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Nevirapine Resistance Mutation at Codon 181 of the HIV-1 Reverse Transcriptase Confers Stavudine Resistance by Increasing Nucleotide Substrate Discrimination and Phosphorolytic Activity*

Giuseppina BlancaDagger , Fausto Baldanti§, Stefania Paolucci§, Alexander Yu Skoblov, Lyubov Victorova, Ulrich Hübscher||, Giuseppe Gerna§, Silvio SpadariDagger , and Giovanni MagaDagger **

From the Dagger  Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche, via Abbiategrasso 207, I-27100 Pavia, Italy, the § Servizio di Virologia, Istituto di Ricovero e Cura a Carattere Scientifico, Policlinico S. Matteo, Piazzale Golgi 2, 27100 Pavia, Italy, the  Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov Street 32, 117984 Moscow, Russia, and the || Institute of Veterinary Biochemistry and Molecular Biology, University of Zürich-Irchel, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland

Received for publication, January 17, 2003, and in revised form, January 29, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

Recombinant HIV-1 reverse transcriptase (RT) carrying non-nucleoside inhibitors (NNRTIs) resistance mutation at codon 181 showed reduced incorporation and high efficiency of phosphorolytic removal of stavudine, a nucleoside RT inhibitor. These results reveal a new mechanism for cross-resistance between different classes of HIV-1 RT inhibitors.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

Anti-human immunodeficiency virus type 1 (HIV-1)1 highly active therapy, based on the combined use of nucleoside (NRTIs) and non-nucleoside (NNRTIs) reverse transcriptase (RT) inhibitors, often select for multidrug resistance (1-3). To date, three molecular mechanisms are known for NRTIs resistance: (i) selective alterations in inhibitor binding and/or incorporation (discrimination), (ii) template/primer repositioning, which influences NRTIs incorporation, and (iii) phosphorolytic removal of an incorporated chain-terminating NRTI from the 3'-end of the nascent DNA strand (4). In particular, the mechanisms of resistance to the NRTI stavudine (d4T) are still elusive. High levels of d4T resistance appear concomitantly with resistance to zacitalbine (ddC) and zidovudine (AZT) and seem to correlate with the acquisition of complex patterns of mutations, suggesting that it would be difficult for a single mutation in the HIV-1 RT gene to decrease the incorporation efficiency for d4T (5-10). In the present study we show, for the first time, that a NNRTI resistance mutation at codon 181 selected by nevirapine (NVP) or other NNRTIs can contribute to a decreased sensitivity to d4T inhibition, by two indepedent resistance mechanisms: increased nucleotide selectivity and specific phosphorolytic removal.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

Chemicals-- NVP and d4T were purchased from Roche Molecular Biochemicals and Bristol-Myers Squibb, respectively. [3H]dTTP (40 Ci/mmol), [gamma -32P]ATP (3000 Ci/mmol), and AZT triphosphate were from Amersham Biosciencies. Poly(rA), oligo(dT), the 66- and 24-mer oligodeoxynucleotides and unlabeled dNTPs were from Roche Molecular Biochemicals. GF/C filters were supplied by Whatman. d4T triphosphate was synthesized in the laboratory of the authors as reported previously (11). All other reagents were of analytical grade and purchased from Merck or Fluka.

