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J Virol, June 1998, p. 4858-4865, Vol. 72, No. 6
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
3'-Azido-3'-Deoxythymidine (AZT) Mediates Cross-Resistance to
Nucleoside Analogs in the Case of AZT-Resistant Human Immunodeficiency
Virus Type 1 Variants
Eric J.
Arts,1,*
Miguel E.
Quiñones-Mateu,1
Jamie L.
Albright,1
James-Paul
Marois,2
Charles
Hough,2
Zhengxian
Gu,2 and
Mark A.
Wainberg2
Division of Infectious Diseases, Department
of Medicine, Case Western Reserve University, Cleveland, Ohio
44106,1 and
McGill AIDS Centre, Lady
Davis Institute-Jewish General Hospital, Montreal, Quebec, Canada
H3T 1E22
Received 20 January 1998/Accepted 20 March 1998
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ABSTRACT |
Difficulties in deciphering the mechanisms of
3'-azido-3'-deoxythymidine (AZT)-resistance by human immunodeficiency
virus type 1 (HIV-1) variants are due in part to an inability to
reconstitute resistance in vitro using AZT-resistant reverse
transcriptases. We decided to characterize mechanisms of AZT resistance
in tissue culture infections by studying the ability of drug-resistant
viruses to synthesize viral DNA in the presence or absence of drug.
Through use of PCR amplifications, we discovered an AZT-mediated
stimulation of reverse transcription by AZT-resistant viruses carrying
the M41L and T215Y mutations that can apparently override the
inhibitory effects of AZT-5'-triphosphate. In addition, the presence of
AZT also causes viruses containing the M41L and T215Y substitutions to
have diminished sensitivity to other nucleoside analogs (i.e., ddC,
ddI, and d4T). This AZT-mediated cross-resistance may help to explain
the virological failure of treatment regimens that included ddI plus
AZT or ddC plus AZT in situations in which the T215Y and/or M41L
mutations were present (F. Brun-Vézinet, C. Boucher, C. Loveday,
D. Descamps, V. Fauveau, J. Izopet, D. Jeffries, S. Kaye, C. Krzyanowski, A. Nunn, R. Schuurman, J. M. Seigneurin, C. Tamalet,
R. Tedder, J. Weber, and G. J. Weverling, Lancet
350:983-990, 1997). Our results suggest that the use of AZT may be
contraindicated in those patients for whom resistance to this compound
(M41L and/or T215Y) has been demonstrated.
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INTRODUCTION |
Resistance to antiretroviral
drugs is a formidable obstacle in the treatment of human
immunodeficiency virus-positive (HIV+) patients. Development of
new antiretroviral drugs, such as HIV type 1 (HIV-1) protease
inhibitors and nonnucleoside reverse transcriptase (RT) inhibitors,
and use of new treatment strategies, e.g., triple-drug-combination therapy, has not eliminated the emergence of drug-resistant isolates (24). In addition, the problems of drug cross-resistance and the potential for transmission of drug-resistant viruses in new infections are of great concern. In the developed world, treatment with
3'-azido-3'-deoxythymidine (zidovudine) (AZT) or transmission of
AZT-resistant virus has resulted in an increase in the HIV+ population
carrying AZT-resistant HIV-1 (4). AZT remains the drug most
commonly included in combination therapies, even though we lack a solid
understanding of the mechanisms responsible for AZT resistance
(4).
AZT, shown in 1985 to inhibit HIV-1 replication (36), was
the first antiretroviral agent administered to HIV-infected patients (13). Even with the extensive development and analysis of
new nucleoside analogs such as 3TC
(2',3'-dideoxy-3'-thiacytidine) and 1592U89
[(
)-(1S,4R)-1-4-[2-amino-6-(cyclo- propyl-amino)-9H-purin-9-yl]-2-cyclopentene-1-methanol], these
drugs are still less effective than AZT in blocking HIV replication in
tissue culture; concentrations responsible for 50% inhibition
(IC50s) for these drugs are at least 10- to 100-fold more
than that for AZT (0.001 to 0.01 µM) (10, 11, 44). However, virological response (e.g., the drop in viral load) is generally more pronounced in HIV+ individuals receiving 3TC or 1592U89
than in those receiving AZT monotherapy (20). In almost all
cases of nucleoside analog monotherapy, drug-resistant HIV-1 variants, which result from specific mutations in the coding region of
HIV-1 reverse transcriptase (RT), will dominate the HIV-1 population during the course of therapy (30, 43).
