The Valine-to-Threonine 75 Substitution in Human Immunodeficiency Virus Type 1 Reverse Transcriptase and Its Relation with Stavudine Resistance*

Boulbaba SelmiDagger , Joëlle BorettoDagger , Jean-Marc Navarro§, Josephine Sire§, Sonia LonghiDagger , Catherine Guerreiro, Laurence Mulard, Simon Sarfati, and Bruno CanardDagger ||

From the Dagger  CNRS and Universités d'Aix-Marseille I and II, UMR 6098, Architecture et Fonction des Macromolécules Biologiques, ESIL-Case 925, § INSERM U-372, 163 Avenue de Luminy, 13288 Marseille Cedex 9, and the  Institut Pasteur, 28 Rue du Docteur Roux, 75724 Paris Cedex 15, France

Received for publication, October 27, 2000




    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The amino acid change V75T in human immunodeficiency virus type 1 reverse transcriptase confers a low level of 2',3'-didehydro-2',3'-dideoxythymidine (stavudine, d4T) resistance in vivo and in vitro. Valine 75 is located at the basis of the fingers subdomain of reverse transcriptase between the template contact point and the nucleotide-binding pocket. V75T reverse transcriptase discriminates 3.6-fold d4T 5'-triphosphate relative to dTTP, as judged by pre-steady state kinetics of incorporation of a single nucleotide into DNA. In addition, V75T increases the DNA polymerization rate up to 5-fold by facilitating translocation along nucleic acid single-stranded templates. V75T also increases the reverse transcriptase-mediated repair of the d4TMP-terminated DNA by pyrophosphate but not by ATP. The V75T/Y146F double substitution partially suppressed both increases in rate of polymerization and pyrophosphorolysis, indicating that the hydroxyl group of Thr-75 interacts with that of Tyr-146. V75T recombinant virus was 3-4-fold d4T-resistant and 3-fold resistant to phosphonoformic acid relative to wild type, confirming that the pyrophosphate traffic is affected in V75T reverse transcriptase. Thus, in addition to nucleotide selectivity V75T defines a type of amino acid change conferring resistance to nucleoside analogues that links translocation rate to the traffic of pyrophosphate at the reverse transcriptase active site.




    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Antiretroviral therapy directed against the human immunodeficiency virus (HIV)1 is currently increasing the life expectancy of infected individuals. To replicate in infected cells, HIV relies on reverse transcriptase (RT), an essential RNA- and DNA-dependent DNA polymerase encoded by the viral pol gene (1). RT is a major target for drugs acting as inhibitors of retroviral replication. Because dNTPs are natural substrates for RT, more than 20 nucleoside analogues are currently used in vitro, in clinical trials, and in the clinic to inhibit retroviral replication (2, 3). To act as specific RT inhibitors, nucleoside analogues must be converted into 5'-triphosphate nucleosides upon phosphorylation by intracellular nucleoside/nucleotide kinases. Most nucleotide analogues do not possess a 3'-OH group required for phosphodiester bond formation during DNA synthesis. Therefore, they act as DNA chain terminators once incorporated into the nascent viral DNA.

The efficacy of antiretroviral chemotherapy is limited by at least three factors. First, nucleoside analogues have to cross the cellular membrane to be activated to the 5'-triphosphate form by cellular kinases. This series of phosphorylation reactions is generally not efficient enough to produce adequate levels of the active nucleoside 5'-triphosphate able to compete efficiently with natural dNTPs. Second, RT incorporates nucleotide analogues with, at best, equal efficiency to that of their natural counterparts, i.e. dTTP for both AZTTP and d4TTP. The consequence of these two points is that retroviral replication is not completely suppressed. Third, mutant viruses escaping replication inhibition arise and are resistant to nucleoside analogues due to amino acid substitutions in the pol gene.

AZT has been the first approved drug used in antiretroviral therapy (4). In cultured cells infected by HIV-1, AZT is a potent drug active in the nanomolar range. In the cell, AZT is efficiently converted into AZTMP. The intracellular concentration of AZTMP is in the millimolar range, whereas AZTTP levels are in the micromolar range (5). Both thymidylate kinase and nucleoside diphosphate kinase represent a "bottleneck" in the activation of AZTMP and AZTDP, respectively. AZTMP and AZTDP are 2 × 102- and 104-fold less efficient substrates for thymidylate kinase and nucleoside diphosphate kinase than their thymidine counterparts, respectively (6, 7).

d4T is another thymine analogue showing antiretroviral properties similar to those of AZT, but unlike AZT, it is poorly converted into d4TMP by thymidine kinase (8). Consequently, intracellular levels of d4TMP are low (8, 9). The activation of d4TMP into d4TDP by thymidylate kinase is poorly characterized in vitro. At the following step of activation toward the triphosphate, the presence of an intramolecular CH···O bond in d4TDP makes this nucleotide a 10-fold better substrate for nucleoside diphosphate kinase than AZTDP. However, d4TDP is still converted 103-fold less efficiently than its natural counterpart dTDP by nucleoside diphosphate kinase (6, 10, 11). This 10-fold enhancement relative to AZTDP does not compensate for the low levels of d4TMP; d4TTP levels are even lower (about 20-fold) than those observed for AZTTP (8). The comparison of the activation properties of AZT and d4T illustrates well that the chemical nature of the modifications in the ribose moiety is critical for antiretroviral potency. For example, ddTTP is a potent inhibitor of RT in vitro (12), but ddT has limited therapeutic value.

Interestingly, ribose modifications also specify their own pattern of nucleoside resistance in the viral pol gene. Indeed, when AZT is given as the sole drug, a pattern of up to 6 amino acid changes involving M41L/D67N/K70R/L210W/T215F/T215Y/K219Q is observed in RT and confers a >100-fold AZT resistance to HIV-1 (3, 13). The resistance pattern is different for d4T. First, moderate (<10-fold) resistance to d4T is observed in the clinic as well as in cultured cells infected by HIV-1 (14, 15). Unlike AZT, d4T does not induce a specific pattern of mutations in the pol gene. In the clinic, the most important d4T resistance mutations are in fact AZT resistance mutations such as those described above (15, 16). These mutations have thus been termed thymidine-associated mutations (TAMs). It is, however, important to note that the use of AZT in the clinic has predated that of d4T, thus facilitating the diffusion, selection, and persistence of these mutations among HIV-1 isolates. Second, the d4T therapeutic usefulness is not short lived, and the definition of d4T resistance is elusive (17). A single substitution seems to be associated specifically to the use of d4T, however. When d4T is given as the sole drug in cultured cells infected by HIV-1, RT bearing a valine to threonine substitution at position 75 can be selected (14). This substitution confers a 2-3-fold increase in IC50 for d4T to HIV-1, and this level of resistance is generally considered close to the limit of significance. In the clinic, the V75T substitution is observed at low frequency (up to 10%) following d4T therapy, the highest frequency being on AZT naive patients (18-20). The V75T substitution does not appear on RT having a pre-existing AZT resistance background (20).

