From the Department of Virology and Institut d'Etude
et de Transfert de Gènes, Franche-Comté University, F-25030
Besançon, France, the ¶ Université Libre de Bruxelles,
Institut de Biologie et de Médecine Moléculaires (IBMM),
Chimie Biologique, 6041 Gosselies, Belgium, and the
§ Cytokine Research Section, Department of Bioimmunotherapy,
Cytokine Research Section, The University of Texas M. D. Anderson
Cancer Center, Houston, Texas 77030
Received for publication, September 19, 2002, and in revised form, October 29, 2002
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ABSTRACT |
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The human immunodeficiency virus (HIV) Nef
protein plays a critical role in AIDS pathogenesis by enhancing
replication and survival of the virus within infected cells and by
facilitating its spread in vivo. Most of the data obtained
so far have been in experiments with endogenous Nef protein, so far
overlooking the effects of exogenous soluble Nef protein. We used
recombinant exogenous Nef proteins to activate nuclear transcription
factors NF- Nef is a 27-kDa HIV1
protein that is produced early during infection and translated from
multiply spliced viral mRNAs (1). Information is beginning to
emerge that suggests that endogenous Nef may have evolved a number of
different, independent functional activities to enhance the replication
and survival of the virus within infected cells and to facilitate its
spread in vivo (2, 3). Nef enhances virion infectivity (4,
5) and increases viral replication in primary lymphocytes and
macrophages (6, 7). Nef can mediate down-regulation of CD4 cell surface
expression, a phenomenon shown to be important for the release of HIV-1
from the cell (2, 3). Nef can also downregulate the cell surface expression of major histocompatibility complex class I (MHC-I) molecules, (8), an effect found to protect infected cells from killing
by cytotoxic T cells (9). Nef prevents apoptosis of HIV-1-infected T
cells (10-12). Nef expression within macrophages has been reported to
favor the recruitment of resting T cells via the secretion of C-C
chemokines and to subsequently favor their activation, suggesting a
role for Nef in lymphocyte recruitment and activation at sites of viral
replication (13). In vivo, several studies have demonstrated
the importance of Nef for the efficiency of viral replication and for
the maintenance of high viral loads (14, 15).
Nef can alter T-cell signaling pathways (16). Nef has been found to
interact with several signaling molecules: with a serine kinase (17),
with a distinct serine/threonine kinase, the Nef-associated kinase
(NAK) identified as a member of the p21-activated kinase (PAK) family
(18-21), with members of the Src-family of tyrosine kinases, notably
Lck (17, 22-24), Hck (25), Lyn, (26), Fyn (27), and with
mitogen-activated protein kinase (MAPK) (23), c-Raf-1 (28), p53 (29),
and protein kinase C HIV replication is tightly regulated at the transcriptional level
through the specific interaction of viral regulatory proteins, namely,
Tat and cellular transcription factors binding to a variety of
cis-acting DNA sequences in the HIV long terminal repeat (LTR) (reviewed in Ref. 31). One of the main mediators of HIV LTR transcription is nuclear factor- Since the identification of NF- The activity of activator protein-1 (AP-1), a transcription factor,
consisting of a homodimer and heterodimers of members of the Jun family
(c-Jun, JunB, and JunD) and heterodimers of the Jun and Fos (c-Fos,
FosB, Fra1, and Fra2) families, is regulated, at least in part, by the
activation of c-Jun N-terminal kinase (JNK) (45). It has also been
suggested that the activation of NF- Exogenous Nef protein is detected in the serum of HIV-infected subjects
(48). Both antibodies and cytotoxic T lymphocytes (CTLs) directed
against Nef have been found in a large proportion of infected
individuals (49, 50). This suggests that in vivo Nef is
processed and presented by antigen-presenting cells, as the result of
uptake of extracellular Nef possibly released by infected apoptotic
cells (51). Exogenous Nef protein has been shown to enter the cell by
adsorptive endocytosis following nonspecific binding to the surface of
CD4+ T cells, primary macrophages, and U937 promonocytic
cells (51) and to activate the signal transducer and activator of
transcription 1 (STAT-1) in human monocytes/macrophages (52). Confocal
microscopy indicates that the intracellular distribution of
internalized FITC-labeled recombinant Nef is identical to that of
endogenously produced Nef, localizing both in an intracytoplasmic
punctate pattern and at the cell margin (51, 53). Although
most of the results reported so far in regard to signaling were
obtained following expression of endogenous Nef protein, exogenous Nef protein could also be involved in the modulation of cell signaling, especially in promonocytic cells and primary macrophages. Since activation of NF- Reagents--
Recombinant SIVmac Nef protein (AIDS Research and
Reference Reagent Program, National Institutes of Health cat. 2999, kindly provided by Jose Torres) and recombinant Nef protein derived
from HIV-1 strains, SF-2, BH10 (kindly provided by M. Harris, Leeds University, UK) and NL4-3 (kindly provided by U. Mahlknecht, Frankfurt University, Germany) were used to treat U937 cells and U1 cells. Anti-SIV Nef antibody was provided by the AIDS Research and Reference Reagent Program, National Institutes of Health (cat. 2659). Anti-HIV-1 Nef antibody was provided by AbCys (Paris, France). Antibody against I Cell Lines--
Most of the studies were performed with the
promonocytic cells U937 obtained from the American Tissue Cell Culture
Collection (ATCC, Manassas, VA). The promonocytic cell line U1, derived
from cells surviving acute infection of the U937 cell line, contains two integrated HIV copies per cell (57). U1 cells were a gift from Dr.
