(Received for publication, November 5, 1996, and in revised form, March 6, 1997)
From the Department of Clinical and Experimental
Medicine, Medical School, University of Reggio Calabria, 88100 Catanzaro, Italy and the § Department of Biochemistry and
Biomedical Technology, Medical School, University "Federico II,"
Naples 80131, Italy
Human immunodeficiency virus type 1 (HIV-1)
infection is associated with severe psoriasis, B cell lymphoma, and
Kaposi's sarcoma. A deregulated production of interleukin-6 (IL6) has
been implicated in the pathogenesis of these diseases. The molecular
mechanisms underlying the abnormal IL6 secretion of HIV-1-infected
cells may include transactivation of the IL6 gene by HIV-1. Here we report the molecular mechanisms of Tat activity on the expression of
the IL6 gene. By using 5 deletion mutants of pIL6Pr-CAT and using
IL6:HIV-1-LTR hybrid constructs where discrete regions of the IL6
promoter replaced the TAR sequence in HIV-1 LTR, we identified a short
sequence of the 5
-untranslated region of the IL6 mRNA that is
required for Tat to trans-activate the IL6 promoter. This sequence
acquires a stem-loop structure and includes a UCU sequence that binds
to Tat and is necessary for full trans-activation. In addition, we
provide the evidence that Tat can function by enhancing the CAAT
enhancer-binding protein (C/EBP) DNA binding activity and is able to
complex with in vitro translated C/EBP
, which is a major
mediator of IL6 promoter function. By using the yeast two-hybrid system
and immunoprecipitation, we observed that the interaction of Tat with
C/EBP proteins also occurred in vivo. The data are
consistent with the possibility that Tat may function on heterologous
genes by interacting with RNA structures possibly present in a large
number of cellular and viral genes. In addition, Tat may function by
protein-protein interactions, leading to the generation of heterodimers
with specific transcription factors.
Human immunodeficiency virus type 1 (HIV-1)1 is the etiologic agent for acquired immunodeficiency syndrome (AIDS) and causes various clinical and immunological abnormalities, including activation of polyclonal B cells that manifests as hypergammaglobulinemia and autoantibody production, lymphadenopathy, Kaposi's sarcoma, and lymphoma of the B cell phenotype (1-3). Studies on small cohorts of subjects who were exposed to HIV-1 and did not develop HIV-1 infection and individuals who harbored HIV-1 but remained disease-free for long periods (4, 5) strongly suggest that the development of AIDS may depend on a dynamic interplay between viral and host cellular gene products. Accordingly, in HIV-1-infected subjects there is a deregulated production of cytokines, including the proinflammatory interleukin-6 (IL6) (6), which affects the growth and differentiation of lymphoid and mesenchymal cells (7) and may contribute to the development of the clinical features of AIDS. Accordingly, IL6 gene transcription is induced in cells infected by HIV-1 (8), and increased levels of IL6 have been reported in serum and cerebral spinal fluid of HIV-1-infected patients (9).
The Tat protein of HIV-1 is required for efficient viral gene
expression (10-15). Tat increases the initiation of transcription from
the HIV-1 LTR (14) and affects RNA processing and utilization by
interacting with a transactivating responsive element (TAR) located
between nucleotides +1 and + 44 with respect to the initiation site
(+1) of viral transcription (16, 17). TAR contains a 6-nucleotide loop
and a 3-nucleotide pyrimidine bulge that are essential for Tat activity
(18-21). Tat binds to the bulge and appears to require cellular
factors binding to the loop sequence to efficiently transactivate the
HIV-1 LTR (22-24). In addition, Tat interacts with upstream regulatory
DNA sequences circumscribed within the NF-B/Sp1 sites of the HIV-1
promoter (25) and with host cell proteins (12, 24). The 86-amino
acid-long Tat contains a highly conserved cysteine-rich region, which
mediates the formation of metal-linked dimers in vitro and
is essential for Tat function (16-18). A conserved basic region with 6 arginines and 2 lysines in nine residues, stretching from amino acid 47 to 58, is crucial for nuclear localization, mediates the specific
binding of Tat to TAR RNA, and is required for the full activity of Tat
(26-29).
In addition to its role in HIV-1 transcription, Tat may participate in
the development of AIDS by modulating the expression of heterologous
genes. In support of this possibility, Tat has been shown to increase
the expression of cellular genes, such as the IL6 (30) and tumor
necrosis factor- genes (31, 32), and to activate the life cycle of
some AIDS-associated viruses (33). The mechanisms of the Tat-mediated
activation of non-HIV-1 genes are obscure. Here, we describe the
mechanisms for Tat-mediated induction of the IL6 gene expression. We
find that Tat is tethered to the IL6 transcription start site by
specific binding to a UCU sequence present in the stem-loop structure
of IL6 leader RNA. Tat physically interacts with C/EBP
and increases
selectively the nuclear pool of C/EBP factors binding to the C/EBP
cis sequence in the IL6 promoter. This interaction was
confirmed to occur in vivo by immunoprecipitation and by
using the yeast two-hybrid system.
pILIC-CAT (34), a
HIV-LTR-CAT plasmid was obtained from A. Rabson (MBCL, Piscataway, NJ).
