From the Howard Hughes Medical Institute, Program in Molecular Medicine, University of Massachusetts Medical Center, Worcester, Massachusetts 01605
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ABSTRACT |
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The hepatitis B virus pX protein is a potent
transcriptional activator of viral and cellular genes whose mechanism
of action is poorly understood. Here we show that pX dramatically
stimulates in vitro DNA binding of a variety of cellular
proteins that contain basic region/leucine zipper (bZIP) DNA binding
domains. The basis for increased DNA binding is a direct interaction
between pX and the conserved bZIP basic region, which promotes bZIP
dimerization and the increased concentration of the bZIP homodimer then
drives the DNA binding reaction. Unexpectedly, we found that the DNA binding specificity of various pX-bZIP complexes differs from one
another and from that of the bZIP itself. Thus, through recognition of
the conserved basic region, pX promotes dimerization, increases DNA
binding, and alters DNA recognition. These properties of pX are
remarkably similar to those of the human T-cell lymphotrophic virus
type I Tax protein. Although Tax and pX are not homologous, we show
that the regions of the two proteins that stimulate bZIP binding
contain apparent metal binding sites. Finally, consistent with this
in vitro activity, we provide evidence that both Tax and pX
activate transcription in vivo, at least in part, by
facilitating occupancy of bZIPs on target promoters.
Hepatitis B virus (HBV)1
is the major cause of acute and chronic hepatitis and has been
implicated in the initial stage of HBV-related hepatocarcinogenesis
(1-5). The HBV genome is 3.2 kb in length and contains four open
reading frames: pre-S/S, which encodes the surface antigens; core/e,
which encodes the core protein; pol, which encodes the viral reverse
transcriptase; and the X protein (pX) (for review, see Ref. 6). pX is
required for viral infection (7, 8), can transform rodent cells (3, 4, 9, 10), and can cause hepatocellular carcinoma in transgenic mice
(11).
HBV pX, a 154-aa protein, is a potent transcriptional activator
required for efficient viral gene expression. Like several other viral
activators, pX activity is promiscuous; in addition to the cognate HBV
promoter, pX can activate transcription from a diverse set of cellular
and viral promoters that appear to have little in common (12-24). The
mechanism by which pX activates transcription is controversial; it has
been proposed that pX acts through various protein kinase signal
transduction pathways (25-28), is itself a protein kinase (29), has a
ribo/deoxy ATPase activity (30), and is a protease inhibitor (31).
We have noted several similarities between HBV pX and the Tax protein
of human T-cell lymphotrophic virus, type I (HTLV-I). First, like pX,
Tax activates transcription from its own promoter as well as
heterologous promoters (for review, see Ref. 32). Second, the HTLV-I
long terminal repeat and the HBV enhancer contain binding sites for ATF
proteins (ATFs) (33-35), an extensive family of cellular transcription
factors that contain homologous basic region/leucine zipper (bZIP) DNA
binding domains (36). These viral ATF binding sites have been directly
implicated in the Tax (reviewed in Ref. 37) and pX (33) transcriptional
responses. Third, like Tax, pX is present in the nucleus of expressing
cells (10, 38). Finally, and of particular significance, Tax can substitute for pX in transcriptional activation of the HBV enhancer (2,
39, 40). Taken together, these observations suggest that pX and Tax may
stimulate transcription by a common mechanism.
Recent studies have helped clarify the mechanism by which Tax
functions. Tax can dramatically increase the in vitro DNA
binding of proteins containing bZIP DNA binding domain (41-45). bZIP
domains comprise a leucine-rich dimerization motif and a basic region that mediates DNA contact (46-49). This DNA binding increase occurs by
a mechanism in which Tax promotes dimerization of the bZIP domain in
the absence of DNA and the elevated concentration of the bZIP homodimer
then drives the DNA binding reaction (43, 44). Here we show that pX
functions in a manner remarkably similar to Tax.
