ABSTRACT
The hepatitis B virus X protein is a promiscuous transcriptional transactivator.
Transactivation by the X protein is most likely mediated through binding to
different cellular factors. Using the yeast two-hybrid method, we have isolated a clone that encodes a novel X-associated cellular protein: XAP2. X and XAP2 interactions also
occur
in vitro.
Antiserum raised against XAP2 recognizes a cytoplasmic protein with an apparent
molecular mass of 36 kDa. The interaction between X and XAP2 requires a small
region on X containing amino acids 13-26. From Northern blot analyses, XAP2 is ubiquitously expressed in both
liver- derived and non-liver-derived cell lines as well as in normal non-liver tissues. In contrast, XAP2 is expressed in very low level in the normal human liver. In transfection assays, overexpression of XAP2 abolishes transactivation by the X protein. Based on these
results, we suggest that XAP2 is an important cellular negative regulator of
the X protein, and that X-XAP2 interaction may play a role in HBV pathology.
The hepatitis B virus (HBV) is a small, human DNA virus that causes acute and
chronic hepatitis and is strongly associated with the development of primary hepatocellular carcinoma. In addition to the genes that encode viral structural proteins, and a gene that encodes the
viral polymerase, the HBV genome contains an open reading frame that encodes a
protein of 154 amino acids, termed X. The X open reading frame is conserved
among various HBV strains and among different hepadnaviruses. Although the X
gene does not appear to be essential for virus replication in culture, it does appear to play a major role in viral replication in animals (
1
-
4
). Furthermore, in spite of the fact that there is little evidence to suggest
that X is a direct agent in tumorigenesis, there is good reason to speculate
that the X gene product may contribute to HBV-induced tumorigenesis. This is reinforced by findings that the X protein
is capable of inducing transformation of NIH3T3 cells (
5
) and mouse hepatocytes (
6
), and causes liver tumors in some transgenic mice (
7
).
The X gene product is expressed during HBV infection. Two mRNAs of 0.7 and 3.9
kb that potentially encode X have been detected in mammalian cells transfected
with the complete HBV genome (
4
,
8
-
10
). Direct evidence for the ability of the X gene to encode a protein product has
been provided by its expression in both prokaryotic and eukaryotic cells (e.g.,
11
,
12
). Antibodies to the X protein have been detected in sera of HBV infected
patients (e.g.,
11
,
13
-
15
), and antisera raised against the X protein have been used to detect cross-reacting proteins in HBV infected livers (
14
,
16
,
17
).
Many studies have demonstrated that the X protein is a transcriptional activator
(reviewed in ref.
18
). Based on the experimental results from these studies, several non-mutually exclusive models have been proposed to explain the promiscuous transactivational activity of X. In one model, X stimulates the activity
of a number of different transcription factors. For example, it has been reported that the expression of X coincides with an elevated level
of active TFIIIC (
19
). In a second model, X uses a complex signal transduction pathway for
activation (
20
-
22
). A third model proposes that X is a protease inhibitor and functions by modulation of serine protease activity, giving rise to quantitative or qualitative changes of cellular transcription factors (
23
). Alternatively, a fourth model proposes that X acts as a coactivator and potentiates potent activation domains (
24
). Finally, it has been proposed that X binds to transcription factors, which in turn modulate upstream
promoter elements found in X-responsive promoters (
25
-
27
).
Despite the different proposed mechanisms for X transactivation, overwhelming evidence supports the hypothesis that transactivation by the X protein is mediated by cellular factors. First, investigation of the DNA sequences susceptible to transactivation by X revealed that no
single recognition sequence is present in all of the targets (reviewed in ref.
18
). Second, transactivation by X is cell type but not species-specific and that only a subset of promoters are transactivated in any
particular cell type (
28
). Finally, although X functions in the vicinity of the promoter, it does not
directly bind to DNA under conditions in which other known DNA-binding proteins bind DNA efficiently (
25
,
29
-
31
). Instead, X may belong to a class of transcriptional activators believed to be
brought to DNA by binding to a second protein.
Previously, several groups found that cellular transcription factors such as ATF-2, CREB, RPB5, TATA-binding protein and p53 can directly bind to X
in vivo
and
in vitro
(
25
,
32
-
34
). In addition, the SV40 large T antigen and a serine protease tryptase TL2 can
also bind X under various conditions (
23
,
35
). Recently, using the yeast two-hybrid system, Lee
et al.
(
36
) identified an X-associated protein (XAP1) that is a human homolog of a monkey UV-damaged DNA-binding protein. The physiological significance of the X-XAP1 interaction, as well as other X-cellular factors interaction, remains to be determined.
