From the Lady Davis Institute for Medical Research
and Departments of ¶ Microbiology and § Medicine,
McGill AIDS Center, McGill University, Montreal,
Quebec H3T 1E2, Canada
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ABSTRACT |
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Human immunodeficiency virus (HIV-1) utilizes the
NF-B/Rel proteins to regulate transcription through NF-
B binding
sites in the HIV-1 long terminal repeat (LTR). Normally, NF-
B is
retained in the cytoplasm by inhibitory I
B proteins; after
stimulation by multiple activators including viruses, I
B
is
phosphorylated and degraded, resulting in NF-
B release. In the
present study, we examined the effect of tetracycline-inducible
expression of transdominant repressors of I
B
(TD-I
B
) on
HIV-1 multiplication using stably selected Jurkat T cells. TD-I
B
was inducibly expressed as early as 3 h after doxycycline addition and
dramatically reduced both NF-
B DNA binding activity and LTR-directed
gene activity. Interestingly, induced TD-I
B
expression also
decreased endogenous I
B
expression to undetectable levels by
24 h after induction, demonstrating that TD-I
B
repressed
endogenous NF-
B-dependent gene transcription.
TD-I
B
expression also sensitized Jurkat cells to tumor necrosis
factor-induced apoptosis. De novo HIV-1 infection of Jurkat
cells was dramatically altered by TD-I
B
induction, resulting in
inhibition of HIV-1 multiplication, as measured by p24 antigen, reverse
transcriptase, and viral RNA. Given the multiple functions of the
NF-
B/I
B pathway, TD-I
B
expression may interfere with HIV-1
multiplication at several levels: LTR-mediated transcription,
Rev-mediated export of viral RNA, inhibition of HIV-1-induced
pro-inflammatory cytokines, and increased sensitivity of HIV-1-infected
cells to apoptosis.
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INTRODUCTION |
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NF-B/Rel transcription factors serve essential roles in the
regulation of the immunomodulatory genes and activate genes including cytokines, cell surface receptors, and acute phase proteins, as well as
viral genes including the
HIV-11 LTR (for review, see
Refs. 1-3). NF-
B activity is regulated in part at the level of
subcellular localization. In unstimulated cells, NF-
B heterodimers
are retained in the cytoplasm by inhibitory I
B proteins (4, 5), also
a family of proteins that include I
B
(6), I
B
(7), I
B
(8), I
B
(9, 10), and Bcl-3 (11, 12), as well as the precursor
proteins p105 (13) and p100 (14). Upon stimulation by many activating
agents, including cytokines (TNF and interleukin-1), viruses, and
double-stranded RNA, I
B
is rapidly phosphorylated on Ser-32 and
Ser-36; N-terminal phosphorylation of I
B
represents a signal for
ubiquitination and degradation via the 26 S proteasome pathway
(15-19). Substitution of alanine for Ser-32 and Ser-36 completely
abolished signal-induced phosphorylation and degradation of I
B
,
and blocked the activation of NF-
B (20-22). These mutations also
blocked in vitro ubiquitination of the I
B
protein (19,
23, 24). Although the N terminus of I
B
is necessary for
signal-induced degradation, degradation of I
B
also requires the
C-terminal PEST domain of the protein (25-30). Once released,
NF-
B is able to activate target genes until new I
B
is
synthesized. Since I
B
contains NF-
B binding sites in
its promoter, NF-
B autoregulates the transcription of its own
inhibitor (31-34).
The intracellular efficiency of HIV-1 gene expression and replication
is due in part to the ability of HIV-1 to utilize host signaling
pathways to mediate its own transcriptional regulation. In this regard,
the NF-B/Rel pathway plays a central role in HIV-1 LTR-driven
transcription. The HIV-1 LTR contains two adjacent high affinity
NF-
B binding sites in the enhancer region of its LTR (
109 to
79)
(35). Transient transfection studies using HIV LTR or HIV enhancer
reporter constructs demonstrate that HIV gene expression increases upon
induction of NF-
B DNA binding activity with stimulators such as
TNF
and interleukin-1 or upon co-infection with other pathogens
(reviewed in Refs. 1 and 2).
The dependence of HIV-1 on its NF-B sites in the LTR for virus
replication has been examined in several studies (see Refs. 36-38, and
references therein). HIV-1 infection of primary macrophages or myeloid
cell lines leads to constitutive NF-
B expression (1, 39-41),
increased proteasome-mediated turnover of I
B
, and elevated expression of NF-
B1, NF-
B2, and c-Rel proteins (42, 43). This
modulation of intracellular NF-
B levels may contribute to enhanced
NF-
B-directed gene expression and increased HIV-1 replication. Recently, Chen et al. (37) measured virus multiplication in T cell lines with different basal levels of NF-
B and in primary T
cells by infecting with wild type virus or virus containing NF-
B
enhancer site mutations. These studies demonstrated that NF-
B sites
play a central role in enhancing HIV-1 growth, although the virus was
still able to grow more slowly in the absence of
B sites. In primary
T cells, an intact enhancer element contributed to an earlier and
higher peak titer of virus, thus reflecting the selective advantage
conferred by the maintenance and utilization of functional NF-
B
sites (37). Interestingly, pathogenic viral isolates with NF-
B site
deletions have been described that contain a duplication of a putative
TCF-1 site in the U3 region of the LTR (44); compensatory mutations
such as the TCF-1 repeat may decrease the dependence of HIV-1 on intact
NF-
B sites in vivo (37).