These results give to d4T a very favorable profile in terms of avoiding the drug resistance problem. However, a number of questions need to be addressed to use the full therapeutic potential of d4T. Why does d4T elicit only a moderate (<10-fold) resistance as compared with AZT (>100-fold)? What is the molecular mechanism by which V75T would confer d4T resistance? The V75T substitution comes from a 2-base pair mutation involving a GTN right-arrow ACN codon change. Although a 2-base pair mutation occurs at a lesser frequency than a single mutation, the classical AZT resistance-associated T215Y change also involves a 2-base pair mutation and is found at a greater frequency than V75T. T215Y confers a <2-fold resistance to d4T, but subsequent AZT resistance mutations provide a higher level of resistance than V75T alone (3), explaining why V75T does not occur in an AZT resistance background. Recently, Ross et al. (20) have found a certain degree of exclusion between AZT resistance and d4T resistance mutations since an increased phenotypic resistance to d4T was observed in a group of patients who had either at least one TAM or the V75T substitution. This observation suggests that the mechanism by which V75T mediates d4T resistance might be different from that of AZT resistance.

The main AZT resistance mechanism involves repair of the AZTMP-terminated primer either by ATP (21) or pyrophosphate (PPi) (22). These repair reactions have in common the nucleophilic attack of a hydroxyl group from either the gamma -phosphate of ATP or the PPi on the terminal phosphodiester bond of the AZTMP-terminated DNA primer. The molecular mechanism underlying d4T resistance in general is poorly defined. In the case of V75T, Lennerstrand et al. (23) found that ATP had no effect on the repair of d4TMP-terminated primer. They concluded d4TTP might be discriminated against dTTP or in other words that the V75T substitution might be involved in the decreased binding of RT to d4TTP independently from ATP-mediated repair.

In this paper, we examine the effects of the V75T substitution in RT on d4T resistance. We show that V75T changes both nucleotide selectivity and repair of the d4TMP-terminated DNA chain by PPi but not by ATP.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmids, Genes, and Proviral DNA Constructions-- The wild-type RT gene construct p66RT7 has been described (24). This plasmid was further modified and used to construct the mutant RT gene at codon 75 (V75T) and the double mutant V75T/Y146F using synthetic oligonucleotides and a strategy described previously (24). The same RT gene mutated at codon 75 was used in conjunction with the recombinant HIV-1 proviral AD8 DNA (HIV-1AD8 (25)) to construct V75T HIV-1AD8. The latter recombinant infectious clone differs from wild-type AD8 DNA only at codon 75 (V75T). The construction of V75T HIV-1AD8 will be described elsewhere.2 All constructs were verified by restriction enzyme analysis and nucleotide sequencing.

Gene Expression and Protein Purification-- All recombinant RTs were expressed and purified as p66/p51 heterodimers. Briefly, p66 subunits carrying a His6 tag at the C terminus were coexpressed with the HIV-1 protease in Escherichia coli, and the resulting p66/p51 heterodimer was purified to homogeneity.2

Reagents-- DNA oligonucleotides were obtained from Life Technologies, Inc. Oligonucleotides were 5'-32P-labeled using T4 polynucleotide kinase (New England Biolabs, Beverly, MA). All 32P-labeled nucleotides were from Amersham Pharmacia Biotech. Adenosine 5'-triphosphate, 3'-deoxy- and 2',3'-dideoxynucleoside 5'-triphosphates (ddNTPs and dNTPs) were from Amersham Pharmacia Biotech. Both d4TTP and alpha -borano-(Rp)-2',3'-didehydro-2',3'-dideoxythymidine 5'-triphosphate (alpha -BH3-d4TTP) were synthesized and purified as described (11).

Reverse Transcriptase Assays-- Wild-type and V75T RT were titrated so as to determine the proportion of active enzyme using active-site titration as described (26) and the primer/template system described previously (11). Active-site titrations yielded higher values when the p66 subunit was coexpressed with the HIV-1 protease than when the p66/p51 heterodimer was obtained by mixing E. coli supernatants containing p66 and p51 subunits expressed separately.2 Thus, RT was prepared following the former protocol exclusively. The concentration of RT was determined using UV spectroscopy and the extinction coefficients used previously (26). For both wild-type and V75T RT, the value of active enzyme was 55% of the value determined using UV. All RT concentrations are expressed as active-site concentrations.

Standard RT activity was assayed using poly(rA)/oligo(dT)19 or poly(rC)/oligo(dG)19 and DE81 ion-exchange paper discs as described (24). To determine the KM for a dNTP, the primer/template was kept at a saturating concentration of 200 nM, and the concentrations of the corresponding dNTP were varied from 2 to 50 µM. The RT concentration was 10 nM. Filter 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.

Assay of RT DNA Polymerization Rate-- The rate of DNA polymerization was measured using a 5'-32P-labeled oligo(dT)21 primer annealed to poly(rA). Extension products were analyzed using a gel assay. The template average length was 612 nucleotides and was annealed to primer using a 1:1 molar ratio calculated using nucleotide concentration. The primer/template (25 nM, 15 µl) was incubated with RT (50 nM) in RT buffer (50 mM Tris-HCl, pH 8.0, 50 mM KCl, 0.05% Triton X-100) at 37 °C. The reaction was initiated by the addition of 15 µl of 500 µM dTTP in 6 mM MgCl2 at 37 °C, and quenched at various times by 80 µl of 0.3 M EDTA using a fast-quench apparatus (KinTek Austin, TX). The quenched reaction was diluted with 300 µl of gel loading buffer made of 90% formamide, 0.1% bromphenol blue, 0.1% xylene cyanol. Electrophoretic analysis was performed on a 14% denaturing polyacrylamide gel, and the gel was analyzed using an imaging plate and a FujiImager.

Nucleotide Analogue Inhibition at Steady State-- The inhibition of reverse transcription was measured as described previously (11, 24). Inhibition was expressed as the concentration of inhibitor producing a 50% inhibition (IC50) using the standard reverse transcription assay described above. Each aliquot was spotted in duplicate, and the values of IC50 are the average from at least two independent experiments.

