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Journal of Virology, October 2000, p. 9532-9539, Vol. 74, No. 20
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
3'-Azido-3'-Deoxythymidine (AZT) and AZT-Resistant
Reverse Transcriptase Can Increase the In Vivo Mutation Rate of
Human Immunodeficiency Virus Type 1
Louis M.
Mansky* and
Lisa C.
Bernard
Department of Molecular Virology, Immunology,
and Medical Genetics, Center for Retrovirus Research, and Comprehensive
Cancer Center, Ohio State University Medical Center, Columbus, Ohio
43210
Received 11 May 2000/Accepted 28 July 2000
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ABSTRACT |
How antiretroviral drug resistance influences human
immunodeficiency virus type 1 (HIV-1) evolution is not clear. This
study tested the hypothesis that antiretroviral drugs such as
3'-azido-3'-deoxythymidine (AZT) can influence the in vivo mutation
rate of HIV-1. It was observed that AZT can increase the rate of HIV-1
mutation by a factor of 7 in a single round of replication. In
addition, (
)2',3'-dideoxy-3'-thiacytidine (3TC) was also found to
increase the mutation rate of HIV-1 by a factor of 3. It was also found
that HIV-1 drug-resistant reverse transcriptase (RT) variants
can influence the in vivo mutation rate. Replication of HIV-1 with
AZT-resistant RTs increased the mutation rate by as much as a factor of
3, while replication of HIV-1 with a 3TC-resistant RT (M184V)
had no significant effect on the mutation rate. It was observed that
only high-level, AZT-resistant RT variants could influence the in vivo
mutation rate (i.e., M41L/T215Y and M41L/D67N/K70R/T215Y). In
total, these observations indicate that both antiretroviral drugs and
drug resistance mutations can influence the in vivo mutation rate of
HIV-1.
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INTRODUCTION |
The emergence of drug-resistant
variants of human immunodeficiency virus type 1 (HIV-1) has frustrated
efforts to effectively block virus replication at a clinically
meaningful level (4, 16). These variants are not only
resistant to antiretroviral drugs such as 3'-azido-3'-deoxythymidine
(AZT) and ()2',3'-dideoxy-3'-thiacytidine (3TC) but can also have
altered tropism and virulence properties (29). Reverse
transcriptase (RT) is thought to play a role in the generation of
retrovirus diversity (38).
Antiretroviral drugs have been previously shown to influence the in
vivo mutation rate of spleen necrosis virus (SNV) and murine leukemia
virus (MLV). First, 5-azacytidine, which is a nucleoside analog that is
incorporated into RNA and inhibits protein synthesis, was found to
increase the in vivo SNV mutation rate by a factor of 13 (30). Second, AZT was found to increase the SNV mutant
frequency by a factor of 10, while it increased the MLV mutant
frequency by a factor of 3 (10). It has been shown that
deoxynucleoside triphosphate (dNTP) pool imbalances can influence the
SNV and MLV mutation rates but that AZT influences the SNV and MLV
rates by a mechanism not involving alterations in dNTP pools
(11).
A tractable genetic system has been developed to measure the forward
mutation rate of HIV-1 with a vector containing the lacZ
peptide gene as a reporter for mutations (21, 23, 24). This system allows for the study of mutations that occur during a
single round of HIV-1 replication. The mutation rate of HIV-1 in this system was determined to be 3 × 10
5 mutations per
target base pair per cycle in HeLa cells (24) and 4 × 105 mutations per target base pair per cycle in a
T-lymphoid cell line (20).
Three issues were addressed in the present study. First, we tested the
hypothesis that the antiretroviral drugs AZT and 3TC could influence
the HIV-1 mutation rate. Second, we tested whether specific
mutations in HIV-1 RT that conferred resistance to either AZT or 3TC
could influence the rate of HIV-1 mutation. Third, we tested whether
there was a correlation between increased AZT drug resistance and the
HIV-1 mutation rate. We found that replication of HIV-1 in the
presence of either AZT or 3TC can increase the HIV-1 mutation rate. In
addition, we found that two AZT-resistant variants, but not a 3TC
variant, increased the mutation rate. Finally, we observed that
high-level AZT resistance in HIV-1 correlated with significant
increases in the in vivo mutation rate.
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MATERIALS AND METHODS |
Retroviral vectors and expression plasmids.
The HIV shuttle
vector used in these studies is shown in Fig.
1A and has been described previously
(21, 23). Included in the vector is a cassette containing
the simian virus 40 (SV40) promoter driving expression of the neomycin
phosphotransferase gene (neo), an origin of replication from
pACYC 184, and the lacZ peptide gene.
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FIG. 1.
(A) HIV-1 vector used in the in vivo mutation rate
studies. The vector is shown in the proviral DNA form and has been
described previously (21, 23). (B) Protocol for one cycle of
HIV-1 vector virus replication. The steps, going from a parental
shuttle vector provirus in the step 2 cell to a vector provirus in the
step 3 cell, constitute a single cycle of replication.
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The HIV-1
gag-pol expression plasmid used was
pSVgagpol-rre-r (
35), which was a gift from David Rekosh,
University of Virginia.
This expression plasmid contains the SV40
promoter driving expression
of the HIV-1
gag-pol genes. The
amphotropic MLV
env expression
plasmid used, pSV-A-MLV-env
(
18), was provided by Dan Littman,
New York University. The
vector used for expression of wild-type
Vpr, pCMV-Vpr, has been
described previously (
21).