In the present study, we generated Jurkat T cell lines that inducibly
express transdominant repressors of IB
(TD-I
B
), or I
B
super-repressors, and examined the effect of Tet-inducible expression
of (TD-I
B
) on NF-
B DNA binding activity,
NF-
B-dependent gene expression and de novo
HIV-1 multiplication. The time course of de novo HIV-1
infection in TD-I
B
-expressing Jurkat cells was altered by
doxycycline (Dox) induction of TD-I
B
, resulting in a dramatic
transcriptional inhibition of HIV-1 multiplication, as measured by p24
antigen, reverse transcriptase, and viral RNA analyses.
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MATERIALS AND METHODS |
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Generation of Plasmids--
CMVt-rtTA contains the
Moloney murine leukemia virus-based pBABE vector backbone, which
contains a puromycin resistance gene under the control of the CMV
promoter. Plasmid construction consisted of the consecutive insertion
of three components into the polylinker site: the
doxycycline-responsive promoter CMVt from the
CMVtBL vector (a kind gift from A. Cochrane), the rtTA gene
from the pUHD172-1neo plasmid (45), and the poly(A) fragment from the pSVK3 vector. neo CMVt BL was constructed in two steps.
First, an intermediary plasmid (neo BL) was generated by ligation of a
3-kilobase pair XhoI/EcoRI fragment from the pMV7
vector (contains the neomycin (neo) resistance gene) to a 3.8-kilobase
pair XhoI/EcoRI fragment from the CMV BL vector
(contains the poly(A) site and the ampicillin (Amp) resistance gene).
Second, a 450-bp XhoI (blunt)/NotI fragment of
CMVt BL (contains the CMVt promoter) was cloned
into the EcoRI (blunt)/NotI sites of neo BL.
CMVt-IB2N and I
B2N
4 were constructed by cloning an
EcoRV (blunt)/BamHI I
B
-2N mutant cDNA
fragment downstream of CMVt at the EcoRI (blunt,
filled with Klenow enzyme)/BamHI site of neo
CMVt BL.
Cell Culture and Generation of IB
-expressing Cell
Lines--
Jurkat cells were transfected with CMVt-rtTA
plasmid by the DEAE-dextran method. The precipitated
CMVt-rtTA plasmid (15 µg) was resuspended in TS solution
(8 mg/ml NaCl, 0.38 mg/ml KCl, 0.1 mg/ml
Na2HPO4·7H2O, 3.0 mg/ml Tris, 0.1 mg/ml MgCl2, 0.1 mg/ml CaCl2, pH 7.4) and
subsequently DEAE-dextran (Amersham Pharmacia Biotech) was added. For
transfection, 1 × 107 cells in exponential phase were
washed once in TS, resuspended with the DNA solution and incubated at
room temperature for 20 min, and then incubated at 37 °C for 30 min
in 10 ml with RPMI 1640 medium supplemented with 10% fetal bovine
serum (FBS), 2 mM glutamine, 10 µg/ml gentamicin
(Schering Canada, Pointe Claire, Quebec, Canada), and 0.1 mM chloroquine (Sigma), after which they were centrifuged
and resuspended in fresh medium. Cells were selected beginning at
24 h after transfection in RPMI 1640 medium containing 10% FBS, 2 mM glutamine, 10 µg/ml gentamicin, and 2.5 µg/ml
puromycin (Sigma). Resistant clones carrying the CMVt-rtTA
plasmid (rtTA-Jurkat cells) were then transfected with
CMVt-Neo, CMVt-2N, and CMVt-2N
4 plasmids by DEAE-dextran. Cells were selected beginning at 24 h
after transfection for approximately 4 weeks in RPMI containing 10%
FBS, 2 mM glutamine, 10 µg/ml gentamicin, 2.5 µg/ml
puromycin, and 400 µg/ml G418 (Life Technologies, Inc.). Initially,
pools of transformants corresponding to rtTA-, rtTA-neo-,
rtTA-I
B-2N-, and rtTA-I
B-2N
4-expressing Jurkat cells were
analyzed for inducible I
B expression; subsequently, 6-10 individual
clones from each transformant pool were selected for further analysis.
To analyze growth kinetics, rtTA-neo-, rtTA-I
B-2N-, and
rtTA-I
B-2N
4-expressing Jurkat cells were cultured in the presence
of 1 µg/ml Dox for various times at an initial cell density of 1 × 105cells/ml and then counted every other day. All cell
lines grew well in the above medium with doubling times of 50 ± 4 h. Values obtained were the average of two experiments.