Oligonucleotide Sequences for Gel-shift Assays--
Sequences
were as follows: collagenase TRE, 5'-AGCTTTGACTCATCCGGA-3'; CRE,
5'-TCCTAAGTGACGTCAGTGGAA-3'; C/EBP binding site, 5'-TGCAGATTGCGCAAT
CTGCA-3'; OR1 site of the Purification of pX Protein--
pX cDNA was cloned in the
pRSET-C vector (Invitrogen) and expressed as a His-tagged derivative in
the BL21(pLysE) Escherichia coli strain. BL21 cells were
grown at 30 °C in the presence of 50 µM
ZnCl2, and pX expression was induced with 0.5 mM IPTG for 2 h. Cells were harvested and resuspended
in lysis buffer (1 M NaCl, 20% glycerol, 0.1% Tween 20, 20 mM Tris-HCl, pH 8, 20 µM ZnCl2, 1 mM PMSF, 10 mM
pX and Tax Deletion Mutants--
pX was first cloned in the
pGEX-CS vector (50) and subsequently digested with the appropriate
enzymes to generate C DNA-binding Proteins--
cDNA sequences of CREB (aa
23-341), ATF-1 (aa 57-271), and ATF-2 (aa 144-505) were cloned in
pGEX vectors and expressed as GST fusion proteins in E. coli. The C/EBP DNA Mobility Shift Assays--
Each reaction mixture contained 1 µg of BSA, 1 µg of poly(dI-dC), 0.5× buffer D, and 0.1 ng of a
32P-labeled CRE oligoduplex. The amount of DNA-binding
protein and pX used is indicated in the figure legends. The reaction
products were analyzed on a 5% polyacrylamide, 0.5× TBE gel except
where otherwise noted.
Chemical Cross-linking--
The indicated amounts of GCN4
polypeptide were incubated with 200 ng of BSA or 100 ng of pX.
Cross-linking was performed by adding glutaraldehyde (Fisher) to a
final concentration of 0.02% and incubated for 30 min at room
temperature. Reactions were terminated by addition of 0.5 µl of 0.5 M Tris, pH 8.0. Monomers and dimers were separated on a
15% SDS-polyacrylamide gel and transferred to a nitrocellulose
membrane. The filter was probed with an anti-GCN4 polyclonal serum
(kindly provided by Peter Kim). 20 ng of H6-T7tag-pX were incubated for
10 min with glutaraldehyde to a final concentration of 0.01%.
Multimers were separated on a 10% SDS-polyacrylamide gel, transferred
to a nitrocellulose membrane, and probed with an anti-T7tag monoclonal
antibody (Novagen).
Far-Western Analysis--
Purified proteins were separated on an
SDS-polyacrylamide gel and transferred to an Immobilon membrane
(Millipore). The filter was preincubated in far-Western buffer (150 mM KCl, 20 mM Hepes, pH 7.5, 5 mM
MgCl2 0.5 mM EDTA, 0.05% Nonidet P-40, 1 mM dithiothreitol, 1 mM PMSF, and 5% BSA).
After 2 h of incubation, [35S]methionine-labeled pX
was added and incubation continued for 4 h. The membrane was
washed twice with far-Western buffer for 15 min, dried, and autoradiographed.
Cell Cultures and Transfections--
HepG2 cell were maintained
in Dulbecco's modified Eagle's medium containing nonessential amino
acids (Life Technologies, Inc.), 2 mM
L-glutamine (Life Technologies, Inc.), antibiotics, and
10% FBS (HyClone). Cell transfections were carried out by using
Opti-MEM reduced serum medium and LipofectAMINE reagent as described by the manufacturer (Life Technologies, Inc.).
pX Increases DNA Binding of Diverse bZIP Proteins--
To
investigate the mechanism of pX action a series of recombinant bZIP
proteins were tested for DNA binding in the presence and absence of pX.
pX was expressed in E. coli as a histidine-tagged derivative
and purified according to a nondenaturing procedure (see "Materials
and Methods"). For reasons described below, DNA binding was performed
at several protein concentrations, which, in the absence of pX, gave
rise to a low level of DNA binding. Fig.
1 shows that, under these conditions,
addition of purified pX greatly increased DNA binding of proteins of
the ATF family (CREB, ATF-1, ATF-2), c-Jun family (c-Jun, GCN4), and
C/EBP family (C/EBP). Significantly, however, pX did not stimulate DNA
binding of all bZIPs (e.g. ATF-4; Fig. 5B). pX
cannot enhance the DNA binding activity of the ATF-4 bZIP transcription
factor (36). Neither an E. coli crude extract nor unrelated
recombinant proteins stimulated DNA binding (data not shown; and see
Ref. 43). pX comparably stimulated DNA binding of bZIP derivatives
containing or lacking a GST moiety. Consistent with previous studies
(33, 39), we could not detect an interaction between purified pX and
DNA (data not shown).
The pX-mediated DNA Binding Increase Is Dependent on bZIP
Concentration--
To investigate the possible role of bZIP
concentration in the pX-mediated DNA binding increase, we measured the
effect of pX at GCN4 concentrations ranging from 0.05 to 20 nM. Fig. 2 shows that maximal
stimulation of DNA binding was observed at peptide concentrations below
4 nM and, at higher protein concentrations, pX did not
significantly increase DNA binding. The failure to increase DNA binding
at higher bZIP concentrations was not due to a limiting amount of pX;
DNA binding was again not affected when the concentration of pX was
increased (data not shown). These results indicate that pX overcomes a
concentration-dependent step that normally limits the
extent of DNA binding.
pX Increases Formation of bZIP Homodimers in the Absence of
DNA--
Dimerization of bZIP proteins occurs in the absence of DNA
and is a prerequisite for DNA binding (51-54). pX could therefore stimulate DNA binding by increasing either dimerization or the subsequent interaction between the bZIP homodimer and DNA.
