Nuclear I
B
Maintains Persistent NF-
B Activation in
HIV-1-infected Myeloid Cells*
Carmela
DeLuca
§¶,
Louisa
Petropoulos
,
Dana
Zmeureanu
, and
John
Hiscott
§
**
From the
Lady Davis Institute for Medical Research,
Sir Mortimer B. Davis Jewish General Hospital and the Departments of
Microbiology & Immunology and § Medicine and the
** McGill AIDS Centre, McGill University, Montreal, Quebec H3T 1E2,
Canada
 |
ABSTRACT |
Monocytic cells exhibit constitutive NF-
B
activation upon infection with human immunodeficiency virus-1 (HIV-1).
Because I
B
has been implicated in maintaining NF-
B·DNA
binding, we sought to investigate whether I
B
was involved in
maintaining persistent NF-
B activation in HIV-1-infected monocytic
cell lines. I
B
was present in the nucleus of HIV-1-infected cells
and participated in the ternary complex formation with NF-
B and DNA.
In contrast to uninfected cells, the addition of recombinant
glutathione S-transferase-I
B
protein to preformed
NF-
B·DNA complexes from HIV-1-infected cell extracts did not
completely dissociate the complexes, suggesting that I
B
may
protect NF-
B complexes from I
B
-mediated dissociation. Immunodepletion of I
B
resulted in an NF-
B·DNA binding
complex that was sensitive to I
B
-mediated dissociation, thus
demonstrating the protective role of I
B
. In addition,
co-transfection studies with an NF-
B-dependent reporter
construct demonstrated that I
B
co-expression partially alleviated
inhibition of NF-
B-mediated gene expression by I
B
, implying
that I
B
can maintain transcriptionally active NF-
B·DNA
complexes. Furthermore, constitutive phosphorylation of I
B
was
observed. Immunoprecipitation of the I
B kinase (IKK) complex
followed by in vitro analysis of kinase activity
demonstrated that IKK was constitutively activated in HIV-1-infected
myeloid cells. Thus, virus-induced constitutive IKK activation, coupled with the maintenance of a ternary NF-
B·DNA complex by I
B
,
maintains persistent NF-
B activity in HIV-1-infected myeloid cells.
 |
INTRODUCTION |
HIV-11-infected cells of
the myeloid lineage serve as intracellular reservoirs for virus
dissemination (1-4). Infection of monocytic cells leads to the
deregulation of numerous immunoregulatory functions and aberrant
expression of inflammatory cytokines (5, 6), which may further their
ability to spread virus and cause disease progression. The NF-
B/Rel
family of transcription factors participates in the activation of a
number of host immunoregulatory cytokine genes (reviewed in Refs. 7 and
8), and its perturbation by HIV-1 infection leads to altered gene
expression. In HIV-1-infected myeloid cell lines that express
constitutive NF-
B·DNA binding activity (9-12), NF-
B strongly
induces HIV long terminal repeat-driven gene expression (9, 13, 14) and
maintains cell viability (15).
NF-
B consists of five family members including RelA and c-Rel, which
contain transcriptional activation domains p100 and p105 precursor
proteins, which are cleaved to the active members, p52 and p50,
respectively, and RelB (reviewed in Refs. 7, 8, and 16). In most cells,
NF-
B is found sequestered in the cytoplasm by inhibitory I
B
proteins. Several I
B proteins have been identified including
I
B
, I
B
, and most recently, I
B
(17). The precursor proteins p100 and p105 can also serve as functional I
B molecules, retaining NF-
B in the cytoplasm although their regulation is less
well understood. Serine phosphorylation of I
B
at Ser-32 and
Ser-36 represents the critical regulatory signal leading to ubiquitin-dependent, proteosome-mediated degradation of
I
B, which allows NF-
B to translocate to the nucleus and activate
NF-
B-dependent genes. Recently, several groups have
identified the I
B kinase complex (IKK) (18-23), a multisubunit
complex that phosphorylates both I
B
and I
B
(24, 25).
Several pathways of NF-
B activation are thought to converge at the
level of IKK activation, implicating this complex as a critical
regulator of NF-
B transcriptional regulation.
The multimeric IKK complex includes two subunits, IKK
and IKK
,
which are responsible for phosphorylating I
B molecules. Several
other components of the IKK complex have been identified including the
regulatory subunit NEMO (NF-
B essential modulator, also called
IKK
) (26, 27) and a scaffolding protein, IKAP (IKK-associated
protein) (28), which binds the IKK subunits and, together with
NF-
B-inducing kinase (NIK), assembles them into an active kinase
complex. The IKK complex is rapidly stimulated by TNF
, IL-1, and
PMA, although the mechanism of activation requires further elucidation.
Recently, NIK was found to activate IKK
directly (29), confirming
earlier reports that NIK co-expression leads to IKK
phosphorylation
(19). MEKK-1 has also been found tightly associated in the IKK complex
(21) and has been shown to stimulate IKK activity (30). Further studies
are required to determine whether these kinases are essential upstream
regulators of IKK activity.