Analysis of Apoptosis-- To identify apoptotic cells, cells were treated with TNF after 24 h culture in the absence or presence of 1 µg/ml Dox, and stained using the TUNEL assay (Boehringer Mannheim) and Hoechst dye 33258. The mixture was then viewed under UV illumination using a Leica fluorescent microscope. To calculate percent apoptosis, a minimum of 200-400 cells were counted. Apoptosis was also analyzed by DNA fragmentation assay. ~A total of 3 × 106 cells were collected, resuspended in 0.25 ml of TBE containing 0.25% Nonidet P-40 and 0.1 mg/ml RNase A, and incubated for 30 min at 37 °C. Extracts were then treated with 1 mg/ml proteinase K for 30 min at 37 °C. DNA preparations (30 µl) were loaded on 1.8% agarose gel; DNA fragmentation was visualized under UV light.
Western Blot Analysis--
To characterize kinetics of
expression, rtTA-neo, rtTA-IB-2N, and rtTA-I
B-2N
4 Jurkat T
cells were cultured in the presence of 1 µg/ml Dox (Sigma) for
various times. Cells were then washed with phosphate-buffered saline
and lysed in Western Lysis Buffer (WLB) containing 10 mM
Tris-Cl, pH 8.0, 60 mM KCl, 1 mM EDTA, 1 mM dithiothreitol (DTT), 0.5% Nonidet P-40, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml
leupeptin, 10 µg/ml pepstatin, and 10 µg/ml aprotinin. Equivalent
amounts of whole cell extract (20 µg) were subject to
SDS-polyacrylamide gel electrophoresis in a 10% or 15% polyacrylamide
gel. After electrophoresis, the proteins were transferred to Hybond
transfer membrane (Amersham Pharmacia Biotech) in a buffer containing
30 mM Tris, 200 mM glycine, and 20% methanol
for 1 h. The membrane was blocked by incubation in phosphate-buffered saline (PBS) containing 5% dried milk for 1 h
and then incubated overnight with N-terminal I
B
monoclonal antibody MAD 10B (29), anti-NF-
B antibodies (46), p24 (ID Laboratories), or anti-actin antibody (Sigma) in 5% milk/PBS, at
dilutions of 1:500 or 1:1000. These incubations were done at 4 °C
overnight. After four 10-min washes with PBS, membranes were reacted
with a peroxidase-conjugated secondary goat anti-rabbit or anti-mouse
antibody (Amersham Pharmacia Biotech) at a dilution of 1:1000. The
reaction was then visualized with the enhanced chemiluminescence
detection system (ECL) as recommended by the manufacturer (Amersham
Pharmacia Biotech).
RT-PCR Analysis of HIV RNA--
Total RNA was isolated from
rtTA-Neo and rtTA-2N4 Jurkat cells infected with HIV in the presence
or absence of Dox using the RNeasy Mini Kit (QIAGEN) and treated with 1 unit of RNase-free DNase (RQ1 DNase; Promega Biotech, Madison, WI) for
30 min at 37 °C, phenol:chloroform:isoamyl alcohol-extracted, and
ethanol-precipitated. RT was performed on 2 µg of RNA and 0.2 pmol of
random hexamers using 200 units of Moloney murine leukemia virus
reverse transcriptase (Life Technologies, Inc., Burlington, Ontario,
Canada) in buffer containing 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 10 mM dithiothreitol, 3 mM
MgCl2, 500 nM dNTP, 0.1 mg/ml bovine serum
albumin, 272.5 units/ml RNase inhibitor (Amersham Pharmacia Biotech).
PCR assays were performed using 7 µl of RT product, in PCR buffer
containing 100 mM Tris-HCl, pH 8.4, 500 mM KCl,
15 mM MgCl2, 200 mM dNTP, 15 pmol
of
-32P-labeled primers, and 1.25 units of
Taq DNA polymerase (Amersham Pharmacia Biotech).
Nucleotide sequences of primers used are as follows: M667,
5'-GGCTAACTAGGGAACCCACTG-3'; M668, 5'-CAGGTCCCTGTTCGGGCGCC-3'; LA45,
5'-GCCTTAGGCATCTCCTATGGC-3'; LA41, 5'-TGTCGGGTCCCCTCGTTGCTGG-3'; M669,
5'-GTGTGCCCGTCTGTTGTGTGACTCTGGTAAC-3'; LA23,
5'-GCCTATTCTGCTATGTCGACACCC-3'.