U. Mahlknecht (University of Frankfurt, Germany). U937 and U1 cells
were cultivated in RPMI 1640 supplemented with 10% fetal bovine serum.
Electrophoretic Mobility Shift Assay--
To measure NF- Western Blot Analysis--
Cytoplasmic extracts of U937 cells
treated for different times with exogenous SIV Nef protein were used to
examine I c-Jun Kinase Assay--
The c-Jun kinase (JNK) assay was
performed according to the method of Manna and Aggarwal (58).
Reporter Gene Expression Assays--
To examine SIV Nef-induced
NF-
To examine NF- p24 Assay--
U1 cells were treated with different
concentrations of exogenous HIV-1 Nef or TNF. Culture supernatants were
collected every day and assessed for p24 antigen using a microELISA
assay (Organon Teknika).
Exogenous Nef Activates NF-
The degradation of I
Since SIV Nef and HIV-1 Nef may differ in regard to some of their
biological functions (62), we tested recombinant HIV-1 Nef proteins
derived from three HIV-1 isolates, NL4-3, SF2, and BH10, in regard to
NF- Exogenous Nef Activates AP-1 and JNK--
Most agents that
activate NF-
Activation of JNK is another early event initiated by many stress
stimuli and is required for AP-1 activation (45). Treatment of U937
cells with exogenous SIV Nef protein led to an increase in JNK activity
in a time- (Fig. 3D) and dose-dependent manner (Fig. 3E). The overall level of JNK activation triggered by
exogenous SIV Nef protein was less than that observed following TNF
treatment (3-fold versus 6-fold) (Fig. 3E).
Since SIV Nef and HIV-1 Nef could differ in regard to their biological
functions (62), we tested recombinant HIV-1 Nef proteins derived from
three HIV-1 isolates, NL4-3, SF2, and BH10 in regard to AP-1
activation in U937 cells. Exogenous BH10 Nef protein activated AP-1 in
a time-dependent (Fig.
4A) and
dose-dependent manner (data not shown). The gel shift band
was specific, as formation of the complex was blocked with an unlabeled
AP-1 oligonucleotide and with an unlabeled consensus AP-1
oligonucleotide, but not with a heterologous NF- Exogenous Nef Stimulates HIV-1 LTR--
We assessed whether
NF-
Then we assessed the effect of exogenous HIV-1 Nef on
NF- Exogenous Nef Stimulates HIV-1 Replication in Chronically Infected
Promonocytic U1 Cells--
NF- Exogenous Nef protein has been detected in serum from HIV-infected
subjects (48). However its significance in regard to HIV pathogenesis
has not been studied. Here we have demonstrated that exogenous Nef
proteins derived from SIV and HIV-1 isolates activate NF- Increased NF- Activation of transcription factors NF- The amounts of exogenous Nef detected in the serum of HIV-infected
subjects is around 10 ng/ml (48). At 10 ng/ml, we observed that
recombinant Nef protein activates NF-kB, AP-1, and JNK (Figs. 1 and 3).
In agreement with our results, it has been reported that
recombinant Nef protein activates STAT1 in monocyte-derived macrophages
starting at a concentration of 10 ng/ml (52), pointing out the
possibility that our observations reflect phenomena actually occurring
in vivo. Also, we cannot rule out that higher amount of
exogenous Nef, e.g. 100 ng/ml, could be present in tissue
compartments such as lymph nodes where macrophages and infected
lymphocytes tightly interact (67, 68). The low amounts of exogenous Nef detected in the serum of HIV-infected subjects could also result from
the presence of immune complexes Nef-antiNef as reported previously
(48).
The features observed in promonocytic cells and primary macrophages
following exposure to exogenous Nef are very similar to those observed
following TNF treatment. Both exogenous Nef and TNF activate NF- Although exogenous Nef binds to the surface of macrophages, the
molecular mechanisms underlying this interaction are not yet unveiled
(70). Exogenous Nef enters the cell by adsorptive endocytosis following
nonspecific binding to the surface of U937 cells (51, 70). No specific
receptor for exogenous Nef protein has been described (51). However, we
cannot exclude possible low-level expression of a potential cell
surface Nef receptor that could not be revealed by
fluorescence-activated cell sorting (FACS) analysis on
rNef-FITC-treated cells. Through cytofluorometric analyses, the
internalization of FITC-conjugated recombinant Nef into primary
macrophages was observed (51). Confocal microscopy indicated that the
intracellular distribution of internalized recombinant Nef was
identical to that of endogenously produced Nef, localizing both in an
intracytoplasmic punctate pattern and at the cell margin (51, 53). The
identical intracellular distribution of internalized recombinant Nef
and of endogenously produced Nef could explain why exogenous Nef
protein activates NF-kB, AP-1, and JNK and stimulates the HIV-1 LTR.
