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Journal of Virology, August 2001, p. 7202-7205, Vol. 75, No. 15
Virco UK, Ltd., Cambridge CB4
0GA,1 and Structural Biology Division,
The Wellcome Trust Centre for Human Genetics, University of Oxford,
OX3 7BN Oxford,3 United Kingdom, and
Virco N.V., B-2800 Mechelen, Belgium2
Received 14 September 2000/Accepted 28 April 2001
Using a large panel of human immunodeficiency virus type 1 site-directed mutants, we have observed a higher correlation than has
previously been demonstrated between zidovudine (AZT)-triphosphate resistance data at the reverse transcriptase (RT) level and
corresponding viral AZT resistance. This enhanced-resistance effect at
the RT level was seen with ATP and to a lesser extent with
PPi when ATP was added at physiological concentrations. The
ATP-dependent mechanism (analogous to pyrophosphorolysis) appears to be
dominant in the mutants bearing the D67N and K70R or 69 insertion
mutations, whereas the Q151M mutation seems independent of ATP for
decreased binding to AZT-triphosphate.
Resistance to zidovudine (AZT)
commonly develops during human immunodeficiency virus type 1 (HIV-1)
therapy, because there is rapid virus turnover and the HIV-1
reverse transcriptase (RT) is an error-prone polymerase (3,
13, 15). The selection of resistant virus is seen for all HIV-1
inhibitors currently in clinical use (25), and in
addition, multidrug resistance has been documented during combination
therapy (14, 21). Analysis of AZT-resistant HIV-1
strains from patients has resulted in the identification of
several mutations in the RT coding sequence (6, 9, 15).
For example, changes at codons 41, 67, 70, 210, 215, and 219 result in
specific resistance to AZT (6, 9, 15). These mutations can
cause the virus to become highly resistant to AZT (in some
combinations, they produce a >100-fold increase in the 50% inhibitory
concentration [IC50]). However, the biochemical
mechanism of resistance conferred by these mutations has remained a
puzzle for a long period. This is because only minor or no differences
were seen by comparing the kinetic properties of mutant enzymes with
those of the wild-type enzyme in the presence of different
AZT-triphosphate (AZT-TP) concentrations (4, 10, 11, 17).
However, a different set of RT mutations, i.e., A62V, V75I, F77L,
F116Y, and Q151M, which confer resistance to multiple nucleoside
analogs (where Q151M is the key mutation), did show decreased
binding of AZT-TP that correlated with virus data (24).
Recently, novel biochemical mechanisms for AZT resistance have been
proposed. Arion et al. (1) showed that removal of
AZT-monophosphate (AZT-MP) from blocked primers was enhanced for an
AZT-resistant mutant compared to what occurred with wild-type RT
and that this was due to pyrophosphate
(PPi)-dependent pyrophosphorolysis, the reverse
reaction of DNA synthesis. Likewise, Meyer et al.
(19) have demonstrated a ribonucleotide ATP- or
GTP-dependent primer-unblocking reaction that produces dinucleoside
polyphosphate. Both of these studies were performed with HIV-1 RT
having mutations at codons D67N, K70R, T215F, and K219Q. An
increase in AZT-TP resistance of up to fivefold was demonstrated
with this mutant using physiological concentrations of either
PPi (1) or ATP (19).
Thus, it is likely that a nucleoside triphosphate (NTP),
PPi, or both are involved in the AZT resistance mechanism.
In the present study, an RT assay was established to enable
discrimination of AZT-TP resistance when ATP, GTP, or
PPi was added in physiological concentrations.
The aim was also to identify the particular mutations involved, by
comparing the level of binding of AZT-TP to those of a variety of
mutant RTs containing combinations of different mutations. The choice
of mutations was based on those seen in patient samples treated with
AZT alone or together with other nucleoside analogs (14).
Further, mutations were chosen to study the potential involvement of
the residues in the RT finger domain as well as those causing
resistance to multiple nucleoside drugs.
We performed site-directed mutagenesis with single- or
multiple-nucleotide changes being introduced into the RT coding region of a wild-type HXB2-D EcoRI-NdeI restriction
enzyme fragment cloned into the expression vector pKK233-2
(Pharmacia Biotech) (16). Mutations were generated
using commercial site-directed mutagenesis kits: ExSite and
QuikChange (Stratagene). The mutated cloned expression vectors
were transformed into Escherichia coli strain XL1-Blue, and
the genotypes were verified by DNA sequence analysis
(12). Using the HIV-1 drug susceptibility assay, the
phenotypic susceptibilities of these viable recombinant viruses to AZT
was determined as described previously (7). Briefly,
the proviral molecular clone pHIV?RTBstEII (8) was cotransfected with the RT-PCR product into
MT-4 cells (5, 7). The fold level of resistance was
calculated by dividing the mean IC50 for a
recombinant virus from a mutant clone by the mean
IC50 for the recombinant wild-type virus (HXB2).