We measured the effect of pX on bZIP dimerization in the absence of DNA
using a chemical cross-linking assay. Previous studies have shown that
the two subunits of bZIP dimers can be cross-linked to one another with
glutaraldehyde, a bifunctional cross-linking reagent (53). In Fig.
3A, GCN4 was incubated in the
presence or absence of pX, and following addition of glutaraldehyde the products were fractionated on an SDS-polyacrylamide gel and analyzed by
immunoblotting. At a concentration of 0.1 µM, GCN4 is
predominantly a monomer (lane 2) and as the
concentration of GCN4 was raised, homodimer formation increased
(lane 5). Significantly, addition of pX
dramatically increased the amount of GCN4 homodimer (compare lanes 2 to 3 and 5 to
6). An additional band with an approximate size of 46 kDa
(arrow) appeared only when pX and GCN4 were both present. We
suspect that this product results from a pX dimer cross-linked to a
GCN4 dimer, but further experimentation would be required to confirm
this supposition.
To confirm that bZIP dimerization was required for the pX-mediated DNA
binding increase, we analyzed the ability of pX to stimulate DNA
binding of a "pre-dimerized" protein. The synthetic peptide br1s
(52) contains two GCN4 basic regions joined by a disulfide linkage.
Fig. 3B shows that pX did not stimulate DNA binding of br1s,
under the same conditions in which DNA binding of GCN4 was markedly
enhanced. Thus, pX function requires a protein with an appropriate
dimerization domain, consistent with the ability of pX to promote dimerization.
pX Does Not Recognize the Leucine Zipper--
The fact that pX can
stimulate DNA binding of proteins containing only a bZIP (GST-JUN bZIP,
GST-ATF2 bZIP, GCN4) implies that this domain is the target of pX
action. To delineate the portion of the bZIP required for pX
recognition, we analyzed a series of bZIP derivatives. Because pX
increases dimerization, we began by examining the role of the leucine
zipper. The synthetic peptide BR-CC contains the basic region of the
yeast GCN4 bZIP protein attached to an idealized coiled-coil
dimerization motif (51). BR-CC contains the canonical leucine repeat,
but otherwise the dimerization domains of GCN4 and BR-CC bear no
similarity (Fig. 4, bottom).
BR-CC dimerizes and binds an AP-1 site with an affinity similar to GCN4
and Fig. 4 shows that pX stimulated DNA binding of BR-CC and GCN4
comparably.
We next asked whether the leucines were important for pX recognition.
The DNA binding domain of Zta, an Epstein-Barr virus protein, is a
highly divergent member of the bZIP family (55, 56). Although the Zta
basic region is similar to that of other bZIP proteins, the
dimerization domain is not a typical leucine zipper. Nevertheless, this
domain assumes a coiled-coil structure that supports dimerization and
DNA binding. Fig. 4 (right) shows that pX efficiently
stimulated binding of ZTA to the collagenase promoter AP-1 site.
Similarly, pX increased DNA binding of a chimeric protein containing
the human c-Fos basic region fused to the ZTA dimerization domain (Fig.
4, right). The combined data of Fig. 4 indicate that no
specific sequence of the leucine zipper is required for pX function.
pX Recognizes the bZIP Basic Region--
To test whether the
conserved basic region is the target for pX, we first analyzed a hybrid
protein,
To confirm that pX interacted directly with the basic region, we
performed a far-Western blotting experiment. bZIP derivatives were
fractionated by SDS-polyacrylamide gel electrophoresis, transferred to
nitrocellulose, and probed with 35S-labeled pX. The results
show that pX efficiently bound to proteins that contain a complete bZIP
(Fig. 5C). In addition, pX interacted with a GST-ATF2
derivative containing the ATF2 basic region alone but not with ATF2 or
c-Jun derivatives containing only the leucine zipper. Moreover, pX did
not interact with ATF-4, consistent with its inability to stimulate
ATF-4 DNA binding. These results provide strong evidence that pX binds
to the bZIP basic region.
pX Alters DNA Recognition--
The fact that pX interacts with the
basic region, which mediates DNA contact, prompted us to investigate
the DNA binding specificity of pX-bZIP complexes. We analyzed the
ability of pX to increase DNA binding of several bZIP proteins to four
different DNA binding sites. These DNA probes contained an AP1 or an
ATF consensus flanked by sequences derived from either the collagenase
promoter (AP1-C, ATF-C) or a synthetic polylinker (AP1-S, ATF-S) (Fig.