HIV-1 RT is a heterodimer composed of the polymerase-RNase H active
66-kDa subunit and the inactive but structurally significant 51-kDa
subunit (4). High-resolution crystal structures of HIV-1 RT
have modeled the p66 subunit as a human right hand (27). The
"fingers" and "thumb" subdomains are involved in substrate binding (i.e., deoxynucleosidetriphosphate [dNTP] and
primer-template), whereas the "palm" subdomain contains the
polymerase active site (27). With most nucleoside analogs,
resistance is conferred by a single mutation in the RT coding region of
HIV-1 that is stably maintained throughout therapy. A cluster of
nucleoside analog-resistant mutations are found in a region (residues
65 to 77) of the fingers subdomain (
3-4 hairpin) thought to be involved in dNTP and/or primer-template binding (4, 7, 47). We and others have previously shown that resistance to ddI
(2',3'-dideoxyinosine) and ddC (2',3'-dideoxycytidine)
conferred by mutations L74V and K65R, respectively, is the result
of decreased binding by the mutant RT to the analog and
increased selectivity for the native nucleoside (17, 19,
33). This effect may be related to possible alterations in the
primer-template binding site. With an extended template modeled
into the original crystal structure of HIV-1 RT complexed with an
18/19-nucleotide (nt) template/primer, it appears that residues L74V
and E89G may interact with the template strand (7).
Treatment with AZT will also select for a mutation (K70R) in this
region of RT, but for reasons yet unclear, the K70R HIV-1 is not
stable and is eventually outgrown by another AZT-resistant HIV-1 containing a T215Y/F change (12). This mutation and
the rarer M41L, K219E/Q, and L210W AZT-resistant mutations (21, 26, 29) are found outside the polymerase-active site in the palm subdomain, a region of unknown function. With the exception of a
modest increase in processivity during DNA synthesis by an AZT-resistant RT (9), no differences have been confirmed
between the wild-type or AZT-resistant RT in competitive inhibition by AZT-5'-triphosphate (AZT-TP), incorporation of AZT-TP, RNase H activity, and fidelity (28).
In this study, we have employed a tissue culture infection system to
study AZT-resistant mechanisms, rather than an in vitro assay, so as
not to omit any cellular or viral factors. Amounts of proviral
DNA synthesized by various AZT-resistant clinical isolates,
AZT-resistant clones, and wild-type HIV-1 were measured in the presence or absence of AZT. An obvious trend was
apparent from these analyses, i.e., AZT appears to stimulate reverse
transcription in cells infected with AZT-resistant viruses
containing the M41L and/or T215Y mutations. No such stimulation was
observed in cells exposed to wild-type virus or to HIV-1 carrying
only the K70R mutation. Thus, we hypothesize that an AZT anabolite
(e.g., AZT-5'-monophosphate) (AZT-MP) may mediate a stimulation
of reverse transcription by AZT-resistant, M41L and T215Y HIV-1
to overcome any inhibitory effects of AZT-TP. Considering that all
triphosphorylated nucleoside analogs inhibit HIV-1 reverse
transcription in a similar fashion, we examined whether AZT could also
mediate cross-resistance by AZT-resistant viruses to other
nucleoside analogs. We found that M41L and T215Y HIV-1 was
resistant to each of 2',3'-didehydro-2',3'-deoxythymidine (d4T), ddI,
and ddC, but only in the presence of low concentrations of AZT.
Only the AZT-resistant T215Y HIV-1 or a multinucleoside
analog-resistant HIV-1 (but not the K70R virus or viruses resistant to ddI or ddC) was isolated from HIV+ patients treated with AZT plus
ddI or AZT plus ddC (8, 23, 25, 38, 42). Thus, it is
possible that the T215Y mutation, in the presence of AZT, may confer
multinucleoside analog resistance. It is important that we understand
these mechanisms of AZT resistance, since a large proportion of HIV+
individuals, many of whom are AZT experienced, are now being treated
with multiple-drug regimens that commonly include a protease inhibitor
as well as AZT.
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MATERIALS AND METHODS |
Cell culture.
Jurkat (CD4+ T lymphocytic tumor cell line)
and MT4 (human T-cell leukemia virus type 1 transformed CD4+ T
lymphocytes) cells, obtained from the American Type Culture Collection
(Rockville, Md.), were grown in 1× RPMI 1640 supplemented with 10%
fetal calf serum, L-glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 µg/ml) at 37°C and 5%
CO2.
Viruses and infection studies.