Rapid resynthesis of I
B
establishes an autoregulatory loop
whereby NF-
B activation is self-limited. Unlike I
B
, I
B
is not an NF-
B-regulated gene and is not rapidly resynthesized after inducer-mediated degradation (31). Several inducers, however, result in
persistent NF-
B activation, which has been associated with the
additional release of NF-
B from I
B
complexes and its resynthesis in a hypophosphorylated form that sustains NF-
B
activation (32, 33). Hypophosphorylated I
B
can bind NF-
B
without masking its nuclear localization signal (32), thus acting as a
chaperone for NF-
B nuclear entry and preventing its sequestration by
I
B
. Recently, two isoforms of I
B
that differ in their
C-terminal PEST domain as a consequence of alternative splicing have
been identified in human cells (34). The larger protein, approximately 43 kDa, is homologous to the murine I
B
, whereas the 41-kDa form is unique to human cells. The 43-kDa protein degrades upon stimulation and enters the nucleus when hypophosphorylated, whereas the 41-kDa protein is resistant to degradation by several inducers and is found
only in the cytoplasm (34).
In this report, the mechanism underlying constitutive activation of
NF-
B in HIV-1-infected myeloid cells has been examined. We
demonstrate that the association of nuclear I
B
with NF-
B·DNA complexes maintains persistent NF-
B·DNA binding activity, and we
show that the IKK complex is constitutively activated in HIV-1-infected cells. Constitutive IKK activity and protection of NF-
B·DNA
activity from I
B
-mediated dissociation by nuclear I
B
may
explain the persistent NF-
B-dependent gene activation
observed in HIV-1-infected cells.
 |
MATERIALS AND METHODS |
Cell Culture--
Promonocytic U937 and HIV-1-infected U9-IIIB
cells, as well as myelomonoblastic PLB-985 and HIV-1-infected PLB-IIIB
cells (infected with HIV strain IIIB), were maintained in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 5% Fetal Clone I
(Hyclone), 2 mM L-glutamine, and 20 µg/ml
gentamicin (Schering Canada). Human embryonic kidney 293 cells were
cultured in modified Eagle's medium (
-MEM) plus 10% fetal bovine
serum (Biomedia Canada), 2 mM L-glutamine, and
20 µg/ml gentamicin.
GST Fusion Proteins--
I
B
human cDNA bearing a
22-amino acid C-terminal truncation in the PEST domain (I
B-
4,
which inhibits NF-
B binding as efficiently as wild type) was
subcloned into pGEX 2T as described previously (35).
GST·I
B
-(1-55) and GST·I
B
-(1-55) (S32A/S36A) were a kind
gift from Antonis Koromilas. DH5
bacteria expressing GST·proteins
were grown in Luria broth, washed with phosphate-buffered saline,
resuspended in 10% Triton in phosphate-buffered saline, and sonicated.
Protein was recovered using Sepharose 4B glutathione beads (Amersham
Pharmacia Biotech) and eluted with 20 mM GSH (Calbiochem) in 50 mM Tris, pH 8.0. Isolation purity and quantity were
confirmed by SDS-polyacrylamide gel electrophoresis, Coomassie Blue
staining, and a visual comparison with bovine serum albumin standards.
Electromobility Shift Assay--
Nuclear extracts were prepared
from untreated cells or cells treated for varying times with one of the
following inducers: TNF
(10 ng/ml, R & D Systems), PMA (50 ng/ml,
Sigma), or IL-1
(5 ng/ml, R & D Systems). An electrophoretic
mobility shift assay was carried out using an NF-
B
32P-labeled probe corresponding to the PRDII region of the
IFN-
promoter (5'-GGAAATTCCGGGAAATTCC-3') as described (5). In
supershift experiments, the antibody (36) and its corresponding peptide where indicated (Santa Cruz Biotechnology), were incubated with 5 µg
of nuclear extract for 20 min. Poly(dI·dC) (5 µg) was added for an
additional 10 min followed by incubation with labeled probe for 20 min.
All steps were carried out at room temperature. In other experiments in
which GST fusion proteins were used, nuclear extracts were incubated
with poly(dI·dC) for 10 min and then incubated with labeled probe for
20 min. Increasing amounts of GST·I
B
4 (10 ng/µl) were
added for an additional 20 min. The resulting protein-DNA complexes
were resolved by a 5% Tris/glycine gel and exposed to x-ray film. To
demonstrate the specificity of protein-DNA complex formation, 125-fold
molar excess of unlabeled oligonucleotide was added to the nuclear
extract before adding the labeled probe.
Immunoblot Analysis--
Whole cell extracts were prepared by
resuspending the cells in Nonidet P-40 lysis buffer and examined by
Western blot analysis as described previously (12). I
B
antibodies
C20 (recognizes the 43-kDa isoform) and G20 (recognizes both the 43- and 41-kDa forms) were purchased from Santa Cruz Biotechnology. The
phosphoserine 32 I
B
antibody was purchased from New England
Biolabs, and the monoclonal I
B
antisera MAD-10B was a kind gift
from Ron Hay (37). The
-tubulin antibody was obtained from ICN and
the actin antisera from Sigma. The ECL-Western blotting detection
system (NEN Life Science Products) was used according to
manufacturer's instructions to visualize the specific signals.