Analysis of HIV-1 LTR- and NF-B-dependent Gene
Expression--
Cells were transiently transfected with HIV LTR CAT or
HIV enhancer containing CAT reporter plasmids (15 µg) by the
DEAE-dextran method. In some experiments, pSVexTat plasmid (wtTat) (48)
was also cotransfected together with the reporter plasmids. At 24 h after transfection, cells were incubated with or without Dox in the
presence of 10 ng/ml TNF-
(Boehringer Mannheim) or PMA (100 ng/ml,
ICN) plus PHA (1 µg/ml, ICN). At 24 h after induction, cells
were harvested and lysed. Extracts (200-400 µg) were assayed for CAT
activity for 4-8 h, depending on the experiment. The percent acetylation was determined by ascending thin layer chromatography as
described previously (49) and quantified using the Bio-Rad Gelscan
phosphor imager and the Molecular Analyst (Bio-Rad) software program.
Electrophoretic Mobility Shift Assay--
Following the addition
of 1 µg/ml Dox to the culture medium for times ranging from 1 to
48 h, nuclear extracts were prepared from rtTA-neo, rtTA-IB-2N,
and rtTA-I
B-2N
4 Jurkat T cells after induction with TNF-
(10 ng/ml) or PMA (100 ng/ml)/PHA (1 µg/ml) for 4 h. Cells were
washed in Buffer A (10 mM HEPES, pH 7.9, 1.5 mM
MgCl2, 10 mM KCl, 0.5 mM
dithiothreitol (DTT), and 0.5 mM phenylmethylsulfonyl fluoride (PMSF)) and were resuspended in Buffer A containing 0.1% Nonidet P-40. Cells were then chilled on ice for 10 min before centrifugation at 10,000 × g. Pellets were then
resuspended in Buffer B (20 mM HEPES, pH 7.9, 25%
glycerol, 0.42 M NaCl, 1.5 mM
MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, 5 µg/ml leupeptin, 5 µg/ml pepstatin, 0.5 mM spermidine, 0.15 mM spermine, and 5 µg/ml
aprotinin). Samples were incubated on ice for 15 min before being
centrifuged at 10,000 × g. Nuclear extract
supernatants were diluted with Buffer C (20 mM HEPES, pH
7.9, 20% glycerol, 0.2 mM EDTA, 50 mM KCl, 0.5 mM DTT, and 0.5 mM PMSF). Nuclear extracts were
subjected to EMSA by using a 32P-labeled probe
corresponding to two copies of the PRDII region of the IFN-
promoter
(5'-GGGAAATTCCGGGAAATTCC-3'). The resulting protein-DNA complexes were
resolved by 5% Tris-glycine gel and exposed to x-ray film. To
demonstrate the specificity of protein-DNA complex formation, a
125-fold molar excess of unlabeled oligonucleotide was added to the
nuclear extract before adding labeled probe.
Analysis of HIV-1 Multiplication--
rtTA-neo-, rtTA-IB-2N-,
and rtTA-I
B-2N
4-expressing Jurkat cells after preincubation with
or without Dox for 24 h were infected with HIV-IIIB, derived from
the HXB2D molecular clone of HIV-1 (50) in serum-free medium for 2 h at a m.o.i. of 0.01 pfu/ml and then grown in complete medium for 36 days. Cell supernatants (precleared by centrifugation at 3000 rpm for
30 min at 4 °C) were collected every 4 days and analyzed for virus
RT activity as described previously (51). The relative amount of virion protein p24 present in the medium was determined by ELISA (52). Proteins were extracted from a portion of the collected cells by
resuspending them in WLB. Proteins were analyzed by immunoblotting as
described above using I
B
MAD10B monoclonal antibody (29), p24-specific antibody (ID Laboratories), anti-RelA(p65) antibody (46),
or actin monoclonal antibody (ICN).
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RESULTS |
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Dox-inducible Expression of TD-IB
Down-regulates Endogenous
I
B
Expression--
To examine the consequences of overexpression
of transdominant repressors of I
B
(TD-I
B
) on
NF-
B-dependent gene expression and virus multiplication,
Jurkat T cell lines were generated that inducibly expressed two forms
of mutant I
B
(TD-I
B
). One form was a full-length I
B
mutated at Ser-32 and Ser-36 (termed I
B
-2N), two sites present in
the signal response domain of I
B
required for inducible
phosphorylation and degradation of I
B
(20-22). The other form of
I
B
(I
B
-2N
4) also contained the S32A/S36A mutations, as
well as a 22-amino acid deletion of the C-terminal portion of I
B
(25, 27), a region of the PEST domain that is dispensable with regard
to binding of NF-
B subunits but is important in I
B
degradation
(Fig. 1A). Starting with
Jurkat T cells selected for expression of the reverse tetracycline
transactivator protein rtTA (53), expressed under the control of the
CMVt autoregulatory promoter (Fig. 1A), we
isolated pools and clones of rtTA-Jurkat cells expressing I
B
-2N
and I
B
-2N
4. The first pool of I
B
-2N
4 cells was
disappointing because of the high level of leakiness of the transgene
(Fig. 1B, lane 6), compared with endogenous
I
B
(Fig. 1B, lanes 1-4). However, analysis
of I
B
expression revealed an interesting modulation of endogenous
I
B
protein level when I
B
-2N
4 was Dox-induced for 24 h. The level of endogenous I
B
was decreased about 4-fold in
Jurkat cells expressing I
B
-2N
4 compared with the I
B
levels in either Jurkat or rtTA-Jurkat cells (Fig. 1B,
lanes 1-4 and 6). Dox induction resulted in a 5-fold increase in I
B
-2N
4 expression after 24 h and an
almost complete inhibition of endogenous I
B
expression (Fig.