Pre-steady State Kinetics of a Single Nucleotide Incorporation into DNA-- Pre-steady state kinetics were performed using d4TTP, alpha -BH3-d4TTP, and dTTP in conjunction with wild-type and V75T RT as described (11). The formation of product (P) over time was fitted with a burst Equation 1(26),


(<UP>P</UP>)=A · (1−<UP>exp</UP>(<UP>−</UP>k<SUB><UP>app</UP></SUB> · t))+k<SUB>SS</SUB>t (Eq. 1)
where A is the amplitude of the burst; kapp is the apparent kinetic constant of formation of the phosphodiester bond; and kSS is the kinetic constant of the steady state, linear phase. Although curve fitting was better with an equation containing two exponential terms (27, 28) than with Equation 1, either equation gave the same dependence of kapp on dNTP concentration, described by the following hyperbolic Equation 2,
k<SUB><UP>app</UP></SUB>=k<SUB><UP>pol</UP></SUB> · (<UP>dNTP</UP>)/(K<SUB>d</SUB>+(<UP>dNTP</UP>)) (Eq. 2)
where Kd and kpol are the equilibrium and the catalytic constant of the dNTP for RT, respectively. Curve fitting was performed using the Kaleidagraph software.

Pyrophosphorolysis and ATP-mediated Repair Assays-- The primer/template system used for both pyrophosphorolysis and ATP-mediated repair assays was a 5'-32P-enlabeled 21-mer DNA primer (5'-ATACTTTAACCATATGTATCC-3') annealed to a 35-mer (5'-GGTCCGTTGCATGCGGATACATATGGTTAAAGTAT-3') DNA template. The single template A specifying a single thymidine insertion site four bases away from the 3' end of the primer is shown in bold. DNA polymerization was initiated by the addition of wild-type or V75T RT (100 nM) and nucleotides (25 µM each of dATP, dCTP, and dGTP, and 5 µM analogue TP (d4TTP)) for 15 min at 37 °C in RT buffer. The repair reaction was started by adding dTTP to reach a final concentration of 25 µM in the presence of either PPi or ATP, as indicated. Aliquots were withdrawn during the time course of the reaction, and products were analyzed by denaturing gel electrophoresis (11). The percent repair of blocked primer is the ratio × 100 of extension products larger than 25 nucleotides over those larger than 24 nucleotides.

Cell Culture, Transfections, and Infections-- HeLa cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The C8166R5 cell line (a gift from M. Malim) stably transfected with CCR5 coreceptor was used to monitor the replication of the macrophage-tropic AD8 viral strain. Cells were maintained in RPMI supplemented with 10% fetal bovine serum in the presence of 1 µg/ml of puromycin to select for the CCR5 coreceptor expression. Peripheral blood mononuclear cells (PBMC) were obtained from a normal donor sample that was aliquoted and stored in liquid nitrogen. After thawing, PBMCs were stimulated with phytohemagglutinin at a final concentration of 2 mg/ml (Difco) and cultivated in RPMI supplemented with 15% fetal calf serum. Three days after stimulation, interleukin-2 (Chiron) was added in the medium at a final concentration of 0.01 µg/ml, and cells were allowed to grow for 3 days before being infected. To obtain wild-type and V75T viral stocks, HeLa cells were transiently transfected using the FuGENE 6 kit as recommended by the manufacturer (Roche Molecular Biochemicals). Cell-free supernatant containing viruses were titrated by assaying the fusogenic properties on C8166 cells (29) infected with serial 5-fold dilutions to determine the 50% tissue culture infective dose of viral stocks. Virus stocks from transfected cells were calibrated either to infect 1.5 × 106 primary cells at a multiplicity of infection (m.o.i.) of 0.02 and 0.002 or to infect 2 × 106 C8166R5 cells at an m.o.i. of 0.01, 0.0025, and 0.0005. Virus production was monitored by measuring twice a week RT activity in infected cell-free supernatant.

Inhibition of Viral Growth on Wild-Type and V75T HIV-1-infected Cells-- C8166R5 cells (1 × 106 cells) were pretreated for 24 h with different amounts of d4T or PFA before being infected. Cells were then infected for 3 h with wild-type or V75T viruses calibrated for similar m.o.i., washed twice in serum-free RPMI, and then incubated for 15 min with 15 µg of trypsin type XIII (Sigma) per ml at room temperature to remove viral particles bound at the membrane surface. Cells were then washed in serum-free RPMI before being grown in RPMI supplemented with 10% fetal calf serum in the presence of inhibitors. One day post-infection, cells were again washed in serum-free RPMI and then allowed to grow in the presence of inhibitors. Day two post-infection, cell-free supernatant was removed and monitored for RT activity. Media were then replaced by fresh inhibitor-containing media, and infected cells were then incubated for 2 additional days. On day 4 post-infection, RT activity was measured in the cell-free supernatant.

Three-dimensional Computer Modeling of RT-- The coordinates of RT in complex with DNA and nucleotide were obtained from the Brookhaven Protein Data Bank. Their Protein Data Bank accession numbers are indicated in the text and the legend of Fig. 1. The crystal structure models were displayed using INSIGHT II and the TURBO graphics program (30).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have investigated the role of the V75T substitution observed in HIV-1 RT. This substitution is associated with viruses isolated from patients under stavudine treatment. The low level of resistance conferred by V75T suggests that the changes in biochemical properties of stavudine 5'-triphosphate incorporation by V75T RT relative to wild-type RT might be subtle. To date, two types of mechanisms of nucleoside resistance observed using purified RT and the corresponding 5'-triphosphate analogue have been reported. In the first type, substitutions leading to drug resistance can affect binding of the triphosphate analogue by the resistant RT. In this case, a decrease in binding affinity of RT for the analogue can be measured, such as for the M184V substitution and 3TC triphosphate (31). In the second type, the analogue can be incorporated without being significantly discriminated against its natural counterpart but be repaired preferentially by the drug-resistant RT. This is the case for AZT resistance mutations, where the terminal AZT-MP of the chain-terminated primer is excised by either ATP or PPi (21, 22). We investigated these two possibilities for the V75T substitution.