Expression and Purification of Recombinant Wild Type and Mutated (Y181I, Y188L, and K103N) HIV-1 RT Enzymes-- The coexpression vectors pUC12N/p66(His)/p51 with the wild type or the mutant (Y181I, Y188L, and K103N) forms of HIV-1 RT p66 were kindly provided by Dr. S. H. Hughes (NCI-Frederick Cancer Research and Development Center) and purified as follows (2). The Escherichia coli strain JM109(DE3) harboring the corresponding expression plasmids was grown at 37 °C in 1 liter Luria Broth to an A600 of 0.6. Isopropylthiogalactoside was added to a final concentration of 1 mM, and growth was continued for 4 h. Cells were harvested by centrifugation, resuspended in 10 ml of buffer A (40 mM Tris (pH 8.0), 0.5 M NaCl, 1 mM PMSF), and subjected to a French press. The mixture was cleared by centrifugation at 30,000 × g for 30 min. The supernatant was loaded on a 1-ml HiTrap Chelating column (Amersham Biosciences) complexed with Co2+ ions and equilibrated in buffer A. The enzyme was eluted from the column with a 0-0.5 M imidazole HCl gradient in buffer A. Pooled fractions were dialyzed against buffer B (50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM 2-mercaptoethanol, 0.4 mM PMSF, 10% (v/v) glycerol) and loaded onto a Mono S column (Amersham Biosciences) equilibrated in buffer B. The HIV-1 RT proteins were eluted from the column with a 50-500 mM NaCl gradient in buffer B. Pooled fractions were dialyzed against 50 mM Tris-HCl (pH 7.5), 20% (v/v) glycerol, 1 mM EDTA, 1 mM dithiothreitol, 0.4 mM PMSF. All enzymes were purified to >95% purity and had a specific activity on poly(rA)/oligo(dT) of: HIV-1 p66(His)/p51, 75,670 units/mg; p66(K103N)/p51, 96,415 units/mg; p66(Y181I)/p51, 65,770 units/mg; p66(Y188L)/p51, 79,050 units/mg. 1 unit of DNA polymerase activity corresponds to the incorporation of 1 nmol of dNMP into acid-precipitable material in 60 min at 37 °C. All enzymes were purified to >95% purity, as judged by silver-stained SDS-polyacrylamide gels.

Evaluation of HIV-1 RT DNA Polymerase Activity-- RNA- and DNA-dependent DNA polymerase activities were assayed as described in the presence of 0.5 µg of poly(rA)/oligo(dT)10:1 (0.3 µM 3'-OH ends), 10 µM [3H]dTTP, and 2-4 nM RT. When the 5'-32P-labeled d24:d66-mer template was used, a volume of 10 µl contained 0.05 µM (3'-OH ends) of the DNA template and nucleotides as indicated in the figure legends. After incubation at 37 °C, the products of the reaction were separated on a 7 M urea/20% polyacrylamide sequencing gel. Quantification of the products was performed by scanning the gel with a Amersham Biosciences PhosphorImager and integrating with the program ImageQuant.

Steady-state Kinetic Measurements-- Time-dependent incorporation of radioactive nucleotides into the different template-primers at different nucleotide substrate concentrations was monitored by removing 25-µl aliquots at 2-min time intervals. Initial velocities of the reaction were then plotted against the corresponding substrate concentrations. When the 5'-32P-labeled d24:d66-mer template was used, initial velocities after 10 min of incubation at 37 °C in the presence of different substrate concentrations were calculated from the integrated gel band intensities (see also below). For determination of the Km and kcat values, an interval of substrate concentrations from 0.2 Km to 10 Km was used. For Ki determination, an interval of inhibitor concentrations between 0.2 Ki and Ki was used in the inhibition assays.

Kinetic Parameters Calculation-- All values were calculated by non-linear least squares computer fitting of the experimental data to the appropriate rate equations. Km, Vmax, and kcat values were determined according to the Michaelis-Menten equation. Ki values were calculated according to the equation for competitive inhibition. The values of integrated gel band intensities in dependence of the nucleotide substrate concentrations were fitted to the equation: I*T/IT-1 Vmax[dNTP]/(Km + [dNTP]), where T = target site, the template position of interest and I*T = the sum of the integrated intensities at positions T, T+1 ... T+n.

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

Recombinant HIV-1 RT Carrying Mutated Codon 181 Shows Reduced Sensitivity to d4TTP-- The sensitivity of recombinant HIV-1 RTs carrying NNRTIs resistance-associated mutations to the inhibition by the dideoxynucleoside triphosphate analogs ddTTP, ddCTP (the active form of zalcitabine), AZTTP (the active form of zidovudine), and d4TTP (the active form of stavudine) was tested in comparison with the wild type enzyme. As can be seen from the results summarized in Table I, only the mutation at codon 181 significantly reduced the affinity of the enzyme for the dTTP substrate, with an increase of >2.5-fold in the measured Km values with respect to wild type RT. The same mutation was also associated with an increase in the Ki values for ddCTP, ddTTP, and d4TTP, with respect to wild type RT. In contrast, no significant differences were found for AZTTP inhibition.


