown).
DNA/Protein Cross-linking Assays Using Photoprobe-coupled
Oligonucleotide--
The p-azidophenacyl-modified primer
was labeled and annealed in a fashion similar to that of unmodified
primers (see below). RT (250 nM) was mixed (1:1 v/v) with
an equimolar amount of 5' 32P-labeled
p-azidophenacyl-modified oligonucleotide annealed to a DNA
or RNA template on parafilm laid on a flat, cold (4 °C) surface in a
10-µl final volume in 10% glycerol, 5 µg/ml bovine serum albumin,
5 mM MgCl2, 25 mM potassium
phosphate buffer, pH 7.4, 1 mM DTT, and KCl as indicated.
The mixture was irradiated ( = 365 nm) with a two-bulb 15-watt UV
lamp at a distance of 2 cm to yield a UV irradiation dose of 0.1-0.2
watt/cm2. The mixture was heated in SDS-PAGE loading buffer
containing 80% formamide, 4% SDS, and 1 mM
-mercaptoethanol at 95 °C for 3 min and loaded onto a
SDS-polyacrylamide gel containing 7 M urea. Gels were
covered with cellophane wrap, radioactive signals were measured using a
Fuji imaging apparatus, and the gel was exposed to x-ray films.
Measurements of radioactive label incorporated into protein products
indicated that about 10% of the input protein was labeled.
Exonuclease III Footprinting of RT/Primer/DNA Template Complexes-- Duplex DNA substrates were prepared by annealing the labeled primer (5'-ATACTTTAACCATATGTACC-3', 2.5 µM) with a 31-mer DNA template (5'-TTTTTTTTTAGGATACATATGGTTAAAGTAT-3') at a 1:1.5 primer/template molar ratio (determined spectrophotometrically) in 10 mM Tris-HCl, pH 8.0, 50 mM KCl by heating to 80 °C and slow cooling to room temperature over a period of at least 1 h. Immediately prior to the assay, the primer/template was diluted in RT buffer (50 mM Tris-HCl, pH 8.0, 50 mM KCl, 10 mM MgCl2, 0.05% Triton X-100). RT (100 nM) was allowed to bind to the 5' 32P-labeled primer/DNA template (100 nM), and 25 units of Exo III were added at time 0. When present, AZTTP (10 µM) was incorporated into the primer/template prior to Exo III digestion to yield a 3'-AZTMP-terminated 22-mer primer annealed to the 31-mer template. Aliquots were withdrawn at indicated times, and the reaction was quenched by the addition of formamide gel loading buffer. Products were analyzed by electrophoresis through a 12% polyacrylamide denaturing gel. Autoradiography and measurements of products using photostimulable plates were as described above.
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RESULTS |
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We have compared binding of wild-type and AZT-resistant RT to various primer/templates in which the 3' end of the DNA primer is either a hydroxyl or an azido group, and the template is either DNA or RNA.
Active-site Titration and Affinity of RT for Primer/DNA Templates-- The binding affinities of wild-type and of AZT-resistant RT for a primer/DNA template have been determined using active-site titration procedures (17, 20, 22). Since the rate-limiting step is the release of the enzyme from the primer/template, the ternary complex accumulates much faster than it undergoes catalysis, and a burst of product is observed in the early course of the reaction. This burst is related to the concentration of the ternary complex. Hence, at early times during the reaction course, one can measure the ternary complex and obtain the equilibrium constant KD of RT for its primer/template as well as the proportion of enzyme E0, which is catalytically productive in the ternary complex. When the enzyme is allowed to bind to the primer/template and this binary complex is rapidly mixed with an excess of a single dNTP, the amplitude of the burst can be described as a function of the total enzyme concentration (E0), the dissociation constant (KD) of enzyme from its nucleic acid substrate and total nucleic acid concentration (D0). Consequently, we assayed the polymerase activity of RT in this manner with a single dNTP present in the reaction in order to determine the equilibrium constant of dissociation (KD) of wild-type and AZT-resistant RT from their primer/templates. Single turnover incorporation products were analyzed and measured by denaturing gel electrophoresis.