The 5 deletion mutants of IL6 promoter, pIL6(
596/+15)-CAT,
pIL6(
225/+15), and pIL6(
112/+15)-CAT plasmids, were generated as
reported (30). To generate the HIV-1-LTR:(
112/+15) IL6 promoter
fusion plasmid, the TAR-deleted EcoRI-BglII
fragment of pILIC-CAT was isolated, filled in, and inserted at the
SstI site (filled) of pIL6(
112/+15). The resulting
p
ILIC:IL6(
112/+15)-CAT plasmid carries the IL6 promoter region
from
112 to +15 that substitutes for TAR. To generate
p
ILIC:IL6(
112/
67)-CAT plasmid, the EcoRI fragment of
p
ILIC:IL6(
112/+15)-CAT was filled in and digested with
SspI. The EcoRI-SspI fragment,
containing the TAR-deleted LTR fused to the
112/
67 region of IL6
promoter, was cloned in pEMBL-CAT digested with
BamHI-HindIII (filled). The
SspI-EcoRI fragment, formed by the
67/+15
region of IL6 promoter fused to a part of the cat gene, was
recovered and HindIII-digested. The
67/+15 region of the
IL6 promoter, the SspI-HindIII fragment, was
recovered and cloned in p
ILIC:IL6(
112/+15)-CAT from which the
112/+15 IL6 fragment was removed by
KpnI-HindIII digestion. pIL6(
596/+15) mutants
were produced with the TransformerTM site-directed
mutagenesis kit, as instructed by manufacturer (CLONTECH Laboratories, Inc., Palo Alto, CA), with
minor modifications. In fact, one oligonucleotide was used to introduce
the desired mutations in IL6 promoter and to create the site for
PstI. The following oligonucleotides were used (the mutated
bases are underlined): 5
-CTGAGGCTCATTGGGCCCTCGACCTGCAGGCAT-3
for
pIL6(
596/+15) MI-CAT (bulge mutant);
5
-ATTCTGCCCTAGGCCCGCAGGCATGC-3
for pIL6(
596/+15) M2-CAT (stem mutant); and
5
-CTGAGGCTCATTCTGAGCTCGACCTGCAGGCAT-3
for pIL6(
596/+15)M3-CAT (loop mutant).
The pSVT8 and pSVT10 plasmids, expressing the tat gene in a
sense or antisense orientation, respectively (35), were obtained from
A. Caputo. pCMV-TAT plasmid, expressing the first exon of the
tat gene, and pCMV-TAT 49 were a gift of K. T. Jeang
(Laboratory of Molecular Microbiology, NIAID, NIH, Bethesda, MD). The
pGEX-TAT plasmid was obtained from M. Giacca (International Center for Genetic Engineering and Biotechnology, Trieste, Italy). In this plasmid, the first exon of the tat gene is cloned in
pGEX-2T, an isopropyl-1-thio--D-galactoside-inducible
expression vector (Pharmacia, Uppsala, Sweden), which allows the
production of GST-Tat fusion proteins. The pBlue610 plasmid expressing
the C/EBP
(obtained from S. Akira, Institute for Molecular and
Cellular Biology, Osaka University, Osaka, Japan) was used for in
vitro transcription and translation of C/EBP
. The pSP6:BSF2.5
plasmid, which allows for the in vitro production of IL6,
was obtained from T. Kishimoto (Institute for Molecular and Cellular
Biology, Osaka University, Osaka, Japan). The pGAL4-TAT plasmids, used
in yeast transfections, were a gift of B. Cullen (36). In these
plasmids, the expression of GAL4-Tat fusion sequences, consisting of
wild-type or truncated Tat protein fused to GAL4 DNA-binding domain
(amino acids 1-117), is directed by the yeast alcohol dehydrogenase
promoter, while the yeast selectable marker is HIS3. To generate
pGAD424-C/EBP
, the c/EBP
cDNA was cloned downstream the GAL4
activation domain (amino acids 768-881). The C/EBP
cDNA was
excised from pBlue610 by SalI-EcoRI digestion and
cloned in compatible sites of pGAD-424 vector (37). In this yeast
expression vector, the selectable marker is LEU2, while the production
of the fusion protein is driven by the alcohol dehydrogenase
constitutive promoter. The correct insertion of the plasmids was
verified by multiple restriction digestion and by sequencing using the
Sanger method (38).
HeLa-T8 and HeLa-T10,
expressing the tat gene in a sense or antisense orientation,
respectively, have been described (30). Cells were cultured in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum (Flow Laboratories, Milan, Italy), 3 mM glutamine,
and 10 mM Hepes buffer, pH 7.2 (Life Technologies, Inc.,
Milan, Italy). For transient expression experiments, cells were
transfected by electroporation using a Bio-Rad apparatus (Bio-Rad,
Milan, Italy). 3 × 106 cells were resuspended in 0.3 ml of RPMI 1640 supplemented with 20% fetal calf serum and subjected
to a double electrical pulse (0.2 V, 960 microfarads) in the presence
of the indicated amounts of plasmid DNA. After electroporation, cells
were washed and plated in complete medium. Transfection efficiency was
monitored by cotransfecting the cells with 5 µg of pnls-LacZ plasmid.
-Galactosidase activity was assayed using 50 µg of protein
extracts as described (39).
48 h after transfection, cells were harvested and washed once with PBS. Cell extracts were prepared by three cycles of freeze-thawing in 0.25 M Tris, pH 7.8, and CAT assays were performed as described previously (39). Proteins were measured in each cell extract with the Bio-Rad protein assay kit, and equal amounts of proteins were analyzed for each sample. Each assay contained 50 µg of cell extract, 20 µl of 4 mM acetyl-coenzyme A (Boehringer Mannheim), 1 µl (0.5 µCi) of D-threo-[1,2-14C]-chloramphenicol (DuPont NEN) in a final volume of 150 µl of 0.25 M Tris, pH 7.8. Reactions were incubated for 3 h at 37 °C, extracted with ethyl acetate, dried, and spotted on Polygram Sil G silica gel plates (Macherey-Nagel, Düren, Germany). Plates were run in a TLC tank containing a mixture of chloroform:methanol (95:5). After a 16-h autoradiography, the TLC plates were cut, and samples were counted in a Beckman LS5000TD scintillation counter.