Expression and purification of RT have been described previously
(22). Briefly, RT was expressed in E. coli
XL1-Blue grown with 50 mg of kanamycin per liter and ampicillin in 2×
TY medium (1.6% Bacto Tryptone, 1% yeast extract, 0.5% NaCl) for
18 h at 36°C and in presence of 100 µg of
isopropyl- First we studied the kinetics of AZT-TP inhibition with wild-type RT
toward one mutant RT (Table 1). The
degree of resistance shown by mutant 41/67/70/215 (bearing the
mutations M41L, D67N, K70R, and T215Y) is expressed as an increase
in the Ki/Km
value compared to the corresponding wild-type ratio. In the absence of
added ATP or PPi, mutant 41/67/70/215's
Ki/Km ratio
was 0.6 when divided by the ratio derived for wild-type RT (Table 1). This apparent lack of biochemical resistance is consistent with the
results of a previous study of AZT-resistant mutants using conventional
RT assays (11). However, the inclusion of physiological concentrations of ATP (3.2 mM) dramatically increased this resistance 10.3-fold. This degree of resistance was within a factor of 2 of the
24-fold resistance seen with the HIV drug susceptibility assay (Table
2). We also tested the effect of
PPi (150 µM) on AZT-TP inhibition. The level of
resistance of mutant 41/67/70/215 relative to that of the wild type was
4.4-fold (Table 1). A control experiment which ensured that the
previous effect seen by ATP was not influenced by
PPi contamination was performed. This was done by
pretreating the ATP reagent with thermostable pyrophosphatase (Boehringer Mannheim) (data not shown). The ATP effect was, however, dependent on the concentration used (see results with 1.5 and 6 mM ATP
in Table 1). The highest resistance level of mutant 41/67/70/215 was
found with 3.2 mM ATP. When ATP (3.2 mM) combined with
PPi (150 µM) was studied, both the wild type
and mutant 41/67/70/215 showed an increase in
Ki and Km
values, but the level of resistance of mutant 41/67/70/215 (6.0-fold)
was lower than the level with 3.2 mM ATP alone (10.3-fold). However,
ATP per se was slightly inhibiting, as seen by decreased relative
Vmax values in Table 1. Also, in a
separate experiment with this assay system, ATP seemed to behave as a
noncompetitive inhibitor (data not shown).
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.15.7202-7205.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Correlation between Viral Resistance to Zidovudine and Resistance
at the Reverse Transcriptase Level for a Panel of Human
Immunodeficiency Virus Type 1 Mutants
ABSTRACT
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-D-thiogalactopyranoside (IPTG) per
liter for 6 h. After sonication of extracts containing the enzyme,
purification was performed by ion-exchange chromatography in several
steps using both 50 HQ and 50 HS columns (PerSeptive Biosystems). The final RT products, in a heterodimeric form
(p66-p51), had a purity of >90% by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (data not shown). The
purified RTs were assayed for inhibition of AZT-TP using separate kit
components from the Lenti RT assay (Cavidi Tech, Uppsala,
Sweden). The principle, performance, and composition of this
nonradioactive microtiter plate RT assay have been described previously
(4, 20). The assay components used in this study for the
RT activity step were (i) RT reaction buffer [HEPES, 10 mM, pH 7.6;
oligo(dT)22, 40 pM;
MgCl2, 10 mM; dextran sulfate, 0.05 g/liter;
spermine, 2 mM; Triton X-100, 0.5% (vol/vol); EGTA, 0.2 mM; bovine
serum albumin, 0.5 mg/ml], (ii) the template [i.e., 96-well
microtiter plates were filled with solid-phase-conjugated
poly(A) at 40 ng/well], and (iii) the substrate deoxy-NTP,
namely, separately lyophilized bromodeoxyuridine TP (BrdUTP). Extra
chemicals for novel use in this assay system were ATP and GTP
(Pharmacia Biotech) and sodium PPi (Sigma). The
RT reaction was started by addition of purified RT (mutant or wild-type
enzyme) at 20 to 60 pM (1 to 4 mU/well). The RT reaction proceeded for
3 h at 33°C and was terminated by washing; in the next step,
alkaline phosphatase-conjugated anti-BrdU antibody was incubated for 90 min at 33°C and the reaction was stopped with a new wash. The RT
activity was measured by adding alkaline phosphatase substrate and
reading the color change at 405 nm. The Km
values were determined using BrdUTP at six different concentrations
from 30 nM to 16 µM. Within this substrate range, the enzyme reaction
was linear for the RT assay step (3 h) and, thereby, steady-state
kinetics were assumed. The Ki values were determined for AZT-TP (Moravek, Brea, Calif.) using three different concentrations (ranging from 1.5 to 100 nM) of the nucleotide, adjusted
optimally for the expected Ki value of
each mutant. Competitive inhibition can be assumed since the
Vmax values did not change with these
different AZT-TP concentrations (data not shown). This finding makes it
relevant to use the mutant
Ki/Km ratio
relative to the wild-type ratio as a measurement of fold resistance.