From the data obtained from the experiments shown in Fig. 1 (A and B), the KD for binding DNA by the wild-type and AZT-resistant RT was 6.2 and 4.1 nM, respectively. This result is in agreement with the value that has been reported previously for a similar primer/template of defined sequence (17, 22). Depending on the enzyme preparation, 0.4-0.6 active site/dimer were found for the wild-type and 0.8-1 for the AZT-resistant RT, as judged by active-site titration regardless of the template used (Fig. 1B).
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Determination of the Catalytic Constant kcat-- For wild-type RT, kcat represents the rate-limiting step of nucleotide polymerization when a single nucleotide is added to a primer. kcat is equal to the off-rate constant koff from the primer/template. It is thus important to determine if kcat still represents the off-rate koff for the AZT-resistant RT. We have measured the catalytic constant kcat of the two RTs under steady-state conditions with various primer templates using assay 2.
The values of kcat are shown in Table I. For the wild-type enzyme, kcat values for the incorporation of various dNTPs are in the vicinity of 0.05 s
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Cross-linking of the Primer/Template to Wild-type and AZT-resistant RT-- kcat values are 4-25-fold lower for AZT-resistant compared with wild-type RT. In order to determine if kcat equals koff for the AZT-resistant RT also, we have examined both the binding of RT to a primer/template and the kinetics of release of RT from the RT/primer/template complex. If kcat equals koff, it would indicate that primer/template binding properties differ for the two enzymes. Such a difference could either originate from a different manner of binding to the primer/template, or binding in a similar manner but with different kinetic properties. In order to address these issues, we have examined the binding of RT to a primer/template by cross-linking the enzyme to the primer. In this system, a 21-mer DNA primer has a photoreactive group at nucleotide position 15 relative to the 3' end (26). This primer, labeled at its 5' end with 32P, is first annealed to a 31-mer DNA or RNA template, and then RT is allowed to bind. Upon UV irradiation, the primer will be cross-linked to the p66 and p51 subunits of RT at amino acid residues sufficiently close to the photoreactive group. The nucleotide position 15 was chosen because the photoreactive group cross-links efficiently to either p66 when RNA is the template or p51 when DNA is the template (26). The extent and ratio of cross-linking of the labeled primer to p66 and p51 can be determined by denaturing polyacrylamide gel electrophoresis, where the 51- and 66-kDa subunits of RT cross-linked to the primer have apparent molecular masses of 57 and 72 kDa, respectively. It has been shown that both KCl concentration and template excess can affect primer/template binding by RT (27, 28). Therefore, we assessed the effect of excess template and KCl concentrations on the outcome of our cross-linking experiments.
Comparison of cross-linking patterns using wild-type and AZT-resistant RT are shown in Fig. 2 (A and B, respectively). On DNA templates, 75% of the labeled primer was cross-linked to the p51 subunit of the wild-type RT For wild-type RT (Fig. 2A, lanes 1-4), whereas on RNA templates p66 is the predominantly labeled subunit (lanes 5-8). The presence of a 20-fold molar excess of template in the cross-linking reaction decreased the overall labeling efficiency 2-fold on DNA templates in the absence of KCl for wild-type RT (lanes 1 and 2). The distribution of the label between subunits was not dependent on the template concentration (compare even and odd lanes in Fig. 2A). KCl had no effect on the efficiency of cross-linking of the primer to the wild-type RT, but when RNA was the template it did suppress labeling of the p51 subunit (Fig. 2A, lanes 5-8).