The RT variants analyzed in these experiments were constructed by
introducing mutations coding for RT amino acid substitutions
into
pSVgagpol-rre-r by a primary/combinatorial two-step PCR protocol
(
9,
21).
Transfections, infections, and cocultivations.
The HeLa and
COS-1 cell lines used were obtained from the American Type Culture
Collection (Manassas, Va.) and were maintained in Dulbecco's modified
Eagle's medium containing 10% calf serum or 10% fetal bovine serum,
respectively. HIV-1 vectors and expression plasmids were transfected
into HeLa cells by use of dimethyl sulfoxide (DMSO)-Polybrene
(12) or with Superfect (Qiagen). HeLa cells were infected in
the presence of Polybrene (7). Infection of HeLa target
cells was also done by cocultivation of virus-producing cells with
target cells (22, 25). Briefly, virus-producing cells
(typically 2.5 × 105 cells in a 60-mm petri dish)
were treated with mitomycin C (10 µg/ml), an inhibitor of host cell
DNA synthesis, for 2 h at 37°C. The cells were then washed three
times with fresh medium, and 2.5 × 105 HeLa target
cells were added. Two days after cocultivation, selective medium
containing G418 was added. Control experiments were done with each
cocultivation experiment to ensure that mitomycin C-treated, virus-producing cells did not proliferate and no longer adhered to the
surfaces of culture dishes.
The influence of the antiretroviral drugs AZT and 3TC on the HIV-1
mutation rate was determined by either pretreatment, posttreatment,
or
both pre- and posttreatment of cells with drug. Pretreatment
refers to
maintaining the virus-producing cells in medium supplemented
with
either AZT or 3TC for 24 h before cocultivation. AZT or 3TC
pretreatment could influence the HIV-1 mutation rate by altering
the
accuracy of the transcription of the provirus into RNA by
cellular RNA
polymerase II. Posttreatment refers to maintaining
HeLa target cells in
medium supplemented with various concentrations
of AZT or 3TC for
2 h before cocultivation and continuing until
24 h after
cocultivation. Posttreatment with AZT or 3TC may influence
the HIV-1
mutation rate only during reverse
transcription.
Experimental protocol for a single cycle of HIV-1
replication.
The experimental protocol developed to obtain a
single cycle of HIV-1 shuttle vector replication is shown in Fig. 1.
The protocol contains three steps. In step 1, the HIV-1 shuttle vector
was introduced into COS-1 cells by transfection and placed under G418 selection. Cell clones were then transiently transfected with the
helper plasmids pSVgagpol-rre-r, pSV-A-MLV-env, and pCMV-Vpr. In step
2, vector virus was harvested from step 1 cells at 48 h
posttransfection and was used to infect fresh HeLa cells. Step 2 clones
were tested by Southern analysis to ensure that only a single vector
proviral DNA was present. The lacZ peptide gene in the
vector proviral DNA of step 2 clones was sequenced to confirm that no
mutations had been introduced. G418-resistant cell clones were
transiently transfected with the helper plasmids (step 2 cells). The
step 2 clones used in these studies have been used previously in
mutation rate analyses (21, 23). In step 3, vector virus was
transferred to fresh HeLa target cells by cocultivation; cells were
then placed under G418 selection (step 3 cells). Cocultivation was used
to produce step 3 cells because it was desirable to obtain the largest
number of step 3 cells for analysis of mutant frequency.
Proviral DNA recovery.
Purified genomic DNA (34)
from pools of step 3 clones was digested with the restriction enzymes
StuI and XhoI to release the neo,
pACYC origin of replication, and lacZ peptide gene
sequences from the HIV-1 shuttle vector proviral DNA (Fig. 1). Proviral DNA was purified with the Lac repressor protein as previously described
(25). The Lac repressor protein was purified from Escherichia coli strain HB101/lac pIQ (kindly
supplied by Tom Record, University of Wisconsin, Madison) as previously
described (17). The purified proviral DNA was filled in
using the Klenow fragment of DNA polymerase and was ligated and used to
electroporate competent E. coli XLI Blue cells (Stratagene).
Kanamycin-resistant bacterial colonies were selected in the presence of
the isopropyl-
-D-thiogalactoside (IPTG) inducer. The
ratio of white plus light-blue bacterial colonies to total bacterial
colonies observed provided a forward mutation rate for a single
retroviral replication cycle. Plasmid DNA was purified (34)
and sequenced in the lacZ peptide gene region.
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RESULTS |
HIV-1 vector replication.
The experimental protocol developed
to assay a single cycle of HIV-1 shuttle vector replication is shown in
Fig. 1. The step 1 cells and the cell clones transfected with helper
plasmids to make step 2 cells have been described previously
(21). The HIV-1 shuttle vector used was a previously used
HIV-1 vector (Fig. 1), which contains a deletion in the
gag-pol and env genes with an insertion, in the
env gene, of a cassette containing the neo gene, the pACYC origin of replication, and the lacZ peptide
gene. In addition, a mutation which prevents expression of Vpr was
introduced (21). This vector can replicate in mammalian
cells as a virus and can be selected with the neomycin analog G418. The
vector can replicate in E. coli as a plasmid and is selected
by using the drug kanamycin. To be packaged into a virus particle, the vector is complemented in trans by transient transfection of
cells with an HIV-1 gag-pol expression plasmid, the
amphotropic MLV env expression plasmid, and the wild-type
vpr expression plasmid.
Vector virus produced from either COS-1 or HeLa cells was used to
infect fresh HeLa target cells (Fig.