The Km and Ki
values were obtained by fitting the data to the Michaelis-Menten competitive inhibition equation using the Grafit 4.10 program (Erithacus Software).
TABLE 1.
Comparison of the AZT susceptibilities of a strain with
wild-type RT and the RT mutant M41L/D67N/K70R/T215Y in AZT-TP
inhibition assay systemsc
TABLE 2.
AZT resistance of RT mutants and the wild type measured
by the AZT-TP-RT inhibition assay with or without 3.2 mM ATP and
by the HIV-1 drug susceptibility assaya
As ATP appeared predominant for the AZT-TP resistance mechanism, the system of AZT-TP inhibition of RT was next used to investigate which mutation sites were involved and the results were compared with results for the same mutations in an HIV-1 drug susceptibility assay. Ten site-directed mutant RTs together with mutant 41/67/70/215 and wild-type RT were studied with and without the addition of ATP. These data are illustrated in Table 2. In the absence of ATP, all the mutants except the Q151M mutant (mutant 151) behaved in a way similar to that of the strain with wild-type RT (0.7- to 1.6-fold resistance), and no correlation with the virus inhibition data was seen. By contrast, mutant 151 showed similar fourfold levels of resistance both with and without added ATP. For the remaining mutants, a reasonable correlation was observed between results of the virus assay and results of the AZT-TP inhibition assay when 3.2 mM ATP was included. However, there was one apparent exception. Only 2.6-fold resistance was seen for mutant 41/215 compared to its level of resistance in the drug susceptibility assay (26.2-fold). The levels of resistance of mutants with T69S-XX insertions, known to be associated with multinucleoside resistance (14, 26), all matched well their levels of resistance in the virus assay, and in some cases the biochemical resistance data were even higher than the corresponding viral resistance data. The behavior of the mutants having the M184V mutation indicated in certain cases that there was a reduction of resistance at the enzyme level that matched the virus data. When intracellular concentrations of GTP (0.5 mM) and PPi (150 µM) were studied for two other mutants, 41/215 and 69S-AG, similar or lower-fold resistance data were found (data not shown) as seen in Table 1 for mutant 41/67/70/215 using GTP or PPi. These data further confirmed that ATP has the dominant effect when it is used in this assay system.
Our aim was to set up a simple AZT-TP-RT inhibition assay that better reflected the AZT sensitivities of mutants and therefore reproduced the cell culture data more accurately. As ATP- and PPi-dependent excision of the incorporated AZT-MP has been shown by other investigators (1, 19), an alternative explanation for how ATP or PPi stimulates excision for specific mutants in our assay system seems less likely. Thus, we have demonstrated using our assay system a PPi-dependent pyrophosphorolysis and, even more, an ATP-dependent mechanism for AZT-TP resistance in a wide range of RT mutants. The reason that our assay seems to yield higher magnitudes of resistance may be that it enables the measurement of multiple chain termination events. This should reflect the in vivo situation better and may explain why Meyer et al. (19) did not find a similar magnitude of resistance with 3.2 mM ATP and no effect with PPi (although only the effects of a 50 µM concentration were examined) using their mono-chain-terminating system. However, the real influence of PPi (together with that of ATP) in vivo obviously depends on intracellular concentrations. In this regard, the intracellular concentrations of NTPs are well established at 3.2 ± 1.5 mM for ATP and 0.5 ± 0.2 mM for GTP (23). The intracellular concentration of PPi has been reported to be 130 µM (2).
