From the 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
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.
Chemicals--
NVP and d4T were purchased from Roche
Molecular Biochemicals and Bristol-Myers Squibb, respectively.
[3H]dTTP (40 Ci/mmol), [ 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 5 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 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.
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.
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.
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
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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
-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.
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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
Inhibition by nucleoside analogs of RNA-dependent DNA
synthesis catalysed by HIV-1 RT wt and mutants
View larger version (30K):
[in a new window]
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).
Kinetic parameters of deoxy- and dideoxynucleoside triphosphate analogs
incorporation catalyzed by wild type and mutated HIV-1 RT
Relative incorporation efficiencies of dideoxynucleoside triphosphates
analogs by wild type and mutated HIV-1 RT
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).
![]() |
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Loveday, C. (2001) J. Acquir. Immune Defic. Syndr. 26 Suppl. 1, S10-S24[Medline] [Order article via Infotrieve] |
2. | Maga, G., Amacker, M., Ruel, N., Hübscher, U., and Spadari, S. (1997) J. Mol. Biol. 274, 738-747[CrossRef][Medline] [Order article via Infotrieve] |
3. | Maga, G., and Spadari, S. (2002) Curr. Drug Metab. 3, 73-95[Medline] [Order article via Infotrieve] |
4. | Sluis-Cremer, N., Arion, D., and Parniak, M. A. (2000) Cell. Mol. Life Sci. 57, 1408-1422[Medline] [Order article via Infotrieve] |
5. | Iversen, A. K. N., Shafer, R. W., Wehrly, K., Winters, M. A., Mullins, J. I., Chesebro, B., and Merigan, T. C. (1996) J. Virol. 70, 1086-1090[Abstract] |
6. |
Larder, B. A.,
Bloor, S.,
Kemp, S. D.,
Hertogs, K.,
Desmet, R. L.,
Miller, V.,
Sturmer, M.,
Staszewski, S.,
Ren, J.,
Stammers, D. K.,
Stuart, D. I.,
and Pauwels, R.
(1999)
Antimicrob. Agents Chemother.
43,
1961-1967 |
7. | Mouroux, M., Descamps, D., Izopet, J., Yvon, A., Delaguerre, C., Matheron, S., Coutellier, A., Valantin, M. A., Bonmarchand, M., Agut, H., Massip, P., Costagliola, D., Katlama, C., Brun-Vezinet, F., and Calvez, V. (2001) Antivir. Ther. 6, 179-183[Medline] [Order article via Infotrieve] |
8. | Ross, L., Henry, K., Paar, D., Salvato, P., Shaefer, M., Fisher, R., Liao, Q., and St Clair, Q. (2001) J. Hum. Virol. 4, 217-222[Medline] [Order article via Infotrieve] |
9. | Ross, L., Scarsella, A., Raffanti, S., Henry, K., Becker, S., Fisher, R., Liao, Q., Hirani, A., Graham, N., St Clair, M., and Hernandez, J. (2001) AIDS Res. Hum. Retroviruses 17, 1107-1115[CrossRef][Medline] [Order article via Infotrieve], (NZT40012 Study Team) |
10. | Shirasaka, T., Kavlick, M. F., Ueno, T., Gao, W.-Y., Kojima, E., Alcaide, M. L., Chokekijchai, S., Roy, B. M., Arnold, E., Yarchoan, R., and Mitsuya, H. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2398-2402[Abstract] |
11. | Dyatkina, N. B., von Janta-Lipinski, M., Minasyan, S. K., Kukhanova, M. K., Krayevsky, A. A., Chidgeavadze, Z. G., and Beabealashvilli, R. S. (1987) Bioorg. Khim. (Russian) 13, 1366-1374 |