1). Cocultivation was
used to
produce step 3 cells from step 2 cells because it was
desirable to
obtain the largest number of step 3 cells for analysis
of the mutant
frequency. Cocultivation of mitomycin C-treated
step 2 cells (typically
2.5 × 10
5 cells per 60-mm petri dish, 5 × 10
5 cells per 100-mm petri dish, or 7.5 × 10
5 cells per 150-mm petri dish) with fresh HeLa target
cells led
to 8 × 10
2 to 3 × 10
3
CFU/2.5 × 10
5 HeLa target cells. The steps going from
a parental shuttle vector
provirus in the step 2 cells to a vector
provirus in the step
3 cells constitute a single cycle of replication
(Fig.
1). These
steps include transcription of the proviral DNA by the
cellular
transcription machinery, packaging of the viral RNA, release
of
viral particles, infection of target cells, reverse transcription,
and integration of newly synthesized viral DNA to generate a vector
provirus.
The influence of AZT or 3TC on the rate of HIV-1 mutation was
determined by maintaining the HeLa target cells in medium supplemented
with various concentrations of AZT or 3TC. The target cells were
treated for 2 h before infection as well as 24 h after
infection.
The AZT or 3TC treatments were initiated 2 h before
infection
to ensure that dNTP pools were altered by the drugs at the
time
of infection, when the process of reverse transcription initiates.
The target cells were maintained in medium with the drug for 24
h
after infection to ensure that the dNTP pool imbalance was present
throughout HIV-1 replication and establishment as a provirus (~8
h
after infection). The choice of drug concentrations was based
both on
physiological relevance and on the experimental requirement
to avoid
greatly diminishing the level of infection of target
cells.
At increased concentrations of AZT and 3TC, it was found that virus
transfer to target cells was reduced to 20 and 9% of that
in the
absence of drug treatment, respectively. Mutant frequencies
increased
in a concentration-dependent manner for both AZT and
3TC. AZT increased
mutant frequencies by a factor of 7, whereas
3TC increased mutant
frequencies by a factor of 3. These observations
indicated that
concentrations of AZT and 3TC that significantly
influenced HIV-1 virus
transfer also led to statistically significant
increases in mutant
frequencies (see
below).
Trypan blue exclusion was used to assess whether decreased virus
transfer was due to killing of target cells by drug treatment.
For each
drug, 5 × 10
5 HeLa cells were plated on three 100-mm
dishes per group treated.
After 24 h, the cell culture medium was
replaced with a drug-containing
medium for another 24 h. Numbers
of viable cells were then determined.
The results indicated that
treatment with AZT or 3TC decreased
the fraction of viable cells by 10 or 20% relative to that for
untreated controls, respectively. This
indicates that the cytotoxic
effects observed with AZT and 3TC
treatment do not account for
the reductions in virus
transfer.
AZT treatment of cells increases HIV-1 mutant frequencies.
To
determine whether treatment of cells with AZT would influence the
mutant frequency of HIV-1, the effects of postinfection AZT treatment
of infected target cells were determined. In parallel experiments,
target cells infected with the HIV-1 vector were grown in the presence
of AZT, ranging from 0 to 0.4 µM (Table 1). In the absence of AZT, the average
mutant frequency was 0.005 (35 of 6,427) mutation/cycle. Posttreatment
of cells with 0.1 µM AZT resulted in a mutant frequency of 0.007 (60 of 8,150) mutation/cycle, which was comparable (chi square = 2;
P > 0.1) to the control mutant frequency. In contrast,
posttreatment of cells with either 0.2 or 0.4 µM AZT resulted in a
mutant frequency of 0.019 (127 of 6,579) or 0.038 (217 of 5,712)
mutation/cycle, respectively. Therefore, posttreatment with 0.02 or
0.04 µM AZT significantly increased the mutant frequency to either 3 (chi square = 50; P < 0.001) or 7 (chi
square = 151; P < 0.001) times that of the control, respectively. These observations indicate that AZT treatment leads to an increase in the mutant frequency of HIV-1 in a
dose-dependent manner. The relative amount of virus transfer to target
cells compared to that for controls was reduced to 0.8 with 0.1 µM
AZT, and to 0.4 and 0.2 with 0.2 and 0.4 µM AZT, respectively. This indicates that concentrations of AZT that reduced titers to 60 and 80%
significantly increased the mutant frequencies to 3 and 7 times that of
the control, respectively.
To determine if both pre- and posttreatment with AZT would lead to a
significant increase in mutant frequency, both the virus-producing
cells and the virus target cells were treated with 0.2 µM AZT
(Table
2). The mutant frequency with AZT
pretreatment was 0.012
(47 of 3,960) mutant/cycle, while AZT
posttreatment led to a mutant
frequency of 0.023 (83 of 3,595)
mutant/cycle. Pre- plus posttreatment
with 0.2 µM AZT led to a mutant
frequency of 0.029 (110/3,788)
mutant/cycle, which is not significantly
different from that with
AZT posttreatment alone (chi square = 2.4;
P > 0.1). These data
indicate that AZT
pretreatment does not significantly increase
the mutant frequency and
that the majority of the mutations identified
occurred during reverse
transcription. The significant increase
in mutant frequency observed
with AZT posttreatment was interpreted
as due to increases in the error
rate of reverse transcription.
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TABLE 2.
Effects of AZT pretreatment, posttreatment, and combined
pre- and posttreatment on the mutant frequency in recovered
HIV-1 proviruses
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3TC treatment of cells increases the mutation frequency of HIV-1 in
a dose-dependent manner.