                              
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Table I
Inhibition by nucleoside analogs of RNA-dependent DNA synthesis catalysed by HIV-1 RT wt and mutants

Mutation at Codon 181 Specifically Reduces the Incorporation Efficiency of Deoxy- and Dideoxynucleoside Triphosphate Analogs by HIV-1 RT-- Recombinant wild type and mutant RTs were analyzed for their ability to incorporate dTTP, dCTP, ddCTP, AZTTP, and d4TTP on a heteropolymeric DNA substrate, corresponding to nucleotides 1006-1071 (codons 169-190 of the RT coding sequence) of the HIV-1 pol gene (HXB2 isolate). Fig. 1A shows the incorporation of d4TTP by HIV-1 wild type RT and for the 181 mutant. Quantification of the gel bands intensities clearly showed a reduced efficiency of the 181 mutant RT in utilizing d4TTP as the substrate with respect to wild type RT (Fig. 1B). As summarized in Table II, mutations at codons 103 and 188 did not alter the incorporation efficiencies (kcat/Km values) for AZTTP, d4TTP, and dTTP with respect to wild type RT. In contrast, the mutation at codon 181 decreased the incorporation efficiency for dTTP by 3-fold and for d4TTP by about 15-fold. On the other hand, the 181-mutated RT was able to incorporate AZTTP with efficiency similar to wild type RT.


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Fig. 1.   HIV-1 RT with mutation at codon 181 is less susceptible to d4T. A, sequencing gel analysis shows a reduced accumulation of the products at position +2 (arrow) of the standard substrate (d24:d66-mer, see "Materials and Methods"), due to d4TTP incorporation, for the mutant Y181I (lanes 2-7) in comparison with the wild type enzyme (lanes 8-13). B, quantification of the data from the experiment in A shows a reduced affinity for d4TTP for the mutant (squares) compared with wild type enzyme (circles). C, effects of increasing concentrations of ATP on the inhibition potency (Ki) values for d4TTP for wild type (squares) or Y181I HIV-1 RT (circles) on the RNA-dependent DNA synthesis activity (see "Material and Methods"). The ATP-dependent increase of the Ki values was plotted against the ATP concentrations. From the curves shown, a maximum decrease in inhibition potency (indicated by the increase of the Ki values) of 11 (± 1)-fold and 2 (±0.5)-fold was calculated for Y181I and wild type HIV-1 RT, respectively. D, sequencing gel analysis shows a more efficient ATP-dependent phosphorolytic removal of the incorporated d4TMP, reflected by the disappearance of the products at position +2 (arrow) of the d24:d66-mer template-primer by HIV-1 RT Y181I (lanes 7-12) with respect to the wild type enzyme (lanes 1-6).


                              
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Table II
Kinetic parameters of deoxy- and dideoxynucleoside triphosphate analogs incorporation catalyzed by wild type and mutated HIV-1 RT

Mutation at Codon 181 Increases the Ability of RT to Discriminate between dTTP and d4TTP-- As summarized in Table III, comparison of the incorporation efficiencies kcat/Km for the natural substrates dTTP or dCTP and the cognate dideoxy analogs AZTTP, d4TTP, or ddCTP indicated that the dideoxy analogs were preferred as substrates to the corresponding deoxynucleotides. However, compared with wild type enzyme, discrimination between d4TTP and dTTP and between ddCTP and dCTP was 5-fold and 2-fold, respectively, more efficient with the 181-mutated RT. None of the other NNRTIs-associated mutations significantly affected the specificity of the RT for the nucleotide analogs studied, with the exception of the 188 mutation, which induced a 2.5-fold higher preference for d4TTP incorporation over dTTP than the wild type enzyme.