Structural Analysis of the V75T Substitution-- Polymerases share a common fold to catalyze phosphodiester bond formation during synthesis of RNA or DNA. Their most salient feature is the presence of three domains called the thumb, fingers, and palm because they resemble a right hand. This right hand holds the double-stranded nucleic acid in the palm between fingers and thumb. The 3'-OH of the primer is positioned in the palm, but the template makes a sharp turn upon encountering the basis of the fingers and does not extend between fingers and thumb (32). Valine 75 is located at the basis of the fingers subdomain of RT, >6 Å away from the catalytic triad of the polymerase active site. Valine 75 is located where the single-stranded templates makes its sharp turn but does not contact directly the template (Fig. 1A). Instead, the Val-75 amino acid side chain is directed away from the solvent, toward the inner part of the fingers. A change from valine to threonine involves replacing either one of the methyl groups of the Val-75 side chain by a hydroxyl group. The neighboring side chains located less than 4 Å away from these two methyl groups (labeled 1 and 2 in Fig. 1B) are listed in Table I. We examined the potential loss and gain of interaction with these neighboring amino acid side chains (Fig. 1B and Table I). If an OH substitutes methyl group 1, four hydrophobic interactions involving Val-60 and Phe-77 are potentially lost with no compensating interactions brought by the presence of the OH (Table I). If an OH substitutes methyl group 2, one hydrophobic interaction is lost with Val-60, but two new hydrogen bonds are potentially created with Lys-73 and Tyr-146. The epsilon -NH2- of Lys-73 and the para OH-group of Tyr-146 are located 3.70 and 3.71 Å away from the modeled hydroxyl group of threonine 75, respectively. Given that the resolution of the crystal structure is 3.2 Å and the typical distance involved into a hydrogen bond is 2.7 Å, the possibility of such new hydrogen bonds can be neither confirmed nor excluded using structural models. Gln-151 is another amino acid found in the vicinity of Val-75. Although Gln-151 is located slightly more than 4 Å away from methyl group 2, Q151M is an important substitution found in multidrug resistance for which a biochemical characterization has been described (33). Gln-151 interacts directly with the 3'-group of incoming nucleotides, and the Q151M substitution might provide discrimination of nucleotide analogues relative to their natural counterparts (33). Thus, it is possible that minor conformational modifications induced by V75T had an indirect effect on Gln-151 that would increase discrimination against d4TTP relative to wild-type RT.



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Fig. 1.   Crystal structure of RT in complex with a double-stranded DNA primer/template and a nucleotide. A, location of valine 75. The atomic coordinates of Huang et al. (32) were used to visualize the complex using the program INSIGHT II. Valine 75 (red) and dNTP (pink) are shown using ball-and-stick and stick-only representations, respectively. RT (degraded color from N to C terminus) and primer/template (red) are shown using a ribbon representation. B, the environment of valine 75. Amino acid side chains located at less than 4 Å from either methyl group 1 (dashed lines) or 2 (solid lines) of the Val-75 side chain are represented.


                              
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Table I
Structural analysis of the Val-75 side chain environment

From this examination of the structure of RT, we formulate two hypotheses concerning the biochemical changes brought by the V75T substitution. First, V75T might indirectly alter the positioning of Gln-151 in such a way that d4TTP would be discriminated against dTTP during incorporation into DNA. This should be measurable using steady state or pre-steady state kinetic methods. The second hypothesis is that V75T might alter the interaction of the finger domain with the single-stranded template and have an effect on polymerization or repair of the terminal analogue, unrelated to nucleotide analogue selectivity. We tested these two hypotheses.

Steady State Kinetics of RT Carrying the V75T Substitution-- Wild-type and V75T RTs were compared for their RT activities using standard homopolymeric templates. KM and kcat were measured for dTTP and dGTP, and they are reported in Table II. No significant differences are found between the two enzymes. The ability of d4TTP to inhibit reverse transcription using poly(rA)/oligo(dT)19 as primer/template and dTTP as the nucleotide substrate was also measured (Table II). d4TTP is a potent inhibitor of reverse transcription in vitro; IC50 values in the vicinity of 150 nM were measured for both enzymes. Thus, no significant difference was found to explain convincingly V75T-mediated d4T resistance. As a control, PFA resistance was also evaluated. Wild-type RT has an IC50 of 0.8 µM, whereas V75T confers a 3-fold PFA resistance to RT (IC50 = 2.4 µM). We conclude that V75T does not confer d4TTP resistance when assayed using steady state kinetics but does confer a 3-fold resistance to PFA.


                              
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Table II
Steady-state kinetic constants and inhibitory constants of wild-type (WT) and V75T RT for primed homopolymeric RNA templates

Pre-steady State Kinetics of RT Carrying the V75T Substitution-- Steady state kinetics are often insufficient to describe kinetic properties such as substrate affinity or catalytic efficiency. In particular, the Michaelis constant Km(dNTP) does not faithfully reflect Kd(dTTP), the affinity of the dNTP for RT. Rather, KM reflects several constants such as Kd(dTTP), kcat, and the kinetic constant of the catalytic step kpol (12, 26). Therefore, DNA polymerization by wild-type and V75T RT was assayed using pre-steady state kinetics. We tested the possibility that either Kd(d4TTP) or kpol(d4TTP) was different between wild-type and V75T RT. When a single nucleotide is incorporated by RT into DNA, the rate-limiting step is the release of the enzyme from the primer/template after creation of the phosphodiester bond (26). Therefore, the ternary complex accumulates and adds one nucleotide into DNA much faster than it dissociates. Consequently, a burst of product is observed in the early times of the reaction, followed by a slower phase of product formation corresponding to the dissociation of the complex and cycling of RT on yet unextended primer/templates. The burst rate of product formation follows saturation kinetics and allows the measure of both the binding affinity constant Kd of the dNTP for RT and the kinetic constant of formation of the phosphodiester bond kpol (12, 26). This biphasic kinetic scheme was verified for d4TTP and allowed us to measure these constants. Substitution of the (Rp)-oxygen of a nucleotide with a borano group (BH3) has been shown to increase the catalytic constant kpol of nucleotide incorporation and presents enhanced inhibition properties of drug-resistant RTs (11). Thus, BH3-d4TTP was included in our pre-steady state kinetic analysis.

Fig. 2 presents a typical data set from which these constants are measured. The constant values of stavudine analogues are reported and compared with those of dTTP in Table III. Wild-type RT binds dTTP and d4TTP equally well, as judged by similar binding affinities in the vicinity of 20 µM for both analogues. Catalytic constants of 13.2 and 10.8 s-1 are measured for dTTP and d4TTP, respectively. These constants lead to incorporation efficiencies for dTTP and d4TTP of 0.75 and 0.51 s-1 µM-1, respectively. Thus, wild-type RT does not discriminate d4TTP. Unlike wild-type RT, V75T RT binds d4TTP (Kd(d4TTP)= 36.3 µM) with a 3-fold lower affinity than dTTP (Kd(dTTP)= 13.3 µM), whereas the catalytic constant for both nucleotides is almost unchanged around 10 s-1. These values were used to calculate an overall efficiency of incorporation of d4TTP (0.25 s-1 µM-1) that was 3.6-fold lower than that of dTTP (0.92 s-1 µM-1). Thus, d4TTP is discriminated against by V75T RT. When BH3-d4TTP is tested as a substrate for both enzymes, a 2-fold increase in kpol is found as expected (21.6 s-1). However, the presence of the BH3 also has an effect on the binding of d4TTP to RT, decreasing 2-fold the affinity of d4TTP for V75T RT (from 36.3 to 64.5 µM). Interestingly, the presence of V75T yielded a 3.4-fold increase in Kd when BH3-d4TTP is used as the substrate, from 18.7 to 64.5 µM. Thus, V75T is sensitive to a modification at the alpha -phosphate of the incoming nucleotide. The presence of a BH3 group does not provide a significant improvement at the incorporation step as it does with other RT however. It should be noted that the kinetic constant kpol might represent a rate-limiting step before creation of the phosphodiester bond rather than the true kinetic constant of the chemical step (26), but this was not investigated further. We conclude that V75T discriminates against d4TTP by binding it with a lower affinity than that observed for its natural counterpart dTTP.