To determine whether treatment of cells
with 3TC would increase the mutant frequency of HIV-1, the effects of
postinfection 3TC treatment of infected target cells were determined.
In parallel experiments, target cells infected with the HIV-1 vector
were grown in the presence of 3TC, ranging from 0 to 0.6 µM (Table 3). In the absence of 3TC, the average
mutant frequency was 0.006 (37 of 6,551) mutation/cycle. Posttreatment
of cells with 0.1 µM 3TC resulted in a mutant frequency of 0.007 (46 of 7,013) mutation/cycle, which was comparable (chi square = 0.46;
P > 0.1) to the control mutant frequency. However,
posttreatment of cells with either 0.3 or 0.6 µM 3TC resulted in a
mutant frequency of 0.014 (96 of 6,746) or 0.017 (81 of 4,909)
mutant/cycle, respectively. Therefore, posttreatment with 0.3 or 0.6 µM 3TC increased the mutant frequency to either 2 (chi square = 24; P < 0.001) or 3 (chi square = 32; P < 0.001) times that of the control. These
observations indicate that 3TC treatment can increase the mutant
frequency of HIV-1 in a dose-dependent manner. The relative amount of
virus transfer to target cells compared to that for controls was
reduced to 0.9 with 0.1 µM 3TC, and to 0.5 and 0.09 with 0.3 and 0.6 µM 3TC, respectively. This indicates that concentrations of 3TC that
reduced titers to 50 and 91% significantly increased the mutant
frequencies to 2 and 3 times that of the control, respectively.
To determine if both pre- and posttreatment with 3TC would
lead to a significant increase in mutant frequency, both the
virus-producing
cells and the virus target cells were treated with 0.3 µM 3TC
(Table
4). The mutant frequency
with 3TC pretreatment was 0.007
(39 of 5,379) mutant/cycle, while 3TC
posttreatment led to a mutant
frequency of 0.013 (69 of 5,450)
mutant/cycle. Pre- plus posttreatment
with 0.3 µM 3TC led to a mutant
frequency of 0.017 (81/4,907)
mutant/cycle, which is not significantly
different from that with
3TC posttreatment alone (chi square = 2.6;
P > 0.1). As was observed
with AZT pretreatment,
these data indicate that 3TC pretreatment
does not significantly
increase the mutant frequency and that
the majority of the mutations
identified occurred during reverse
transcription. The increase in
mutant frequency observed with
3TC posttreatment was interpreted to be
due to an increase in
the error rate of reverse transcription.
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TABLE 4.
Effects of 3TC pretreatment, posttreatment, and combined
pre- and posttreatment on the mutation frequency in recovered
HIV-1 proviruses
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Sequence analysis of HIV-1 vector mutants following AZT or 3TC
pretreatment, posttreatment, or pre- and posttreatment.
To
determine whether AZT or 3TC treatment of cells led to significant
changes in the spectrum of mutations in HIV-1 from that for HIV-1
replicating in the absence of drug, the lacZ peptide gene
region was sequenced in a sampling of mutant clones. Table 5 shows that following either AZT
pretreatment, posttreatment, or combined pre- and posttreatment, the
general spectrum of mutations observed was base pair substitution
mutations, frameshift mutations, deletion mutations, and mutants
containing multiple mutations (hypermutants). The predominant types of
base pair substitution mutations observed were G-to-A and C-to-T
transition mutations. In addition, frameshift mutations were observed
as primarily 1 frameshifts in runs of T's and in A's. Deletion
mutations included simple deletion mutations and two deletions with an
insertion of a nucleotide sequence of unknown origin. The
hypermutants characterized were predominantly G-to-A and C-to-T
hypermutants (Table 5). Interestingly, one hypermutant was identified
with two frameshift mutations in runs of A's.
A general comparison of the characterized mutants from AZT
pretreatment, posttreatment, or combined pre- and posttreatment
with
mutants of the HIV-1 vector in the absence of AZT revealed
that the
spectrum and general types of mutations characterized
were similar
(Table
5). In addition, the general locations of
mutations within the
lacZ peptide gene region were similar to
those identified
in the absence of the drug (data not shown).
This indicates that the
increase in the overall rate of mutations
observed with AZT
posttreatment and with AZT pre- and posttreatment
was due to a general
increase in substitution, frameshift, and
deletion mutations. These
results suggest that AZT increased the
propensity to mutations, but not
the specific types of mutations
or locations within the
lacZ peptide gene
region.
Analysis of the spectrum of mutations identified from a sampling of
mutants following 3TC pretreatment, posttreatment, or
combined pre- and
posttreatment revealed base pair substitutions,
frameshifts, and
deletion mutations (Table
6). The general
trends
indicated that the mutations identified for 3TC treatment were
similar in type and rate to those observed when the HIV-1 vector
was
replicated in the absence of 3TC. The characterized substitution
mutations were predominantly G-to-A and C-to-T mutations, while
the
frameshift mutations were mostly
1 frameshifts. Deletion
mutations
were observed and included both simple deletions and
deletions with an
insertion. Several G-to-A hypermutants were
identified (Table
6). The
general location of mutations after
3TC treatment was similar to that
seen in the absence of drug
(data not shown). This suggests that 3TC
increased the frequency
of mutations but not the mutation type or
location in the
lacZ peptide gene region.
An AZT-resistant HIV-1 RT variant can increase mutant frequencies
and the in vivo HIV-1 mutation rate.