Peripheral blood was drawn
from HIV+ patients treated with AZT for 3 to 12 months. Virus was
propagated from patient peripheral blood mononuclear cells (PBMCs) in
the presence of low AZT concentrations (0.01 µM) as described
previously (15). These AZT-resistant isolates were used
to infect MT4 cells in soft agar-containing, RPMI-supplemented media.
Individual AZT-resistant isolates were purified from single plaques
(identified by syncytium formation and cytopathic effects) and
propagated on MT4 cells. The RT-coding region was PCR amplified
from proviral DNA found in infected MT4 lysates, cloned into M13, and
sequenced. Almost all plaques, produced by infectious AZT-resistant
viruses isolated from each patient sample, harbored HIV-1 isolates
with the same AZT-resistance mutations. The AZT-resistant
HIV-1 clones containing the K70R or M41L and T215Y mutations,
kindly provided by Brendan Larder (Glaxo-Wellcome, Beckenham, United
Kingdom), were also propagated on MT4 cells. Culture fluids containing
purified AZT-resistant HIV-1 clones or patient isolates (ARI-1,
ARI-2, ARI-3, ARI-4, ARI-5, and ARI-6) were passed through a
0.45-µm-pore-size filter, DNase I-treated, and stored at
70°C. The absence of viral DNA in the supernatant after DNase I
treatment was verified by PCR analysis (1). HIV DNA was
found in less than 0.1% of virions used in this infection study
(2).
The same 50% tissue culture infective dose for each virus was used to
infect Jurkat or MT4 cells, pretreated for 8 h with AZT (0.001 to
0.1 µM) (Moravek Biochemicals, Brea, Calif.). After 2 h of viral
adsorption, the cultures were washed twice in phosphate-buffered saline
and resuspended in RPMI 1640 containing 10% fetal calf serum. Cultures
were incubated for a further 12 h in this medium containing drug
as indicated, following which 106 cells were washed four
times in phosphate-buffered saline and then lysed in Hirt buffer (0.6%
sodium dodecyl sulfate; 10 mM Tris-HCl, pH 7.4; 10 mM EDTA, pH 8.0)
(1, 3). These experiments were repeated three times in
Jurkat cells to obtain the standard deviation shown in Fig.
1B.
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FIG. 1.
Synthesis of minus-strand strong-stop DNA by wild-type
and AZT-resistant HIV-1 isolates. Jurkat cells were exposed to
wild-type or AZT-resistant isolates (ARI-1 through ARI-6; Table 1)
in the presence or absence of AZT (0.1 µM). Minus-strand strong-stop
HIV-1 DNA and mitochondrial DNA were PCR amplified from cellular
extracts of LMW DNA and then electrophoresed on 5% denaturing
polyacrylamide gels (A). Using phosphor-imaging analysis, amount of
minus-strand strong-stop DNA produced in the presence of AZT with one
viral isolate is presented relative to the amount of mitochondrial DNA
in that sample and to the amount of minus-strand strong-stop DNA
produced in the absence of drug (B). PCR-amplified minus-strand
strong-stop DNA from cells exposed to K65R HIV-1 or M184V HIV-1
are not shown in panel A, but quantitations of three independent
experiments with these viruses as well as the ARI viruses are presented
in panel B.
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DNA isolation, purification, and PCR amplification.
Low-molecular-weight (LMW) DNA was isolated as described previously
(1). The LMW DNA (5 µl) was then added to a 100-µl reaction mixture containing 100 pmol of unlabeled sense primer and
[
-32P]-end-labelled antisense primer, 0.2 mM
concentrations of the four dNTPs, 50 mM KCl, 10 mM Tris-HCl (pH 8.3),
2.5 mM MgCl2, and 2 U of Taq polymerase
(Boehringer Mannheim, Indianapolis, Ind.) and subjected to 27 amplification cycles as described previously (1, 3). The
following primer pairs were employed: A13 (635 to 614)-S1 (496 to 516)
to amplify a 140-bp segment in the U5 region of the long terminal
repeat; A2 (410 to 389)-S2 (221 to 242) to amplify a 207-bp segment in
the U3 region of the long terminal repeat; and AG4 (804 to 784)-SG4
(709 to 730) for a 95-bp segment upstream of the gag gene
(Fig. 2A) (1). The MTA (390 to
370)-MTS (260 to 280) primer pair was used to amplify a 130-bp segment
in the noncoding region of human mitochondrial DNA (1). The
plasmid pH
HXB2 (approximately 13 kbp) was linearized by restriction endonuclease digestion with XhoI and serially diluted
(1:10); each dilution was subjected to PCR amplification as a
quantitation control (Fig. 1C). The addition of Hirt LMW extracts from
107 cells had no effect on the amplification efficiency of
linearized plasmid and was therefore not included as a control. The PCR
amplifications (1:20) were electrophoresed on 7% denaturing
polyacrylamide gels, which were dried, autoradiographed, and analyzed
by phosphor-imaging (Molecular Dynamics Inc., Sunnyville, Calif.).