Primer extension was carried out as described (38). 20 µg of total
RNA was annealed to the oligonucleotides 5-CAACGGTGGTATATCCAGTG-3
(for cat RNA) and 5
-CAGATACTACACTTG-3
(for U2 RNA). RNA
was elongated with reverse transcriptase, digested with RNase A, and separated over a 6% denaturing 7 M urea-acrylamide gel.
Nuclear and
cytosolic extracts were isolated as described elsewhere (39-41). Cells
were harvested, washed once in cold PBS, and transferred to 1.7-ml
microcentrifuge tubes for a second wash. The supernatant was removed,
and the cell pellet was resuspended in lysing buffer (10 mM
HEPES, pH 7.9, 1 mM EDTA, 60 mM KCl, 1 mM dithiothreitol (DTT), 1 mM
phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml aprotinin, 10 µg/ml
leupeptin, and 0.2% (v/v) Nonidet P-40). After a 5-min incubation on
ice, nuclei were collected by centrifugation (500 × g,
5 min). The supernatant (cytosolic proteins) was recovered and stored
at 80 °C. Nuclei were rinsed with Nonidet P-40-free lysing buffer,
resuspended in 300 µl of Nonidet P-40-free lysing buffer, and layered
on the top of 300 µl of the same buffer containing 30% sucrose.
After centrifugation at 2,900 × g for 10 min, the
pelletted nuclei were resuspended in 150 µl of buffer containing 250 mM Tris-HCl, pH 7.8, 60 mM KCl, 1 mM DTT, 1 mM PMSF, 10 µg/ml aprotinin, and 10 µg/ml leupeptin. Nuclei were then subjected to three cycles of
freezing and thawing. The suspension was cleared by centrifugation
(135,000 × g, 15 min), and aliquots were immediately
tested in a gel retardation assay or stored at
80 °C until
use.
For EMSAs, the following oligonucleotide probes were used:
5-GATCGGACGTCACATTGCACAATCTTAATAAT-3 (IL6 C/EBP binding site), 5-GGACGTCACACTACAAACTCTTAATAA-3
(mutant IL6 C/EBP binding site), 5
-TCGAGTTGCCTGGACTTGCCTGGCCTTGCCTTTTC-3
(p53 binding site), 5
-CATTCTGCCCTCGAG-3
(IL6, nucleotide +1/+15 coding strand), and
5
-CTCGAGGGCAGAATG-3
(IL6, nucleotide +1/+15 minus strand). Each
oligonucleotide was annealed to its complementary strand and
end-labeled with [
-32P]ATP (Amersham Life Science,
Inc.) by using polynucleotide kinase (New England Biolabs, Beverly,
MA). Equal amounts of nuclear extracts were incubated in a reaction
mixture consisting of 20 µl of buffer containing 10% glycerol, 60 mM KCl, 1 mM EDTA, 1 mM DTT, 2 µg of poly(dI-dC) (Boehringer Mannheim), and 5 µg of extract for 5 min
on ice. 1 µl of
-32P-labeled double-stranded probe
(0.2 ng, 4-6 × 104 cpm) was then added with or
without a 100-fold molar excess of competitor wild-type or mutant
oligonucleotide. Where indicated, 2 µg of antibody to C/EBP (Santa
Cruz Biotechnology Inc., Santa Cruz, CA), or preimmune serum were
added. The reactions were incubated at room temperature for 20 min and
run on a 5% acrylamide/bisacrylamide (30:1) gel in 22.5 mM
Tris borate, 0.5 mM EDTA, and 3.7 mM
-mercaptoethanol. Gels were dried and autoradiographed.
To produce the IL6 RNA
[-32P]UTP-labeled probes, we used oligonucleotides
encompassing the sequence of T7 promoter and either the wild type or
mutant IL6 untranslated sequences in sense or antisense orientation.
These were as follows:
5
-GATCTAATACGACTCACTATAGGGCATTCTGCCCTCGAGCCACCGGGAAC-3
(IL6 wild type
sense),
5
-GATCTAATACGACTCACTATAGGGCATTGGGCCCTCGAGCCACCGGGAAC-3
(IL6 MI sense),
5
-GATCTAATACGACTCACTATAGGGCATTCTGCCCTAGGGCCACCGGGAAC-3
(IL6 M2 sense), and
5
-GATCTAATACGACTCACTATAGGGCATTCTGAGCTCGAGCCACCGGGAAC-3
(IL6 M3 sense). For antisense transcripts, the following
oligonucleotides were used:
5
-GATCTAATACGACTCACTATAGGGCAAGGGCCACCGAGCTCCCGTCTTAC-3
(IL6 wild type antisense),
5
-GATCTAATACGACTCACTATAGGGCAAGGGCCACCGAGCTCCCGGGTTAC-3
(IL6 M1 antisense),
5
-GATCTAATACGACTCACTATAGGGCAAGGGCCACCGGGATCCCGTCTTAC-3
(IL6 M2 antisense),
5
-GATCTAATACGACTCACTATAGGGCAAGGGCCACCGAGCTCGATCTTAC-3
. TAR RNA was obtained by transcribing the
HindIII-linearized pTAR plasmid with T7 RNA polymerase.