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Stability of a RT/Primer/Template Complex-- The results presented in the previous section indicated that kcat values are different between wild-type and AZT-resistant RT, and that the presence of the AZT resistance mutations alters binding properties of RT to its primer/template. In order to determine if these differences are related, we designed an alternate method to measure the off-rate constant koff of the enzyme from the primer/template and to compare koff to kcat. In these experiments, wild-type or AZT-resistant RT was bound to a 5' 32P-labeled primer annealed to a template. At time 0, a large excess of DNA was added to serve as a trap for the free enzyme, and the decay of the concentration of the RT/primer/template complex was measured over time. We used a primer/template in which the 5'-labeled primer has a photoreactive probe attached at nucleotide position 15 on the internucleotidic phosphodiester bond (26). At various times after addition of the DNA trap, aliquots were UV-irradiated; the photoreactive probe of the 5'-labeled can potentially cross-link to both p66 and p51 RT subunits, and the resulting amount of radioactivity present in both subunits is proportional to the RT/primer/template complex concentration. The amount of radioactivity in each aliquots was measured after electrophoresis of the products through a denaturing gel.
The stability of a RT/primer/RNA template complex is shown in Fig. 3. On RNA templates, both wild-type and AZT-resistant RT bound in a similar manner to their primer/template. Both enzymes bound relatively tightly as judged by the slow decrease of label over the 10-min period. For the wild-type enzyme (Fig. 3A, left panel), there was no difference when the 3' end of the primer was either AZTMP or dCMP. When dCMP terminated the primer, the decay of wild-type RT from the RT/primer/template complex was biphasic (Fig. 3B), with an initial phase too fast to be measured precisely in this experiment, followed by a slower phase with an off-rate constant around 0.0008 s
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Mapping the Interactions of the 3' End of the Primer with RT-- In order to examine the basis of the enhanced binding of the AZT-resistant RT to a primer/DNA template, we determined the footprint of RT on a RT/primer/DNA template complex. In this experiment, RT was bound to a primer/DNA template in which the 5' 32P-labeled primer bears either a hydroxyl (dCMP) or an azido (AZTMP) group at its 3' end. In order to introduce an AZTMP at the 3' end of the primer, the primer/template/RT complex was first incubated in the presence of 10 µM AZTTP. In this manner, more than 90% of the 21-mer primer gave rise to an AZTMP-terminated 22-mer product. At time 0, an excess of Exo III was added to the reaction. Exo III degrades double-stranded DNA in a distributive fashion in the 3' to 5' direction, thus eliminating instantaneously any free primer/template. Therefore, any primer/template remaining after the addition of Exo III is a measure of the primer/template protected by RT. Consequently, any additional contact made by the AZT-resistant RT with the primer should increase protection of the primer from degradation by Exo III.
The results of such an experiment are shown in Fig. 5. As a control, when RT was not present, no protection occurred, and the 5' 32P-labeled primer was degraded rapidly (Fig. 5A). At 0.16 min after addition of Exo III, 4 nucleotides have been removed from the 3' end of the 21-mer primer as judged by the appearance of products corresponding to a 17-mer or less. This 17-mer product was further degraded over time to yield oligonucleotides under 10 nucleotides in length. When wild-type RT was present, degradation of the primer was retarded, as judged by the persistence of a product corresponding to the full-length primer. In addition, there was a slower progressive degradation of the primer with products of 6-20 nucleotides remaining after 10 min (Fig. 5B). In the presence of wild-type enzyme, some of the 21-mer primer and 19-mer remained over time (Fig. 5B, arrows). In contrast to the wild-type enzyme, a greater amount of the 21-mer primer persisted over time when the AZT-resistant enzyme was present (Fig. 5C, arrows). When AZTMP was present at the 3' end of the 22-mer primer, the same pattern of degradation as when the primer had a 3'-OH was observed for the wild-type enzyme (Fig. 5D, arrows). Comparatively, AZT-resistant RT showed an enhanced protection of the AZTMP-terminated primer as judged by the persistence over time of the 22-mer and 21-mer products (Fig. 5E, arrows). Control experiments indicated that Exo III was not inhibited by 10 µM AZTTP (data not shown). It is noteworthy that the 19-mer product was still predominant during the early time of the reaction (Fig. 5E, lower arrow). Since this 19-mer product was present with either a 21-mer or a 22-mer primer, it might indicate that protection of the 3' end of the primer occurred without translocation of RT on DNA.