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Fig. 2.   Pre-steady state kinetics of d4TMP incorporation into DNA using V75T RT. A, pre-steady state kinetics of a single d4TMP addition into DNA. A 5'-32P-labeled 21-mer primer was annealed to a 31-mer template (100 nM) specifying a single thymidine insertion site immediately adjacent to the 3' end of the primer, and RT (50 nM active sites) was allowed to bind. The reaction was initiated by the addition of 200 µM d4TTP, 6 mM MgCl2 in rapid quench apparatus and quenched at various times with 0.3 M EDTA as described under "Experimental Procedures." Products were analyzed by denaturing gel electrophoresis and quantitated using photo-stimulated plates and a FujiImager. Data were fit to the burst equation (Equation 1). B, complete data set of pre-steady state rates determined as described under "Experimental Procedures" using 2 (), 5 (), 10 (open circle ), 20 (×), 40 (+), 60 (black-square), 100 (Delta ), 150 (diamond ), and 200 µM (black-triangle) d4TMP, 6 mM MgCl2. Data were fitted to the burst equation (Eq. 1). C, determination of the equilibrium constant Kd for d4TTP using the first-order rates determined from B. Apparent polymerization rate constants determined from B were plotted against d4TTP concentration to determine KdM) and kpol (s-1) using hyperbolic fitting of the data (Equation 2).


                              
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Table III
Pre-steady state kinetic constants of wild-type and V75T RT for primed heteropolymeric DNA templates

ATP- and PPi-mediated Repair of the d4TMP-terminated Primer by RT Carrying the V75T Substitution-- TAMs involved in AZT resistance provide cross-resistance to d4T. Since TAMs have been involved in a mechanism of excision of the terminal AZTMP from the primer DNA (21), it was of interest to determine whether V75T belonged to such a class of substitution. Therefore, RT-mediated repair of d4TMP-terminated DNA by ATP was investigated. In this assay, a 5'-32P-labeled primer is annealed to a template containing a single dTMP (or d4TMP) insertion site. RT is allowed to bind, and the primer is extended by the addition of a mix of nucleotides in which dTTP had been replaced with d4TTP. The primer is extended by RT up to the point where d4TMP is incorporated into DNA and chain termination occurs. The repair reaction is then started by the addition of ATP, in the presence of dTTP. In the case of AZT, ATP unblocks the DNA chain with the concomitant release of AppppAZT (21). In the case of d4T, ATP should unblock the DNA chain with the concomitant release of Appppd4T. The 3'-OH group of the primer DNA is now free and can be extended by all four dNMPs present in the reaction up to a product corresponding to the length of the template DNA. To mimic conditions found under physiological conditions, millimolar concentrations of ATP are generally used (21). Fig. 3A shows the result of such an experiment when ATP (3.2 mM) is used as the unblocking agent. V75T RT exhibits a lower (~20%) ability to unblock the terminal d4TMP than wild-type RT. As a control, Fig. 3B shows the same experiment in which AZTTP is used instead of d4TTP. Unblocking of the AZTMP-terminated primer is a much faster process than unblocking of the d4TMP-terminated primer. As expected (Fig. 3B), an AZT-resistant RT bearing the D67N/K70R/T215F/K219Q substitutions shows an increased ability to unblock the AZTMP-terminated primer, about 2-fold under these experimental conditions (21). Therefore, we conclude that ATP is not involved in the d4T resistance mechanism and that V75T does not belong to the same class of resistance substitutions as the so-called TAMs.



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Fig. 3.   Repair of incorporated d4TMP or AZTMP by wild-type, V75T, and AZT-resistant RT. A, ATP-mediated repair of d4TMP using wild-type RT and V75T RT. A 5'-32P-labeled 21-primer was annealed to a 35-mer DNA template (50 nM) specifying a single thymidine insertion site (d4TMP arrow) 4 bases away from the 3' end of the primer, as described under "Experimental Procedures." Polymerization was initiated by addition of wild-type RT or V75T RT (100 nM) nucleotides (25 µM of each dATP, dCTP, dGTP, and 5 µM d4TTP) for 15 min at 37 °C. The repair reaction was initiated by the addition of 25 µM dTTP and 3.2 mM ATP. Aliquots were withdrawn during the time course of the reaction, and products were analyzed by denaturing gel electrophoresis. Polymerization products were visualized and quantitated using photo-stimulated plates and a FujiImager. B, ATP-mediated repair of AZTMP using wild-type RT and AZT-resistant RT. The experimental conditions and quantitation are the same as in A.

Another possibility is that PPi serves as the unblocking agent once DNA synthesis has been terminated by d4TMP. Pyrophosphorolysis is the reversal of the polymerization reaction in which PPi attacks the last 3'-phosphodiester bond in DNA to produce a DNA shortened by one nucleotide and a dNTP. In so doing, PPi and RT unblock the analogue-terminated DNA chain that can be extended again by canonical dNTPs. The role of PPi in AZT resistance-mediated by TAMS is unclear. AZT-resistant RT has been reported to exhibit the same (34), a higher (11, 22), or lower (21) pyrophosphorolysis activity than wild-type RT. During DNA synthesis, the incorporation of regular dNTPs is favored about 100-fold over pyrophosphorolysis (28), and the presence of potent pyrophosphatases maintaining an intracellular concentration of PPi around 150 µM impedes pyrophosphorolysis (35). However, when DNA polymerization is stopped by the incorporation of a chain terminator, PPi present in the medium is able to restore a functional 3'-OH and rescue the blocked DNA. It was thus of interest to see whether V75T had any effect on pyrophosphorolysis.

When PPi is used as the unblocking agent, pyrophosphorolysis occurs to a greater extent with V75T RT than with wild-type RT (Fig. 4A). After the repair reaction, a futile cycle occurs because either d4TTP or dTTP can be re-incorporated into DNA. In the case of V75T, our pre-steady state kinetic data indicate that dTMP has a 3.6-fold better chance of being incorporated than d4TMP. Therefore, the increased pyrophosphorolytic repair potentiates the slight discrimination against d4TTP at the RT active site. After repair, incorporation of dTMP is 3.6-fold more likely than another chain termination event mediated by the incorporation of d4TMP.