1B, lane 5). To characterize this inhibition
further, a representative clone of I
B
-2N
4 Jurkat cells was
Dox-induced for 3-48 h and then treated with TNF
(10 ng/ml) for 5 min as an inducer of I
B
phosphorylation, following a 30-min
pretreatment with calpain inhibitor I (100 µM) to block
inducer-mediated degradation of I
B
(27, 54). The different forms
of I
B
were resolved by 15% SDS-polyacrylamide gel
electrophoresis; both endogenous I
B
and the slower migrating
phosphorylated form of I
B
(P-I
B
) were detected by
immunoblotting (Fig. 1C, lanes 1 and
2). In the I
B
-2N
4-Jurkat clone without Dox
addition, the three forms of I
B
were detected (Fig.
1C, lane 3); Dox addition for 3-6 h resulted in
a 3-5-fold increase in I
B
-2N
4 (Fig. 1C,
lanes 4-6), and a progressive decrease in the endogenous
I
B
forms such that, by 24 and 48 h after Dox addition,
endogenous I
B
was undetectable (Fig. 1C, lanes
4-8). Because of the S32A/S36A mutation in I
B
-2N
4, this
form of I
B
was not phosphorylated as a consequence of TNF addition (20-22). Additionally, a clone of I
B
-2N Jurkat cells was analyzed that demonstrated properties similar to the
I
B
-2N
4 Jurkat cells, although it was not possible to
distinguish the endogenous from the transfected I
B
forms (Fig.
1D, lanes 1 and 2). With this clone,
the level of leakiness of the TD-I
B
appears to be significantly
lower than that observed with I
B
-2N
4, since the amounts of
I
B
in the control rtTA-Jurkat and I
B
-2N-Jurkat cells in the
absence of Dox were similar (Fig. 1D, lanes 1 and 2). Nevertheless, Dox induction resulted in a 20-25-fold
increase in the overall level of I
B
in these cells (Fig.
1D, lanes 2-7). Based on these results, we have
isolated clones of Jurkat cells inducibly expressing TD-I
B
under
the control of the Tet-responsive promoter. Furthermore, Dox induction
of TD-I
B
resulted in the inhibition of endogenous I
B
,
consistent with the fact that the I
B
gene is tightly regulated by
an NF-
B-dependent mechanism (32).
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Growth of TD-IB
-expressing Cells and Induction of
Apoptosis--
All stably transfected TD-I
B
-expressing
clones grew well in 10% serum, and growth was not dramatically
retarded as a consequence of Dox addition or, in the case of
I
B
-2N or I
B
-2N
4-Jurkat cells, by up-regulation of
TD-I
B
expression (doubling time of 50 ± 4 h). Based on
recent observations that NF-
B may play a protective role in
apoptotic cell death (55-57), the response of control and
TD-I
B
-expressing Jurkat cells to TNF-induced apoptosis was
examined by TUNEL assay. At 16 h after TNF (100 ng/ml) treatment in Dox-induced cells, 65-80% of the TD-I
B
-expressing cells were undergoing apoptotic cell death, whereas in Dox-induced
TD-I
B
-expressing cells without TNF treatment, less than 2% of
the cells were apoptotic (Table I).
Similarly, TNF treatment alone resulted in 2% apoptosis in
TD-I
B
-expressing cells and 6% apoptotic cells in rtTA-Neo Jurkat
cells (Table I). Dox induction of TD-I
B
expression resulted in an
increased sensitivity to TNF-induced apoptosis, as detected by DNA
fragmentation analysis at 8 or 16 h after TNF addition in the
TD-I
B
-expressing cells but not in the control rtTA-Neo cells
(data not shown). It appears that Dox induction of TD-I
B
expression does not lead to apoptosis per se but, instead,
dramatically sensitizes Jurkat cells to TNF-induced apoptosis. This
observation may explain the observed selection against cells that
constitutively express TD-I
B
(see below).
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Inhibition of NF-B DNA Binding Activity and Gene
Expression by TD-I
B
--
The induction of NF-
B DNA binding
activity in control and TD-I
B
-expressing cells was examined
following treatment of cells with several different inducers (Fig.
2). Treatment of rtTA-neo-Jurkat (Fig.
2A) or rtTA-Jurkat (data not shown) with TNF
or PMA/PHA for 4 h resulted in a strong induction of NF-
B DNA binding
activity, irrespective of Dox treatment for 6-48 h (Fig.
