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Journal of Virology, August 1999, p. 6700-6707, Vol. 73, No. 8
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Reverse Transcriptase Inhibitors Can Selectively Block the
Synthesis of Differently Sized Viral DNA Transcripts in Cells
Acutely Infected with Human Immunodeficiency Virus Type 1
Yudong
Quan,1
Liwei
Rong,1
Chen
Liang,1 and
Mark A.
Wainberg1,2,*
McGill University AIDS Centre, Lady Davis
Institute-Jewish General Hospital, Montreal, Quebec, Canada H3T
1E2,1 and Department of Microbiology and
Immunology, McGill University, Montreal, Quebec, Canada H3A
2B42
Received 7 January 1999/Accepted 14 May 1999
 |
ABSTRACT |
We have recently reported that the in vitro inhibition of human
immunodeficiency virus type 1 (HIV-1) reverse transcription by
inhibitors of reverse transcriptase (RT) occurred most efficiently when
the expected DNA products of RT reactions were long (Quan et al.,
Nucleic Acids Res. 26:5692-5698, 1998). Here, we have used a
quantitative PCR to analyze HIV-1 reverse transcription within acutely
infected cells treated with RT inhibitors. We found that levels of
minus-strand strong-stop DNA [(
)ssDNA] formed in acutely infected
MT2 cells were only slightly reduced if cells were infected with
viruses that had been generated in the presence of either
azidothymidine or nevirapine (5 µM) and maintained in the presence of
this drug throughout the viral adsorption period and thereafter.
Control experiments in which virus inoculation of cells was performed
at 4°C, followed directly by cell extraction, showed that less than
1% of total ()ssDNA within acutely infected cells was attributable
to its presence within adsorbed virions. In contrast, synthesis of
intermediate-length reverse-transcribed DNA products decreased
gradually as viral DNA strand elongation took place in the presence of
either of these inhibitors. This establishes that nucleoside and
nonnucleoside RT inhibitors can exert similar temporal impacts in
regard to inhibition of viral DNA synthesis. Generation of full-length
viral DNA, as expected, was almost completely blocked in the presence
of these antiviral drugs. These results provide insight into the fact
that high concentrations of drugs are often needed to yield inhibitory
effects in cell-free RT assays performed with short templates, whereas
relatively low drug concentrations are often strongly inhibitory in
cellular systems.
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INTRODUCTION |
Several reports have shown that the
nucleoside analog chain-terminating drug azidothymidine (AZT) can
efficiently block the reverse transcriptase (RT)-mediated formation of
full-length human immunodeficiency virus (HIV) viral DNA but not of
minus-strand strong-stop DNA [()ssDNA] during acute infection
(3, 13, 24, 25). However, the reasons for this discrepancy
remain unclear, in spite of the fact that the inhibitory potential of RT inhibitors is dependent on the length of the template being copied
and the number of sites at which inhibitors might exert their effect
(15, 23).
We have previously used cell-free reactions to study the relationship
between the inhibitory effects of the active form of AZT, i.e., AZT
5'-triphosphate, and the lengths of products generated in cell-free
assays performed with recombinant RT. We have also studied the
nonnucleoside RT inhibitor nevirapine in this regard. The results
showed that the inhibitory effects of these molecules were directly
related to the expected lengths of the RT products generated
(19). Similar findings were obtained with endogenous RT
reactions performed with purified virus particles (18),
consistent with theoretical predictions (8). However, it is
not known whether intracellular reverse transcription follows the same
pattern, and the failure of AZT to affect the intracellular synthesis
of ()ssDNA is complicated by the fact that HIV type 1 (HIV-1) can initiate reverse transcription prior to infection and can carry ()ssDNA product into target cells (14, 22, 26-28).
Conceivably, in fact, such DNA might contribute to the efficiency of
infection (26-28). We now report on the intracellular
effects of both AZT and nevirapine in the synthesis of specific RT products.