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Fig. 4.   Pyrophosphorolytic repair of incorporated d4TMP by wild-type and V75T RT. A, unblocking the d4TMP-terminated primer using PPi and wild-type RT or V75T RT. The experimental settings and quantitation are as described under Fig. 3 except that the unblocking reaction was performed using a PPi concentration of 2 mM. B, comparative pyrophosphorolytic repair of incorporated d4TMP by wild-type and V75T RT. The rates of pyrophosphorolytic repair were measured as described under "Experimental Procedures", and the ratio of V75T over wild-type RT rate is represented for several PPi concentrations. C, unblocking the d4TMP-terminated primer using 500 µM PPi and either wild-type, V75T, or V75T/Y146F RT. The experimental settings and quantitation are the same as those described in A for wild-type RT (), V75T RT (black-square), and V75T/Y146F RT (open circle ).

Because the 2 mM PPi is a high, unphysiological concentration, the effect of PPi concentration on the repair reaction was tested. As expected for both enzymes, the efficiency of repair of the terminal d4TMP varies with PPi concentration (data not shown). V75T RT exhibits a better ability to repair the d4TMP-terminated primer than wild-type RT at all PPi concentrations tested (Fig. 4B). The difference in efficiency between V75T RT and wild-type varies from 1.2-fold at 150 µM PPi to 3.7-fold at 2 mM PPi (Fig. 4, A and B). As a control, we tested the influence of the PPi concentration on the pyrophosphorolytic repair provided by the D67N/K70R/T215F/K219Q AZT-resistant RT. The increase of PPi-mediated repair by the latter RT relative to wild-type was not observed at any PPi concentration (data not shown).

Structure-based Design of Suppressive Variants-- We next investigated how the pyrophosphorolytic reaction could be enhanced by the V75T substitution. As shown above in our structural analysis, Val-75 does not contact the leaving PPi of the nucleotide. Rather, Val-75 is located in the fingers subdomain close to the single-stranded template contact point. Our structural study of V75T identified two putative additional contacts for the hydroxyl group of threonine 75, namely lysine 73 and tyrosine 146. Lys-73 belongs to the finger domain of RT in a region critical for nucleotide binding and/or catalysis. As yet, no mutation has been found among natural or drug-resistant variants at Lys-73. Several substitutions are possible at this position, but the corresponding RTs exhibit an impaired ability to complement polA E. coli mutants, indicating that they might have an altered polymerase activity (36). Therefore, this residue was not included in our mutagenesis analysis. Instead, we constructed the double variant V75T/Y146F to indicate the presence of this putatively novel hydrogen bond. This double variant was tested for its ability to repair the d4TMP-terminated primer by PPi as described in Fig. 4, A and B, and the results are described in Fig. 4C. The presence of the V75T/Y146F substitution decreases the pyrophosphorolytic activity toward d4TMP-terminated primers to a level that is intermediate between those of wild-type and V75T RT. This indicates that the hydroxyl group of Tyr-146 might well be involved in a hydrogen bond with the hydroxyl group of Thr-75 and that this additional contact might influence pyrophosphorolysis.

Polymerization Rate of RT Carrying the V75T Substitution-- PPi is a product of the polymerization reaction. Thus, the number of PPi molecules produced at the polymerase active site is directly related to the number of nucleotides incorporated into DNA. Although the PPi concentration is regulated in the cell, we reasoned that the micro-environment of the RT active site might experience fluctuations in PPi concentration depending on the whether or not RT is actively incorporating nucleotides into DNA. In that case, one can see a positive relationship between DNA polymerization rate and pyrophosphorolysis. Due to the location of V75T in the fingers subdomain, V75T would either change the angle where the single-stranded template leaves the active site or modify the mobility of the fingers, leading in either case to an increase of the DNA polymerization rate.

We examined the DNA polymerization rate of V75T RT and wild-type RT comparatively. In this assay, a 5'-32P-labeled oligo(dT)21 primer is annealed to a poly(rA) template, and RT is allowed to bind. The amount of active RT is measured using an active-site titration procedure so as to use a defined ratio of primer:active RT and allow precise comparison between different RTs. The polymerization reaction is started by the addition of a mix of dTTP and Mg2+ using a rapid mixing apparatus, quenched at various times, and analyzed using denaturing polyacrylamide gel electrophoresis and autoradiography. The average size of the extension product synthesized per s is a measure of the polymerization rate. The results are shown in Fig. 5. V75T exhibits a faster polymerization rate than wild-type RT both at the initiation and elongation step of DNA synthesis (lanes 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, and 17). This is mostly apparent after 10 s where the average product length in nucleotides is 15 and 75 for wild-type RT and V75T RT (lanes 10 and 11), respectively. This yields a 5-fold increase in polymerization rate by V75T relative to wild-type RT. This increase in polymerization rate was also apparent using bacteriophage M13 single-stranded DNA templates but to a lower extent (about 2-fold, data not shown).



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Fig. 5.   Comparative polymerization rate of wild-type, V75T, and V75T/Y146Y RT. Wild-type RT (WT), V75T RT (75), or V75T/Y146F RT (S) was bound to 5'-32P-end-labeled oligo(dT)21 primer annealed to poly(rA) template as described under "Experimental Procedures." At time 0, DNA synthesis was initiated by the addition of 500 µM dTTP, 6 mM MgCl2. After 1, 2, 4, 10, 20, or 30 s, the reaction was quenched by 80 µl of 0.3 M EDTA using a fast-quench apparatus. Electrophoretic analysis was performed on a 14% denaturing polyacrylamide gel, and products of the reaction were visualized using an image plate and a FujiImager.

The polymerization rate of V75T/Y146F is shown in Fig. 5 (lanes 3, 6, 9, 12, 15, and 18) in comparison with wild-type RT and V75T RT. The absence of hydroxyl group in Phe-146 suppresses the increase in polymerization rate brought by the V75T substitution. This suppression is observed at the initiation of polymerization, i.e. after the insertion of ~20 nucleotides, but V75T/Y146F RT exhibits a comparable rate of DNA polymerization than wild-type RT once >20 nucleotides are incorporated, a rate becoming faster than that of V75T eventually (lanes 14, 15, 17, and 18). We conclude that the hydroxyl group of threonine 75 interacts with the hydroxyl group of Tyr-146 and that this novel contact is responsible, in part, for the novel biochemical properties of V75T RT.