Transfections and CAT Assays--
Human embryonic kidney 293 cells were transfected in 100-mm plates by the calcium phosphate DNA
precipitate transfection method. Each plate was transfected with 7 µg
of NF-
B CAT (PRDII element of the IFN
gene linked to CAT) and
either 4 µg of pSVK3-I
B
and 4 µg of pSVK3 empty vector, with
4 µg of pSVK3-I
B
and 4 µg of pSVK3-I
B
, or 8 µg of
empty vector. Cells were incubated with precipitate for 12 h after
which time they were washed with phosphate-buffered saline and fed with
fresh medium. Cells were maintained for an additional 36 h and
stimulated with PMA (50 ng/ml) for the last 24, 16, or 8 h of the
transfection. CAT activity was determined using 100 µg of total cell
extract assayed for 2 h at 37 °C. Quantitation of activities
was performed using NIH Image 1.60 software. The fold activation
reported is the average of a minimum of three experiments with error
bars representing the standard deviation.
IKK Assay--
Cells were pelleted by centrifugation and washed
with ice-cold phosphate-buffered saline. Pellets were resuspended in
TNN buffer (20 mM Tris, pH 7.5, 200 mM NaCl,
0.5% Nonidet P-40, 0.5 mM phenylmethylsulfonyl fluoride, 5 µg/ml each of aprotinin, leupeptin, and pepstatin, 0.5 mM
spermine, 0.15 mM spermidine, 1 mM NaF, and 1 mM NaVO4) and incubated on ice for 10 min.
Supernatants were removed after centrifugation (14,000 rpm, 10 min,
4 °C) and assayed for protein concentration. 150-300 µg of
protein extract was incubated with 5 µl of IKK
antibody (Santa
Cruz Biotechnology) or 2 µl of normal rabbit serum for 2 h while
rotating at 4 °C. 20 µl of protein A-conjugated Sepharose beads,
washed three times with TNN buffer, were added to the mixture and
incubated with rotation at 4 °C for an additional hour. Beads were
pelleted (1000 rpm) and washed once with TNN and twice with kinase
buffer (50 mM Tris, pH 8.0, 100 mM NaCl, 2 mM MgCl2, 0.5 mM
phenylmethylsulfonyl fluoride, 4 µg/ml chymostatin, 5 µg/ml each of
pepstatin, leupeptin, and aprotinin, and 1 mM each
dithiothreitol, NaF, and NaVO4). Beads were resuspended in
20 µl of kinase mix containing 2 µg of GST·I
B
-(1-55) or
I
B
-(1-55) (S32A/S36A) and 0.5 µCi of
-ATP and incubated for
30 min at 30 °C. 20 µl of 2× SDS loading dye was added to each
reaction, boiled for 5 min, and separated on a 10% SDS-polyacrylamide
gel. The gel was fixed and Coomassie-stained (5% acetic acid, 10%
ethanol, Coomassie Blue dye). It was subsequently destained (5% acetic
acid, 10% ethanol), dried, and exposed to film for 1-3 h at
80 °C.
 |
RESULTS |
I
B
Is Part of the NF-
B·DNA Binding Complex in
HIV-1-infected Cells--
Previous studies demonstrated that myeloid
cell lines PLB-985 and U937 acquire constitutive NF-
B·DNA binding
activity upon HIV-1 infection (9-11, 15). Analysis of this complex
revealed that the DNA binding activity was composed predominantly of
RelA and p50 heterodimers with a minor contribution by c-Rel and p50 heterodimers (10-12). To investigate the possibility that I
B
may
be involved in maintaining this persistent activation, nuclear extracts
prepared from HIV-1-infected PLB-IIIB and U9-IIIB cells were analyzed
for DNA binding levels by electrophoretic mobility shift assay.
Analysis of NF-
B·DNA binding activity, using an I
B
-specific
antibody that recognized the 43-kDa isoform of I
B
, demonstrated
that I
B
protein was a part of the DNA binding complex (Fig.
1A, lanes 2 and
10) and could be detected in cells stimulated with TNF
or
IL-1
for 6 h (Fig. 1A, lanes 5 and
7). Preincubation with the cognate peptide recognized by the
I
B
antibody demonstrated the specificity of antibody recognition
(Fig. 1A, lanes 3 and 11), whereas
incubation with excess unlabeled NF-
B probe competed the
NF-
B-specific complexes (Fig. 1A, lane 8).
Similar experiments using I
B
did not produce a shifted complex
(data not shown), suggesting that I
B
was uniquely present in
HIV-1-infected cells. Uninfected PLB-985 and U937 cells stimulated with
TNF
or PMA for 18 h (Fig. 1B, lanes 9,
14, and 16), but not cells treated for shorter
times (Fig. 1B, lanes 3 and 5),
likewise exhibited an NF-
B·DNA binding complex that could be
supershifted with I
B
antibody. Induction of PLB-IIIB cells with
TNF
or PMA for 0, 2, 4, 8, 12, or 18 h revealed that I
B
remained part of the DNA binding complex over the course of induction
(Fig. 1C and data not shown).

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Fig. 1.
Electrophoretic mobility shift assay of
HIV-1-infected myeloid cells for
I B -containing
NF- B·DNA binding complexes.