Exogenous Nef might interfere with intracellular signaling pathways
downstream of TNF receptors and thereby could mimic the effect of TNF
on primary macrophages. Exogenous Tat has been shown to activate
NF- Our results indicate that exogenous Nef protein and exogenous Tat
protein might share similar biological functions. Thus, like exogenous
Nef protein, exogenous Tat protein activates NF-kB, AP-1 and JNK,
transactivates the HIV-1 LTR when added exogenously to U937 cells
growing in culture and stimulates HIV-1 replication in U1 cells
(73-77). Also, like exogenous Nef protein, exogenous Tat protein is
detected in the serum of HIV-infected subjects (78) and can be released
from HIV-infected cells (79). Similarly, exogenous Tat protein has been
reported to bind nonspecifically to the cell surface and to enter U937
promonocytic cells by adsorptive endocytosis in vitro (74,
80) and in vivo (81), contributing to the transcellular
activation of HIV-1 LTR promoter in latently infected cells (82, 83).
Also, fluorescent experiments with rhodamine-conjugated Tat showed
punctate staining on the cell surface and then localization to the
cytoplasm and nucleus (74). Thus, both exogenous Nef protein and
exogenous Tat protein can enter target cells and activate HIV-1 LTR.
Similar effects of Nef protein and Tat protein on T cell apoptosis have
been reported. Both endogenous Tat and endogenous Nef have been shown
to block apoptosis in HIV-infected T cells, while both exogenous Tat
and exogenous Nef have been shown to deliver proapoptotic signals to
uninfected T cells (10, 12, 29, 84). All together these data indicate a
redundancy between the effects of exogenous Nef and exogenous Tat on
several biological functions including transcription activation.
Since exogenous Nef activates AP-1, NF- In conclusion, our results show that exogenous Nef activates NF-B and AP-1 in the promonocytic cell line U937. Exogenous
SIV and HIV-1 Nef proteins activated NF-
B and AP-1 in a dose- and time-dependent manner. Activation of NF-
B by exogenous
Nef was concomitant to the degradation of the inhibitor of NF-
B,
I
B
. In agreement with increased AP-1 activation, a time- and
dose-dependent increase in JNK activation was observed
following treatment of U937 cells with exogenous Nef. Since exogenous
Nef activates the transcription factors NF-
B and AP-1, which bind to
the HIV-1 long terminal repeat (LTR), we investigated the effect of
exogenous Nef on HIV-1 replication. We observed that exogenous Nef
stimulated HIV-1 LTR via NF-
B activation in U937 cells and enhanced
viral replication in the chronically infected promonocytic cells U1. Therefore, our results suggest that exogenous Nef could fuel the progression of the disease via stimulation of HIV-1 provirus present in
such cellular reservoirs as mononuclear phagocytes in HIV-infected patients.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(30).
B (NF-
B) (32). An inducible transcription factor, NF-
B is composed of homo- and heterodimers of
Rel family proteins (reviewed in Ref. 33). All of the NF-
B proteins
contain an N-terminal Rel homology domain, which mediates DNA binding,
dimerization, and interaction with the inhibitory proteins, or I
Bs.
In addition, c-Rel, p65 (RelA), and RelB contain a C-terminal
transactivation domain (reviewed in Ref. 33). The classic NF-
B
complex (p50/p65) is sequestered in the cytoplasm by interaction with a
family of inhibitory proteins, or I
Bs, including I
B
, I
B
,
I
B
, I
B
, and the proto-oncogene Bcl-3 (34). Following cell
activation by a variety of extracellular stimuli, such as tumor
necrosis factor
(TNF), I
B
is phosphorylated at the N-terminal
residues Ser-32 and Ser-36 by the I
B kinase (IKK) complex, leading
to ubiquitination and subsequent proteasome-mediated degradation, which
allows NF-
B to translocate to the nucleus, where it activates gene expression.
B elements in the HIV LTR (32),
multiple studies have assessed the effect of this family of
transcription factors on the transcriptional regulation of the HIV LTR
and its impact on HIV reactivation from latency (35-38). Study of the
interaction between NF-
B and HIV in both human monocytic cells and
transformed human macrophages has mainly focused on how monocyte
differentiation may lead to HIV expression (39, 40) and how HIV
infection leads to NF-
B activation. In the promonocytic cell line
U937, HIV activates the inducible pool of NF-
B as a result of
enhanced I
B
degradation, which is believed to be secondary to IKK
activation (41-44).
B is regulated by some upstream
MAP kinases that also regulate JNK activation in the cells (46).
Induction of AP-1 in macrophages by endogenous HIV-1 Nef has been
reported to be a cell type-specific response that requires both Hck and
MAPK signaling (47).
B and AP-1 results in stimulation of both HIV-1 and
SIV replication (41, 42, 54-56), we investigated the role of exogenous
Nef protein in this process. We observed that exogenous Nef protein
activates NF-
B, AP-1, and JNK in promonocytic cells U937 and
enhances HIV-1 replication in chronically infected U1 cells.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
and the double-stranded oligonucleotide having the AP-1
consensus sequence were obtained from Santa Cruz Biotechnology (Santa
Cruz, CA). Phospho-I
B
(Ser32) antibody was purchased from New
England BioLabs (Beverly, MA). TNF was purchased from R&D Systems.