2A, lanes 5-14). In contrast, TNF
induction
of NF-
B activity in I
B
-2N
4 Jurkat cells (Fig.
2B, lanes 5 and 6) was completely
blocked by the induction of I
B
-2N
4 for 6 h or longer
(Fig. 2B, lanes 6-9); the strong induction of
NF-
B binding activity by PMA/PHA was also more than 95% inhibited
by Dox activation of I
B
-2N
4 (Fig. 2B, lanes
10-14). Similarly, Dox induction of I
B
-2N also inhibited
completely the induction of NF-
B binding activity by TNF
(Fig.
2C, lanes 2-6) and PMA/PHA (Fig. 2C,
lanes 7-12). In a subsequent experiment, the kinetics of
activation of I
B
-2N
4 and the inhibition of NF-
B binding
activity were examined (Fig. 2, D and E).
Surprisingly, as early as 1 h after Dox addition, expression of
I
B
-2N
4 was up-regulated (Fig. 2E, lanes
1 and 2) and NF-
B DNA binding activity was inhibited
(Fig. 2D, lanes 1 and 2).
Interestingly, comparison of the levels of I
B
-2N
4 and
endogenous I
B
revealed that constitutive expression of
I
B
-2N
4 was dramatically decreased with time in culture
(compare lane 6 in Fig. 1B and lane 1 in Fig. 2E), whereas the cells remained highly inducible in
response to Dox addition (Fig. 2E, lanes 2-9).
Low background, high inducibility of I
B
-2N was also observed in
I
B
-2N-Jurkat cells, based on the resistance of the induced
I
B
to TNF
-induced degradation (data not shown). TD-I
B
-expressing Jurkat cells were also resistant to induction of
NF-
B binding activity by double-stranded RNA (poly(I·C)) treatment (data not shown). Thus, overexpression of TD-I
B
blocks the
induction of NF-
B DNA binding activity by multiple inducers.
Additionally, continued growth in culture selects for Jurkat cells that
display low constitutive expression of TD-I
B
, possibly due to an
increased sensitivity of TD-I
B
-expressing cells to apoptosis.
|
|
Inhibition of de Novo HIV-1 Multiplication in
TD-IB
-expressing Jurkat Cells--
Given the involvement of
NF-
B in the early transcriptional control of HIV-1 LTR gene
expression (reviewed in Ref. 1), the impact of TD-I
B
expression
on the course of de novo HIV-1 protein synthesis and virus
production was next examined. Control and TD-I
B
-expressing Jurkat
cells after preincubation with or without Dox for 24 h were
infected with the HIV-1 strain IIIB (derived from the molecular clone
HXB2D) at an m.o.i. of 0.01 pfu/ml and HIV-1 infection was monitored by
RT assay, p24 ELISA, and p24 antigen accumulation over periods varying
from 16 days to 36 days depending on the infection. Both RT (Fig.
4A) and p24 ELISA analyses
(Fig. 4B) demonstrated that continuous Dox-induced expression of I
B
-2N
4 resulted in a dramatic delay in the onset of HIV-1 multiplication. In control rtTA-Neo-Jurkat cells, RT and p24
expression were detected as early as 8 days post-infection (p.i.),
whereas in I
B
-2N
4 cells infection was first detected at day
12; addition of Dox at different times during the infection delayed the
onset of detectable HIV-1 multiplication until 16-20 days (Fig. 4,
A and B). The most effective inhibitory regimen was Dox addition 24 h before infection (day
1) and subsequent Dox addition to the medium at days 8, 18, and 24. One addition of Dox
at day
1 or addition of Dox at day 12 also effectively blocked the
onset of HIV-1 replication. However, with these treatments, breakthrough of HIV-1 multiplication was observed at day 36, as detected by RT and p24 ELISA. On the other hand, intermittent replenishment of Dox in the medium dramatically repressed HIV-1 RT and
p24 expression throughout the course of infection (Fig. 4, A
and B).
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|
Decreased Levels of HIV-1 mRNA in TD-IB-expressing Jurkat
Cells--
To link the inhibition of HIV-1 multiplication with NF-
B
inhibitory effects of TD-I
B, the effects of TD-I
B induction on HIV-1-induced NF-
B binding activity were evaluated by mobility shift
analysis. Virus-induced activation of NF-
B was blocked by TD-I
B
expression (data not shown), thus complementing the results described
in Fig. 2, and confirming that TD-I
B interfered with HIV-induced
NF-
B binding. To further characterize the block in HIV-1
multiplication, accumulation of HIV-1 viral RNA species was evaluated
by semi-quantitative RT-PCR, using the primers and procedures described
previously (58). The primer pair M667/M668 detected total viral RNA
with a fragment length of 161 bp; M669/LA23 identified a fragment of
214 bp, which represented singly spliced (env) and doubly
spliced tat/rev RNA; and LA41/LA45 amplified a fragment of
123 bp, corresponding to doubly spliced tat/rev RNA (Fig.