A, nuclear extracts from PLB-IIIB cells were subjected to
supershift analysis using an I B -specific antibody
(Ab). Extracts were either unstimulated (control
(CON), lanes 1-3) or stimulated for 6 h
with IL-1 (lanes 4 and 5) or TNF
(lanes 6 and 7). I B supershifted bands are
identified by the upper arrow. Antibody specificity was
confirmed by preincubating the antibody with cognate peptide
(lane 3). Competition of TNF -stimulated extract with
excess unlabeled probe demonstrated specificity of binding (lane
8). Unstimulated U9-IIIB nuclear extract (lanes 9-11)
was incubated with I B antibody (lane 10) or antibody
preincubated with peptide (lane 11) and analyzed as above.
B, uninfected cells exhibit I B NF- B·DNA binding
complexes upon stimulation. Nuclear extracts from PLB-985 cells or U937
cells were unstimulated (lanes 1 and 12),
stimulated with TNF for 2 h (T2, lanes
2-3) or 18 h (T18, lanes 6, 7, 13, 14, 17, and 18), or stimulated with PMA for 2 h
(P2, lanes 4 and 5) or 18 h
(P18, lanes 8-11, 15, and 16).
Extracts were incubated alone (lanes 1, 2, 4, 6, 8, 11-13,
15, and 18), with I B antibody (lanes 3, 5, 7, 9, 14, and 16), or with I B antibody and
peptide (lanes 10 and 17). PMA-stimulated extract
(lane 11) or TNF -induced extract (lane 18)
were competed with cold probe. C, I B -NF- B·DNA
binding complex is not disrupted by stimulation with TNF or PMA.
PLB-IIIB cells were stimulated with TNF (lanes 1-6) or
PMA (lanes 7-12) for 0 or 18 h. Extracts were
incubated alone (lanes 1, 3, 7, and 9) or with
I B antibody (lanes 2, 4, 8, and 10) or
I B antibody and peptide (*, lanes 5 and
11). I B -containing complexes are identified with an
arrow. Specificity was confirmed by incubating with excess
unlabeled probe (*, lanes 6 and 12).
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|
The presence of I
B
in the nuclear compartment of HIV-1-infected
myeloid cells was confirmed by biochemical fractionation. Cytoplasmic
and nuclear extracts from PLB-IIIB and PLB-985 cells stimulated with
TNF
or PMA were separated by SDS-polyacrylamide gel electrophoresis
and immunoblotted for I
B
. Whereas I
B
was present in the
cytoplasm and nucleus of HIV-1-infected cells (Fig. 2A, lanes 1 and
6), I
B
was predominantly cytoplasmic in noninfected PLB-985 cells (Fig. 2B, lanes 1 and
6). I
B
was present in the nucleus of HIV-1-infected
cells after stimulation with TNF
or PMA (Fig. 2A,
lanes 7-10), and low levels were also detected in stimulated PLB-985 cells (Fig. 2B, lanes 7-10).
Nuclear extracts were shown to be free of cytoplasmic contamination by
reprobing with
-tubulin antibody (Fig. 2, A and
B, lower panels). Similar results were obtained
with U937 and U9-IIIB cells (data not shown).

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Fig. 2.
I B is found in
the nucleus of HIV-1-infected cells. PLB-IIIB (A) or
PLB-985 cells (B) were treated with TNF (lanes 2, 3, 7, and 8) or PMA (lanes 4, 5, 9, and
10) for 0, 2, or 18 h. Cytoplasmic extract (40 µg)
(lanes 1-5) or nuclear extract (40 µg) (lanes
6-10) was separated by SDS-polyacrylamide gel electrophoresis and
immunoblotted for I B (upper panels). The blots were
stripped and reprobed for -tubulin (lower panels).
|
|
I
B
Protects NF-
B·DNA Complexes from I
B
-mediated
Dissociation--
Because previous in vitro studies have
demonstrated that NF-
B·DNA complexes are sensitive to dissociation
by I
B
(38, 39), the possibility that I
B
protects
NF-
B·DNA complexes from I
B
dissociation was evaluated. The
NF-
B·DNA binding complex from HIV-1-infected U9-IIIB cells was
resistant to GST·I
B
-mediated dissociation (Fig.
3A, lanes 1-3).
Furthermore, GST·I
B
did not reduce NF-
B binding to levels
lower than those observed in unstimulated HIV-1-infected cells (Fig.
3A, lanes 4-15). Similar results were obtained
with PLB-IIIB cells (Fig. 3B), suggesting this phenomenon was a property of HIV-1-infected myeloid cells. In contrast,
NF-
B·DNA complexes from TNF
- or PMA-stimulated PLB-985 and U937
cells were completely dissociated by GST·I
B
(Fig. 3,
C and D, lanes 2, 3, 5, and
6).

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Fig. 3.
Recombinant
I B does not
dissociate preformed NF- B·DNA complexes in
HIV-1-infected cells. U9-IIIB (A) and PLB-IIIB
(B) cells were untreated (control (CON),
lanes 1-3), treated with TNF for 2 h
(T2, lanes 4-6) or 18 h (T18,
lanes 7-9) or with PMA for 2 h (P2,
lanes 10-12) or 18 h (P18, lanes
13-15). Nuclear extracts were incubated with labeled PRDII probe
followed by incubation with increasing amounts of GST·I B .