The RT products that have been specifically monitored include
()ssDNA, intermediate-length viral DNA, and full-length viral DNA and
were detected by quantitative PCR. Our results show that more than 99%
of the ()ssDNA made during acute infection of MT2 cells was formed
after infection and was not carried into cells by infecting virions.
However, ()ssDNA was made to the same extent in acutely infected
cells that had been exposed to virus particles derived either from
untreated, chronically infected H9 cells or from H9 cells that had been
continuously treated with either AZT or nevirapine. However, the number
of intermediate viral DNA products made in acutely infected cells
treated with these drugs diminished gradually as a function of DNA
strand elongation, and the synthesis of full-length viral DNA product
was almost completely blocked by these RT inhibitors, as previously
demonstrated (3, 13, 24, 25).
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MATERIALS AND METHODS |
Generation of infectious virus.
MT2 and H9 cells were
routinely maintained in RPMI 1640 medium (GIBCO Laboratories,
Mississauga, Canada) supplemented with 10% fetal bovine serum (Flow
Laboratories, Toronto, Canada), 2 mM L-glutamine, 100 U of
penicillin/ml, and 100 µg of streptomycin/ml. HIV-1 wild-type virus
stocks were prepared by transfection of 2 µg of proviral DNA (HxB2D)
into 5 × 105 MT2 cells with Lipofectin according to
the manufacturer's recommendations (GIBCO BRL, Montreal, Canada).
To minimize the amount of virion-carried DNA as well as to obtain
enough HIV for study, all viruses used in subsequent infection experiments were harvested from chronically infected H9 cells in the
absence or presence of RT inhibitors and were purified, whenever
appropriate, to eliminate the inhibitor. Recombinant wild-type viruses
were harvested from H9 cells that were chronically infected with the
progeny of the MT2 cell transfections. The IIIb strain of HIV-1
(HIV-IIIb) was similarly harvested from chronically infected H9 cells
(a gift of R. C. Gallo, Bethesda, Md.).
Chronically infected H9 cells were pelleted, and equal numbers of cells
were then incubated in the absence or presence of
either 5 µM AZT or
5 µM nevirapine. After 10 h, the cells were
again pelleted and
resuspended in fresh drug-free medium or medium
containing 5 µM AZT
or 5 µM nevirapine; in the case of drug-containing
culture fluids,
the intent was to limit the formation of any (
)ssDNA
in viral
particles generated from cells that contained integrated
viral DNA.
Viral particles in culture fluids were harvested after
a further
16 h of incubation. Viruses were ultracentrifuged (at
80,000 ×
g for 1 h at 4°C) to eliminate RT
inhibitors in culture
fluids and to limit any influence of such
molecules during a subsequent
round of infection, although, in some
cases, fresh drug was included
in these subsequent rounds both during
the adsorption period and
thereafter. Pelleted viruses were resuspended
in fresh drug-free
medium, evaluated for p24 CA content (Abbott
Laboratories, North
Chicago, Ill.) and RT activity, and kept frozen at
70°C until
further
study.
Wild-type (HxB2D) viruses harvested from H9 cells grown in the absence
of drug or in the presence of either AZT or nevirapine
were termed
H9-wt, H9-wt-AZT, and H9-wt-Nev, respectively. The
corresponding IIIb
viruses were termed H9-IIIb, H9-IIIb-AZT, and
H9-IIIb-Nev,
respectively.
De novo infection.
A total of 106 MT2 cells were
infected with HxB2D or IIIb viruses (equivalent to 10 ng of p24),
derived from either drug-treated or untreated H9 cells, as described
above. Following a 2-h adsorption at 37°C, the cells were pelleted at
4°C and were resuspended in fresh medium containing either no drug, 5 µM AZT, or 5 µM nevirapine. Prior to infection, the cells had
either been left untreated or treated with 5 µM AZT or 5 µM
nevirapine for 3 h so as to ensure that the drugs would act
against HIV-1 reverse transcription from the time of viral adsorption.