B and
AP-1 activation, EMSA were carried out as previously described by Manna
and Aggarwal (58) and by Van Lint et al. (59). Briefly,
nuclear extracts prepared from cells treated with exogenous Nef protein
were incubated with 32P-end-labeled 45-mer double-stranded
NF-
B oligonucleotide,
5'-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-3' (bold indicates NF-
B binding sites), and the DNA-protein complex formed was resolved from free oligonucleotide on a 6% native
polyacrylamide gel. A double-stranded mutated oligonucleotide
5'-TTGTTACAACTCACTTTCCGCTGCTCACTTTCCAGGGAGGCGTGG-3', was used to examine the specificity of binding of NF-
B to the DNA. The specificity of binding was also examined by competition with
the unlabeled oligonucleotide and a heterologous unlabeled oligonucleotide. To measure AP-1 activation, nuclear extracts, prepared
as described above, were incubated with the 32P-end-labeled
AP-1 consensus oligonucleotide 5'-CGCTTGATGACTCAGCCGGAA-3' (bold indicates AP-1 binding site) and analyzed on a 6% native polyacrylamide gel. The specificity of the binding was examined by
competition with unlabeled oligonucleotide, with a heterologous unlabeled oligonucleotide and with a consensus unlabeled
oligonucleotide. The dried gels were visualized and radioactive bands
quantified by a PhosphorImager (Molecular Dynamics, Sunnyvale, CA)
using ImageQuant software.
B
degradation by Western blot procedure as described
(58).
B-dependent gene expression, cells were transfected
with the secreted alkaline phosphatase (SEAP) expression plasmid for
10 h before treatment with SIV Nef. After 24 h, cell culture
conditioned-medium was harvested and analyzed (25 µl) for alkaline
phosphatase activity, as described in the manufacturer's protocol
(Clontech, Palo Alto, CA). SEAP activity was
assayed on a 96-well fluorescence plate reader (Fluoroscan II, Lab
Systems) with excitation set at 360 nm and emission at 460 nm. This
reporter system was specific since TNF-induced NF-
B SEAP activity
was inhibited by overexpression of the I
B
mutant, I
B
-DN,
which lacks Ser-32 and Ser-36 (60).
B LTR-driven gene expression by exogenous HIV Nef,
107 U937 cells were transfected with 750 ng of pLTR-Luc or
750 ng of pLTRmut-NFkB-Luc using the DEAE-dextran procedure (61).
Twenty-four hours later, the cells were stimulated with different
concentrations of exogenous HIV-1 Nef or with TNF. At 48-h
post-transfection, luciferase activity was measured in cell lysates
using a luminometer (TD-20/20; Promega, Madison, WI) as previously
reported (11). Values normalized to protein concentrations were
expressed in fold increase over unstimulated control values.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B--
Treatment of U937 cells with
exogenous SIV Nef for 90 min revealed a dose-dependent
activation of NF-
B by EMSA (Fig.
1A), with maximum activation
at ~50-100 ng/ml, but to a lesser extent than TNF, the most potent
activator of NF-
B (3.5-fold versus 5.2-fold induction).
Exogenous SIV Nef activated NF-
B in a time- and
dose-dependent manner reaching a peak by 90 min (Fig.
1B). The gel shift band was specific as formation of the
complex was blocked with an unlabeled oligonucleotide and was
supershifted by either anti-p50 or anti-p65 antibody alone, and also by
a mixture of anti-p50 and anti-p65 antibodies (Fig. 1C),
indicating that it is composed of p50 and p65 subunits. To rule out the
possibility that a TNF inducer, such as lipopolysaccharide (LPS),
produced the activity, we treated exogenous SIV Nef protein with 1%
trypsin or boiled it at 100 °C (Fig. 1D). Both treatments
abolished Nef-induced NF-
B activity, indicating that exogenous Nef
protein, but not LPS contamination, was responsible for NF-
B
activation. The pretreatement of exogenous Nef protein with a
neutralizing anti-Nef monoclonal antibody blocked NF-
B activation
(Fig. 1D), thereby indicating the effect was
Nef-specific.
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Fig. 1.
NF- B activation in
U937 cells treated by exogenous SIV Nef. A, dose response of
exogenous SIV Nef-induced NF-
B activation. U937 cells (2 × 106/ml) were treated with different concentrations of
exogenous SIV Nef for 30 min at 37 °C and then assayed for NF-
B
by EMSA as described under "Experimental Procedures." B,
time response of exogenous SIV Nef-induced NF-
B activation. U937
cells were treated with 50 ng/ml exogenous SIV Nef for different times
at 37 °C, and then NF-
B activation was measured. C,
supershift and specificity of NF-
B activation by exogenous SIV Nef.