A series of HIV-1 RT variants
that confer AZT resistance was tested for their ability to influence
the HIV-1 mutant frequency. The RT variants selected for analysis
correspond to AZT-resistant RT variants that appear during the course
of AZT therapy. Specifically, three different variants, corresponding
to RT variants with increasing levels of AZT resistance, were tested:
T215Y, M41L/T215Y, and M41L/D67N/K70R/T215Y. The HIV-1 vector was
replicated in one round of replication with each RT variant, and then
proviral DNA from pools of infected target cells was purified and
introduced into E. coli in order to determine mutant
frequencies. The mutant frequency of HIV-1 replicated with the T215Y RT
variant led to an average mutant frequency of 0.005 (35 of 7,087)
mutant/cycle (Table 7). The mutant
frequency of HIV-1 replicated with T215Y was not significantly different (chi square = 0.33; P > 0.5) from that
of HIV-1 replicated with wild-type RT, 0.004 (33 of 7,690) mutant/cycle
(Table 7). Replication of HIV-1 with the M41L/T215Y RT variant resulted
in an average mutant frequency of 0.013 (63 of 5,005) mutant/cycle, which is 2 times higher (chi square = 27; P < 0.001) than that for virus replication with wild-type RT. When
HIV-1 was replicated with the M41L/D67N/K70R/T215Y variant, the
resulting average mutant frequency was 0.017 (84 of 4,943)
mutant/cycle, which is 3 times higher (chi square = 52;
P < 0.001) than the mutant frequency of HIV-1
replicated with wild-type HIV-1 RT (Table 7). HIV-1 was also replicated
with M184V RT, which confers resistance to 3TC. The average mutant
frequency was found to be 0.003 (14 of 4,846) mutant/cycle. This
frequency is not significantly different (chi square = 1.6;
P > 0.1) from that obtained when HIV-1 was replicated
with wild-type RT (Table 7).
Sequence analysis from a sampling of mutants was done to characterize
the types of mutations that had occurred during replication.
The
spectrum of mutations observed subsequent to the replication
of HIV-1
containing the T215Y alteration was found to be similar
to that seen
when HIV-1 was replicated with wild-type RT (Table
8). The mutations characterized from
proviruses that had been
replicated with either the M41L/T215Y or the
M41L/D67N/K70R/T215Y
alteration in RT also revealed trends in mutations
that were comparable
to that identified by replication with T215Y or
with wild-type
RT. The locations of the mutations identified when HIV-1
was replicated
in the presence of AZT were similar to those obtained
when HIV-1
was replicated in the absence of the drug (data not shown).
This
indicates that the AZT resistance mutations did not alter the
type
or location of mutations but increased their frequency.
Replication of HIV-1 with M184V RT resulted in a pattern of mutations
that suggests a trend different from that for wild-type
RT. Fifty
percent of the mutations (7 of 14) characterized from
vectors
replicated with M184V RT were substitution mutations,
while 50% (7 of
14) were frameshift mutations (Table
8). No deletion
mutations were
observed in vectors replicated with M184V RT. For
the control, 88% (24 of 28) of the characterized mutants had substitutions,
7% (2 of 28)
had frameshifts, and 7% (2 of 28) had deletion
mutations.
| |
DISCUSSION |
AZT can increase the HIV-1 in vivo mutation rate.
The data
presented here indicate that the antiretroviral drug AZT can increase
the rate of HIV-1 mutation. Mutant frequencies were determined in a
single cycle of replication with an HIV-1 vector containing the
lacZ peptide gene as a mutational target. Postinfection
treatment of target cells revealed that the mutant frequency increased
by a factor of 3 or 7 with an AZT concentration of 0.2 or 0.4 µM,
respectively. AZT pretreatment led to a mutant frequency that was not
significantly different from that of HIV-1 replicated in the absence of
the drug, and AZT pre- and posttreatment led to a mutant frequency that
was not significantly different from the mutant frequency with AZT
posttreatment alone. These data indicate that AZT treatment
significantly influences reverse transcription.
Potential mechanisms for the AZT-mediated increase in the HIV-1
mutation rate.
Sequencing analysis of a sampling of HIV-1 vector
mutants recovered after AZT treatment indicated that the general
location and spectrum of substitution, frameshift, and deletion
mutations observed with AZT pretreatment, posttreatment, and combined
pre- and posttreatment were comparable to the spectrum and location of
mutants observed in the absence of drug. This indicates that the
mechanisms by which mutations occurred were similar but that the rate
had increased.
The HIV-1 vector used in these studies cannot readily be used to
determine whether mutations occurred during minus-strand
or plus-strand
DNA synthesis, but the locations of the mutations
suggest particular
mechanisms for their creation. The majority
of the G-to-A transitions
characterized, including those from
the G-to-A hypermutants, were in
GpA dinucleotides. This has been
previously observed with HIV-1
hypermutants (
6,
39). Transition
mutations adjacent to runs
of a single nucleotide have been suggested
to occur by the mechanism of
dislocation mutagenesis (
1,
14).
According to this model,
dislocation of the primer with respect
to the template produces an
unpaired nucleotide base; realignment
occurs between the primer and the
template, resulting in a mismatch,
followed by elongation beyond the
mismatch. Most of the G-to-A
transition mutations occurred at sites
adjacent to a run of nucleotides,
suggesting that these mutations may
have resulted from dislocation
mutagenesis.