32P-labeled transcripts were resolved in an 8 M
urea, 10% polyacrylamide gel and eluted from the gel slides by a
37 °C overnight incubation in TE buffer (10 mM Tris/Cl,
pH 8.0, 1 mM EDTA, pH 8.0). Eluted RNAs were extracted with
phenol-chloroform and precipitated with ethanol. The standard binding
reaction was performed in 20 µl of reaction mixture containing 1 × binding buffer (25 mM Tris/HCl, pH 8.0, 1 mM
MgCl2, 0.5 mM DTT, 50 mM NaCl, 5%
glycerol), 1 µg of sonicated salmon sperm DNA, 0.4 µg of tRNA, 15 units of RNase inhibitor, 2.5 µg of recombinant Tat (AIDS Research
and Reference Reagents) or of GST or GST-Tat, and competitors. After a
10-min incubation in ice, 1 µl of 32P-labeled probe
(about 0.3 ng, 70,000 cpm) was added. The samples were incubated for 20 min at room temperature and run on 6% polyacrylamide gel, 0.5 × TBE at 4 °C, and 180 V. Gels were dried and autoradiographed.
To produce GST-Tat
proteins, the plasmid pGEX-TAT was introduced in Escherichia
coli strain SF8. Bacteria containing the plasmid were
grown to 0.4 A600 and induced with 0.5 mM isopropyl-1-thio--D-galactoside, Inalco,
Milan, Italy) for 2 h. Cells were then collected by centrifugation at 4 °C (3,000 × g for 15 min) and resuspended in
RE buffer containing 50 mM Tris/HCl, pH 8.0, 120 mM NaCl, 0.5% Nonidet P-40, 5 mM DTT, 10 µg/ml aprotinin, 10 µg/ml leupeptin and 1 mM PMSF.
Cells were broken with a French press apparatus, and the lysates were
clarified by centrifugation at 4 °C and 27,000 × g
for 30 min. Proteins were recovered and added to glutathione-Sepharose
beads (Pharmacia), previously equilibrated in RE buffer. After an
overnight incubation, the beads were extensively washed, and GST-Tat
was eluted in RE buffer with 10 mM glutathione. The
35S-labeled C/EBP
proteins were in vitro
translated by using TNTTM coupled reticulocyte lysate
systems (Promega, Madison, WI) according to the instructions of the
manufacturer. For protein interaction studies, 10 µg of GST and
GST-Tat proteins were incubated with 15 µl of translation mixture in
buffer A (20 mM Hepes, pH 7.9, 10 mM
MgCl2, 0.2 mM EDTA, 1 mM DTT, 150 mM NaCl, 5% (v/v) glycerol, and 0.05% Nonidet P-40). The
samples were incubated for 2 h at room temperature. At the same
time, the glutathione-Sepharose beads were washed, blocked in buffer A
with 1 mg/ml BSA for 2 h, and washed again. These beads were added
to the samples. After 3 h, the beads were collected by
centrifugation (2,000 × g for 10 s) and washed 10 times with buffer A. The pellets were then resuspended in sample buffer
(70 mM Tris/HCl, pH 6.8, 7 mM EDTA, 0.01%
bromphenol blue, 13% sucrose, 1% SDS, 7 M urea, and 10% (v/v)
-mercaptoethanol) and resolved on 12% SDS-polyacrylamide gel.
Gels were treated with the Entensify kit (DuPont NEN), dried, and
exposed.
Total cell
extracts were prepared as described (38). Briefly, 2 × 107 transfected cells were harvested, washed twice with
cold PBS, and resuspended in 0.5 ml of lysis buffer containing 50 mM Hepes, pH 7.5, 150 mM NaCl, 10% glycerol,
1% Triton X-100, 1 mM EGTA, 1.5 mM
MgCl2, 100 mM NaF, 10 mM sodium
pyrophosphate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 5 mM DTT. Samples were incubated for 1 h in ice and
lysed by gently passing through a 21-gauge needle. After centrifugation
at (500 × g, 15 min), the proteins were recovered and
stored at 80 °C. For immunoprecipitation, 1 mg of protein was
incubated overnight at 4 °C with 20 µg of mouse anti-Tat
monoclonal antibody (ABT, Milan, Italy). The immunocomplexes were
precipitated with protein A-Sepharose (Sigma-Aldrich, Milan, Italy) by
centrifugation at 720 × g for 10 min. The
immunoprecipitates were washed several times in buffer containing 20 mM Hepes, pH 7.5, 150 mM NaCl, 10% glycerol,
0.1% Triton X-100, 1 mM sodium orthovanadate, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 5 mM DTT. Immunoprecipitates were resuspended in sample
buffer, run on 12% SDS-polyacrylamide gel, and analyzed by
immunoblotting with anti-C/EBP
polyclonal antibody (Santa Cruz
Biotechnology), or anti-Tat antibody (AIDS Research and Reference
Reagents).
Immunoblotting analysis was performed as described (39). Total cell extracts, nuclear proteins, or cytosolic proteins (10 µg) were separated by SDS-10% polyacrylamide gel electrophoresis, transferred onto a membrane filter (Cellulosenitrate, Schleicher & Schuell), and incubated with the indicated first antibody in PBS plus 5% dry milk for 2 h at room temperature. Filters were washed three times in PBS and incubated with peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (Boehringer Mannheim) at a 1:2000 dilution for 1 h. The proteins were revealed by using the enhanced chemiluminescence system (ECL) (Amersham).