6, bottom). Remarkably, the
requisite combination of consensus binding site and flanking sequence
differed for each bZIP protein tested. For example, pX stimulated DNA
binding of CREB only to the AP1-C, ATF-C, and ATF-S sites. For GCN4,
the pX-mediated DNA binding increase occurred with AP1-C, AP1-S, and
ATF-C but was very modest with ATF-S. The effect of pX on FOS-ZTA DNA
binding was maximal only with collagenase flanking sequences and
independent of the core binding sites. In contrast, pX-mediated
stimulation of ATF2 DNA binding depended only on the ATF core site.
To exclude that the diverse bZIP response to pX was not due to
differences in their intrinsic binding activity, we analyzed the DNA
binding of the four bZIPs alone to the AP-1 and ATF sites. The results
of this experiment, summarized in Fig. 6B, show that CREB,
ATF2, GCN4, and F-ZTA bind the four sites with comparable affinities.
Taken together these results suggest that pX- mediated stimulation of
bZIP binding requires an appropriate combination of core site and
flanking sequences, and that DNA recognition of the pX-bZIP complex
differs from that of the bZIP alone.
Delineation of the Regions of pX and Tax That Facilitate bZIP
Binding--
To identify the region of pX required to enhance bZIP DNA
binding, we constructed and analyzed several pX deletion mutants (Fig.
7A). The results show that the
region between amino acids 13 and 105 is necessary and probably
sufficient to stimulate bZIP DNA binding. Interestingly, the region
between amino acids 49 and 85 is extremely conserved among human HBV
isolates (58) and contains several histidines and cysteines suggestive
of a metal binding domain.
HTLV-I Tax possesses an N-terminal zinc binding domain previously shown
to be required for interaction with CREB (59) and stimulation of
transcription in vivo (60-62). We investigated whether this
region of Tax was also sufficient to enhance bZIP DNA binding. The
results of Fig. 7B show that the Tax activity was abolished upon deletion of this N-terminal metal binding site.
Evidence That Tax and pX Activate Transcription in Vivo by
Promoting bZIP DNA Binding--
We next designed an experiment to
examine whether Tax and pX activated transcription in vivo
by stimulating binding of bZIPs to the promoter. We reasoned that if
Tax and pX function by promoting bZIP binding in vivo, their
ability to stimulate transcription should decrease under conditions of
increased promoter occupancy (i.e. high bZIP concentration).
To test this prediction, we took advantage of the human HepG2 cell
line, which contains relatively low levels of certain bZIP proteins,2 enabling the
intracellular bZIP concentration to be controlled by cotransfection.
Fig. 8 shows that transcriptional
stimulation of the somatostatin promoter by Tax decreased upon
cotransfection of increasing amounts of CREB expression vector.
Similarly, transcriptional stimulation by pX decreased upon
co-transfection of increasing c-Jun expression vector. Thus, in both
cases a significant reduction of Tax- and pX-mediated stimulation
occurred at increased intracellular bZIP concentrations. These in
vivo results recapitulate the in vitro DNA binding data
in which the effects of Tax and pX were found to be inversely
proportional to bZIP concentration (see Fig. 2 and Ref. 43).
In this report, we have demonstrated that pX increases the DNA
binding of diverse bZIP proteins. Our results confirm and extend several previous studies (33, 38, 63-65) and define novel aspects of
the pX mechanism of action, which are discussed in greater detail
below. Specifically, we show for the first time that: (i) pX increases
bZIP dimerization through specific recognition of the basic region;
(ii) pX itself can dimerize and/or multimerize, suggesting that the
pX-bZIP complex contains at least two pX molecules; (iii) pX appears to
increase promoter occupancy in vivo; (iv) pX alters bZIP DNA
binding selectivity; (v) the region of pX required to stimulate bZIP
DNA binding is highly conserved among HBV serotypes and resembles that
of metal-binding proteins; (vi) pX resembles HTLV-I Tax, both
structurally and functionally, despite lacking sequence similarity,
supporting the idea that different viruses may have independently
evolved promiscuous activators to target the same class of cellular
transcription factors.
Stimulation of DNA binding requires only a minimal bZIP domain, and the
two subdomains contribute different but essential functions; pX
recognizes the basic region but requires a leucine zipper dimerization
domain to increase DNA binding. The strong conservation of the basic
region explains how pX can act upon a variety of bZIP proteins. Whereas
previous studies have suggested that ATF-2 (33, 65), CREB, and other
bZIPs (63, 64) might be the target of pX action, we have found that pX
increases DNA binding of multiple bZIPs by promoting their
homodimerization. The diverse bZIP proteins upon which pX can act are
likely relevant to its ability to activate transcription promiscuously.