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DISCUSSION |
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In HIV-1-infected cells, replication of the viral genome is dependent on the virus-encoded RT. For this reason, considerable efforts have been devoted to identifying inhibitors of RT. Since nucleotide analogues usually act as competitive inhibitors, most attempts to understand the mechanism of drug resistance have logically focused on nucleotide recognition. Previously, no obvious differences had been found for dTTP and AZTTP in nucleotide incorporation parameters, such as Km and Ki values, sufficient to explain a >100-fold resistance to the analogue of the AZT-resistant virus in cultured cells (13, 14). Pre-steady state kinetic studies have found that AZT-resistant RT is able to discriminate only 4-fold AZTTP when RNA is the template (15, 16). Consequently, it would appear that either undetectable changes in biochemical properties of RT in vitro translate into large phenotypic changes in AZT resistance in vivo, or as yet unknown cellular factor(s) are involved in AZT resistance (13).
The results presented here show that a clear difference exists in primer/template binding of the AZT-resistant RT compared with the wild-type enzyme. In addition, the nature of the template, either DNA or RNA, influences the binding of the RT to the primer/template. Wild-type and AZT-resistant RT show a similar binding to primer/RNA template tighter than to a primer/DNA template. Although this is in agreement with the results of Kerr and Anderson (15), these authors report values of kcat representing the dissociation rate constant significantly higher than the values reported here. These lower values might be due to the fact that our experiments were performed at 23 °C instead of 37 °C. However, RT used in the experiments of Kerr and Anderson (15) carries a polyhistidine extension at the N terminus of the p66 subunit. Examination of the 3D-structure of RT (29) shows that the p66 N-terminal peptide chain runs in the direction of the domain carrying mutations at amino acid 215 and 219, and proline 1 in the peptidic sequence of RT is located only 7.4 Å from threonine 215. A peptide made of 6 histidines would cover 15 Å when fully extended, and 9 Å when under a hypothetical helical conformation. Thus, it remains possible that the histidine tag on the RT used by Kerr and Anderson (15) interfered with the binding properties of RT to its primer/template.
The release of RT from the RT/primer/template complex is biphasic except when a 3'-azido group is present at the 3' end of the primer. Therefore, the half-life of the RT/primer/template complex, which refers to a first order, monophasic decay, is not appropriate for describing the kinetics of decay of the complex. Biphasic binding of wild-type RT to its primer/template is in agreement with results presented by others (27, 30, 31). We show here that the presence of an AZTMP-terminated DNA primer converts the biphasic pattern of decay to a monophasic one. The presence of the next correct dNTP does not increase the stability of the complex (data not shown), indicating that the AZTMP-terminated DNA primer is sufficient to induce this switch from a biphasic to a monophasic decay.