Nuclear extracts from U937 cells treated with exogenous SIV Nef (50 ng/ml) were incubated for 15 min with anti-p50, anti-p65, anti-p50 + anti-p65, anti-cyclin D1, anti-c-Rel, preimmune serum, cold NF-
B
oligonucleotide probe, and mutated NF-
B oligonucleotide and then
assayed for NF-
B DNA binding activity. D, assessment of
the specificity of NF-
B activity by exogenous SIV Nef. Left
panel, effect of trypsinization and boiling on the ability of
exogenous SIV Nef protein to activate NF-
B in U937 cells. U937 cells
were left untreated or were treated with exogenous SIV Nef (50 ng/ml),
exogenous SIV Nef treated with 1% trypsin for 1 h at room
temperature, or exogenous SIV-Nef boiled at 100 °C for 10 min. U937
were also trypsinized before addition of exogenous SIV Nef (lane
3). Right panel, effect of anti-SIV Nef monoclonal
antibody on NF-
B activity induced by exogenous SIV Nef in U937
cells. 50 µg of exogenous SIV Nef were preincubated with 10 µl of
anti-SIV Nef antibody before addition to U937 cells. E, time
course of I-
B degradation (upper panel) and I
B
phosphorylation (lower panel) in U937 cells treated by
exogenous SIV Nef (50 ng/ml).
B
in U937 cells treated with exogenous SIV
Nef protein for different periods of time was also examined using
Western blot analysis. We observed that I
B
started to degrade at
15 min, degraded maximally by 30-60 min, and started to be
resynthesized at 90 min (Fig. 1E, upper panel).
The presence of a slow migrating band of I
B
in samples prepared
from SIV Nef-treated cells suggested the appearance of the
phosphorylated form of I
B
, which is required for I
B
degradation. The induction of the phosphorylated form of I
B
by
exogenous SIV Nef was detected by using an antibody directed against
the phosphorylated form of I
B
, with a maximum detection by 30 min
(Fig. 1E, lower panel).
B activation in U937 cells. We observed that exogenous SF2 Nef
protein activated NF-
B in a time-dependent manner (Fig.
2A). The gel-shift band was
specific as formation of the complex was blocked with an unlabeled
NF-
B oligonucleotide, but not with a mutated NF-
B oligonucleotide
or with a heterologous oligonucleotide (Fig. 2B). The gel
shift band was supershifted by either anti-p50 or anti-p65 antibody
alone (Fig. 2C), indicating that it is composed of p50 and
p65 subunits. Nef-induced NF-
B activation was also observed
following treatment of U937 cells with exogenous HIV-1 BH10 and NL4-3
Nef proteins (Fig. 2D), indicating that NF-
B activation
mediated by exogenous Nef is viral isolate-independent.
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Fig. 2.
NF- B activation in U937 cells treated by
exogenous HIV-1 Nef. A, time course of NF-
B activation
induced by exogenous HIV-1 Nef in U937 cells. U937 cells (1.5 × 106/ml) were treated with 1000 ng/ml exogenous HIV-1 SF2
Nef for 30 min and 2 h, and then NF-
B activation was measured
by EMSA, as described under "Experimental Procedures."
B, specificity of NF-
B activation by exogenous HIV-1 Nef.
Nuclear extracts from U937 cells treated with exogenous HIV-1 SF2 Nef
were incubated for 20 min with increasing concentrations of unlabeled
NF-
B oligonucleotide, unlabeled mutated NF-
B oligonucleotide, and
unlabeled heterologous oligonucleotide and then assayed for NF-
B
activation. C, supershift of NF-
B activation by exogenous
HIV-1 Nef. Nuclear extracts from U937 cells treated with exogenous
HIV-1 SF2 Nef were incubated for 30 min with anti-p50, anti-p65,
anti-p-52, anti-cRel, and anti-RelB and then assayed for NF-
B DNA
binding activity. Untreated U937 cells, used as a negative control, are
also represented. D, NF-
B activation in U937 cells
treated by exogenous Nef derived from different HIV-1 isolates.
Following treatment of U937 cells with exogenous HIV-1 Nef derived from
NL4-3, BH10, and SF2 isolates (1000 ng/ml), NF-
B binding was
detected by EMSA at the indicated times post-treatment and measured as
fold induction versus untreated cells, using a
PhosphorImager.
B also activate the transcription factor AP-1 (45).
Therefore, we investigated the ability of exogenous SIV Nef protein to
activate AP-1 in U937 cells. Exogenous SIV Nef protein activated AP-1
in a dose-dependent manner, but to a lesser extent than TNF
(3.5-fold versus 5-fold induction) (Fig.
3A). AP-1 activation by
exogenous SIV Nef was time-dependent, with optimum
activation occurring at ~90 min (Fig. 3B). Supershift analysis with specific antibodies against c-Fos and c-Jun indicated that AP-1 activation induced by exogenous SIV Nef consisted of Fos and
Jun (Fig. 3C). Lack of supershift by unrelated antibodies and disappearance of the AP-1 band by competition with unlabeled oligonucleotide indicate that the interaction was specific (Fig. 3C).
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Fig. 3.
AP-1 and JNK activation in U937 cells treated
with exogenous SIV Nef. A, dose response of AP-1 activation
induced by exogenous SIV Nef. U937 cells (2 × 106/ml)
were treated with indicated concentrations of exogenous SIV Nef for 30 min at 37 °C, and then AP-1 activation was measured by EMSA, as
described under "Experimental Procedures." B, time
course of AP-1 activation induced by exogenous SIV Nef. U937 cells were
treated with 50 ng/ml exogenous SIV Nef for different periods of time
at 37 °C, and then AP-1 activation was measured. C,
supershift and specificity of AP-1 activation by exogenous SIV Nef.