In Vivo Protein Interaction: Two-hybrid SystemSaccaromyces cerevisiae CTY2 strain was grown
in YPD medium (20 g/liter peptone, 10 g/liter yeast extract, 20 g/liter
glucose, pH 5.8) and transformed by electroporation. Yeast were
inoculated at 3 × 106 cells/ml and grown up to 3 × 107 cells/ml. Cells were harvested by centrifugation at
room temperature (3,000 × g, 15 min) and rinsed with
H2O. Yeast pellet was resuspended in prepulse buffer (10 mM Tris/HCl, pH 7.5, 1 M sorbitol) and left for
30 min at room temperature. Cells were collected and resuspended in
YPD, 1 M sorbitol at 6 × 109 cells/ml.
3 × 108 cells were added to the DNA mix consisting of
20 µg of each plasmid in YPD, 1 M sorbitol to a 10-ml
final volume. After 10 min, the mixture was electroporated with a
Bio-Rad apparatus set at 1,100 V, 600 ohms, and 25 microfarads in
0.2-cm gap cuvettes. Cells were transferred in 1 ml of 1 M
sorbitol and plated on 5-bromo-4-chloro-3-indolyl -D-galactoside (x-gal)-selective minimal medium
containing 6.7 g of yeast nitrogen base, 20 g of agar,
20 g of glucose, 1 M sorbitol, 1 × amino acids
minus leucine and histidine, 0.1 M KPO4, pH 7, 20 µg/ml of x-gal and H2O to 1 liter. Colonies were
visible in 3 days and blue-positive after 4-5 days. The expression of
fusion proteins was assayed by immunoblotting, as previously reported (39). For this purpose, 50 µg of cell extracts of transfected yeast
cells were probed with polyclonal antibodies to Tat or to C/EBP
proteins, which were obtained from the National Institutes of Health
AIDS Research and Reference Reagent Program (Bethesda, MD) and from
Santa Cruz Biotechnology, respectively.
We have recently reported that tat expression in
epithelial HeLa cells and in MC3 lymphoblastoid cells resulted in the
activation of endogenous IL6 gene transcription, as well as in the
transcriptional induction of pIL6-CAT plasmid, an IL6 promoter-CAT
construct (30). To gain further insight into the molecular mechanisms
of the Tat-mediated activation of the IL6 gene, we constructed 5
deletion mutants of pIL6-CAT in which the region from
596 to +15,
225 to +15, or
112 to +15 (29, 41), was inserted 5
to the
cat gene (shown in Fig. 1). These plasmids,
hereafter referred to as pIL6(
596/+15)-CAT, pIL6(
225/+15)-CAT, and
pIL6(
112/+15)-CAT, respectively, were transiently transfected in HeLa
cells stably expressing the tat gene in a sense (HeLa-T8) or
antisense (HeLa-T10) orientation. Results from these experiments showed
that pIL6(
596/+15)-CAT and pIL6(
225/+15)-CAT plasmids were
efficiently transactivated by Tat, while the pIL6(
112/+15)-CAT
construct was unresponsive to Tat (Fig. 1). This suggested that
Tat-induced activation of the IL6 promoter required a region located
between
225 and
112 bp. Indeed, this region harbors a C/EBP
(NF-IL6) enhancer necessary for efficient IL6 promoter function (30,
43). Next, we generated a plasmid where the
112/+15 base pair region
of the IL6 promoter was inserted downstream to a TAR-deleted HIV-1 LTR
sequence (p
ILIC-CAT). The resulting p
ILIC:IL6(
112/+15)-CAT
plasmid (shown in Fig. 1) was transiently expressed in Tat-positive or
Tat-negative HeLa cells. The
112/+15 sequence of the IL6 promoter,
(see pIL6(
112/+15)-CAT in Fig. 1), conferred Tat responsiveness to
the TAR-deleted HIV-1 LTR promoter (compare p
ILIC-CAT and
p
ILIC:IL6(
112/+15)-CAT plasmids in Fig. 1). This indicated that
the
112/+15 region, which was unresponsive to Tat in the context of
the IL6 promoter (see pIL6(
112/+15)-CAT in Fig. 1), could act as a
TAR-like element when placed in the context of the HIV-1 promoter.
A primer extension analysis of cat mRNA transcribed from
pIL6(596/+15)-CAT revealed a protected band of 98 nucleotides (Fig. 2A), corresponding to the major transcription
start site of the IL6 gene (42). The p
ILIC:IL6(
112/+15)-CAT
generated a major band of 248 nucleotides, corresponding to the
transcription start site of the HIV-1 LTR (shown in Fig.
2A). Moreover, we observed the presence of a 98-nucleotide
additional cat band in cells transfected with the
p
ILIC:IL6(
112/+15)-CAT, indicating that the start sites of the IL6
promoter and of the HIV-1 LTR were both functional (Fig.
2B). A densitometric analysis of the cat bands
confirmed that the HIV-1 LTR start site was preferentially utilized,
with a minimal transcription originating from the IL6 promoter start site (not shown). The amount of cat mRNA in
tat-expressing cells was 8-10-fold higher than the
cat mRNA transcribed by anti-tat-transfected cells. In fact, both the 98-nucleotide cat band generated by
transfecting pIL6(
596/+15)-CAT and the 248-nucleotide cat
band generated by the p
ILIC:IL6(
112/+15)-CAT were stronger in
Tat-positive than in Tat-negative cells (Fig. 2A). These
results identified the sequence of
112/+15 as the minimal region of
the IL6 promoter required for Tat to transactivate the
TAR
HIV-1-LTR. This suggested that the
112/+15 base pair region could
function as a Tat-responsive sequence, possibly allowing Tat to be
directed close to the TATA box of the IL6 promoter.