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FIG. 2.
Synthesis of minus-strand DNA in AZT-treated MT4
cells infected by wild-type HIV-1 or AZT-resistant HIV-1
clones. MT4 cells were untreated or treated with AZT (0.001 and 0.1 µM) and then exposed to wild-type HIV-1, K70R HIV-1, or M41L
plus T215Y HIV-1. Minus-strand strong-stop DNA (immediately
upstream of initiation of reverse transcription), first template
switched DNA (200 nt upstream of initiation), and complete minus-strand
DNA (~9,000 nt upstream of initiation) was PCR amplified from LMW DNA
extracts. These products are schematically illustrated in panel A. Each
bar (B) represents the amount of PCR-amplified minus-strand DNA product
relative to the amount of PCR-amplified mitochondrial DNA in the same
sample and the amount of PCR-amplified minus-strand DNA product in the
absence of drug (adjusted to 1).
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Drug sensitivity assays.
MT4 cells, untreated or treated for
8 h with 0.001, 0.01, or 0.1 µM AZT, were exposed to wild-type
HIV-1, ddC-resistant (K65R) HIV-1, K70R HIV-1, or M41L and
T215Y HIV-1 for 2 h. Washed cells were then plated into
microtiter plates containing 10-fold dilutions (0.0001 to 100 µM) of
different nucleoside analogs (AZT, ddI, ddC, d4T, and 3TC; Sigma
Chemical Co., St. Louis, Mo., or Moravek Biochemicals). Upon initial
detection of cytopathic effects, supernatants were harvested and used
for RT assays (10). RT activity was then plotted against
drug concentration for determination of IC50s directly from
the graph or by using the PROBIT program (46).
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RESULTS |
Effects of AZT on reverse transcription by AZT-resistant
clinical isolates of HIV-1.
Although resistance to AZT
has been well documented in tissue culture infections by using
virus isolated from AZT-treated HIV+ patients (4), no
one has examined proviral DNA synthesis by AZT-resistant
viruses in the presence of this drug. We, therefore, determined the
amount and extent of proviral DNA synthesis in CD4+ T lymphocytic cells
exposed to wild-type HIV-1 or to various AZT-resistant clinical
isolates in the presence or absence of AZT. AZT-resistant clinical
isolates were propagated on PBMCs and plaque purified. Sequencing
of the RT coding region of these isolates revealed several AZT
resistance mutations (e.g., M41L, K70R, and T215Y) (Table
1) as well as other nonspecific changes not associated with any drug-resistant phenotype.
Sensitivity to AZT varied with each clinical isolate but virus
containing both the M41L and T215Y mutations (ARI-1, ARI-4, and ARI-6)
showed consistently higher IC50s for AZT than did viruses
containing only the K70R mutation (ARI-3) (Table 1).
In this set of experiments, untreated or AZT (0.1 µM)-treated
Jurkat cells were exposed to virus for 2 h and then incubated
for
a further 20 h prior to cell harvest and lysis. LMW DNA was
subjected to PCR amplification by using HIV-1 DNA-specific and
mitochondrial DNA-specific primer pairs (see Materials and Methods).
As
previously described, nucleoside analogs (e.g., AZT) did not
inhibit
synthesis of minus-strand DNA in Jurkat cells exposed
to wild-type
HIV-1 (Fig.
1A and B), due to high intracellular
dNTP
concentrations (
1). Instead, nucleoside analogs were
preferentially
incorporated after the first template switch, a
significant pause
site during reverse transcription (
1).
Similar results were
obtained in experiments employing
phytohemaglutinin-stimulated
PBMCs. However, the total amount of
reverse-transcribed HIV-1
DNA was at least 10-fold less in
PBMCs than in Jurkat cells (
3).