The frameshift mutations characterized were mainly
1 frameshifts in
runs of T's and A's. These general trends are in agreement
with those
found for frameshift mutations made by purified HIV-1
RT
(
1). Plus-one frameshift mutations in runs of T's and A's
occurred with SNV in vivo (
3,
32). The frameshift mutations
in homo-oligomeric runs suggest that these result from template
primer
slippage (
2,
13,
36,
37). The +1 frameshift mutations
may
have occurred during minus-strand DNA synthesis (
3), while
the
1 frameshift mutations could have occurred during either
minus-
or plus-strand DNA synthesis. Simple deletion mutants and
deletion-with-insertion mutants have been previously identified,
and
the mechanisms by which they could have occurred have been
proposed
(
31,
33).
Increases in the SNV and MLV mutation rate have been observed
previously when these viruses were replicated in the presence
of AZT
(
11). Several potential mechanisms have been proposed
for
this effect on the mutation rate. One proposed mechanism is
direct
interaction of AZT with RT in a noncatalytic manner, which
could induce
a conformational change that alters enzyme fidelity.
Although the
influence of AZT on the SNV and MLV mutation rates
has been shown to be
due to a mechanism not involving alterations
in dNTP pools
(
11), it is possible that this is not the case
with HIV-1
(
15,
26,
28,
40).
3TC increases the in vivo mutation rate of HIV-1.
It was found
that another antiretroviral drug, 3TC, could also increase the HIV-1
mutation rate. The mutant frequency was observed to increase by
postinfection treatment of target cells with 0.3 and 0.6 µM 3TC by
factors of 2 and 3, respectively. Pretreatment of virus-producing cells
with 0.3 µM 3TC did not significantly influence the mutant frequency,
and pre- and posttreatment together (a factor-of-2 increase) did not
significantly increase the mutant frequency over that of just
posttreatment alone (a factor-of-3 increase). This indicates, as was
observed with AZT, that 3TC influences the reverse transcription step
of the HIV-1 life cycle. Sequencing analysis of a sampling of mutants
recovered after 3TC treatment indicated that the spectrum and location
of mutations observed were similar to those seen in the absence of the drug.
Increased AZT drug resistance correlates with an increase in the
HIV-1 in vivo mutation rate.
The mutant frequencies for several
AZT-resistant RTs (i.e., T215Y, M41L/T215Y, and M41L/D67N/K70R/T215Y)
were characterized in order to determine if increasing levels of AZT
drug resistance were associated with higher mutation rates. T215Y did
not influence the mutant frequency compared to that of wild-type RT,
but M41L/T215Y and M41L/D67N/K70R/T215Y resulted in mutant frequencies
that were increased by factors of 2 and 3, respectively. This indicates that there is a correlation between increased drug resistance and the
ability to influence mutant frequency. However, this relationship is
not linear, as the drug resistance levels of T215Y, M41L/T215Y, and
M41L/D67N/K70R/T215Y RT are about 16, 70, times, and 180 times that of
wild-type RT, respectively (19). Sequencing analysis of a
sampling of mutants indicated that the observed mutations were similar
in type and location to those observed with wild-type RT, indicating
that the AZT-resistant RTs made errors more frequently, but by the same
mechanisms as wild-type RT.
A mechanism of AZT resistance has been recently reported in a cell-free
reaction (
27). An AZT-resistant RT (containing the
D67N,
K70R, T215F, and K219Q amino acid substitutions) was found
to have an
increased ability compared with wild-type RT to extend
the primer past
several potential termination sites in the presence
of AZTTP when ATP
was added to the cell-free reaction mixture.
It was also observed that
transfer of the AZTMP residue from the
primer terminus to ATP to form
dinucleoside polyphosphate and
unblocked primer was enhanced in the
mutant enzyme. The inhibition
of this activity by the next
complementary dNTP was found to be
reduced compared to that for the
wild type. The crystal structure
of a covalently trapped catalytic
complex containing HIV-1 RT,
chain-terminated primer and template, and
dTTP has been determined
(
8). The majority of the AZT
resistance mutations are in the
neighborhood of the incoming
nucleotide, which appears to provide
structural support for this
mechanism of AZT
resistance.
A 3TC-resistant RT variant does not influence the mutation
rate.
Replication of the HIV-1 vector with an M184V RT
variant did not lead to a significant change in the mutant frequency.
This indicates that the threefold increase in fidelity by M184V in nucleotide insertion assays (41) does not have a phenotype
that can be distinguished from wild-type RT in the in vivo mutation rate assay. It has previously been shown that M184V does not affect the
overall cell-free error rate of HIV-1 RT by use of the
lacZ peptide gene as a mutational target (5).
Sequencing analysis of the mutations in recovered mutants indicated a
pattern in which there were an approximately equal number of base
substitution and frameshift mutations recovered. This trend is
different from that for wild-type RT, where there are about twice as
many substitution mutations observed as frameshift mutations.
| |
ACKNOWLEDGMENTS |
We thank R. Cherian, M. Mauck, S. Uchida, S. Webb, and A. Waggoner for outstanding technical assistance. We also thank M. Williams for comments on the manuscript.
This work was supported by the Public Health Service (GM56615), the
American Cancer Society, and the Ohio Cancer Research Associates.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Virology, Immunology, and Medical Genetics, 2078 Graves Hall, 333 W. 10th Ave., Columbus, OH 43210. Phone: (614) 292-5525. Fax: (614)
292-9805. E-mail: mansky.3{at}osu.edu.
| |
REFERENCES |
| 1.
|
Bebenek, K.,
J. Abbotts,
J. D. Roberts,
S. H. Wilson, and T. A. Kunkel.
1989.
Specificity and mechanism of error-prone replication by human immunodeficiency virus-1 reverse transcriptase.