The viruses used for infection were diluted into drug-free medium or
medium supplemented with 5 µM AZT or nevirapine, depending on the
individual experiment. Figure 1A
summarizes the process of infection and analysis.
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FIG. 1.
(A) Schema of experimental procedures used; (B)
schematic representation of DNA oligonucleotide primer pairs used to
amplify regions of the HIV-1 genome by quantitative PCR. The direction
of a sense or antisense primer is indicated by an arrow pointing to the
3' or 5' terminus. LTR, long terminal repeat.
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MT2 cells, exposed to virus for periods between 2 and 12 h, were
pelleted, washed once with medium, and resuspended in lysis
buffer
containing 0.5% sodium dodecyl sulfate and 1 mg of protease
K/ml for
6 h (
12,
16), and extracted DNA was analyzed by
quantitative
PCR with primer pairs able to detect both full-length and
partially
elongated viral DNA products (HIV genomic sites amplified by
these
primer pairs are listed in Table
1
and diagrammed in Fig.
1B).
For example, (
)ssDNA and full-length DNA
were detected by primer
pairs PS-A55 and PS-PA, respectively
(
12,
13,
24,
25).
The shortest products after the first
template switch (
300 bases)
were amplified by primer pair pUT-A55.
Template-switched DNA strands
that were elongated to lengths of about
1, 2, 4, and 8 kb were
amplified by primer pairs p1k-p1kA, p2k-p2kA,
p4k-p4kA, and p8k-p8kA,
respectively.
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RESULTS |
Virus production by chronically infected H9 cells treated with AZT
or nevirapine.
H9 cells that were chronically infected with
recombinant HxB2 virus were left untreated or treated with either 5 µM AZT or 5 µM nevirapine for 5 h to minimize the extent of
synthesis of ()ssDNA in harvested viral particles. The cells were
pelleted and resuspended in drug-containing or drug-free medium. The
results (Table 2) show that after 16 h, levels of both viral p24 CA and RT activities were similar in the
drug-treated and control cultures. This shows, as expected, that these
drugs, both of which affect RT preintegrationally, had not affected the
production of viral proteins.
Inhibition of synthesis of ()ssDNA and full-length viral DNA in
acutely infected cells grown in the presence of RT inhibitors.
A
number of reports have shown that AZT treatment of acutely infected
cells had little effect on the formation of ()ssDNA while blocking
the production of full-length viral DNA (3, 13, 24, 25). To
further address this issue, we have performed a more-extensive series
of experiments that are better controlled than those previously carried
out. We adopted two strategies to control the influence of
virion-carried DNA that may interfere with subsequent PCR analysis.
First, the viruses used were harvested from cells that had been
subjected to treatment with RT inhibitors. For this purpose, we used
viruses derived from chronically infected H9 cells that had themselves
been grown in the presence of drug (i.e., 5 µM AZT or nevirapine).
The inhibitors were subsequently removed from virions by the
purification step. We then used these viruses to acutely infect MT2
cells in the presence of the same compounds; drugs were present during
the pretreatment period, during the adsorption period, and for various
times thereafter, following which DNA was extracted from the cells as
described above. As an additional control, viral adsorption was carried out for 2 h at 4°C in order to allow viral attachment but not internalization and initiation of reverse transcription, and DNA was
extracted immediately thereafter (6).
Figure
2A shows that levels of (
)ssDNA
were similar whether MT2 cells had been infected with the HxB2D clone
of HIV derived
from untreated H9 cells or with that from H9 cells that
had been
treated with either 5 µM AZT or 5 µM nevirapine, and that
treatment
of the MT2 target population with either AZT or nevirapine (5
µM) had no significant effect on the levels of (
)ssDNA that were
generated. The control lanes show the much lower levels of (
)ssDNA
that were present within virus particles that had attached to
MT2 cells
at 4°C. These results are presented in graphic form
on the basis of
molecular imaging analysis in Fig.