PLB-985 (C) and U937 (D) cells were stimulated
with TNF for 2 h (T2, lanes 1-3) or
18 h (T18, lanes 4-6) and treated as
described in A.
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Next, nuclear extracts were immunodepleted of I
B
using an
I
B
-specific antibody and analyzed for NF-
B·DNA binding.
Incubation of PMA-stimulated U9-IIIB nuclear extracts with
GST·I
B
reduced the amount of NF-
B·DNA binding complexes
but did not completely dissociate NF-
B binding activity (Fig.
4A, lanes 1-6). In
contrast, extracts immunodepleted for I
B
were sensitive to
GST·I
B
-mediated dissociation (Fig. 4A, lanes
9-14). In this case, levels of NF-
B binding were reduced to
the levels observed in uninfected U937 cells (Fig. 3D),
indicating that I
B
played a role in maintaining NF-
B·DNA
binding activity in infected cells. Furthermore, the use of control
serum for the immunodepletion step did not increase the sensitivity of
the protein-DNA complex to I
B
-mediated dissociation (Fig.
4A, lanes 7 and 8), indicating that
the effect was specific. Similar results were obtained with untreated
and PMA-induced PLB-IIIB cells (Fig. 4B). Like U9-IIIB
cells, I
B
-depleted PLB-IIIB extracts were sensitive to
GST·I
B
-mediated dissociation of NF-
B·DNA binding activity
(Fig. 4B, compare lanes 2 and 4 with
lanes 6 and 8), indicating that nuclear I
B
maintained I
B
-insensitive NF-
B·DNA binding activity in
HIV-1-infected cells. Supershift analysis further revealed that similar
NF-
B·DNA binding complexes were present before and after I
B
immunodepletion in PMA-stimulated (Fig. 4C, lanes 1-4 and
9-12) and TNF
-stimulated (Fig. 4C,
lanes 5-8 and 13-16) U9-IIIB (Fig.
4C) and PLB-IIIB (data not shown) cells, arguing against the
specific immunodepletion of an I
B
-insensitive NF-
B
complex.

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Fig. 4.
Depletion of
I B -containing
complexes sensitizes HIV-1 nuclear extracts to dissociation by
I B .
A, DNA binding activity in U9-IIIB cells stimulated with PMA
for 0, 2, or 18 h and depleted for I B by immunoprecipitation
(I B IP, lanes 9-15) were compared with
undepleted nuclear extracts (NE, lanes 1-6).
Extracts were incubated with labeled probe only (lanes 1, 3, 5, 7 9, 11, and 13) or with labeled probe and GST·I B
(lanes 2, 4, 6, 8, 10, 12, and 14).
Immunoprecipitation with normal rabbit serum was used as a control for
specificity (lanes 7 and 8). B,
PLB-IIIB NF- B·DNA binding activity in cells stimulated with PMA
for 0 or 2 h and depleted for I B (I B IP,
lanes 1-4) was compared with the activity in undepleted,
similarly treated nuclear extracts (NE, lanes
5-8). Extracts were incubated with only labeled probe
(lanes 1, 3, 5, and 7) or with labeled probe and
GST·I B (lanes 2, 4, 6, and 8).
C, the NF- B subunit composition of U9-IIIB nuclear
extracts stimulated with PMA or TNF for 18 h and immunodepleted
of I B (lanes 9-16) was compared with that of
undepleted control extracts (lanes 1-8) by supershift
analysis. RelA (lanes 2, 6, 10, and 14), p50
(lanes 3, 7, 11, and 15), or c-Rel (lanes
4, 8, 12, and 16) antibodies were utilized to identify
the NF- B·DNA binding subunits present.
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I
B
Co-expression Increases NF-
B Transcriptional
Activation--
Next, a series of co-transfection experiments were
performed to determine whether I
B
co-expression affected
NF-
B-mediated transcription and to examine whether I
B
blocked
the inhibitory effect of I
B
. Human embryonic kidney 293 cells
were transfected with an NF-
B-driven CAT reporter plasmid and empty
vector or reporter plasmid and pSVK3-I
B
and/or pSVK3-I
B
expression plasmids. PMA (50 ng/ml) was added for 0, 8, 16, or 24 h, and transactivation was assessed by comparing CAT activity levels.
PMA-stimulated cells transfected with both I
B
and I
B
partially alleviated the I
B
-mediated repression of transcription
(Fig. 5, compare 24-h I
B
, I
B
and I
B
, and pSVK3 levels) and exhibited a 50% increase in
NF-
B-dependent expression compared with cells
transfected with I
B
alone (Fig. 5). This result indicated that
I
B
expression partially reversed the inhibitory effects of
I
B
and increased NF-
B-mediated transcription.

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Fig. 5.
I B protects
NF- B transcriptional activity from inhibition
by I B . Human
embryonic kidney 293 cells were transfected with an NF- B CAT (7 µg) reporter construct and pSVK3 control vector (8 µg) or
pSVK3-I B (4 µg) and/or pSVK3-I B (4 µg) and stimulated
with PMA (50 ng/ml) for 0, 8, 16, or 24 h. Equal amounts of
protein were assayed for CAT activity. Results are the average of a
minimum of three experiments ± S.E.