HIV-1 Tat Interacts with the IL6 Leader RNA
The primer
extension results shown in Fig. 2, A and B,
indicated that two transcription start sites were active in
pILIC:IL6(
112/+15)-CAT hybrid plasmid. This allowed the
construction of the p
ILIC:IL6(
112/
67)-CAT plasmid, where the
region of
67/+15, encompassing both the transcription start site and
the 5
-untranslated region of the IL6 gene, was deleted (shown in Fig.
1). The resulting p
ILIC:IL6(
112/
67)-CAT plasmid was transiently
transfected in HeLa-T10 (Tat-negative) and HeLa-T8 (Tat-positive) cells
to address the question of whether the IL6 leader RNA was required for
Tat-induced activation. As shown in Fig. 1, the
p
ILIC:IL6(
112/
67)-CAT plasmid was unresponsive to Tat,
indicating that a discrete region of IL6 leader RNA is strictly
required for Tat. Accordingly, the
67/+15 region, encompassing the
transcription start site and the 5
-untranslated region of the IL6
gene, restored the responsiveness of the Tat-deleted p
ILIC-CAT plasmid (see p
ILIC:IL6(
67/+15)-CAT in Fig. 1). A secondary
structure analysis of this region according to the energy-minimizing
algorithm of Zuker (44) defines an RNA stem-loop structure at the
5
-untranslated region of the IL6 mRNA (shown in Fig.
3). This RNA contains a UCU stretch that fulfills the
sequence requirements for Tat binding to an RNA structure (45) and is
potentially able to bind to Tat. To test this possibility, point
mutations affecting the secondary RNA structure of the IL6 leader RNA
at the bulge, stem, or loop were introduced into pIL6(
596/+15)-CAT.
The resulting mutant plasmids (shown in Fig. 3) were tested for
responsiveness to Tat in transient expression experiments. As shown in
Fig. 3, mutations that affect the bulge and the stem RNA (mutant M1 and
M2, respectively) led to a drastic decrease in Tat responsiveness,
while mutations of the loop were ineffective (mutant M3). In these
experiments, the pIL6(
596/+15)M1-CAT and pIL6(
596/+15)M2-CAT
plasmids did show a significant activation in Tat-positive (HeLa-T8)
cells, suggesting that Tat can function, albeit at lower efficiency, in
the absence of an RNA tethering structure. Indeed, Tat is able to
activate the transcription of HIV-1 genes in a TAR-independent way, as
recently reported (46-47).
To test for the physical binding of Tat to the leader IL6 RNA,
oligonucleotides corresponding to the wild-type IL6 leader RNA and to
the relative mutants M1, M2, and M3 (shown in Fig. 3), were placed
under the transcriptional control of the T7 promoter in a sense or
antisense orientation and in vitro transcribed. Labeled RNAs
were then tested for binding to Tat in an RNA-protein EMSA. Results
shown in Fig. 4 indicate that Tat is able to
specifically bind to the wild-type IL6 leader RNA. The Tat RNA binding
was not displaced by the M1-RNA or M2-RNA (affecting the bulge and stem
IL6 RNA, respectively), while M3-RNA, affecting the loop IL6 RNA, was
able to compete for the binding to Tat. In these experiments, wild-type
TAR RNA competed specifically with IL6 RNA for the binding to Tat (Fig.
4A). Accordingly, the binding of Tat to TAR RNA was
displaced by wild-type IL6 RNA and by M3-RNA, while M1-RNA and M2-RNA
were substantially ineffective (Fig. 4B). In parallel
experiments, the antisense RNA sequences were unable to bind to Tat
(not shown). Moreover, Tat did not bind to either the single or to the
double-stranded oligonucleotides corresponding to the IL6 leader RNA
(shown in Fig. 4C).
The Basic Region of Tat Is Required for Tat-mediated Expression of the IL6 Gene
A basic domain of Tat, encompassing the amino acid
residues 47-58, has been shown to significantly contribute to the
capacity of Tat to bind to HIV-1 TAR RNA through an arginine fork (16, 17, 28, 49). To test whether the arginine-rich domain of Tat is
required to transactivate the IL6 promoter, we transfected HeLa cells
with plasmids expressing either Tat amino acid residues 1-72 or a
truncated form of Tat (residues 1-49, lacking the basic domain),
together with pILIC:IL6(-112/+15)-CAT, p
ILIC:IL6(-112/
67)-CAT, or p
ILIC:IL6(
67/+15)-CAT plasmid. Results from these transient expression experiments showed that the Tat protein lacking the basic
domain was unable to significantly transactivate the IL6 promoter
(compare the results for p
ILIC:IL6(
112/+15)-CAT in Table
I). Consistent with the results shown in Fig. 1,
p
ILIC(
112/
67)-CAT, lacking the IL6 leader RNA, was unresponsive
to both of the tat-expressing plasmids, while
p
ILIC:IL6(
67/+15)-CAT was fully responsive to Tat-(1-72). It is
noteworthy that the residue 1-49-truncated Tat was still able to
activate the wild-type HIV-1 LTR, albeit at a lower level than the
wild-type Tat, suggesting that the amino-terminal domain of Tat can
function as a transcription factor in the absence of TAR binding. Under
these circumstances, Tat is possibly tethered to the HIV-1 LTR by a
strong interaction with transcription factors binding to HIV-1 LTR
cis sequences. This possibility is supported by the
observation that Tat cooperates with transcription factors binding
upstream regulatory DNA sequences circumscribed within the NF-
B/Sp1
region of the HIV-1 promoter and with host cell proteins (12, 24, 25).