Binding of AZT-resistant RT to primer/DNA templates is virtually identical to binding to primer/RNA templates. With either complex a biphasic decay was observed unless a 3'-azido group was present at the 3' end of the primer, in which case the decay was monophasic. This result is in sharp contrast with the wild-type enzyme; 75% of the wild-type RT in the RT/primer/DNA template complex decayed at least 10-fold faster in the initial stages of the reaction than during the second slow phase. Furthermore, the first rapid phase of decay was not suppressed by the presence of AZTMP-terminated DNA. We ruled out an active displacement of 75% of the RT from the RT/primer/DNA template complex during the initial phase by the DNA trap. Indeed, displacement does not occur when AZT-resistant RT is bound to the primer/DNA template (Fig. 2A, lanes 1 and 2). The binding differences between the two RTs when DNA is the template are further demonstrated in a nuclease protection assay. Footprinting experiments indicate that the last 2-4 bases of the AZTMP-terminated DNA primer are bound more tightly to the AZT-resistant enzyme than to the wild-type enzyme. Overall, these results indicate that the wild-type enzyme and the AZT-resistant enzyme binds to the primer differently, depending on whether the template is DNA or RNA. In this scheme, the AZT resistance mutations might add novel contacts to the 3' end of the primer on DNA templates to yield a tighter contact than that observed when the primer is annealed to a RNA template. Since the result shown in Fig. 2 as well as in previous work (26) have shown that primer/DNA templates are not positioned the same as primer/RNA templates at the RT surface, we do not believe that the tighter binding of primer/DNA templates by AZT-resistant RT is due to positioning the primer/DNA template like a primer/RNA template. Boyer et al. (32) and Perach (33) have proposed that the first unpaired 3-4 bases of the DNA template strand are involved in the process of ddI resistance brought by the L74V and E69G amino acid changes. We cannot rule out that the protection of the last 4 nucleotides of the 3' end of AZTMP-terminated primer by AZT-resistant RT from Exo III digestion is mediated by the template strand. In other words, the template strand would make the additional contacts with the amino acid residues arising from AZT resistance, and indirectly mediate protection of the 3'-terminus of the primer. However, it is equally likely that, in the experiments of Boyer et al. (32) and Perach et al. (33), the position of the 3'-terminus may vary according to the overhanging template length. We now know that a biochemical property distinguishes wild-type and AZT-resistant RT. This property is a tighter and more specific binding of AZTMP-terminated DNA chains to AZT-resistant than to wild-type RT. In as much as this property is easily assayed, it would be interesting to determine which of the four amino acid changes found in the AZT-resistant enzyme mediate(s) additional protein-nucleic acid contact(s).
A tighter binding of the 3' end of the AZTMP-terminated primer suggests that repair accounts at least in part for AZT resistance, and argues against any other enzyme than RT itself to mediate this process. In this model, AZTTP would be discriminated against modestly by RT, 1-4-fold (14, 15) to 6-fold.2 Removal of the terminal 3'-AZTMP would provide RT with another chance to incorporate dTMP. Since RT does not have an inherent 3' to 5' exonuclease activity, pyrophosphorolysis, the reversal of nucleotide polymerization, must account for the removal of AZTMP. For wild-type RT, the rate constant for pyrophosphorolytic excision of AZTMP from the 3'-primer terminus is comparable to the rate constant for dissociation (18). Consequently, Reardon (18) has pointed out that millimolar concentrations of intracellular inorganic pyrophosphate would be required for pyrophosphorolytic repair of AZTMP-terminated DNA. Thus, a lower off-rate constant of RT from the 3'-AZTMP terminated primer should increase the likelihood of repair at lower concentrations of pyrophosphate. This is precisely what we observe; the presence of the four mutations decreases koff 25-fold for AZT-resistant RT relative to wild-type RT on AZTMP-terminated primer/DNA templates. Thus, enhanced repair of the AZTMP-terminated DNA chain through an increased stability of the RT/3'-AZTMP primer/template complex and pyrophosphorolysis is an attractive possibility.
Finally, this model of AZT resistance makes several predictions that are fully consistent with existing data regarding HIV-1 chemotherapy. First, pyrophosphorolysis must infer a certain degree of AZT resistance to wild-type RT. This conclusion is consistent with the failure of AZTTP to block completely HIV-1 replication. AZTTP and dTTP are equally good substrates for RT, and AZTTP is present at the micromolar level in the cell (19). Hence, even if dTTP is in excess over AZTTP, an exceedingly small quantity of replicating HIV-1 genomes should avoid premature AZTMP termination during the >4000 events of insertion of a thymine opposite to a template adenine (17). Yet, viral load hardly decreases (<1 log10) after initiation of AZT therapy and then quickly return to its original value prior to the appearance of the first AZT resistance mutations (34). Thus, one can conclude that wild-type HIV-1 has a natural resistance to AZT and that this resistance is not accounted solely by nucleotide selection at the RT active site.