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Turnover of I
B
Isoforms--
The regulation of I
B
protein turnover was investigated next. Antibodies recognizing the
43-kDa isoform or both the 43- and 41-kDa isoforms were used to analyze
the rate of I
B
basal turnover and stimulus-induced degradation.
U937 and U9-IIIB cells were treated with cycloheximide alone or with
different inducers for 0, 2, 6, or 8 h. The 43-kDa isoform of
I
B
exhibited faster constitutive turnover in HIV-1-infected
U9-IIIB cells (compare Fig. 6,
A and C to B and D,
lanes 1-4) and also degraded more quickly following IL-1
(compare Fig. 6, A and C to B and
D, lanes 9-12) or PMA (compare Fig. 6,
A and D, lanes 5-8) stimulation.
TNF
stimulation led to rapid degradation of 43-kDa I
B
in both
infected and uninfected cells (Fig. 6, B and C,
lanes 5-8). The faster migrating 41-kDa form was not
degraded in U9-IIIB or U937 cells (Fig. 6, C and D, respectively, lower bands). Thus, the dynamic
state of degradation and resynthesis of the 43-kDa form of I
B
may
result in the continuous production of hypophosphorylated I
B
, the
form previously shown to shield DNA-bound NF-
B from the effects of
I
B
(32, 40).

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Fig. 6.
I B turnover is
increased in HIV-1-infected cells. A, U9-IIIB cells
were incubated with cycloheximide (CHX) alone, cycloheximide
and PMA (lanes 5-8), or cycloheximide and IL-1
(lanes 9-12) for 0, 2, 6, or 8 h. Whole cell extracts
were separated by SDS-polyacrylamide gel electrophoresis, transferred
to nitrocellulose, and probed with an I B antibody that recognizes
only the 43-kDa isoform. B, U937 cells were incubated with
cycloheximide alone, cycloheximide and TNF , or cycloheximide and
IL-1 for 0, 2, 6, or 8 h. Whole cell extracts were
immunoblotted with anti-I B (43-kDa isoform). C,
U9-IIIB cells were treated as described in B, immunoblotted,
and probed with an antibody that recognizes both the 43- and 41-kDa
I B isoforms. D, U937 cells were treated as described
in A, immunoblotted, and probed with an antibody that
recognizes both isoforms of I B .
|
|
Constitutive Activation of the IKK Complex in HIV-1-infected
Cells--
Because I
B
(12) and I
B
turnover are increased
in HIV-1-infected cells and the constitutive NF-
B·DNA binding in
HIV-1-infected myeloid cells disappears when oxidant signaling pathways
are interrupted (15), we sought to determine whether the I
B kinase
was constitutively active in HIV-1-infected cells. When an antibody
that recognizes the phosphoserine 32 of I
B
was used,
HIV-1-infected U9-IIIB and PLB-IIIB cells, but not their uninfected
counterparts, contained high levels of phosphorylated I
B
in the
presence or absence of inducer (Fig.
7A, top panel, lanes
4-6 and 10-12). Stimulation of uninfected cells with
TNF
or PMA for 10 min resulted in the appearance of phosphorylated
I
B
in U937 (Fig. 7A, top panel, lanes 2 and
3) and PLB-985 (Fig. 7A, lanes 8 and
9) cells. This blot was reprobed with monoclonal I
B
antibody (middle panel) to confirm I
B
turnover. As
expected, TNF
stimulation led to degradation of I
B
in all cell
lines (Fig. 7A, middle panel, lanes 2, 5, 8, and 11). I
B
levels in PMA-stimulated cells
were not reduced (Fig. 7A, middle panel, lanes 3,
6, 9, and 12), although phosphorylated I
B
was detected, reflecting the longer kinetics of PMA induced I
B
degradation. As described previously, an I
B
immunoreactive band
of approximately 30 kDa in size was also detected in PLB-985 cells
(12). The 30-kDa I
B
was also recognized by the
phosphoserine-specific I
B
antibody (Fig. 7A, top
panel, lower arrow) and degraded similarly to I
B
(Fig.
7A, middle panel), suggesting that the regulation
of the 30-kDa form may be similar to that of full-length I
B
.

View larger version (34K):
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|
Fig. 7.
The I B kinase
complex is constitutively active in HIV-1-infected cells.
A, cells were untreated or treated with TNF
(T) or PMA (P) for 10 min. Whole cell extracts
were immunoblotted using an I B antibody that recognizes
phosphoserine 32 of I B (upper panel). The blot was
first reprobed for I B using a monoclonal I B antibody
(middle panel) and then reprobed with actin antibody as a
control for equal loading (lower panel). B,
HIV-1-infected myeloid cells are constitutively activated for IKK.
PLB-985 (PLB, lanes 1, 2, and
5) and PLB-IIIB cells (P-IIIB, lanes 3, 4, and 6) were treated with TNF for 0 or 15 min and
assayed for IKK using GST·I B -(1-55) as a substrate.