Nuclear extracts from U937 cells treated with exogenous SIV Nef (50 ng/ml) were incubated 15 min with anti-c-Jun, anti-c-Fos, and
anti-c-Jun + anti-c-Fos, anti-cyclin D1, anti p-50 antibodies, or
preimmun serum and unlabeled AP-1 oligonucleotide probe, and then AP-1
activity was measured as described under "Experimental Procedures."
D, time course of JNK activation induced by exogenous SIV
Nef. U937 cells (2 × 106/ml) were treated with 50 ng/ml SIV Nef for different periods of time after exposure, and JNK
activation was measured as described under "Experimental
Procedures." E, dose response of JNK activation induced by
exogenous SIV Nef. U937 cells (2 × 106/ml) were
treated with increasing concentrations of exogenous SIV Nef for 2 h, and JNK activity was measured as described under "Experimental
Procedures."
B oligonucleotide
(Fig. 4B). The gel shift band was supershifted by either
anti-c-Fos or anti-c-Jun antibody alone, but also by a mixture of
anti-c-Fos and anti-c-Jun antibodies (Fig. 4C), indicating
that it is composed of c-Fos and c-Jun subunits. AP-1 activation
followed treatment of U937 cells with exogenous HIV-1 Nef proteins
derived from NL4-3, BH10, and SF2 isolates (Fig. 4D),
indicating that AP-1 activation mediated by exogenous HIV-1 Nef was
viral isolate-independent.
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Fig. 4.
AP-1 activation in U937 cells treated by
exogenous HIV-1 Nef. A, time course-dependent
responses of AP-1 activation induced by exogenous HIV-1 Nef in U937
cells. U937 cells (1.5 × 106/ml) were treated with
1000 ng/ml exogenous HIV-1 BH10 Nef for different periods of time, and
then AP-1 activation was measured by EMSA, as described under
"Experimental Procedures." B, specificity of AP-1
activation by exogenous HIV-1 Nef. Nuclear extracts from U937 cells
treated with 1000 ng/ml exogenous HIV-1 BH10 Nef were incubated for 20 min with increasing concentrations of unlabeled homologous or consensus
AP-1 oligonucleotides, or with cold heterologous oligonucleotide, and
then assayed for AP-1 activation. Untreated and treated U937 cells were
negative and positive controls, respectively. C, supershift
of AP-1 activation by exogenous HIV-1 Nef. Nuclear extracts from U937
cells treated with exogenous HIV-1 BH10 Nef were incubated for 30 min
with anti-c-Fos, anti-c-Jun, or anti-c-Fos + anti-c-Jun, and then
assayed for AP-1 DNA binding activity. Untreated U937 cells, used as a
negative control, are also represented. D, AP-1 activation
in U937 cells treated by exogenous Nef derived from different HIV-1
isolates. Following treatment of U937 cells with exogenous HIV-1 Nef
derived from NL4-3, BH10, and SF2 isolates (1000 ng/ml), AP-1 binding
was detected by EMSA at the indicated times post-treatment and measured
as fold induction versus untreated cells, using a
PhosphorImager.
B activation triggers gene expression in U937 cells treated with
exogenous Nef proteins. We examined the effect of exogenous SIV Nef on
NF-
B-driven SEAP gene expression in U937 cells. SIV-Nef
enhanced SEAP gene expression in a
dose-dependent manner, comparable with TNF (Fig.
5A). SEAP gene
expression was NF-
B-specific, since it was abolished by IkBa-DN, an
I
B
mutant lacking Ser-32 and Ser-36 (Fig. 5A).
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Fig. 5.
Exogenous SIV Nef and HIV Nef activate
NF- B-driven reporter gene expression.
A, U937 cells were transiently transfected with the SEAP
expression plasmid for 10 h before treatment with increasing
concentrations of exogenous SIV Nef and TNF. After 24 h, cell
culture-conditioned medium was harvested and analyzed for alkaline
phosphatase activity as described under "Experimental Procedures."
The specificity of the reporter system was confirmed using an I
B
mutant IkBa-DN lacking Ser-32 and Ser-36 residues. B,
exogenous HIV-1 Nef activates HIV-1 LTR via NF-
B stimulation. U937
cells were transiently transfected with 750 ng of p-LTR-Luc or with 750 ng of p-LTR-mut-NF-
B-Luc. Twenty-four hours later, transfected cells
were mock-treated or treated with increasing concentrations of
exogenous HIV-1 NL4-3 Nef or TNF (100 pM), and luciferase
activity was measured in cell lysates. The mock-treated value of the
wild-type LTR reporter construct and of the mutant-LTR reporter
construct were arbitrarily set at a value of 1. Values represent the
means of duplicate samples. A representative experiment of two
independent transfections is shown.
B-dependent LTR stimulation. U937 cells were
transiently transfected with a target plasmid that contains the
luciferase reporter gene under the control of the HIV-1 LTR
promoter, pLTR-Luc (61). Twenty-four hours later,
transfected cells were treated for 24 h with different
concentrations of exogenous HIV-1 NL4-3 Nef and harvested, and
luciferase activity was measured in cell lysates. Exogenous HIV-1 Nef
stimulation doubled LTR activation over untreated control cells (Fig.