Thus, Jurkat cells provide an excellent system in which to study an
AZT-mediated effect, other than inhibition, during minus-strand
strong-stop DNA synthesis. In fact, we observed a nearly fivefold
increase in amounts of minus-strand strong-stop DNA produced by
AZT-resistant clinical isolates (ARI-1, ARI-2, ARI-4, ARI-5,
and
ARI-6) in the presence of AZT (0.1 µM) as compared to the
absence
of drug (Fig.
1A and B). AZT-mediated
stimulation of minus-strand
strong-stop DNA was not observed with
either the ARI-3 AZT-resistant
isolate (containing the K70R), a
ddC-resistant (K65R) HIV-1, a
3TC-resistant (M184V) HIV-1, or
wild-type HIV-1 (Fig.
1B). Interestingly,
there was a slight
AZT-mediated stimulation of minus-strand strong-stop
DNA synthesis
by the ARI-5 virus, found to contain both the K70R
and T215I mutations.
The significance of the T215I mutation is
currently under study.
Figure
1C shows a direct linear correlation
between the amount of
PCR-amplified minus-strand strong-stop DNA
and the copy number of HXB2
proviral DNA. This plot was used to
quantify the amounts of
PCR-amplified sample DNA shown in Fig.
1A.
As discussed earlier, the K70R mutation is located in a region of RT
thought to be involved in dNTP and/or primer-template
binding
(
4). Other drug-resistant mutations in this region
(e.g.,
K65R and L74V), result in decreased binding affinity to
the nucleoside
analog (
15,
16,
30), suggesting that the
K70R mutation may
result in a similar phenotype. In contrast,
the M41L and T215Y
mutations are not found in regions of known
enzymatic or structural
function. Although results from Fig.
1 suggest that the M41L and/or
T215Y mutations are involved in this
AZT-mediated stimulation,
other mutations in the RT coding region
may also contribute to
this effect. In addition, there is no direct
evidence
that the clinical isolates, containing the M41L, K70R,
and/or T215Y
mutations, are resistant to the inhibitory effects
of AZT-TP
since minus-strand strong-stop DNA synthesis is not
affected by AZT
in Jurkat cells. Therefore, we employed AZT-resistant
HIV-1
HXB2 clones (Table
2), containing the K70R mutation or the
M41L and T215Y mutations, to infect MT4 cells (a human T-cell
leukemia virus type 1-transformed T-lymphocyte cell line) in the
absence or presence of AZT (0.001 and 0.1 µM). Unlike Jurkat cells
or
phytohemagglutinin-stimulated peripheral blood lymphocytes
(
1,
3), MT4 cells contain lower dNTP concentrations and
support
nucleoside analog inhibition of early proviral DNA products,
i.e., minus-strand strong-stop DNA (Fig.
2B). In these
experiments,
we examined three successive products of minus-strand DNA
synthesis
by quantitative PCR, i.e., minus-strand strong-stop
DNA, first-template-switched
DNA, and complete minus-strand DNA
by using the PCR primers described
in Materials and Methods (Fig.
2A)
(
1,
2). Similar results
on the activity of nucleoside
analogs have been obtained in quiescent
PBMCs but the total amount
of reverse-transcribed DNA was about
10-fold less than that in MT4
cells (
1,
3). For quantitative
analyses, we have utilized
these tumor cell lines in the following
studies.
Although AZT-mediated stimulation of minus-strand
strong-stop DNA synthesis by the M41L and T215Y HIV-1 was
evident in MT4
cells, this increase was reduced from that observed in
Jurkat
cells (compare Fig.
2B with 1B). In MT4 cells, this reduction
in
stimulation may be related to increased AZT inhibition of minus-strand
strong-stop DNA synthesis by wild-type HIV-1. In the presence
of
AZT, there was nearly a fivefold increase in the amount of
minus-strand
strong-stop DNA produced by an AZT-resistant, M41L
plus T215Y
HIV-1
HXB2 clone over that produced by wild-type
HIV-1
(Fig.
2B). Taking AZT inhibition into account, this
AZT-mediated
stimulation is similar to that observed with
AZT-resistant clinical
isolates (i.e., ARI-1, ARI-2, ARI-4, ARI-5,
and ARI-6) in Jurkat
cells. In addition, only the M41L and/or T215Y
mutations and not
other changes appear to be responsible for this
phenotype. As
will be discussed, AZT-mediated stimulation of
reverse transcription
by M41L plus T215Y HIV-1 may override
inhibition by AZT-TP. This
stimulation effect was maintained
throughout minus-strand DNA
synthesis by M41L and T215Y HIV-1 but
decreased with increasing
AZT concentrations (0.001 to 0.1 µM) (Fig.