6A). Viral RNA species in
rtTA-Neo-Jurkat and rtTA-2N
4-Jurkat cells were detected at different
times after de novo HIV infection in the presence or absence
of Dox-induced TD-I
B (Fig. 6B). In rtTA-Neo Jurkat cells,
HIV mRNA transcripts were detected as early as 2-4 days after
infection (Fig. 6B, lanes 2, 3,
7, and 8) and their levels increased at days 6 and 10 post-infection (Fig. 6B, lanes 4,
5, 9, and 10), regardless of Dox
addition. In rtTA-2N
4 Jurkat cells, without Dox addition, a low
level of full-length viral RNA was detected at day 2 (Fig.
6B, lane 12, upper panel) and by day 4 unspliced and spliced viral RNA was detected (Fig. 6B,
lane 13) and accumulated thereafter at days 6 and 10 (Fig. 6B, lanes 14 and 15). However, with
Dox induction of TD-I
B, viral RNA levels were reduced (Fig.
6B, lanes 17-20). Full-length RNA was detected
at day 2 (Fig. 6B, lane 17, upper
panel), but the overall levels of unspliced and spliced RNA were
decreased at days 6 and 10, relative to the levels detected in the
absence of Dox or in rtTA-Neo cells. Strikingly, doubly spliced
tat/rev mRNA was not detected in rtTA-2N
4 Jurkat
cells treated with Dox (Fig. 6B, lanes 18-20,
lower panel). These results demonstrate a transcriptional
inhibitory effect of TD-I
B expression on HIV mRNA accumulation
in Jurkat cells engineered to express TD-I
B.
|
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DISCUSSION |
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The present studies describe, for the first time, the selection of
Jurkat T clones engineered to inducibly express two transdominant repressor forms of IB
under the control of a Tet-responsive promoter and the first characterization of the impact of TD-I
B repressors on the course of de novo HIV-1 infection. We
demonstrate that: 1) doxycycline-inducible expression of I
B
-2N
and I
B
-2N
4 was detected as early as 1 h after Dox
addition and blocked the induction of NF-
B DNA binding by multiple
inducers including TNF
, PMA/PHA, and double-stranded RNA; 2)
expression of TD-I
B
also repressed the expression of the
endogenous I
B
gene, consistent with the observation that the
I
B
gene contains multiple NF-
B binding sites in its promoter
and is tightly regulated by NF-
B (32); 3)
NF-
B-dependent reporter gene activity was also inhibited following the induction of TD-I
B
; and 4) induced expression of
the TD-I
B
successfully inhibited de novo HIV-1
infection in Jurkat cells as measured by RT assay, p24 antigen
accumulation, and viral RNA analysis by RT-PCR. During these studies,
the levels of CD4 and fusin were also measured by flow cytometry to
determine if TD-I
B
expression repressed cell surface expression
of the HIV-1 co-receptors (59); no modulation of either CD4 or fusin levels was observed after 48-96 h of Dox-induced TD-I
B
expression.2 Thus, HIV-1
infection was not inhibited at the level of virus attachment and
entry.
Our results are complementary to the recent studies of Chen et
al. (37), demonstrating the importance of NF-B sites in enhancing the growth of primary HIV-1 isolates; nonetheless, the virus
was still able to grow slowly in the absence of NF-
B sites. Similarly, conditional inhibition of NF-
B in Jurkat T cells by an
I
B
repressor significantly interfered with virus multiplication, but could not suppress HIV-1 growth indefinitely. Many earlier studies
in lymphoid cells have also shown that mutation of NF-
B motifs
reduced gene expression in the presence or absence of HIV-1 Tat (see
Refs. 36-38, and references therein). Strikingly different
requirements for maximal LTR activation were observed in primary CD4+ T
cells and the J-Jhan T lymphoid cells line (36). In unstimulated CD4+ T
lymphocytes, a low basal level of LTR activity was detected, whereas,
in the lymphoblastoid cell line, a high spontaneous level of LTR
activity was found that was essentially independent of the NF-
B
responsive elements. In lymphoblastoid cell lines, HIV infection
resulted in active replication in the absence of other stimuli,
whereas, in primary T cells, replication was dependent upon T cell
activation for triggering of viral replication (36).
In previous studies, the effect of transdominant repressors of IB
on the synergistic activation of the HIV-1 LTR by TNF
and the HIV-1
transactivator, Tat, was examined in transiently transfected Jurkat T
cells (38). Co-expression of I
B
inhibited Tat-TNF activation of
HIV LTR in a dose-dependent manner, and transdominant
repressor forms of I
B
, mutated in serine or threonine residues
required for inducer-mediated (S32A/S36A-2N) or constitutive phosphorylation (S283A/T291A/T299A-3C) of I
B
, possessed different inhibitory potentials for the HIV-1 LTR. Surprisingly, I
B
-2N (but
not I
B
-2N+3C) was more effective in blocking HIV-1 protein and
RNA synthesis in a single cycle infection than wtI
B
. The observation that mutations in the C-terminal PEST domain of I
B
decreased the inhibitory potential of I
B
-2N suggested that an intact C terminus was required for maximal inhibition of HIV-1 multiplication by I
B
-2N and may reflect a distinct functional activity for the I
B
C terminus (38). Although the above studies suggest a transcriptional role for I
B
in the inhibition of HIV-1 LTR gene expression, I
B
may act at a distinct level in the HIV-1 life cycle, at the post-transcriptional level of Rev function (60, 61).