5B). As a positive control, TNF treatment of transfected
U937 cells increased HIV-1 LTR stimulation by 2.5-fold (Fig.
5B). LTR activation induced by exogenous Nef was not
observed when a plasmid containing a mutated NF-
B site,
pLTR-mut-NFkB-Luc, was used instead of pLTR-Luc (Fig. 5B).
These data indicate that exogenous HIV-1 Nef activated the LTR via
NF-
B stimulation in promonocytic U937 cells.
B and AP-1 DNA-binding sites are
present in the HIV-1 LTR. Therefore, we determined the effect of
exogenous Nef on provirus transcription in the promonocytic cell line
U1, U937 cells that contain two integrated HIV copies per cell (57). We
measured HIV-1 replication in U1 cells following treatment with
exogenous HIV-1 Nef. Exogenous HIV-1 NL4-3 Nef stimulated viral
replication in U1 cells as measured by p24 assay (Fig.
6A). To rule out the possibility that a TNF inducer, such as LPS, enhanced viral replication in U1 cells, we boiled exogenous Nef at 100 °C. Boiling abolished exogenous Nef-induced replication in U1 cells (Fig. 6A),
indicating that Nef protein, but not LPS contamination, was responsible
for enhanced viral replication. Exogenous HIV-1 NL4-3 Nef stimulated HIV-1 replication in a dose-dependent manner (Fig.
6B). The stimulation of HIV-1 replication by exogenous Nef
was significantly diminished by a neutralizing anti-HIV-1 Nef antibody,
and therefore was Nef-specific (Fig. 6B).
View larger version (11K):
[in a new window]
Fig. 6.
Exogenous HIV-1 Nef protein stimulates HIV-1
replication in chronically infected U1 promonocytic cells.
A, time course of HIV-1 replication in U1 cells treated with
exogenous HIV-1 Nef. U1 cells were treated with 1000 ng/ml exogenous
HIV-1 NL4-3 Nef or with 1000 ng/ml exogenous HIV-1 NL4-3 Nef boiled
at 100 °C for 20 min, and p24 was measured at different times
post-treatment in culture supernatants as reported under
"Experimental Procedures." Untreated U1 cells were used as a
negative control (p24 < 50 pg/ml; data not shown). B,
dose response and specificity of viral replication induced in U1 cells
by exogenous HIV-1 NL4-3 Nef. U1 cells were treated with increasing
concentrations of exogenous NL4-3 HIV-1 Nef (0, 100, 1000 ng/ml) in
the absence or presence of neutralizing anti-Nef antibody at 10 µg/ml, and p24 was measured in culture supernatants at day 7 post-treatment. Results are presented as a histogram. For
each point, p24 was quantified from independent duplicates, and the
means of the duplicate samples are presented. A representative
experiment of two independent p24 assays is shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B, AP-1,
and JNK in promonocytic U937 cells. We observed that exogenous HIV-1
Nef stimulates HIV-1 LTR via NF-
B activation. We also found that
exogenous Nef stimulates HIV-1 replication in chronically infected U1
promonocytic cells. These data indicate that exogenous Nef enhances
HIV-1 replication in latently infected promonocytic cells and could
favor the spread of the disease via enhancement of viral replication
from latently infected cellular reservoirs in HIV-infected subjects.
B DNA binding has been described in promonocytic cell
lines infected with HIV (41). Here we report that exogenous Nef
activates NF-
B and induces I
B
phosphorylation and degradation in promonocytic U937 cells. Since phosphorylation of I
B
at serine is induced, exogenous Nef probably activates IKK-
, the only kinase known to phosphorylate I
B
directly (63). IKK-
is regulated by
several upstream kinases and endogenous Nef has been reported to bind
directly to such members of the Src kinase family as Hck, Lyn, Lck, and
Fyn (17, 22, 26, 27, 64). Although Lck has been implicated in NF-
B
activation (60, 65), it is not expressed in primary macrophages and in
U937 cells (66). In agreement with this observation we observed NF-
B
activation in Lck-deficient cells treated with exogenous Nef (data not
shown). Our data also show that exogenous Nef activates AP-1 and JNK in promonocytic cells, indicating that exogenous Nef interferes with the
MAPK pathway. Endogenous Nef has been reported to directly interact
with Lck and MAPK inhibiting their kinase activity in T cells (23).
These results indicate that exogenous and endogenous Nef might have
different effects on the MAPK pathway.
B and AP-1 was observed after
treatment with exogenous Nef derived from SIV and HIV-1 isolates. These
results could indicate that conserved regions between SIV and HIV-1 Nef
proteins are involved in this phenomenon. In fact, SIV Nef is larger
than HIV-1 Nef, and the molecules share 38% amino acid homology (62).
The most homologous regions are the N-terminal myristylation region and
a highly conserved core region (62). Additional studies are required to
determine the regions of SIV and HIV-1 Nef involved in NF-
B and AP-1
activation in promonocytic cells.