J. Biol. Chem.
264:16948-16956[Abstract/Free Full Text].
|
| 2.
|
Bebenek, K., and T. A. Kunkel.
1993.
The fidelity of retroviral reverse transcriptases, p. 85-102.
In
A. M. Skalka, and S. P. Goff (ed.), Reverse transcriptase. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 3.
|
Burns, D. P. W., and H. M. Temin.
1994.
High rates of frameshift mutations within homo-oligomeric runs during a single cycle of retroviral replication.
J. Virol.
68:4196-4203[Abstract/Free Full Text].
|
| 4.
|
Coffin, J.
1995.
HIV population dynamics in vivo: implications for genetic variation, pathogenesis, and therapy.
Science
267:483-489.
|
| 5.
|
Drosopoulos, W. C., and V. R. Prasad.
1998.
Increased misincorporation fidelity observed for nucleoside analog resistance mutations M184V and E89G in human immunodeficiency virus type 1 reverse transcriptase does not correlate with the overall error rate measured in vitro.
J. Virol.
72:4224-4230[Abstract/Free Full Text].
|
| 6.
|
Fitzgibbon, J. E.,
S. Mazar, and D. T. Dubin.
1993.
A new type of G-A hypermutation affecting human immunodeficiency virus.
AIDS Res. Hum. Retrovir.
9:833-838[Medline].
|
| 7.
|
Hu, W.-S., and H. M. Temin.
1990.
Genetic consequences of packaging two RNA genomes in one retroviral particle: pseudodiploidy and high rate of genetic recombination.
Proc. Natl. Acad. Sci. USA
87:1556-1560[Abstract/Free Full Text].
|
| 8.
|
Huang, H.,
R. Chopra,
G. L. Verdine, and S. C. Harrison.
1998.
Structure of a covalently trapped catalytic complex of HIV-1 reverse transcriptase: implications for drug resistance.
Science
282:1669-1675[Abstract/Free Full Text].
|
| 9.
|
Ito, W.,
H. Ishiguro, and Y. Kurosawa.
1991.
A general method for introducing a series of mutations into cloned DNA using the polymerase chain reaction.
Gene
102:67-70[CrossRef][Medline].
|
| 10.
|
Julias, J. G.,
T. Kim,
G. Arnold, and V. K. Pathak.
1997.
The antiretrovirus drug 3'-azido-3'-deoxythymidine increases the retrovirus mutation rate.
J. Virol.
71:4254-4263[Abstract].
|
| 11.
|
Julias, J. G., and V. K. Pathak.
1998.
Deoxyribonucleoside triphosphate pool imbalances in vivo are associated with an increased retroviral mutation rate.
J. Virol.
72:7941-7949[Abstract/Free Full Text].
|
| 12.
|
Kawai, S., and M. Nishizawa.
1984.
New procedure for DNA transfection with polycation and dimethyl sulfoxide.
Mol. Cell. Biol.
4:1172-1174[Abstract/Free Full Text].
|
| 13.
|
Kunkel, T. A.
1990.
Misalignment-mediated DNA synthesis errors.
Biochemistry
29:8003-8011[CrossRef][Medline].
|
| 14.
|
Kunkel, T. A., and P. S. Alexander.
1986.
The base substitution fidelity of eukaryotic DNA polymerases mispairing frequencies, site preferences, insertion preferences, and base substitution by dislocation.
J. Biol. Chem.
261:160-166[Abstract/Free Full Text].
|
| 15.
|
Kunz, B. A., and S. E. Kohalmi.
1991.
Modulation of mutagenesis by deoxyribonucleotide levels.
Annu. Rev. Genet.
25:339-359[CrossRef][Medline].
|
| 16.
|
Kuritzkes, D. R.
1996.
Clinical significance of drug resistance in HIV-1 infection.
AIDS
10(Suppl. 5):S27-S33.
|
| 17.
|
Laiken, S. L.,
C. A. Gross, and P. H. von Hippel.
1972.
Equilibrium and kinetic studies of Escherichia coli lac repressor-inducer interactions.
J. Mol. Biol.
66:143-155[CrossRef][Medline].
|
| 18.
|
Landau, N. R., and D. R. Littman.
1992.
Packaging system for rapid production of murine leukemia virus vectors with variable tropism.
J. Virol.
66:5110-5113[Abstract/Free Full Text].
|
| 19.
|
Larder, B. A.
1994.
Interactions between drug resistance mutations in human immunodeficiency virus type 1 reverse transcriptase.
J. Gen. Virol.
75:951-957[Abstract/Free Full Text].
|
| 20.
|
Mansky, L. M.
1996.
Forward mutation rate of human immunodeficiency virus type 1 in a T-lymphoid cell line.
AIDS Res. Hum. Retrovir.
12:307-314[Medline].
|
| 21.
|
Mansky, L. M.
1996.
The mutation rate of human immunodeficiency virus type 1 is influenced by the vpr gene.
Virology
222:391-400[CrossRef][Medline].
|
| 22.
|
Mansky, L. M.
1994.
Retroviral-vector-mediated gene transfer, p. 27B:5.1-27B:5.10.
In
J. B. Griffiths, A. Doyle, and D. G. Newell (ed.), Cell and tissue culture: laboratory procedures, 6th ed. John Wiley & Sons, New York, N.Y.
|
| 23.
|
Mansky, L. M.,
S. Preveral,
L. Selig,
R. Benarous, and S. Benichou.
2000.
The interaction of Vpr with uracil DNA glycosylase modulates the human immunodeficiency virus type 1 in vivo mutation rate.