It is of interest to determine which mutations are involved in the
ATP-mediated effect. The most important mutations appear to be located
in the finger domain of RT, specifically in the 2-3 loop, as
alterations at positions 67, 69, and 70 were dominant. With respect to
mutants with insertions at position 69, this is the first demonstration
of resistance at the enzyme level for mutants bearing only the 69S-SG
or 69S-AG changes. In contrast, the mutant with the multi-nucleoside
drug-resistant mutation Q151M alone appeared to be involved in
decreased binding to AZT-TP independently of ATP, which is in line with
a previous report (24), although only modest reduced AZT
sensitivity was seen with this key mutation by itself. However, there
was an incomplete correlation between results of the in vitro and cell
culture assays with mutants lacking either mutations at positions 67 and 70 or insertions at position 69. Thus, AZT resistance at the
enzyme level for mutant 41/215 was very low, even with the addition of
ATP, when compared to the susceptibility of the virus with these
mutations (Table 2). It may be possible that different combinations of
mutations may produce differing mechanisms of resistance, some of which
are not analyzable in this assay system. There is clearly a need for additional studies to explain the mechanism of the "non-finger 2-3 loop" mutations (e.g., at codons 41, 215, and 219) in AZT resistance, since it is known that they can contribute considerable AZT
resistance. Using a heteropolymer template primer instead of
poly(A)-oligo(dT) in this assay system might give some insight into
this effect. Although our assay system did not use the natural substrate or sequence of the template primer, this did not appear important for demonstrating a significant effect on AZT resistance. In
our study, the thymidine analog BrdUTP was used as the deoxy-NTP substrate instead of dTTP. However, this has been shown to give similar
Km and
Vmax values for the wild-type RT
enzyme in the Lenti RT assay (4). Although it is possible
that there are differences in the kinetic properties of BrdUTP for
different RT mutants, we believe that these effects are likely to be
small since the ATP-free assay data gave rates similar to that for the wild-type enzyme (with the exception of that for mutant 151). This
finding is understandable since ATP-dependent pyrophosphorolysis involves the removal of AZT-MP from a terminated primer and thus does
not directly have an impact on the BrdUTP substrate. In addition, our
findings related to the ATP-dependent mechanism of resistance for
69S-SS insertion in an AZT mutant background have recently been
confirmed (18).
In conclusion, we have shown a higher correlation than previously has been demonstrated between AZT resistance in the virus and resistance to AZT-TP at the RT enzyme level. This is the first time that this correlation has been studied with a large group of AZT-resistant mutants. With some modifications of our in vitro assay system, one could investigate this potential resistance mechanism with other nucleoside analogs and potentially screen for small molecules that block this mechanism. Furthermore, the study of mutant RT crystals in complex with the primer template and ATP or PPi with an incorporated chain terminator will be of significant interest to confirm this mechanism structurally. These data will be of potential use for the design of novel drugs with greater resilience to common resistance mutations.
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ACKNOWLEDGMENTS |
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We thank C. Nichols for help and advice with the RT protein purification, M. Salim and the Virco UK genotyping team for help with DNA sequencing, and the Virco N.V. phenotyping team for the HIV drug susceptibility data.
This work was supported by grants from the MRC, London, United Kingdom, to D.K.S. and B.A.L. and by a scholarship from Wenner-Gren Stiftelsen, Stockholm, Sweden, to J.L.
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FOOTNOTES |
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* Corresponding author. Present address for Johan Lennerstrand: Division of Clinical Virology, F68, Huddinge University Hospital, 141 86 Stockholm, Stockholm, Sweden. Phone: 46(0)8 58581304. Fax: 46(0)8 58581305. E-mail: johan.lennerstrand{at}impi.ki.se. Mailing address for Brendan A. Larder: Virco UK, Ltd., Cambridge CB4 0GA, United Kingdom. Phone: 44(0)1 223 728 828. Fax: 44(0)1 223 728 801. E-mail: brendan.larder{at}vircolab.com.
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Journal of Virology, August 2001, p. 7202-7205, Vol. 75, No. 15
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.15.7202-7205.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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(2005). Drug Resistance Mutations in the Nucleotide Binding Pocket of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Differentially Affect the Phosphorolysis-Dependent Primer Unblocking Activity in the Presence of Stavudine and Zidovudine and Its Inhibition by Efavirenz. Antimicrob. Agents Chemother.
49: 342-349
[Abstract] [Full Text] -
Meyer, P. R., Smith, A. J., Matsuura, S. E., Scott, W. A.