The second prediction is that any drug-mediated effect on pyrophosphorolysis should interfere with AZT resistance. Precisely such an effect is observed with the pyrophosphate analogue phosphonoformate (PFA). When PFA is used to inhibit HIV-1 in cultured cells, it is possible to select for PFA-resistant isolates of the virus. PFA-resistant mutations map to the pol gene, and include amino acids 88, 89, 90, 92, 113, and 114. Moreover, viruses having these mutations are now hypersensitive to AZT, requiring much less AZT to achieve the same IC50 (11, 35). When these mutations are introduced in the pol gene encoding the four amino acid changes typical of the AZT-resistant enzyme, PFA resistance results but AZT resistance is suppressed (35). Resistance to both PFA and AZT are difficult to achieve and might even be exclusive (35). This finding is also reminiscent of the difficulty for the virus to develop resistance to both AZT and the nucleotide analogue 3'-thiacytidine (3TC) (36). It is interesting to note that the AZT-resistant enzyme has a higher processivity of nucleotide polymerization (37) and primer/template affinity (this work), whereas 3TC-resistant RT (M184L/V) is a less processive enzyme (38). As in the case of PFA and AZT, this suggests that the mechanism of resistance would be different for AZT and 3TC, accounting for the difficulty in obtaining dual resistance to these analogues (36). Consistent with this observation, 3TC resistance can be totally accounted for by a loss of affinity for RT of the 5'-triphosphate form of 3TC (16). It would be interesting to study the pyrophosphorolysis reaction using PFA- and 3TC-resistant or AZT-resistant RTs in order to determine the validity of the proposed model.
The third consequence of the model is the issue of cross-resistance. Amino acid changes altering the selection of nucleotide analogue are expected to be specific and dictated in part by the chemical properties of the analogue. Thus, these amino acids are less likely to yield to cross-resistance to other nucleotide analogues. However, if resistance to two different drugs arises as a result of effects on pyrophosphorolysis, then cross-resistance might occur.
The fourth consequence of the model is related to the pattern of acquisition of the AZT resistance mutations over time. It is clear that this pattern follows a defined order important to obtain full AZT resistance. One logical explanation for this ordered pattern is that the enzyme must acquire at least two new properties, and that acquisition of a single property (e.g. discrimination) might decrease the other (e.g. pyrophosphorolysis). If this explanation is true, then compensatory mutations must occur in order to achieve full AZT resistance. D67N and K219Q amino acid changes do not lead to any obvious AZT resistance phenotype and are thus good candidates to be compensatory mutations of K70R and T215F/Y. In any case, pyrophosphorolysis deserves a closer study using the various intermediate AZT-resistant mutant RTs.
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ACKNOWLEDGEMENTS |
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We thank Kajal Chowdhury, Ingrid Richardson, Hans Huber, and Steve Carroll for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported in part by Grant AI-06045 from the National Institutes of Health, Grant DE-GF88ER60688 from the Department of Energy, and the Agence Nationale de Recherche sur le SIDA.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by CNRS and Agence de Recherche sur le Cancer.
To whom correspondence should be addressed. Tel.:
617-432-1864; Fax: 617-432-3362; E-mail:
ccr{at}bcmp.med.harvard.edu.
1 The abbreviations used are: RT, reverse transcriptase; AZT, 3'-azido-3'-deoxythymidine; AZTMP, 3'-azido-3'-deoxythymidine 5'-monophosphate; AZTTP, 3'-azido-3'-deoxythymidine 5'-triphosphate; dNTP, 3'-deoxynucleoside 5'-triphosphate; RNase H, ribonuclease H; HIV-1, human immunodeficiency virus type 1; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; PFA, phosphonoformate; 3TC, 3'-thiacytidine.
2 B. Canard, S. R. Sarfati, and C. C. Richardson, unpublished results.
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REFERENCES |
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