2B.
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FIG. 2.
(A) PCR analysis of inhibition of synthesis of ()ssDNA
and full-length viral DNA (FL DNA) by AZT or nevirapine (Nev) in
experiments in which MT2 cells were acutely infected either with
wild-type HIV-1 or with viruses grown in chronically infected H9 cells
that had been treated with these drugs as described in Materials and
Methods. PCR products were electrophoresed on 4% acrylamide gels under
nondenaturing conditions. Control lanes, results with MT2 cells that
had been coincubated with virus for 2 h at 4°C. Copy numbers of
proviral DNA are shown on the right side of the gel. (B and C)
Quantification of ()ssDNA (B) and full-length DNA (C) by molecular
imaging under conditions of no drug treatment or exposure to either 5 µM AZT or 5 µM Nevirapine.
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Figure
2A also shows the levels of full-length viral DNA that were
synthesized in acutely infected MT2 cells in the absence
of the two
drugs studied. In this case, it is apparent that both
AZT and
nevirapine (5 µM) had caused significant impairment in
the completion
of viral DNA synthesis and that nevirapine played
a more significant
role in this regard than AZT (Fig.
2C).
Figure
3 reports similar findings with
regard to the IIIb strain of HIV-1. Again, it is apparent that both
pretreatment of
chronically infected virus-producing H9 cells and
pretreatment
of acutely infected MT2 cells had little effect on the
synthesis
of (
)ssDNA in target cells. In contrast, both drugs
impacted
significantly on the generation of full-length viral DNA.
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FIG. 3.
Inhibition of synthesis of ()ssDNA and of full-length
(FL) DNA in MT2 cells acutely infected with IIIb virus in the presence
of 5 µM AZT or nevirapine (Nev). Details are identical to those for
Fig. 2. Control lanes, results obtained with MT2 cells coincubated with
virus for 2 h at 4°C. Copy numbers of proviral DNA are shown on
the right.
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Time course inhibition of synthesis of ()ssDNA and full-length
viral DNA by antiviral drugs.
We next performed time course
studies to determine whether any increased synthesis of either ()ss
viral DNA or full-length viral DNA occurred during infection. The
results of time course experiments show that the extent of synthesis of
()ssDNA increased more than twofold between 2 and 12 h after
infection and that the synthesis of this material was not significantly
affected by the RT inhibitors. The production of full-length viral DNA increased even more significantly over 12 h in the absence of drug; however, both AZT and nevirapine (5 µM) inhibited this process (Fig. 4A). These results also show that
the synthesis of ()ssDNA had largely been completed after 2 h,
when the synthesis of full-length viral DNA had barely started. This
finding is in agreement with other reports that HIV-1 requires more
than 10 h to complete reverse transcription within cells
(11).
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FIG. 4.
Time course of inhibition of synthesis of ()ssDNA and
of full-length (FL) viral DNA by 5 µM AZT or nevirapine (Nev) in
acutely infected MT2 cells. (A) PCR results were obtained on the basis
of gel electrophoresis as in Fig. 2, except that infection proceeded
for either 2 or 12 h. Control lanes, results obtained with MT2
cells coincubated with virus for 2 h at 4°C. (B) Analysis of
results of synthesis of ()ssDNA by molecular imaging.
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|
Quantitation of the (
)ssDNA results on the basis of molecular imaging
is provided in Fig.
4B.
Effects of RT inhibitors on intermediate-length reverse-transcribed
DNA products.
We next used PCR to detect intermediate RT products
generated in acutely infected MT2 cells by probing DNA that had been
extracted at 10 h after infection. Figure
5A shows that synthesis of these intermediate products decreased gradually as viral DNA strands became
further elongated in the presence of either 5 µM AZT or 5 µM
nevirapine. This tendency toward reduction in the amount of product is
displayed in Fig. 5B through F. When products grew larger than 4 kb,
the extent of drug-mediated inhibition was similar to that observed for
the production of full-length viral DNA, with nevirapine exerting an
effect even stronger than that of AZT.
