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INTRODUCTION |
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
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
-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.
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EXPERIMENTAL PROCEDURES |
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
-borano-(Rp)-2',3'-didehydro-2',3'-dideoxythymidine 5'-triphosphate (
-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,
-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),
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(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,
|
(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).
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RESULTS |
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
-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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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
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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
-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 ( ), 20 (×), 40 (+), 60 ( ), 100 ( ), 150 ( ), and 200 µM ( ) 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 Kd (µM) and
kpol (s 1) using
hyperbolic fitting of the data (Equation 2).
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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.
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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 ( ), and V75T/Y146F RT ( ).
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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.
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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.
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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
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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.
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DISCUSSION |
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
-phosphate of d4TTP does not improve incorporation into DNA.
Instead, nucleotide binding is affected, and we interpret that as an
indication that the
-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
3/
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.