(2004). Effects of Primer-Template Sequence on ATP-dependent Removal of Chain-terminating Nucleotide Analogues by HIV-1 Reverse Transcriptase. J. Biol. Chem.
279: 45389-45398
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White, K. L., Chen, J. M., Margot, N. A., Wrin, T., Petropoulos, C. J., Naeger, L. K., Swaminathan, S., Miller, M. D.
(2004). Molecular Mechanisms of Tenofovir Resistance Conferred by Human Immunodeficiency Virus Type 1 Reverse Transcriptase Containing a Diserine Insertion after Residue 69 and Multiple Thymidine Analog-Associated Mutations. Antimicrob. Agents Chemother.
48: 992-1003
[Abstract] [Full Text] -
Diallo, K., Gotte, M., Wainberg, M. A.
(2003). Molecular Impact of the M184V Mutation in Human Immunodeficiency Virus Type 1 Reverse Transcriptase. Antimicrob. Agents Chemother.
47: 3377-3383
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Marchand, B., Gotte, M.
(2003). Site-specific Footprinting Reveals Differences in the Translocation Status of HIV-1 Reverse Transcriptase: IMPLICATIONS FOR POLYMERASE TRANSLOCATION AND DRUG RESISTANCE. J. Biol. Chem.
278: 35362-35372
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Girouard, M., Diallo, K., Marchand, B., McCormick, S., Gotte, M.
(2003). Mutations E44D and V118I in the Reverse Transcriptase of HIV-1 Play Distinct Mechanistic Roles in Dual Resistance to AZT and 3TC. J. Biol. Chem.
278: 34403-34410
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Meyer, P. R., Matsuura, S. E., Zonarich, D., Chopra, R. R., Pendarvis, E., Bazmi, H. Z., Mellors, J. W., Scott, W. A.
(2003). Relationship between 3'-Azido-3'-Deoxythymidine Resistance and Primer Unblocking Activity in Foscarnet-Resistant Mutants of Human Immunodeficiency Virus Type 1 Reverse Transcriptase. J. Virol.
77: 6127-6137
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Meyer, P. R., Lennerstrand, J., Matsuura, S. E., Larder, B. A., Scott, W. A.
(2003). Effects of Dipeptide Insertions between Codons 69 and 70 of Human Immunodeficiency Virus Type 1 Reverse Transcriptase on Primer Unblocking, Deoxynucleoside Triphosphate Inhibition, and DNA Chain Elongation. J. Virol.
77: 3871-3877
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Deval, J., Selmi, B., Boretto, J., Egloff, M. P., Guerreiro, C., Sarfati, S., Canard, B.
(2002). The Molecular Mechanism of Multidrug Resistance by the Q151M Human Immunodeficiency Virus Type 1 Reverse Transcriptase and Its Suppression Using alpha -Boranophosphate Nucleotide Analogues. J. Biol. Chem.
277: 42097-42104
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Ray, A. S., Basavapathruni, A., Anderson, K. S.
(2002). Mechanistic Studies to Understand the Progressive Development of Resistance in Human Immunodeficiency Virus Type 1 Reverse Transcriptase to Abacavir. J. Biol. Chem.
277: 40479-40490
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Chamberlain, P. P., Ren, J., Nichols, C. E., Douglas, L., Lennerstrand, J., Larder, B. A., Stuart, D. I., Stammers, D. K.
(2002). Crystal Structures of Zidovudine- or Lamivudine-Resistant Human Immunodeficiency Virus Type 1 Reverse Transcriptases Containing Mutations at Codons 41, 184, and 215. J. Virol.
76: 10015-10019
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Boyer, P. L., Sarafianos, S. G., Arnold, E., Hughes, S. H.
(2002). Nucleoside Analog Resistance Caused by Insertions in the Fingers of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Involves ATP-Mediated Excision. J. Virol.
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Meyer, P. R., Matsuura, S. E., Tolun, A. A., Pfeifer, I., So, A. G., Mellors, J. W., Scott, W. A.
(2002). Effects of Specific Zidovudine Resistance Mutations and Substrate Structure on Nucleotide-Dependent Primer Unblocking by Human Immunodeficiency Virus Type 1 Reverse Transcriptase. Antimicrob. Agents Chemother.
46: 1540-1545
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Boyer, P. L., Sarafianos, S. G., Arnold, E., Hughes, S. H.
(2002). The M184V Mutation Reduces the Selective Excision of Zidovudine 5'-Monophosphate (AZTMP) by the Reverse Transcriptase of Human Immunodeficiency Virus Type 1. J. Virol.
76: 3248-3256
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