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FIG. 5.
Inhibition of synthesis of intermediate-sized HIV-1
wild-type DNA in acutely infected MT2 cells treated with 5 µM AZT or
nevirapine (Nev). (A) PCR products were electrophoresed as described in
the legend to Fig. 2 and analyzed with primer pairs specifically
designed to amplify intermediate DNA products within the HIV-1 genome.
Control lanes, results obtained with MT2 cells coincubated with virus
for 2 h at 4°C. The shortest DNA products after the first
template switch were amplified by the primer pair pUT-AA55.
Template-switched DNA strands that were elongated to approximately 1, 2, 4, and 8 kb were analyzed with primer pairs p1k-p1kA, p2k-p2kA,
p4k-p4kA, and p8k-p8kA, respectively. (B through F) Analysis of
synthesis of intermediate-length viral DNA by molecular imaging.
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The relationship between the reduction in the amount of the DNA product
and the length of the DNA product in the presence
of RT inhibitor was
further analyzed by using the following equation,
which fits the
results to second-order dynamics (
8,
19):
y = 100 ·
e
kx (where
y
represents the total amount of the product,
x represents
the
product length, and
k is a constant). Our results are
consistent
with those predicted by this equation, as shown in the
normalized
curves of Fig.
6, which
presents a graphic analysis of these data
on the basis of molecular
imaging analysis. No significant effects
on the extent of viral DNA
synthesis were detected when viruses
from drug-treated chronically
infected H9 cells were used to infect
MT2 cells in the presence or
absence of the same RT inhibitors,
suggesting that virion-carried DNA
did not play a significant
role in our assays.
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FIG. 6.
DNA production versus anticipated DNA length. Results
were obtained from Fig. 2 and 5 by dividing the copy number of viral
DNA produced in the presence of RT inhibitors by that produced in the
absence of drugs. MT2 cells were acutely infected with viruses
harvested from H9 cells. Curve annealing was achieved by use of a
Graphpad Prism 2.0 program (Graphpad Software Inc., San Diego, Calif.).
For curve fitting of the synthesis of DNA by wild-type (wt) virus in
the presence of either AZT or nevirapine (Nev), the equations obtained
were yAZT = 100 · e0.000789x + 7.95 and
yNev = 100 · e0.0031x + 5.40.
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DISCUSSION |
Reverse transcription reactions have mostly been studied in vitro
in regard to short template strands. It is known that standard enzyme
values, such as Kis and 50% inhibitory
concentrations (IC50s), are dependent on experimental
conditions such as template length and substrate and inhibitor
concentrations (2, 5, 8, 15, 18, 19, 23).
The relationship between DNA elongation and the inhibitory effects of
anti-RT compounds has been intensively studied in cell-free reactions
(1, 9, 10, 15, 17-21). We have now investigated the
specific production of DNA products of varying lengths in acutely
infected cells grown in the presence of either AZT or nevirapine. The
results show that both drugs caused diminutions in the amounts of DNA
products in infected cells and that this occurred gradually as a
function of DNA chain elongation. Furthermore, nevirapine appeared to
be more active in each case than AZT.
Since RT reactions are processive events, the influence of RT
inhibitors should be amplified during successive cycles of DNA polymerization. Factors that may impact only slightly on RT activity in
regard to work performed with short templates might have more-dramatic effects on the synthesis of full-length products generated from long
templates. We have previously reported a diminution in the synthesis of
RT products alongside the elongation of DNA chains in cell-free
experiments that were shown to follow the equation described above
(19). Our data presented here imply that a second-order dynamic process can also be used to describe reverse transcription under intracellular conditions (Fig. 6).