Indeed, a binding of Tat to Sp1 factors has been reported (50).
|
To gain further
insights into the molecular mechanisms of the Tat-mediated activation
of the IL6 promoter, we tested whether Tat might induce an increased
DNA binding activity of C/EBP (NF-IL6) transcription factors, which are
major stimulants of the IL6 promoter (43). Nuclear extracts from HeLa
cells transfected with pSVT8 (tat-expressing) or with pSVT10
(expressing tat in a antisense orientation) were tested for
binding to an oligonucleotide corresponding to the C/EBP cis
sequence of IL6 promoter. As shown in Fig.
5A, tat expression leads to a
significant increase in C/EBP DNA binding activity. Moreover, an
antiserum to C/EBP supershifted the C/EBP complex, while an
antiserum to C/EBP
was ineffective. In parallel experiments,
cytosolic extracts from tat- or
anti-tat-transfected cells expressed equal levels of C/EBP
DNA binding activity (Fig. 5B). Aliquots of nuclear or
cytosolic extracts were assayed for p53 DNA binding activity to monitor
for protein concentrations (data not shown).
Immunoblot analysis of cell extracts of HeLa cells transfected with
either pSVT8 or pSVT10 plasmid revealed equal amounts of total or
cytosolic C/EBP in both tat- and
anti-tat-transfected cells, while a consistent increase in
C/EBP
proteins was observed in the nuclear fraction of
tat-transfected cells (shown in Fig. 6A). The increase in the nuclear C/EBP
was
detectable at 36 h post-transfection and declined thereafter (not
shown). These data indicate that Tat specifically increases the nuclear
levels of C/EBP
factors, resulting in an enhanced binding activity
to C/EBP cis sequence. Under the same conditions, C/EBP
proteins were undetectable (data not shown).
To address the question of whether Tat could interact with C/EBP
transcription factors, C/EBP was in vitro translated and tested for binding to a GST-Tat fusion protein. As shown in Fig. 7, Tat physically associated with C/EBP
. Under the
same conditions, Tat did not bind to IL6 control protein in
vitro produced from pSP6:BSF2.5 plasmid (not shown).
To test the possibility that Tat-C/EBP complexes could form in
vivo, HeLa cells were transiently transfected with pSVT8 plasmid and subjected to immunoprecipitation with a Tat-specific monoclonal antibody followed by immunoblotting with antibodies to C/EBP proteins. We observed that Tat was readily revealed in transfected cells, and
that C/EBP was specifically detected in immunoprecipitates of
tat-expressing cells (Fig. 8, A
and B).
To test whether Tat could functionally cooperate with C/EBP factors, we
took advantage of the yeast genetic two-hybrid system (37). For this
purpose, the C/EBP cDNA was inserted in frame with the sequence
of GAL4 coding for the GAL4 activation domain (amino acid residues
768-881). The resulting pGAD424-C/EBP
plasmid was cotransfected
with pGAL4-TAT plasmids carrying the full-length tat
sequence or truncated sequences of tat, fused to the GAL4 DNA-binding domain (shown in Fig. 9) in the CTY2 yeast
strain, which carries an integrated copy of lacZ gene. Blue
colonies grown on x-gal-selective medium were evaluated as indicative
of in vivo interaction between C/EBP
and discrete regions
of Tat. Results shown in Fig. 9 indicate that Tat interacted in
vivo with C/EBP
and that this interaction resulted in the
transcriptional activation of the integrated lacZ reporter
gene. This activation was comparable with the transcription induced by
Tat homodimerization (Ref. 36, shown in the first line of
Fig. 9 as the interaction of Tat fused to GAL4 binding domain with Tat
fused to VP16 activation domain). Moreover, this activation also
occurred when Tat-(1-47) was used as a partner of C/EBP
, indicating
that the N-terminal, cysteine-rich, and core regions of Tat represent
the minimal region of Tat required for an efficient heterodimerization,
while the entire protein is required for a full transcriptional
activation. In these experiments, comparable amounts of Tat or C/EBP
proteins were produced by transfected yeast cells, as seen by
immunoblotting of cell extracts, using antibodies to Tat or to C/EBP
(data not shown).
Despite the intensive investigation of the immunopathogenesis of AIDS, many questions concerning the molecular mechanisms of HIV-1 primary infection and progression remain unanswered (5, 51, 52). Recently, the identification of cohorts of HIV-exposed individuals who remain free of infection over a long period of viral exposure (53) as well as the existence of a small subgroup of HIV-1-infected subjects who are long term nonprogressors were described (54). Together with recent reports on viral life cycle (55, 56), the above evidence argues that HIV infection and disease progression may ultimately result from a complex interplay between viral and host cellular factors involved in the immunological response to the viral infection and in the clinical evolution of AIDS.
HIV-1 Tat is a potent transactivator of HIV-1 LTR, acting on nascent
TAR RNA and promoting full-length gene transcription (10-13).
Accordingly, Tat-defective HIV-1 is not viable (57, 58). Emerging
evidence shows that, in addition to its role on HIV-1 gene expression,
Tat may exert additional functions. Tat is released in some extent
extracellularly (20, 59) and can function as a cytokine. In fact, Tat
promotes the growth of endothelial cells and Kaposi's sarcoma cells
directly or synergistically with basic fibroblast growth factor (Ref.