2B). Since AZT inhibition
of minus-strand strong-stop DNA synthesis by
wild-type HIV-1 was
seen in MT4 cells, we could now verify the AZT
resistance phenotype
exhibited by the K70R virus (Fig.
2B). In MT4
cells exposed to
K70R HIV-1, minus-strand DNA synthesis was largely
unaffected
by both inhibitory and stimulatory activities of AZT.
Interestingly,
the K70R HIV-1 exhibits a similar resistance
phenotype to AZT
as does K65R HIV-1 to ddC. In vitro, the K65R
mutation in HIV-1
RT results in increased selectivity for dCTP
and decreased binding
to ddCTP (
17,
19).
AZT mediates resistance by AZT-resistant HIV-1 to other
nucleoside analogs.
The results described above suggest that
two mechanisms, encoded by different RT mutations (e.g.,
K70R versus T215Y), may be responsible for AZT resistance. In HIV+
patients treated with AZT, the T215Y HIV-1, conferring high level
resistance to AZT, is eventually selected over the K70R virus showing
weak AZT resistance (12). However, only the T215Y
virus, and not the K70R HIV-1 or ddC- or ddI-resistant virus, was
isolated from HIV+ patients failing AZT plus ddI or AZT plus ddC
therapy (8, 25, 38). A multinucleoside
analog-resistant HIV-1 was identified in <20% of these patients
(23, 42). These clinical findings suggest that the
AZT-resistant T215Y virus may also be less susceptible to ddI or
ddC, yet previous studies detected no such cross-resistance (4).
Considering that all triphosphorylated nucleoside analogs (ddNTPs)
inhibit HIV-1 reverse transcription in a similar manner
(
4), an AZT-mediated stimulation of reverse
transcription by
M41L and T215Y HIV-1 may override inhibition by
AZT-TP and other
ddNTPs. To test this hypothesis, we treated MT4
cells with 0.001,
0.01, and 0.1 µM AZT prior to infection with
wild-type, K70R,
M41L and T215Y, and ddC-resistant (K65R) viruses.
IC
50s were then
determined for ddC with each virus in the
presence or absence
of AZT. Due to AZT inhibition, IC
50s
for ddC could not be determined
in the case of MT4 cells exposed to
wild-type or K65R HIV-1 in
the presence of AZT (Table
2). Increased
sensitivity to ddC was
observed with the K70R and wild-type HIV-1
when treated with AZT
(Table
2), suggesting an additive inhibition of
AZT and ddC on
these viruses. In contrast, addition of low AZT
concentrations
to MT4 cells resulted in a sixfold decreased sensitivity
to ddC
by M41L and T215Y HIV-1 (Table
2). Resistance to ddC by the
M41L
and T215Y virus diminished with increasing AZT concentrations
suggesting inhibition by AZT-TP and ddCTP may occur independent
of
AZT-mediated stimulation. This decrease in ddC sensitivity
by M41L
plus T215Y HIV-1 in the presence of 0.001 µM AZT was similar
to
that observed with the K65R HIV-1 in the absence of AZT. The
K65R
substitution in HIV-1 is directly associated with ddC failure
in
vivo (
4).
Considering that AZT may mediate cross-resistance to ddC, in the case
of an AZT-resistant (M41L and T215Y) virus, we also
determined if
such cross-resistance extended to other nucleoside
analogs. By using
only the M41L and T215Y virus in the absence
or presence of AZT (0.001, 0.01, and 0.1 µM), IC
50s were calculated
for ddI, 3TC,
and 2',3'-didehydro-2'-deoxythymidine (d4T or stavudine)
(Fig.
3). In the presence of 0.001 µM AZT,
M41L and T215Y HIV-1
showed decreased sensitivity to ddI and d4T
(Fig.
3). Sensitivity
of M41L and T215Y HIV-1 to ddI, d4T, and ddC
increased with increasing
concentrations of AZT (0.001 to 0.1 µM),
suggesting an additive
inhibition by AZT and the other nucleoside
analogs independent
of AZT-mediated cross-resistance (Fig.
3). In
contrast, no significant
cross-resistance to 3TC was found with the
M41L and T215Y virus
in the presence or absence of AZT (Fig.
3). It is
also important
to note that other nucleoside analogs (i.e., ddC, ddI,
or d4T)
or a nucleoside reverse transcriptase inhibitor (i.e.,
nevirapine)
could not mediate stimulation of reverse transcription
or cross-resistance
by the M41L and T215Y virus (data not shown).