Specificity was confirmed using normal rabbit serum as the
immunoprecipitating antibody (lane 5) or by using
GST·I B-(1-55) (S32A/S36A) as substrate (lane 6).
Coomassie Blue staining of the gel (bottom panel) reveals
that equal amounts of recombinant protein were used in each reaction.
C, U937 (lanes 1-3 and 7) and U9-IIIB
(lanes 4-6 and 8) cells were stimulated with
TNF for 1 or 12 h and analyzed for IKK activity using
GST·I B -(1-55) as a substrate. Specificity was confirmed using
normal rabbit serum as the immunoprecipitating antibody (lane
7) and GST·I B -(1-55) (S32A/S36A) as substrate (lane
8).
|
|
Given the crucial role of the IKK in the activation cascade of NF-
B,
the possibility of constitutive IKK activity in HIV-1-infected cells
was also examined. PLB-985 and PLB-IIIB cells were stimulated with
TNF
for 15 min, extracts were immunoprecipitated with anti-IKK antibody, and immunoprecipitates were analyzed for the ability to
phosphorylate N-terminal I
B
-(1-55) in vitro. PLB-985
cells exhibited little or no IKK activity (Fig. 7B,
lane 1) unless stimulated with TNF
(Fig. 7B,
lane 2), whereas HIV-1-infected cells displayed IKK activity
with or without TNF
stimulation (Fig. 7B, lanes 3-4). This activity was specific because immunoprecipitation with normal rabbit serum did not result in detectable kinase activity (TNF
-stimulated PLB-985, Fig. 7B, lane 5) and
mutated I
B
-(1-55) (S32A/S36A) substrate was not phosphorylated
(TNF
-stimulated PLB-IIIB, Fig. 7B, lane 6).
Coomassie Blue staining revealed that equal amounts of I
B
substrate were used in each reaction (Fig. 7B, bottom
panel). Similarly, IKK activity was inducible by TNF
in U937
cells (Fig. 7C, lanes 1-3) but was
constitutively activated in U9-IIIB cells (Fig. 7C,
lanes 4-6).
 |
DISCUSSION |
Previously, we have shown that HIV-1-infected primary monocytes
and myeloid cell lines PLB-985 and U937 exhibit constitutive NF-
B
activation as a consequence of virus infection (10, 11, 15). In
addition, levels of NF-
B subunits p105, p100, and c-Rel are elevated
compared with uninfected cells, and constitutive turnover of I
B
is increased (12). These cells also express a low level of cytokines
such as TNF
and IL-1
, which are able to activate NF-
B (Refs.
5, 6, and 41-44 and references therein). Together these results
suggest that the constitutive activation of NF-
B in HIV-1-infected
myeloid cells is caused by the continuous signal-induced degradation of
I
B.
Because I
B
has been implicated in persistent NF-
B activation
(31-33, 45-47), we sought to characterize its role in maintaining constitutive NF-
B activation. I
B
was found complexed to
NF-
B in nuclear extracts from HIV-1-infected cells and in uninfected cells stimulated with various inducers. NF-
B·DNA complexes could not be completely dissociated by GST·I
B
in HIV-1-infected
cells, whereas complexes induced by TNF
or PMA were readily
dissociated in uninfected cells. Depletion of I
B
from
HIV-1-infected nuclear extracts resulted in NF-
B·DNA binding
complexes that were completely sensitive to inhibition by
GST·I
B
-mediated dissociation, indicating that I
B
protects
DNA-bound NF-
B from dissociation by I
B
. Furthermore,
co-expression experiments demonstrated that I
B
increased
NF-
B-dependent gene activity. Finally, IKK was
constitutively activated in HIV-1-infected myeloid cells.
Interestingly, Sendai virus infection of U937 also leads to prolonged
activation of IKK.2 It is
possible that activation of IKK and formation of I
B
-resistant I
B
·NF-
B·DNA ternary complexes is a common mechanism
exploited by several viruses to regulate host and viral gene expression.
In earlier studies, chronic HIV-1 infection or Sendai virus infection
of PLB-985 cells resulted in the induction of protein DNA complexes
that could not be dissociated with recombinant I
B
or supershifted
with NF-
B antisera (11). Although these proteins could be
specifically competed with unlabeled probe, it was considered that
these NF-
B-like proteins might not specifically belong to the
NF-
B family. The present findings suggest that the NF-
B-like binding activity may in fact be I
B
-bound NF-
B complexes that are protected from I
B
-mediated dissociation.
I
B
was detected in NF-
B·DNA binding complexes of uninfected
U937 and PLB-985 cells after long periods of TNF
or PMA stimulation. In contrast to HIV-1-infected cells, NF-
B complexes from uninfected cells were sensitive to I
B
-mediated dissociation. The reason for
this discrepancy may lie in the additional pathways that are activated
or inhibited in HIV-1-infected cells. I
B
protection of NF-
B
may require additional factors (such as high mobility group proteins)
that are activated in HIV-1-infected myeloid cells. Alternatively,
I
B
-mediated protection in uninfected cells may require longer
induction periods than those used in this study.