Table 3 highlights the relationship
between the production of DNA and DNA chain elongation; it contains
values deduced from the equation given above by using a variety of
constants. These predicted values show the influence of the length of
DNA chain elongation on total DNA synthesis. In the case of
k = 0.1 in Table 3, about 90% of products will have
elongated to a length of 0.2 kb (i.e., ~200 nucleotides [nt]),
while only 35% will have elongated to 2 kb and fewer than 2% to 8 kb,
values similar to the results of our experiments (see the normalized
curves in Fig. 6). Our data also provide an explanation for the fact
that higher drug concentrations are often required to significantly
impede viral DNA synthesis in cell-free RT reactions than in cellular
systems.
The fact that AZT can block formation of full-length DNA but not
()ssDNA can be explained by differences in patterns of DNA chain
elongation. Theoretically, nevirapine should affect every cycle of RT
polymerization while AZT triphosphate should affect the incorporation
of dTMP only, i.e., about one-fourth of the total nucleotides in a DNA
strand. In addition, nevirapine as a nonnucleoside RT inhibitor is
already in its active form when added to cells and needs only to
penetrate into the cytoplasm to be effective. In contrast, AZT must
both enter the target cell and be metabolized to its triphosphate.
Obvious considerations are the levels and function of cellular enzymes
involved in these phosphorylation steps, performed by thymidine kinase
and thymidylate kinase (7).
We have also confirmed that the vast majority of HIV-1 ()ssDNA is
synthesized in infected cells and not in virions, even though the
process of reverse transcription begins before viral entry (14,
22, 26-28). In our studies, viruses grown in chronically infected H9 cells in the presence or absence of drugs were able to
initiate infection with near-equal efficiency, regardless whether the
target cells were treated with antiviral compounds or not. An extensive
series of control studies in which viruses were allowed to adsorb to
MT2 cells at 4°C, followed by extraction of DNA, revealed that the
proportion of total ()ssDNA found in H9 cell-derived virions was low
compared to that produced intracellularly, although it should be
acknowledged that cell types other than H9, when infected by
HIV-1, may conceivably produce greater amounts of ()ssDNA. These data
also provide further verification that virion-carried ()ssDNA is
probably unimportant in regard to de novo infection of actively
dividing cells (4) but may be important in regard to
nondividing cells (26). In this context, it should be noted that the use of 5 µM AZT or 5 µM nevirapine in chronically infected cells, i.e., drug concentrations 500- to 1,000-fold higher than the
IC50s of these drugs for HIV replication, was intended to minimize the amount of ()ssDNA in virions. Indeed, Fig. 2 and 3 (see
the cold-incubation control lanes) demonstrate that some ()ssDNA was
indeed produced within virus particles in this circumstance.
The fact that our data clearly document a relationship between the
observed inhibitory effects of both AZT and nevirapine and the length
of target template makes it unlikely that these drugs have any direct
effect on RT through mechanisms other than direct inhibition of viral
DNA chain elongation. Otherwise, we would likely have observed a
stepwise inhibition of DNA synthesis as a function of increased product
length, rather than the smooth curves depicted in Fig. 6. This figure
also documents that the production of ()ssDNA falls on the same
curves as those predicted by the equation shown above. The shortest
()ssDNA fragment analyzed in Fig. 6 corresponds to about 200 bp. In
acutely infected cells exposed to antiviral drugs, the high levels of
this product are a direct consequence of their short length.
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FOOTNOTES |
*
Corresponding author. Mailing address: McGill AIDS
Centre, Lady Davis Institute/Jewish General Hospital, 3755 Cote
Ste-Catherine Rd., Montreal, Quebec, Canada H3T 1E2. Phone: (514)
340-8260. Fax: (514) 340-7537. E-mail:
mdwa{at}musica.mcgill.ca.
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Journal of Virology, August 1999, p. 6700-6707, Vol. 73, No. 8
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