Instead, these drugs
efficiently inhibited the M41L plus T215Y virus
even in the absence
of other nucleoside analogs. When IC
50s
were calculated for ddI
or d4T with the M41L and T215Y virus, treatment
with low concentrations
of other drugs with the exception of AZT
resulted in additive
inhibition and an increased sensitivity to these
drugs (data not
shown).
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FIG. 3.
Sensitivity of AZT-resistant (M41L and T215Y) virus
to other nucleoside analogs in the presence or absence of AZT. MT4
cells were untreated or pretreated with 0.001, 0.01, and 0.1 µM AZT
prior to addition of the M41L and T215Y virus. IC50s were
then determined for d4T (A), ddI (B), 3TC (C), and ddC (D).
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DISCUSSION |
Although HIV-1 resistance to AZT was first described by
Larder et al. in 1989 (30), mechanisms responsible for
this resistance are still not understood. In contrast, mechanisms
for resistance to other nucleoside analogs (e.g., 3TC, ddI, and
ddC) have been well characterized due to the relative ease in
reconstituting resistance to these drugs in vitro by using
recominant, mutant HIV-1 RTs (17, 23, 33). Most of
these nucleoside analog-resistant mutations (e.g., K65R conferring
resistance to ddC) are found clustered in a region of RT thought to be
involved in both dNTP and primer-template binding (7, 47).
However, it should be noted that the K65R substitution is found
infrequently in patients treated with ddC (18, 48),
whereas the K70R mutation is readily identified in AZT-treated
patients (4, 12, 30). Another amino acid residue at position
184, changed from Met to Val upon selection with 3TC or another
oxathiolated cytosine, is found in the polymerase active site of RT
(5, 15, 40, 45). Nucleoside analog-resistant mutations in
both of these regions result in decreased RT affinity for the analog
and increased selectivity for the native nucleoside (17, 19, 33,
45). Treatment with AZT generally selects for two mutually
exclusive mutations, i.e., the initial K70R mutation located in the
same dNTP or primer-template binding site as described above and the
dominant T215Y mutation found in an RT region of unknown structure or
function (12).
Our studies reveal that K70R HIV-1 displays a similar resistance
phenotype to AZT in tissue culture, as does K65R HIV-1 to ddC,
suggesting a similar mechanism for resistance. Recently, Sharma et al.
have demonstrated that recombinant HIV-1 RT containing the K70R
mutation has a reduced affinity for AZT-TP as well as ddATP
(41). In contrast, reverse transcription by HIV-1
containing the M41L and T215Y mutations was stimulated up to fivefold
in the presence of AZT. This stimulation was reduced with increasing AZT concentrations, suggesting that the AZT-mediated effect may be
independent of inhibition by AZT-TP. Unlike other nucleoside analogs, there is an intracellular accumulation of the monophosphate form of AZT due to a weak affinity by thymidylate kinase for AZT-MP and slow formation of AZT-TP, the active antiretroviral form of AZT
(14, 32). Does this AZT-MP accumulation contribute to a
unique selection of the T215Y mutation in HIV-1? We are currently attempting to answer this question by selecting for AZT-resistant HIV-1 in cells displaying increased efflux of AZT-MP.
Based on our results, it appears that AZT may stimulate reverse
transcription via the M41L and/or T215Y mutations in HIV-1 to
override the inhibitory effects of AZT-TP. Drug-stimulated or
drug-mediated resistance is a common mechanism for microbial drug
resistance. For example, -lactamase expression is induced in many
gram-positive bacteria by -lactam drugs (e.g., penicillin G) to
compensate for the thick peptidoglycan layer and
absence of a periplasmic space (34). Drug-mediated
resistance is also selected by mutants of poliovirus type 3 (Sabin
strain) treated with WIN 51711, an uncoating inhibitor (37).
In fact, some viral mutants became dependent on this drug for efficient
replication (37). However, the drug resistance
mechanism most analogous to that proposed for AZT is found in
Escherichia coli resistance to kirromycin, where selection
occurs for mutants that require kirromycin release from bacterial
elongation factor (EF-Tu) for polypeptide chain elongation during
translation (35). Thus, precedents exist in other microbial
systems for the type of situation described here in which an AZT
anabolite (i.e., AZT-MP, AZT-5'-diphosphate-, or AZT-TP)
may stimulate reverse transcription to overcome the inhibitory effects
of AZT-TP.