J. Virol.
74:7039-7047[Abstract/Free Full Text].
|
| 24.
|
Mansky, L. M., and H. M. Temin.
1995.
Lower in vivo mutation rate of human immunodeficiency virus type 1 than predicted from the fidelity of purified reverse transcriptase.
J. Virol.
69:5087-5094[Abstract].
|
| 25.
|
Mansky, L. M., and H. M. Temin.
1994.
Lower mutation rate of bovine leukemia virus relative to that of spleen necrosis virus.
J. Virol.
68:494-499[Abstract/Free Full Text].
|
| 26.
|
Mathews, C. K., and J. Ji.
1992.
DNA precursor asymmetries, replication fidelity, and variable genome evolution.
Bioessays
14:295-301[CrossRef][Medline].
|
| 27.
|
Meyer, P. R.,
S. E. Matsuura,
A. M. Mian,
A. G. So, and W. A. Scott.
1999.
A mechanism of AZT resistance: an increase in nucleotide-dependent primer unblocking by mutant HIV-1 reverse transcriptase.
Mol. Cell
4:35-43[CrossRef][Medline].
|
| 28.
|
Meyerhans, A.,
J.-P. Vartanian,
C. Hultgren,
U. Plikat,
A. Karlsson,
L. Wang,
S. Eriksson, and S. Wain-Hobson.
1994.
Restriction and enhancement of human immunodeficiency virus type 1 replication by modulation of intracellular deoxynucleoside triphosphate pools.
J. Virol.
68:535-540[Abstract/Free Full Text].
|
| 29.
|
Milich, L.,
B. Margolin, and R. Swanstrom.
1993.
V3 loop of the human immunodeficiency virus type 1 Env protein: interpreting sequence variability.
J. Virol.
67:5623-5634[Abstract/Free Full Text].
|
| 30.
|
Pathak, V. K., and H. M. Temin.
1992.
5-Azacytidine and RNA secondary structure increase the retrovirus mutation rate.
J. Virol.
66:3093-3100[Abstract/Free Full Text].
|
| 31.
|
Pathak, V. K., and H. M. Temin.
1990.
Broad spectrum of in vivo forward mutations, hypermutations, and mutational hotspots in a retroviral shuttle vector after a single replication cycle: deletions and deletions with insertions.
Proc. Natl. Acad. Sci. USA
87:6024-6028[Abstract/Free Full Text].
|
| 32.
|
Pathak, V. K., and H. M. Temin.
1990.
Broad spectrum of in vivo forward mutations, hypermutations, and mutational hotspots in a retroviral shuttle vector after a single replication cycle: substitutions, frameshifts, and hypermutations.
Proc. Natl. Acad. Sci. USA
87:6019-6023[Abstract/Free Full Text].
|
| 33.
|
Pulsinelli, G. A., and H. M. Temin.
1991.
Characterization of large deletions occurring during a single round of retrovirus vector replication: novel deletion mechanism involving errors in strand transfer.
J. Virol.
65:4786-4797[Abstract/Free Full Text].
|
| 34.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
Molecular cloning: a laboratory manual, 2nd ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 35.
|
Smith, A. J.,
M.-L. Cho,
M.-L. Hammarskjold, and D. Rekosh.
1990.
Human immunodeficiency virus type 1 Pr55gag and Pr160gag-pol expressed from a simian virus 40 late replacement vector are efficiently processed and assembled into viruslike particles.
J. Virol.
64:2743-2750[Abstract/Free Full Text].
|
| 36.
|
Streisinger, G.,
Y. Okada,
J. Emrich,
J. Newton,
A. Tsugita,
E. Terzaghi, and M. Inouye.
1966.
Frameshift mutations and the genetic code.
Cold Spring Harbor Symp. Quant. Biol.
31:77-84[Abstract/Free Full Text].
|
| 37.
|
Streisinger, G., and J. Owen.
1985.
Mechanism of spontaneous and induced frameshift mutation in bacteriophage T4.
Genetics
109:633-659[Abstract/Free Full Text].
|
| 38.
|
Temin, H. M.
1993.
Retrovirus variation and reverse transcription: abnormal strand transfers result in retrovirus genetic variation.
Proc. Natl. Acad. Sci. USA
90:6900-6903[Abstract/Free Full Text].
|
| 39.
|
Vartanian, J.-P.,
A. Meyerhans,
B. Asjo, and S. Wain-Hobson.
1991.
Selection, recombination, and G-to-A hypermutation of human immunodeficiency virus type 1 genomes.
J. Virol.
65:1779-1788[Abstract/Free Full Text].
|
| 40.
|
Vartanian, J.-P.,
A. Meyerhans,
M. Sala, and S. Wain-Hobson.
1994.
G-A hypermutation of the human immunodeficiency virus type 1 genome: evidence for dCTP pool imbalance during reverse transcription.
Proc. Natl. Acad. Sci. USA
91:3092-3096[Abstract/Free Full Text].
|
| 41.
|
Wainberg, M. A.,
W. C. Drosopoulos,
H. Salomon,
M. Hsu,
G. Borkow,
M. A. Parniak,
Z. Gu,
Q. Song,
J. Manne,
S. Islam,
G. Castriota, and V. R. Prasad.
1996.
Enhanced fidelity of 3TC-selected mutant HIV-1 reverse transcriptase.
Science
271:1282-1285[Abstract].
|
Journal of Virology, October 2000, p. 9532-9539, Vol. 74, No. 20
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Abstract
Introduction