                              
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Table III
Relative incorporation efficiencies of dideoxynucleoside triphosphates analogs by wild type and mutated HIV-1 RT

The 181 Mutant RT Is More Efficient in Removing d4TMP-terminated Residues than the Wild Type Enzyme-- One important mechanism of NRTIs resistance by HIV-1 RT resides in its ability to unblock a dideoxy-terminated primer by removing the last incorporated nucleotide through a PPi- or ATP-dependent phosphorolytic reaction. In a first series of experiments, the ability of ATP or PPi to reduce the inhibition of either wild type RT or 181-mutated RT by AZTTP and d4TTP was evaluated. The reduction observed with AZTTP and d4TTP was comparable for both enzymes in the case of the PPi-dependent pathway (data not shown). On the other hand, in the ATP-dependent pathway, the inhibition by d4TTP of the 181-mutated RT was reduced more than 10-fold than in the case of wild type enzyme (Fig. 1C). In a second series of experiments, phosphorolytic removal by RT in the presence of ATP of the last incorporated nucleotide from primers terminated by either d4TMP or AZTMP (resulting in a shorter product) was directly visualized by a polyacrylamide gel. As shown in Fig. 1D, the rate of unblocking was higher for the 181-mutated RT (0.15 (±0.01) s-1) than for the wild type enzyme (0.025 (±0.01) s-1) for d4TMP. No significant differences were found for AZTMP (data not shown).

The mutations Y181I/C are involved in high level NNRTIs resistance, especially to nevirapine and delavirdine (1, 2). The results presented in this study show, for the first time, a role of a NNRTIs resistance mutation in contributing to d4T resistance by two different mechanisms: increased nucleotide selection and phosphorolytic removal (Table III and Fig. 1). In addition, we recently showed that d4T could inhibit the replication of a recombinant HIV-1 strain carrying the Y181C substitution, 5-10-fold less efficiently with respect to wt and K103N mutant recombinant HIV-1 strains, thus confirming the impact of mutations at codon 181 on d4T resistance.2 These effects are specific for d4T (since AZT sensitivity was not significantly affected) and for the 181 mutation (since other common NNRTIs resistance mutations such as K103N or Y188L did not show any effect). The specificity observed for the Y181I mutation can be related to the fact that the changed amino acid is adiacent to the conserved catalytic residues Asp185 and Asp186. It is reasonable to hypothesize that this substitution alters the geometry of the active site of HIV-1 RT, resulting in a less favorable interaction with the nucleotide substrate. The fact that only d4T, but not AZT, was affected might be related to the differences in the structures of these two thymidine analogs. In particular, the azido group at the 3'-position of the sugar ring of AZT is known to stabilize binding of the drug to the enzyme. A major difference between AZT and d4T is that the latter drug bears a carbocyclic pentose ring, lacking any substituent at both the 2'- and 3'-positions, thus d4T might be in principle more sensitive to alterations in the nucleotide binding pocket. These findings can have immediate clinical implications; caution should be applied when considering d4T for rescue treatments following NNRTI-containing regimen failure, even in patients not previously exposed to the drug.

    FOOTNOTES

* This work was supported in part by Ministero della Salute, Istituto Superiore di Sanità, Programma Nazionale AIDS (Grants 30D.36 (to G. G.) and 30D.72 (to S. S.)); Ricerca Finalizzata 2001 (Grant 126 (to G. G. and G. M.)) and Ricerca Corrente (Grant 80207 (to F. B.)); by the Kanton of Zurich (to U. H.); and by a grant from the Russian Federation Ministry of Industry, Sciences and Technology (Project 31 (to L. V. and A. Y. S.)).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 should be addressed: Istituto di Genetica Molecolare IGM-CNR, via Abbiategrasso 207, I-27100 Pavia, Italy. Tel.: 39-0382546355; Fax: 39-0382422286; E-mail: maga@igm.cnr.it.

Published, JBC Papers in Press, February 24, 2003, DOI 10.1074/jbc.C300022200

2 Baldanti, F., Paolucci, S., Maga, G., Labò, N., Hübscher, U., Skoblov, A. Y., Victorova, L., Spadari, S., Minoli, L., and Gerna, G. (2003) AIDS, in press.

    ABBREVIATIONS

The abbreviations used are: HIV-1, human immunodeficiency virus type 1; RT, reverse transcriptase; NRTI, nucleoside RT inhibitor; NNRTI, non-nucleoside RT inhibitor; ddC, zacitalbine; AZT, zidovudine; d4T, stavudine; NVP, nevirapine; PMSF, phenylmethylsulfonyl fluoride.

    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

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