Future studies on this AZT-mediated resistance will focus on the
effects of altered AZT metabolism on cross-resistance by AZT-resistant HIV-1 to other nucleoside analogs. AZT
metabolism will be manipulated through the use of thymidine
kinase/thymidylate kinase inhibitors or mutant T-lymphocyte cell
lines with altered nucleoside anabolism (39). We suspect
that the small increases in processivity observed with an
AZT-resistant RT containing the D67N, K70R, T215Y, and K219E
substitutions (9) may be even greater when using an RT
containing the clinically relevant M41L and T215Y mutations and the
correct AZT anabolite. Both the M41L and T215Y substitutions are
located in a region of RT which may be involved in primer-template
interactions. Although the interrelationship between these RT
activities are still being investigated, it appears that an increased
primer-template binding by RT is associated with increased
processivity, increased fidelity, and decreased pausing (3-6,
22). In addition, we have previously shown that increased pausing
by RT during RNA-dependent DNA synthesis contributes to increased
incorporation of triphosphorylated nucleoside analogs (1, 3,
4). A direct or indirect interaction of an AZT anabolite with the
M41L and T215Y RT may switch on this mechanism, i.e., increased
primer-template binding leading to reduced antiviral activity by a
nucleoside analog. An AZT-mediated switch as opposed to
constitutive resistance may be selected by these AZT-resistant viruses due to the possible detrimental effects of this mechanism on
viral fitness. Interestingly, the M41L and/or T215Y substitutions are
quite stable in the virus population in the absence of AZT treatment. In contrast, many drug-resistant viruses (e.g.,
3TC-resistant [M184V] HIV-1) are outgrown by the wild type or
possibly revert when drug pressure is removed (5).
Previous studies have shown that AZT-resistant HIV-1
isolates do not show cross-resistance to other nucleoside analogs
(4). This finding is consistent with our data as
cross-resistance to ddI, ddC, and d4T by AZT-resistant viruses was
only observed in the presence of AZT. Furthermore, an AZT-mediated
cross-resistance was limited to those viruses containing the M41L and
T215Y mutations. As mentioned above, treatment of HIV+ patients with
AZT and ddI or ddC resulted in an eventual failure of both drugs and a
corresponding emergence of AZT-resistant HIV-1 harboring the
M41L and T215Y mutations (8, 38). In contrast, monotherapy
with ddI or ddC selects for specific ddI-resistant (e.g., L74V)
(43) and ddC-resistant (e.g., K65R) (18,
48) viruses, mutations not found when these drugs were
combined with AZT (8, 38). It appears that AZT plus ddI or
AZT plus ddC combination therapy selects for two resistant genotypes: a
multinucleoside analog-resistant virus containing a combination of five
mutations (A62V, V75I, F77L, F116Y, and Q151M) found in only 5 to 20%
of the treated patients (23, 42) and a virus containing the
M41L and/or T215Y mutation found in the vast majority of patients
(8, 38)).
AZT did not mediate cross-resistance to 3TC. Several factors, including
augmented antiviral activity of 3TC over other nucleoside analogs, may
contribute to the success of AZT plus 3TC treatment. For example, the
combination of the M184V and T215Y mutations can restore sensitivity to
AZT in the face of 3TC resistance (31). However, these
findings do not explain why AZT mediates cross-resistance by the M41L
and T215Y HIV-1 to most nucleoside analogs and not to 3TC. Since
3TC appears to be efficiently phosphorylated to 3TC-5'-triphosphate
(3TC-TP) (16), the AZT-mediated stimulation effect may
not suffice to overcome the inhibitory effects of high 3TC-TP
concentrations. With the exception of 3TC, we have shown in this study
that AZT will mediate a cross-resistance by AZT-resistant (M41L and
T215Y) HIV-1 to ddI, ddC, and d4T. These results suggest that prior
treatment with AZT and the appearance of the M41L and/or T215Y
substitutions in patient virus may hamper future combination therapies
involving AZT and another nucleoside analog.
| |
ACKNOWLEDGMENTS |
This research was supported by developmental funds from the
Center for AIDS Research grant (NIH A1-36219) at Case Western Reserve
University (E.J.A.). Research performed in the laboratory of M. A. Wainberg was supported by grants from Health and Welfare Canada, and
Medical Research Council of Canada.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Division of
Infectious Diseases, BRB 1029, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106. Phone: (216) 368-8904. Fax: (216)
368-2034. E-mail: eja3{at}po.cwru.edu.
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Abstract
Introduction