Previous studies have shown that bZIP proteins bind to DNA in a
two-step reaction (54). In the first step, the bZIP dimerizes, followed
by a second step in which the homodimer binds to DNA. We have shown
that, through interaction with the bZIP basic region, pX increases
dimerization in the absence of DNA. Why does binding of pX to the basic
region promote bZIP dimerization? We have shown that pX can form dimers
or multimers. Thus, we propose that a pX dimer (or multimer) interacts
with the basic region of a relatively unstable bZIP dimer, thereby
stabilizing the dimer form. The resulting increased concentration of
the bZIP dimer then shifts the equilibrium toward formation of the
bZIP-DNA complex.
Unexpectedly, we found that pX alters DNA binding site selectivity by a
mechanism that remains to be determined. One possibility is that pX
re-orients the bZIP so that it contacts DNA differently. For example,
amino acids within the basic region, which make backbone contacts or do
not normally interact with DNA (47), might be re-oriented by pX to
contact a base. According to this model, minor differences among bZIP
basic regions could account for the distinct selection of DNA sites by
each bZIP. Alternatively, the pX portion of a pX-bZIP could contact DNA
sequences flanking the consensus binding site.
The portion of pX necessary to stimulate bZIP DNA binding in
vitro includes a 35-amino acid region that is highly conserved (>95%) among human HBV isolates. This region, which is also required for pX-mediated transcriptional activation in vivo (66-68),
contains several histidine and cysteine residues and may comprise a
metal binding domain. Significantly, Tax contains an N-terminal
zinc-binding domain that is essential for stimulation of bZIP DNA
binding in vitro (59) and transcription activation in
vivo (60, 61). Thus, despite lacking conserved primary sequence,
our results indicate that Tax and pX might, in fact, possess
structurally and functionally similar domains. Structural and
functional similarities between Tax and pX are also suggested by the
ability of these viral proteins to facilitate bZIP binding in
vivo, and transcriptionally activate the HTLV-I long terminal
repeat and HBV enhancer (2, 39, 40). It is intriguing that several
other transcriptionally related protein-protein interactions involve
bZIP domain-metal binding site contacts (69, 70).
Our in vivo results support the view that Tax and pX
function, at least in part, by increasing promoter occupancy.
Specifically, activation by Tax and pX was diminished at high bZIP
concentrations; if Tax and pX acted solely at a step subsequent to DNA
binding (e.g. by contributing an activation domain), their
effect should be largely independent of bZIP concentration. However,
our results do not exclude the possibility that Tax and pX also contain
an activation domain, a possibility supported by several previous studies (18, 66). In this regard, pX would be similar to the adenovirus
E1a protein, which has a transcriptional activation domain and
promoter-targeting region both required to activate their viral target
genes (71-73). Furthermore, like pX, E1a's promoter-targeting region
appears to function through interaction with the bZIP DNA binding
domain of factors such as ATF-2 (74).
It has been proposed that pX has a cytoplasmic function that acts in a
signal transduction cascade (26, 27, 38), and our results are not
inconsistent with this possibility. In fact, a series of detailed
experiments have shown that pX is present both in the nucleus and
cytoplasm, and that different portions of pX are involved in nuclear
and cytoplasmic functions (38). The cytoplasmic pX function appears to
involve activation of the Ras, Raf, and mitogen-activated protein
kinase, which ultimately acts through NF- Finally, our results have obvious implications with regard to the role
of pX in viral infection and hepatocarcinogenesis. We have shown that
pX increases DNA binding affinity and alters DNA binding selectivity.
Both of these effects are likely important for the ability of pX to
recruit the appropriate cellular bZIP protein to the HBV enhancer
during viral infection. By altering the activity of cellular bZIP
proteins, pX will also affect cellular gene expression. In particular,
increasing the activity of known oncoproteins, such as Jun and Fos, may
be the basis by which pX transforms cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
phage PRM/PR promoter, 5'-TATCACCGCCAGAGGTA-3'.
-mercaptoethanol, 40 mM imidazole), frozen on dry ice,
and then thawed on ice and sonicated. The bacterial lysate was
centrifuged for 30 min at 12,000 rpm. The supernatant was incubated
with NTA-Ni2+-agarose beads (Qiagen) for 4 h. Beads
were washed four times with lysis buffer. pX protein was eluted with
0.5 M imidazole, pH 7.5, and dialyzed twice against buffer
X (0.2 M NaCl, 20% glycerol, 20 mM Hepes, pH
8, 20 mM ZnCl2, 5 mM
-mercaptoethanol, 0.1% Tween 20, 1 mM PMSF).