60 and references therein) and enhances cell survival in
tat-expressing cells (61). Constitutive expression of
tat in transgenic mice results in tumor development, including Kaposi's-like sarcomas and B cell lymphomas (62). Accordingly, stable expression of tat in
IL6-dependent cells results in growth factor-independent
growth and in tumorigenicity (30). Moreover, data in support of a
nontranscriptional function of Tat in virion infectivity has been
reported (63). The above evidence strongly suggests that Tat may
participate in the establishment of HIV-1 infection and in the
development of AIDS clinical features by promoting the expression of
host cellular genes. In support of this possibility, Tat has been shown
to activate the expression of the proinflammatory cytokines IL6 and
tumor necrosis factor- (30-32) and to increase interleukin-2 and
collagen gene expression (64, 65). Tat was also shown to suppress
promoter activity of major histocompatibility complex class I genes
(66) and to exert immunosuppressive activity on antigen-induced T cell
proliferation (67-69). Moreover, Tat has been shown to promote
apoptosis by up-regulating CD95 ligand expression (70) or by activating
cyclin-dependent kinases (71).
The mechanisms of Tat function on the expression of heterologous genes
are unknown. In this paper, we address in molecular detail the
mechanisms of Tat activity on the expression of IL6, a cytokine with a
broad biological activity (7, 72) whose expression is deregulated in
HIV-infected subjects (6, 8, 9). By using 5 deletion mutants of
pIL6-CAT plasmid, and IL6:HIV-1-LTR hybrid plasmids where discrete
regions of the IL6 promoter replaced the TAR sequence in HIV-1 LTR, we
identified a short sequence of the 5
-untranslated region of IL6
mRNA that is required for Tat to transactivate the IL6 promoter.
This region can acquire a stem-loop structure including a UCU
trinucleotide bulge. Point mutations of the UCU bulge or of the stem
resulted in a drastic decrease in Tat responsiveness (shown in Fig. 3)
and in the inability of Tat to bind to the IL6 leader RNAs (Fig. 4).
The IL6 RNA structure, with an estimated structure energy of
9.1
kcal/mol, is expected to be less stable than the TAR RNA structure.
This suggests that Tat could bind with a low affinity to heterologous
RNA sequences and may account for the ability of Tat to regulate the
expression of multiple genes. Interestingly, Tat was still able to
induce a low but significant activation of the bulge mutant
pIL6(
596/+15)M1-CAT plasmid (shown in Fig. 3), suggesting that Tat
can function, albeit at a lower efficiency, without binding to an RNA
tethering structure. In this case, Tat could be directed to the
transcription start site of IL6 promoter by associating with specific
transcription factors. This possibility is supported by the reports
showing that Tat may associate with Sp1, TFIID factors, RNA polymerase II, and RNA polymerase II-associated factors (50, 73-78). In addition,
we now provide evidence that Tat can function by cooperating with C/EBP
transcription factors. In fact, we observed an increase in the C/EBP
DNA binding activity of tat-expressing cells with a
selective increase in the amounts of nuclear C/EBP
factors (Figs. 5
and 6). This raises the possibility that Tat may increase the nuclear
levels of C/EBP transcription factors by inducing post-translational
modifications of C/EBP factors through the activation of specific
kinases. Indeed, Tat activity on HIV-1 LTR-driven gene expression
requires protein kinase C (79). Moreover, specific interaction of Tat
with a cellular protein kinase has been reported (80), and serine and
threonine phosphorylations of C/EBP
are required for IL6 promoter
activation (81). The above data are consistent with and extend the
recent observation that Tat enhances the tumor necrosis factor-induced
activation of NF-
B binding activity by possibly inducing protein
phosphorylation (82). Since C/EBP and NF-
B factors associate as
heterodimers (83), which are potent activators of HIV-1 LTR (84), the
above data suggest that Tat may also promote HIV-1 gene expression by up-regulating the cellular levels of transcription factors acting on
the viral LTR. Moreover, Tat was able to complex with in
vitro translated C/EBP
, which is a major mediator of IL6
promoter function (Fig. 7). By immunoprecipitation and by taking
advantage of the yeast two-hybrid system, this interaction was proved
to occur also in vivo and to result in transcriptional
activation of a reporter lacZ gene (shown in Figs. 8 and 9).
The Tat association with C/EBP
suggests that Tat may increase the
DNA binding activity of C/EBP dimers by enhancing their affinity for
the target DNA. This mechanism accounts for the Tax activity on
transcription mediated by bZip proteins (86, 87). In the EMSA
experiments shown in Fig. 5A, Tat could not be detected in
the C/EBP-DNA complexes with an anti-Tat antibody (data not shown).
This suggests either that Tat does not directly participate in the
C/EBP-DNA complexes or that Tat dissociates from the DNA-binding
complexes due to the electrical field of EMSAs. These possibilities
warrant further studies.
The data are consistent with the possibility that Tat may function on heterologous genes by interacting with RNA structures possibly present in a large number of cellular and viral genes, as recently reported (30-33). In addition, Tat may function by forming heterodimers with specific transcription factors. These possibilities dramatically enhance the capacity of Tat to modulate the expression of heterologous genes and to play a major role in the pathogenesis of HIV-associated diseases.
We thank A. Rabson for providing pILIC-CAT and A. Caputo for the gift of pSVT8 and pSVT10 plasmids. We also thank K. T. Jeang for the gift of pCMV-TAT plasmids and M. Giacca and S. Akira for providing pGEX-TAT and pBlue610 plasmids, respectively. pSP6:BSF2.5 plasmid was a gift of T. Kishimoto. We are indebted to B. Cullen and R. Sternglanz for pGAL4-TAT and pGAD-424 plasmids, respectively. We acknowledge the careful review of the manuscript by G. Ciliberto and K. T. Jeang. We also thank the AIDS Research and Reagent Program for providing recombinant Tat and antibodies to Tat.