B,
AP-1, and JNK, suggesting that they might modulate the cellular
machinery in a similar way and therefore might have the same effect on
HIV replication in mononuclear phagocytes. We observed that exogenous
HIV Nef, like TNF treatment (69), stimulates HIV-1 replication in the
chronically infected promonocytic cell line U1.
B, AP-1, and JNK and to trigger the release of proinflammatory
cytokines from primary macrophages including TNF, IL-1
, IL-6, and
IL-8 (71, 72). Several features indicate that the activation of
NF-
B, AP-1, and JNK by exogenous Nef is a direct intrinsic effect of
the Nef protein and is not mediated via the release of endogenous TNF. We did not detect increased TNF levels in culture supernatants of U937
cells treated with exogenous Nef (data not shown). The time curve of
NF-
B, AP-1, and JNK activation is similar following treatment with
either exogenous Nef or TNF, with DNA binding starting at 30 min
post-treatment and with a peak at 120 min post-treatment (73). Our
results suggest a redundancy between Nef and TNF in regard to the
activation of NF-
B, AP-1, and JNK in promonocytic cells. Thus,
exogenous Nef mimics TNF biological effects, suggesting that a viral
protein could fuel the progression of the disease even in the absence
of proinflammatory cytokines. This might be of critical importance at
early stages of the disease when chronic immune activation is not yet
predominant and viral factors are needed to establish a productive infection.
B, and JNK, but to a lesser
extent than TNF, exogenous Nef may play a role in macrophage activation
observed during HIV pathogenesis. Nef protein is detected in the serum
of HIV-1 infected patients (48), indicating that exogenous Nef could
participate in the reactivation of the virus from latency, especially
in mononuclear phagocytes. Although endogenous Nef prevents T cell
apoptosis in HIV-infected cells (10-12), the release of soluble Nef
from the infected cells triggers the apoptosis of uninfected
CD4+ T cells present in the vicinity (70). Increased T cell
apoptosis has been reported following treatment with exogenous Nef
(70), and we observed that exogenous Nef triggers T cell apoptosis via activation in poly(ADP-ribose) polymerase
(PARP).2 Thus, exogenous Nef
protein could participate to the immune suppression observed in
HIV-infected subjects via T cell depletion of uninfected bystander
cells. Therefore, exogenous Nef protein could favor the progression of
the disease via both increased HIV-1 replication from latently infected
monocytic cells and enhanced immune suppression due to T cell apoptosis.
B,
AP-1, and JNK, and stimulates viral replication in the chronically
infected promonocytic cells U1 via activation of the HIV LTR. This
observation suggests a critical role for exogenous Nef in AIDS
pathogenesis via enhancement of HIV-1 replication from latently
infected mononuclear phagocytes. A better understanding of the
mechanisms underlying the replication of HIV-1 from latently infected
cellular reservoirs is likely to lead to new therapeutic approaches,
which could help to clear the reservoirs of virions in HIV-infected individuals.
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ACKNOWLEDGEMENT |
---|
We thank the AIDS Research and Reference Program (National Institutes of Health) for reagents used in this study. We thank Mark Harris, Caitriona Dennis, and Sabine Mazaleyrat at the University of Leeds for production of recombinant BH10 and SF2 Nef proteins. We also thank Ulrich Mahlknecht and Ignacio Portero-Robles at the University of Frankfurt for production of recombinant NL4-3 Nef protein.
![]() |
FOOTNOTES |
---|
* This research was funded by The Clayton Foundation for Research (to B. B. A.), by grants from the Franche-Comté University, France (to G. H.), and from the Fonds National de la Recherche Scientifique (FNRS, Belgium), the Télévie-Program, the Université Libre de Bruxelles (ULB), the Internationale Brachet Stiftung, the CGRI-INSERM cooperation, the Région Wallonne-Commission Européenne FEDER, the Agence Nationale de Recherches sur le SIDA (ANRS, France), the Theyskens-Mineur Foundation (to C. V. L.), the Biotechnology and Biological Sciences Research Council (Grant 24/C12902), and the European Union Fifth Framework (Grant QLK2-CT-2000-01630).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.
** Maître de Recherches of the FNRS.
To whom correspondence should be addressed: Cytokine Research
Section; Dept. of Bioimmunotherapy, The University of Texas, M. D.
Anderson Cancer Center; Houston, TX 77030. Tel.: 713-794-1817; Fax:
713-794-1613; E-mail: aggarwal@mdanderson.org.
Published, JBC Papers in Press, November 4, 2002, DOI 10.1074/jbc.M209622200
2 S. K. Manna and B. B. Aggarwal, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
HIV, human
immunodeficiency virus;
AP-1, activator protein-1;
IB, inhibitor of
NF-
B;
JNK, Jun N-terminal kinase;
NF-
B, nuclear factor
B;
NAK, Nef-associated kinase;
PAK, p21-activated kinase;
MAPK, mitogen-activated protein kinase;
LTR, long terminal repeat;
TNF, tumor necrosis factor;
IKK, I
B
kinase;
STAT1, signal transducer
and activator of transcription 1;
SEAP, secreted alkaline phosphatase;
LPS, lipopolysaccharide;
FITC, fluorescein isothiocyanate;
EMSA, electrophoretic mobility shift assay.
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