Several studies have implicated I
B
in maintaining persistent
NF-
B activation (31-33, 45-47). B cells stimulated with
lipopolysaccharide or IL-1 experienced a persistent degradation of
I
B
that correlated with sustained NF-
B activation, whereas
inducers that did not degrade I
B
produced only a transient
activation of NF-
B (31). A similar correlation between I
B
degradation and persistent NF-
B activation was also reported in
human vascular endothelial cells (47). I
B
degradation has been
also implicated in the synergistic activation of NF-
B observed in
TNF
- and IFN-
-stimulated cells (48). Although others have
observed I
B
degradation during transient activation of NF-
B
(49), persistent activation of NF-
B is generally associated with
I
B
degradation. The increased rate of I
B
turnover seen in
our HIV-1-infected cells supports a role for I
B
in maintaining
persistent NF-
B activation.
Several groups (32, 40, 50, 51) have demonstrated that
hypophosphorylated I
B
can bind NF-
B·DNA complexes without inhibiting DNA binding. Hypophosphorylated I
B
did not mask the nuclear localization signal of RelA, permitting NF-
B·I
B
complexes to enter the nucleus and bind DNA (32). Sites important in
regulating the ability of I
B
to chaperone NF-
B into the
nucleus were identified in the C-terminal PEST domain. Phosphorylation
of Ser-313 and Ser-315 by casein kinase II prevented I
B
from
associating with NF-
B·DNA complexes (40), and conversely mutation
of these sites to alanine permitted I
B
to form ternary complexes
with NF-
B and DNA. Other serines in the PEST domain also appear to
be important, because replacing Ser-313 and Ser-315 with a
phosphomimetic amino acid (Glu) was not sufficient to block the ternary
complex formation (40). The I
B
CKII mutant (S313A/S315A) also
blocked the capacity of I
B
to dissociate NF-
B from DNA (40).
Based on these results, it seems likely that the I
B
complexed
with nuclear NF-
B in HIV-1-infected cells is hypophosphorylated.
In accord with studies conducted by Hirano and colleagues (34), two
isoforms of I
B
of 43 and 41 kDa were also detected. The 41-kDa
isoform of I
B
resisted degradation by several inducers in both
infected and uninfected cells, whereas the constitutive protein
turnover of the 43-kDa form was increased in HIV-1-infected cells. One
plausible explanation is that virus infection represents the persistent
activation signal required for the continuous degradation of I
B
.
Similarly, lipopolysaccharide induced a prolonged NF-
B activation in
7OZ/3 pre-B cells as a result of a persistent activating signal that
could be blocked by employing antioxidants (31). Constitutive
activation of NF-
B may also result from decreased cellular
antioxidant levels (52). HIV-1 infection can lead to decreases in
antioxidant levels (53). Increased turnover because of constitutive
stimulation could also explain the decreased steady state level of the
inducer-sensitive 43-kDa I
B
isoform (compared with the 41-kDa
isoform) in HIV-1-infected cells. Other mechanisms likely exist to
maintain I
B
in a hypophosphorylated form, because antioxidant
treatment or proteosome inhibition did not affect the nuclear
localization of hypophosphorylated I
B
in WEHI 231 cells (33).
Maintenance of hypophosphorylated I
B
in these cells may result
from the activation of a phosphatase, because treatment with the
phosphatase inhibitor okadaic acid led to I
B
hyperphosphorylation.
This is the first report demonstrating that I
B
is present in
nuclear NF-
B·DNA complexes in HIV-1-infected cells and contributes to constitutive NF-
B activation. We suggest that the induction of
IKK, arising as a consequence of the low level TNF
production or the
increased pro-oxidant state of HIV-1-infected cells (53), leads to
increased phosphorylation and degradation of I
B
and I
B
.
Both proteins release active NF-
B, which translocates to the nucleus
and transcriptionally activates responsive genes. In addition, newly
synthesized I
B
enters the nucleus and prevents I
B
-mediated
termination of the NF-
B response. This occurrence would create an
environment conducive to viral replication, promoting HIV-1 long
terminal repeat-driven gene transcription and maintaining cell survival
resulting from the antiapoptotic effects of NF-
B (15, 54-56).
Blocking this pathway may be an important strategy in targeting
long-lived HIV-1-infected myeloid cells.
 |
ACKNOWLEDGEMENTS |
We thank Ron Hay for the kind gift of the
I
B
monoclonal antibody and Dmitrios Thanos for supplying I
B
cDNA. We thank members of the McGill Aids Center for helpful
comments and numerous reagents.
 |
FOOTNOTES |
*
This work was supported by the Medical Research Council
(MRC) of Canada and by CANFAR.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.
¶
Recipient of a National Health Research Development Program Fellowship.

Recipient of an MRC Senior Scientist award. 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.
2
C. Heylbroeck and J. Hiscott, unpublished results.
 |
ABBREVIATIONS |
The abbreviations used are:
HIV-1, human
immunodeficiency virus-1;
IKK, I
B kinase;
IL-1, interleukin-1;
PMA, phorbol 12-myristate 13-acetate;
PEST, proline-, glutamic acid-,
serine-, and threonine-rich domain;
TNF
, tumor necrosis factor
;
GST, glutathione S-transferase;
CAT, chloramphenicol
acetyltransferase;
NIK, NF-
B-inducing kinase;
PRD, positive
regulatory domain.
 |
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