Growth of Recombinant Virus Carrying the V75T Substitution in the RT Gene-- V75T RT exhibits increases in both polymerization and pyrophosphorolysis rates. It was thus of interest to determine whether these novel properties would confer any advantage or disadvantage at the virus level. We used a recombinant virus carrying a mutant pol gene encoding the V75T substitution to compare its growth characteristics with that of wild-type virus having the same genomic sequence except for the V75T codon. Wild-type and V75T virus stocks were used to infect either primary activated PBMC or C8166R5 cell lines at different multiplicities of infection (Fig. 6). The relative replication of viruses was assayed by measuring the RT activity in samples of the culture supernatant. No significant difference is measured in viral replication kinetics in either cells (Fig. 6). Thus, the overall balance of effects brought by the V75T substitution is neutral and does not confer any obvious advantage nor disadvantage to HIV-1.



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Fig. 6.   Growth characteristics of recombinant wild-type and V75T HIV-1AD8 in various cell types. A, comparative growth of wild-type and V75T HIV-1AD8 using stimulated PBMC infected at two m.o.i. and followed during 3-4 weeks. Cell-free supernatant was used to measure RT activity as described under "Experimental Procedures." B, comparative growth as in A except that the host cell is C8166R5, a cell line expressing the CCR5 HIV-1 coreceptor.

Stavudine and PFA Resistance of Recombinant Virus Carrying the V75T Substitution in the RT Gene-- To determine how d4T resistance mechanisms described above correlate to viral resistance, wild-type and V75T recombinant viruses were then tested for drug resistance in infected cells. AZT was included as a control, and the results are presented in Table IV. When d4T resistance is tested 2 days post-infection, V75T HIV-1 (IC50 = 0.82 µM) is 3.7-fold resistant to d4T relative to wild-type HIV-1 (IC50 = 0.22 µM), consistent with results obtained by others (23). When PFA is used as the inhibitor, the V75T virus is also 3.9-fold resistant (IC50 = 4.3 µM versus 1.1 µM for wild-type) but is not AZT-resistant (IC50 <2 nM for both viruses). Similar results were obtained when drug resistance was tested 4 days post-infection. IC50 for d4T is slightly lower than when assayed 2 days post-infection, but a 3.6-fold resistance persists. A slight increase in IC50 is observed for PFA, from 1.1 to 5 µM for wild-type RT (5-fold), and from 4.3 to 10 µM for V75T RT (2-fold). It is interesting to note that for wild-type RT, the IC50 for d4T (0.22 and 0.1 µM at 2 and 4 days post-infection, respectively) correlates well with the IC50 in the vicinity of 150 nM obtained using purified RT.


                              
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Table IV
Inhibition of recombinant wild-type and V75T HIV-1 growth by d4T, PFA, and AZT at days 2 and 4 post-infection

Three conclusions can be drawn from these assays. First, V75T confers a low level of d4T resistance to HIV-1, and the 3- to 4-fold resistance levels found in our infected cell experiments are at the limit of significance in terms of drug resistance. Second, the PFA resistance of V75T is consistent with the involvement of PPi in the resistance mechanism. Third, unlike for AZT resistance, the low phenotypic d4T resistance observed using infected cells correlates well with the d4T resistance measured using purified RT.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Nucleoside analogues play a major role in antiretroviral therapy. The thymidine analogue AZT has been the first agent approved in the fight against HIV. The rapid emergence of viral AZT resistance has urged the development of novel antiviral molecules targeting RT as well as other viral proteins. d4T has been introduced in the clinic later than AZT. Two main advantages of d4T over AZT make it widely used to control the course of an HIV-1 infection; it is less toxic than AZT and does not elicit a high level of drug resistance after prolonged periods of treatment (15, 17). So far, V75T seems to be one of the few substitutions in RT typical of d4T resistance arising during d4T therapy. Phenotypic d4T resistance in HIV-1 infected cells is usually low and close to the limit of significance (3-4-fold resistance). The results presented here using both purified RT and a recombinant V75T virus give a molecular basis for the mechanism of the low level of stavudine resistance mediated by the V75T substitution in RT.

Our structural analysis showed that threonine 75 is at the cross-roads of two possible mechanisms potentially involved in drug resistance. One mechanism is direct and involves nucleotide selection through the discrimination of the analogue at the RT active site. The other mechanism is indirect and might involve either a modification of the interaction of the single-stranded template with the fingers of the drug-resistant RT or repair of the d4TMP-terminated primer. Our work shows that V75T mediates d4T resistance by both mechanisms. d4TTP bound with a 3-fold lower affinity to V75T RT than dTTP, whereas this difference was not found using wild-type RT. Thus, V75T has a direct effect on nucleotide selectivity. Since Thr-75 does not contact directly the ribose moiety of the nucleotide in the RT active site, this effect is probably mediated by another amino acid, such as Gln-151. The latter is an amino acid important in drug resistance which has been shown to play a direct role in nucleotide analogue discrimination (32, 33). Our results agree with and extend those of others (37, 38) who showed that wild-type RT used dTTP and d4TTP with equal efficiency. The presence of a BH3 group on the alpha -phosphate of d4TTP does not improve incorporation into DNA. Instead, nucleotide binding is affected, and we interpret that as an indication that the alpha -phosphate is involved in the process of resistance mediated by V75T. This interpretation is consistent with the observed increase in pyrophosphorolytic repair.

Although a 3-fold discrimination might be enough to account for the low level of d4T resistance observed in vivo and in infected cultured cells, we found that repair of the d4TMP-terminated primer is increased by the V75T substitution. In fact, the modest 3-fold increase in discrimination of d4TTP relative to dTTP is further potentiated by the increased repair of the d4TPMP-terminated primer. Surprisingly, this increased repair is not provided by ATP as it is when TAMs are involved (21). The location of V75T in the RT molecule is consistent with this observation. In the crystal structure of RT, V75T is an amino acid change located spatially apart from the classical TAM changes M41L/D67N/K70R/L210W/T215F/T215Y/K219Q. The implication of V75T in any aspect of ATP-mediated primer unblocking is therefore unlikely, a proposition in agreement with our results. Consistent with the latter, Lennerstrand et al. (23) found that the presence of ATP had no effect on d4T resistance using a mutant V75T virus assay. Instead, the enhanced repair is due to an increased pyrophosphorolytic repair of the d4TMP-terminated primer.