1, C
2, and C
3 mutants. To generate
C
1, pX vector was digested with HincII and
HindIII, treated with Klenow, and self-ligated. C
2 and
C
3 were obtained in the same way except that CSP1/HindIII and AatII/HindIII were used, respectively. Ligation products
were transformed in the E. coli BL21 (pLysE) strain. C
4
was instead obtained by PCR amplification of the region between aa 1 and 36. The primers were oligo X5' (CAT GCC ATG GCT GCT CGG GTG TGC
TGC) and oligo X
4 (CGG AAT TCT TAT TAG AGA GTC CCA ACC GGC CCG CA). The PCR product was then digested with NcoI and
EcoRI and cloned in pGEX-CS vector. pX N-terminal deletions
were also obtained by PCR amplification. N
1 sequences were oligo X
N1 (CAT GCC ATG CGT CCC GTC GGC GCT GAA TCC) and oligo X3' (CGG AAT TCT
TAT TTA GGC AGA GGT GAA AAA CAA AC). N
2 sequences were oligo X N2
(CAT GCC ATG CTC TCT TTA CGC GGT CTC CCC) and oligo X3. PCR products were digested with NcoI/EcoRI and cloned in the
pGEX-CS vector. Protein expression was induced with 0.5 mM
IPTG for 2 h at 30 °C. Protein mutants were purified on a
glutathione affinity column and then dialyzed against buffer X. The GST
moiety was removed by cleaving the GST fusion proteins with TEV
protease (Life Technologies, Inc.) and by incubating the protein with
fresh glutathione-agarose beads. Beads were spun down, and the
supernatant containing the pX proteins was dialyzed against buffer X. Tax mutants were obtained by PCR amplification. Oligonucleotides used
to generate Tax constructs were as follows: Tax wt: oligo
T5' (TCC AAC AAC ATG GCC CAC TCC CCA GGG TTT GGA) and oligo T3' (AAA
GGG GGA TCC TCA GAC TTC TGT TTC TCG GAA ATG); Tax C
1:
oligo T5' and oligo TD31 (AAA GGG GGA TCC TCA ATG AAA GGA AGA GTA CTG
TAT GAG); Tax C
2: oligo T5' and oligo TD32 (AAA GGG GGA
TCC TCA GCC ATC GGT AAA TGT CCA AAT AAG); Tax C
3: oligo T5' and
oligo TD33 (AAA GGG GGA TCC TCA CCC TGT GGT GAG GGA AAT TTT ATA); Tax
C
4: oligo T5' and oligo TD34 (AAA GGG GGA TCC TCA GCA GAC AAC GGA GCC TCC CCA GAG); Tax C
5: oligo T5' and oligo TD35 (AAA GGG GGA TCC
TCA GGG TGG AAT GTT GGG GGT TGT ATG); Tax C
6: oligo T5' and oligo
TD36 (AAA GGG GGA TCC TCA CTG ATG CTC TGG ACA GGT GGC CAG). PCR
products were digested with NcoI and BamHI and
cloned in pGEX-CS vector. Proteins were expressed and purified as
described previously (43).
1-2 derivative (a gift from P. Rorth) was
expressed by IPTG induction. After sonication, cell debris was removed
by centrifugation and the supernatant used in gel-shift assays.
Histidine-tagged JUN (aa 225-334) was obtained from T. Curran.
Epstein-Barr virus ZTA peptide (aa 175-229) containing the DNA binding
domain only was a gift from M. Carey. The FOS-ZTA chimera was obtained
from D. S. Hayward. The construct containing the c-Fos basic
region (aa 133-162) and the ZTA coiled-coil domain (aa 197-229) was
PCR-amplified and cloned in the pGEX-2T vector.
-ZIP protein was
obtained by IPTG induction of JH372(pJH370) E. coli (a gift
from R. T. Sauer). Crude extracts were prepared and dialyzed
against 1× buffer D. The synthetic peptide BR-CC (NH-ALKRARNTEAARRSRARKLQRMKQLEDVKELEEKLKALEEKLKALEEKLKALG-COOH; Ref. 51) was kindly provided by W. DeGrado, and the basic region peptide br1s (NH4-ALKRARNTEA-ARRSRARKLQRMKQGGC; Ref. 52) was a
generous gift of P. Kim.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
pX stimulates DNA binding of diverse bZIP
proteins. Purified GST-ATF proteins, GST-JUN, C/EBP, and a GCN4
peptide were assayed for sequence-specific DNA binding. Proteins were
titrated to minimize DNA binding in the absence of pX. Two different
concentrations (1× = 5 ng, and 2×) of each affinity-purified GST
fusion protein were tested in the presence of 1 µg of BSA ( ) or 100 ng of affinity-purified His-tagged pX (+).