HIV Rev contains an RNA binding domain, required for interaction with
HIV-1 RNA, and an effector domain, required for RNA-bound Rev to
function in export. The Rev effector domain contains a nuclear export
signal (NES) and interacts with the nucleoporin Rab/Rip, a protein that
mediate nucleocytoplasmic transport (62-64). I
B
also contains a
consensus NES element at amino acids 264-281, matching the NES
consensus within Rev (65). Furthermore, newly synthesized I
B can
localize to the nucleus, dissociate NF-
B-DNA complexes, and
translocate back to the cytoplasm (66). Thus, an additional function of
I
B is to serve as a shuttle protein to export NF-
B from the
nucleus. One possible explanation for the inhibitory activity of
TD-I
B in HIV-1 infection is that the stable form of I
B competes
effectively for the nuclear export pathway utilized by Rev/Rab.
HIV-1 infection also causes constitutive activation of NF-B
DNA-binding activity in infected cells (1). A direct temporal correlation exists between HIV infection and the appearance of NF-
B
DNA-binding activity in myeloid cells (39-43, 67), which may in turn
prime or stimulate cytokine release (59). HIV-1-induced cytokine
release may account for the elevated levels of multiple cytokines and
chemokines present in the sera of AIDS patients in late stage disease
and may exacerbate symptoms (reviewed in Ref. 1). Preliminary data
suggest that TD-I
B
also interferes with HIV-1 infection at the
level of expression of HIV-1-induced inflammatory
cytokines.3
Recently, NF-B activation was shown to play a protective function in
the response to TNF
-, ionizing irradiation- and daunorubicin-induced apoptosis (55-57). Since apoptosis has been suggested to be one of the
major mechanisms of CD4+ T cell depletion in HIV-1-infected patients
(68-71) and because TNF
plasma levels correlate with disease
progression (1), the role of NF-
B in HIV-1-induced apoptosis was
examined (72). These studies demonstrated
that constitutive NF-
B activation is required to counteract a
persistent apoptotic signal resulting from HIV-1 infection; thus, a
previously unrecognized role for constitutive NF-
B activation in
HIV-1-infected cells is to protect from virus-mediated apoptotic cell
death. Similarly, activation of TD-I
B
sensitized Jurkat cells to
TNF-induced cell death (Table I). Thus, given the multiple functions of
the NF-
B/I
B transcription factors, Dox-induced TD-I
B
expression may interfere with HIV-1 multiplication at several levels:
LTR-mediated transcription, Rev-mediated export of viral RNA,
inhibition of HIV-1-induced pro-inflammatory cytokines, and increased
sensitivity of HIV-1-infected cells to apoptosis. Experiments are in
progress to assess the relative contribution of these processes to the capacity of TD-I
B
molecules to interfere with HIV-1
multiplication.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Ron Hay for the MAD10B antibody and members of the McGill AIDS Center for helpful discussions.
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FOOTNOTES |
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* This work was supported by a grant from the Medical Research Council of Canada (to J. H. and M. A. W.), a grant from the National Cancer Institute (to J. H.), a grant from the Canadian Foundation for AIDS Research (to J. H. and M. A. W.), a FCAR studentship (to H. K.), a NHRDP studentship (to C. D. L.), a Fraser Monat McPherson fellowship from McGill University (to R. L.), and a Medical Research Council Scientist award (to J. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: Lady Davis Institute for Medical Research, 3755 Cote Ste. Catherine, Montreal, Quebec H3T 1E2, Canada. Tel.: 514-340-8222 (ext. 5265); Fax: 514-340-7576; E-mail: mijh{at}musica.mcgill.ca.
1
The abbreviations used are: HIV-1, human
immunodeficiency virus type 1; LTR, long terminal repeat; TNF, tumor
necrosis factor; RT, reverse transcription; PCR, polymerase chain
reaction; ELISA, enzyme-linked immunosorbent assay; pfu, plaque-forming
unit(s); m.o.i., multiplicity of infection; PMA, phorbol 12-myristate
13-acetate; PHA, phytohemagglutinin; Dox, doxycycline; CMV,
cytomegalovirus; FBS, fetal bovine serum; DTT, dithiothreitol; PMSF,
phenylmethylsulfonyl fluoride; PBS, phosphate-buffered saline; CAT,
chloramphenicol acetyltransferase; bp, base pair(s); p.i.,
post-infection; NES, nuclear export signal; TD-IB
, transdominant
repressor of I
B
.
2 H. Kwon, data not shown.
3 H. Kwon and P. Genin, unpublished data.
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REFERENCES |
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