What is the exact mechanism for this enhanced pyrophosphorolytic repair? The concomitant increase in the nucleotide polymerization rate and the d4TMP pyrophosphorolysis rate by V75T RT might not be a coincidence. We propose a two-step mechanism. First, the additional bonds made by V75T with Tyr-146 and potentially Lys-73 could provide an increased "spring" force to the fingers in such a way that the global polymerization rate would increase. Our pre-steady state experiments place this rate enhancement after formation of the phosphodiester bond since no changes in kpol are observed upon incorporation of a single nucleotide. Thus, either PPi elimination or translocation along the single-stranded template (or both) are good candidates to be responsible for the enhanced rates. We observe that V75T polymerizes DNA about 5-fold faster than wild-type RT. Under processive DNA synthesis, a faster polymerization rate might translate into an increased local PPi concentration at the RT active site, which would in turn facilitate pyrophosphorolytic repair when d4TMP is incorporated. Our observation that repair efficiency increases with PPi concentration specifically when V75T is present is consistent with this model. Furthermore, our suppressor variant V75T/Y146F partially restores both wild-type polymerization and pyrophosphorolysis rates.

This mechanism might be relevant to other drug resistance mutations for which no molecular mechanisms have been demonstrated. We noted the presence in the vicinity of V75T of other amino acid substitutions that are involved in drug resistance. A62V, L74V, and V77L are found on multidrug-resistant RT, and they might belong to the same class of substitutions altering the spring force of the finger. They are either located in the beta 3/beta 4 finger subdomain itself or interacting with amino acids belonging to it. Their role in drug resistance is not known, but their location suggests that they may act by the same mechanism as that of V75T. Alternately, if A62V and F77L are not directly involved in pyrophosphorolysis, they could be compensatory mutations responsible for restoring polymerase "fitness" altered by the Q151M change.

Do the results obtained using purified RT in vitro correlate with inhibition studies using infectious clones in cultured cells? A 3- to 4-fold resistance is often considered as the threshold of significance in terms of drug resistance mainly due to the intrinsic reproducibility of the infected cell system. However, more subtle differences in biochemical properties can be detected with confidence using purified RT (20-22, 26, 31, 34, 38, 39). V75T confers to RT an increase in both DNA polymerization and pyrophosphorolysis rates. Whereas an increased polymerization rate could translate into a growth advantage, an increased pyrophosphorolytic rate could have a detrimental effect when RT is stalled, e.g. in a secondary structure of the genomic RNA. The overall balance seems to have no net effect on the virus, since its growth characteristics are indistinguishable from that of wild type. Our results obtained using recombinant viruses show that V75T confers low level resistance to both d4T and PFA consistent with values observed for purified RT in vitro. The PPi analogue PFA is expected to compete for the PPi-binding site or to interfere with PPi elimination. Either using purified RT or live viruses, a 2-3.9-fold PFA resistance is observed upon the presence of the V75T substitution. Thus, the agreement between these two types of PFA-inhibition experiments is excellent. In the case of d4T resistance, results obtained using purified RT- and HIV-1-infected cells indicate that d4T is a 10-100-fold less potent inhibitor than AZT. In addition, the intracellular concentration of d4TTP is at least 1 order of magnitude lower than that of AZTTP (8). The low concentration of intracellular d4TTP and its relatively poor inhibition properties relative to that of AZTTP leave to d4T a tenuous margin of action. Consistent with that, no more than a 10-fold d4T resistance has ever been observed. Perhaps the differences in AZT and d4T resistance mechanisms are related to their wide difference in potency.

To address the question of the comparative efficiencies of AZT and d4T as retroviral inhibitors, we propose a model. Due to the combined low d4TTP concentration and poor efficiency as a RT inhibitor, chain termination by d4TMP should be a rare event. This would leave time for the accumulation of PPi at the RT active site under processive DNA synthesis conditions. When AZT is used, however, chain termination by AZTMP should be more frequent and the PPi-mediated repair less favored than in the case of d4T because PPi cannot accumulate at the RT active site under nonprocessive DNA synthesis. In this context, concentration of ATP in the millimolar range might be critical for ATP-mediated repair of the AZTMP-terminated DNA chain. Thus, a mechanism involving a highly active repair of AZTMP-terminated DNA would lead to high levels of resistance and might explain why a >100-fold resistance can be observed for AZT. Complete viral genomes escaping AZTTP inhibition would have thus gone through many chain termination/repair cycles. In contrast, complete viral genomes escaping d4TTP inhibition would have gone through few DNA synthesis arrests and repairs, explaining why maximum resistance levels observed for d4T are in the vicinity of 10-fold.

Why does d4T exhibit a long lived therapeutic value without immediate selection of d4T resistance substitutions? The sustained potency of d4T over time might point to another cause than RT inhibition. Consistent with this, Hashimoto et al. (40) found that d4T induced apoptosis in HIV-1-infected cells specifically. Both d4T and HIV-1 would kill the host cell specifically, accounting for the prolonged beneficial effect of d4T. This secondary effect would diminish the selective d4T pressure on the virus and explain the low prevalence of V75T as a mutation involved in d4T resistance.

In conclusion, V75T confers novel properties to RT that are consistent with the low level of d4T resistance observed in vivo and using recombinant viruses. It is of interest to determine whether such properties can be found in other drug-resistant HIV-1 isolates for which no detailed resistance mechanisms are proposed.


    ACKNOWLEDGEMENTS

We thank Luis Menéndes-Arias, Dominique Deville-Bonne, and Hélène Dutartre for critical reading of the manuscript and Marie-Pierre Egloff for help with figures.


    FOOTNOTES

* This work was supported in part by the Fond pour la Recherche Médicale (Sidaction) and Ensemble Contre 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.

|| To whom correspondence addressed. Tel.: 33 491 82 86 44; Fax: 33 491 82 86 46; E-mail: bruno@esil.univ-mrs.fr.

Published, JBC Papers in Press, December 27, 2000, DOI 10.1074/jbc.M009837200

2 J. Boretto, S. Longhi, J. M. Navarro, B. Selmi, J. Sire, and B. Canard, manuscript in preparation.


    ABBREVIATIONS

The abbreviations used are: HIV, human immunodeficiency virus; RT, reverse transcriptase; AZT, 3'-azido-3'-deoxythymidine; d4T, 2',3'-didehydro-2',3'-dideoxythymidine; AZTMP, 3'-azido-3'-deoxythymidine 5'-monophosphate; d4TMP, 2',3'-didehydro-2',3'-dideoxythymidine 5'-monophosphate; AZTTP, 3'-azido-3'-deoxythymidine 5'-triphosphate; d4TTP, 2',3'-didehydro-2',3'-dideoxythymidine 5'-triphosphate; dNTP, 2'-deoxynucleoside 5'-triphosphate; ddNTP, 2',3'-dideoxynucleoside 5'-triphosphate; TAM, thymidine-associated mutation; PFA, phosphonoformic acid; m.o.i., multiplicity of infection; BH3, borano group; PBMC, peripheral blood mononuclear cells; AZTDP, 3'-azido-3'-deoxythymidine 5'-diphosphate; d4TDP, 2',3'-didehydro-2',3'-dideoxythymidine 5'-diphosphate.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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