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Fig. 2.
pX overcomes a
concentration-dependent step that limits bZIP DNA
binding. x axis, concentration of GCN4. y
axis, pX-mediated DNA binding increase of GCN4. GCN4 at the indicated
concentration was assayed for DNA binding to a TRE collagen
oligonucleotide probe in the presence or absence of pX. Reaction
products were fractionated on a 5% native polyacrylamide gel. The data
were quantitated on a PhosphorImager.
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Fig. 3.
pX promotes bZIP dimerization and requires an
appropriate bZIP dimerization domain. A, chemical
cross-linking of a GCN4 peptide was performed in the presence of 500 ng
of BSA (lanes 2 and 5) or 100 ng of pX
(lanes 3 and 6) at a final
concentration of 0.02% glutaraldehyde. Reaction products were
fractionated on a 15% SDS-polyacrylamide gel and detected by
immunoblotting with a GCN4-specific polyclonal antiserum. The ~45-kDa
product (arrow) is consistent with cross-linking of a GCN4
dimer to a pX dimer. B, increasing amounts (1× = 0.1 nM, 2×, 4×, 8×, or 16×) of a mixture containing equal
molar concentrations of the GCN4 peptide and a cysteine-linked basic
region peptide (br1s), were incubated together and assayed
for DNA binding in the absence (lanes 1,
3, 5, 7, and 9) or presence
(lanes 2, 4, 6,
8, and 10) of 100 ng of pX. Reaction mixtures
were incubated on ice and fractionated on an 8% polyacrylamide gel,
run at 4 °C. Lower panel, amino acid sequence
and schematic diagram of both peptides.
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Fig. 4.
pX functions on bZIP proteins that contain a
degenerate leucine zipper sequence. Two concentrations (1× = 0.1 nM, and 2×) of a GCN4 peptide, the idealized peptide BR-CC
(51), a GST fusion of the Epstein-Barr virus ZTA protein, or a GST
fusion of a FOS-ZTA chimera (55) were analyzed for DNA binding in the
presence (+) or absence ( ) of pX. Upper panel,
schematic diagrams of the synthetic peptides and chimeric proteins.
Lower panel, sequence alignment of the bZIP
regions. Identical amino acids are marked by asterisks. The
positions of the conserved leucines in the leucine zipper domain are
highlighted and bold.
-ZIP, in which the DNA binding domain of bacteriophage
repressor cI was fused in-frame to the GCN4 leucine zipper (57).
-ZIP efficiently dimerizes through the leucine zipper and binds to
the OR1 site of bacteriophage phage PRM/PR
promoter. Fig. 5A shows that
pX failed to stimulate DNA binding of
-ZIP, indicating that the
leucine zipper is not sufficient for pX responsiveness and suggesting
an essential role for the basic region. Likewise, ATF4 (36), a bZIP
protein with an atypical basic region that diverges at several residues
from the consensus (Fig. 5B, bottom), was also
not pX-responsive (Fig. 5B, top).
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Fig. 5.
pX requires the bZIP basic region to
stimulate DNA binding. A, -ZIP, a chimera containing
the
repressor DNA binding domain (aa 1-101) fused to the GCN4
leucine zipper (aa 250-281) was tested for binding to the OR1 site of
the
phage PRM/PR promoter in the absence (lanes
1 and 3) or presence (lanes
2 and 4) of pX. A schematic diagram of the
-ZIP protein is shown below. B, several concentrations of
GST-ATF4 protein were tested for binding to the ATF-C site (see
"Materials and Methods") in the absence or presence of pX.
Comparison of the ATF4 and GCN4 basic region is shown below.
Non-conserved amino acids are shaded. C,
upper panel, equal amounts of proteins were
fractionated on a SDS-polyacrylamide gel and stained with Coomassie
Blue for protein quantitation. Lower panel,
far-Western analysis; purified GST-bZIP derivatives were fractionated
on an SDS-polyacrylamide gel and transferred to an Immobilon membrane.
The filter was probed with 35S-labeled pX. Excess probe was
removed and bound pX visualized by autoradiography.
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Fig. 6.
A, pX alters bZIP DNA recognition. Two
concentrations (1×, 2×) of GST-CREB (1× = 5 ng), GCN4 peptide (1× = 0.5 ng), GST-ATF2 (1× = 5 ng) and GST-Fos-ZTA (1× = 5 ng) were tested
for DNA binding to a set of four DNA oligo-duplexes in the absence ( )
or presence (+) of pX. DNA-bZIP complexes were separated on a 5%
polyacrylamide gel at room temperature. The sequence of each DNA probe
used is shown below the figure. The oligo-duplexes contain AP-1
(TGACTCA) or ATF (TGACGTCA) sites flanked either by the collagenase
promoter sequence (italics) or the Bluescript polylinker
sequence (shaded). B, intrinsic affinities of
bZIPs to the AP-1 and ATF sites. Increasing concentrations of GST-CREB
(5, 10, 20, and 40 ng), GST-ATF2 (5, 10, 20, and 40 ng), GST-Fos-ZTA
(2.5, 5, 10, and 20 ng), and GCN4 peptide (0.5, 1, 2, and 4 ng) were
tested for binding to the AP1-C, AP1-S, ATF-C, and ATF-S sites in a gel
mobility assay.
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Fig. 7.
Identification of the pX domain required for
bZIP DNA binding stimulation. A, pX deletion mutants
were expressed as GST fusion proteins. The GST moiety was then removed,
and equal molar concentrations of each protein derivative were tested
for stimulation of GCN4 DNA binding. The symbol indicates that
the pX mutant activity is at least 80% lower than that of wild-type
pX. Gray box indicates the pX region that is
highly conserved among several human HBV isolates and harbors a
putative metal binding domain. Below, the conserved pX sequence between
aa 49 and 85 is shown. Cysteine and histidine residues are
shaded. B, Tax deletion mutants were tested for
their ability to stimulate DNA binding of an H6-ATF-bZIP derivative.
The symbol
indicates activity less than 5% of wild-type Tax.
The N-terminal zinc binding domain is indicated (gray
box).
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Fig. 8.
In vivo transcriptional activation
by Tax and pX at various intracellular bZIP concentrations. -Fold
activation was calculated as the ratio between the transcriptional
activity in the presence or absence of the viral activator
(y axis) and plotted as a function of intracellular bZIP
concentration (x axis). Panel A,
hepatitis B pX. In addition to the Rous sarcoma virus-JUN vector, whose
concentration ranged from 0.1 to 3 µg, each transfection mixture
contained 1 µg of 70/coll-luciferase plasmid and 0.5 µg of a
CMV-X vector (a gift of Robert Schneider). Results from the luciferase
assay were normalized based upon the protein concentration of the cell
extract. Panel B, HTLV-I Tax. Tax-mediated stimulation was
tested on the somatostatin promoter, which contains a CREB-responsive
element (CRE). Except for the CREB vector, whose concentration varied
from 0.01 to 0.5 µg, each transfection mixture contained 1 µg of
somatostatin-CRE-CAT reporter (a gift from R. H. Goodman), 1 µg
of Rous sarcoma virus-protein kinase A, 1 µg of CMV-Tax, and 1 µg
of CMV-
-galactosidase. CAT activity was normalized based upon the
-galactosidase assay.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B and AP-1 factors (23, 24,
38, 75). The nuclear pX function is required to activate the HBV
enhancer, most likely through the ATF/CREB bZIP protein family. It is
for these reasons that pX has been referred to as
"dual-specificity" activator (38).
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ACKNOWLEDGEMENTS |
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We thank M. Carey, P. Chirillo, T. Curran, W. DeGrado, R. Goodman, S. D. Hayward, J.-C. Hu, M. Karin, P. Kim, R. Rorth, R. Sauer, and A. Siddiqui for sharing reagents. We also thank M. Melegari, S. Roberts, J. Reese, P. P. Scaglioni, and R. J. Schneider for helpful discussion and assistance.
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FOOTNOTES |
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* This work was supported by a grant from the National Institutes of Health (to M. R. G.).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.
Present address: Abteilung Biochemische Pharmakologie, 37070 Gottingen, Germany.
§ Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Howard Hughes Medical Institute, Program in Molecular Medicine, University of Massachusetts Medical Center, 373 Plantation St., Suite 309, Worcester, MA 01605. Tel.: 508-856-5331; Fax: 508-856-5473; E-mail: michael.green{at}ummed.edu.
2 G. Perini, E. Oetjen, and M. R. Green, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
HBV, hepatitis B
virus;
aa, amino acid(s);
kb, kilobase pair(s);
CMV, cytomegalovirus;
CREB, cyclic AMP response element-binding protein;
CRE, CREB-responsive
element;
HTLV-I, human T-cell lymphotrophic virus, type I;
IPTG, isopropyl-1-thio--D-galactopyranoside;
PMSF, phenylmethylsulfonyl fluoride;
PCR, polymerase chain reaction;
GST, glutathione S-transferase;
BSA, bovine serum albumin;
bZIP, basic region/leucine zipper.
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REFERENCES |
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