As a further step toward understanding the mechanism by which X activates
transcription of many different genes, we have independently used the yeast
interaction trap cloning method to isolate other cellular proteins that may
interact with X. Among the X-interacting clones identified, many of them were found to encode a novel 36 kDa cytoplasmic protein designated X-associated protein 2 (XAP2). XAP2 can form a complex with X
in vitro
, and overexpression of XAP2 interferes with the transcriptional activation
function of X.
Escherichia coli
DH5[alpha] (F
-
[Phi]80d
lac
Z[Delta]M15 [Delta][
lac
ZYA-
arg
F]U169
deo
R
rec
A1
end
[Delta]A1
pho
A
hsd
R17[r
K
-
, m
K
+
]
sup
E44 [lambda]
-
thi
-1
gyr
A96
rel
A1) was the recipient for plasmid transformations and was also used for the
expression of GST fusion proteins.
Escherichia coli
BNN132 (
37
) was used to convert [lambda]ACT phage DNA to plasmid DNA.
Escherichia coli
JA226 (
hsd
R,
hsd
M,
leu
B6,
lop
11,
thi
1,
rec
BC,
str
R) was used to recover expression plasmids from yeast.
Escherichia coli
LE392 (
supE
44
supF
58
hsdR
514
galK
2
galT
222
metB
1
trpR
55
lacY
1) was used to propagate the HeLa [lambda]gt10 recombinant library.
Saccharomyces cerevisiae
Y153 (
38
) was used to screen for HBV X-associated protein clones and to detect for protein-protein interactions.
pAS-X was constructed by subcloning the HBV [subtype adw2 (
39
)] fragment from nucleotide 1375 to 1853 (
Nco
I-
Afl
III) into the Gal4 DNA-binding domain (DBD) tagged plasmid, pAS1 (
38
). This plasmid expresses a fusion protein containing the Gal4DBD and the full-length wild-type X protein. pAS-Rb2, which encodes a Gal4DBD-Rb fusion protein and pAS-SNF1, which encodes a Gal4DBD-yeast SNF1 fusion protein have been
described (
38
). pAS-E12 and pAS-NB encode Gal4DBD fused to transcription factors E12 and Zif268,
respectively, and were kindly provided by B. Christy (University of Texas Health Science Center). pAS-YY1, which contains a 1.2 kb fragment of YY1 cDNA in pAS1 has previously been described (
40
). To construct pGST-X, the
Nco
I-
Sma
I fragment from pAS-X containing the entire HBV-X ORF was subcloned into the pGSTag vector (
41
) in-frame with the GST polypeptide. pGEM-X1-3 was constructed by ligating the
Bgl
II X1-3 insert fragment from pACT-X1-3 with a
Bam
HI-digested pGEM7Zf(+) vector (Promega). pGEM-CyPA was constructed by taking the human cyclophilin A cDNA, an
Eco
RI fragment from pGCyPA (
42
), and ligating it to the
Eco
RI site of pGEM3Z (Promega). pGEM-1-1FL was constructed by ligating the
Eco
RI 1-1FL insert fragment from the 1-1FL [lambda]gt10 clone with a
Eco
RI-digested pGEM7Zf(+) vector (Promega). Plasmid pGST-XAP2 was constructed by subcloning a
Bgl
II fragment from pACTX1-3 into the
Bam
HI site of pGEX3X (Pharmacia). pECE-X, which contains the HBV fragment from nucleotide 1355 to 1987 in the
SV40-derived expression vector pECE, has been described (
43
). pXD1, pXD3, pXD5, pXD8, pXD6, pXD7, pXI5, pXI2, pXI3 and pXI4 were
constructed by first subcloning the X coding sequence (a
Bgl
II fragment from pECE-X) into the
Bam
HI site of a modified pGEM7Zf(+) plasmid (pGEM7Zf-3X) that would provide stop codons in all three reading frames immediately after the X sequence, then using different
restriction enzymes to subdivide the X coding region, and finally subcloning
each individual X mutant into the
Nco
I/
Bam
HI site of pAS1. Reporter plasmids pHIVCAT, pSV2CAT and pRSVCAT have previously
been described (
43
-
45
). pCMV-XAP2, which contains a full-length XAP2 cDNA under the control of the CMV promoter, was
constructed by ligation of an
Eco
RI fragment from the 1-1FL [lambda]gt10 clone into the
Eco
RI site of pcDNAI/Amp (Invitrogen). pTAT-6 expresses the HIV1 transactivating protein TAT under the SV40 promoter/enhancer (
46
). Effector pGal4-VP16 and reporter pG5BCAT have previously been described (
47
,
48
). All plasmid constructions were verified by dideoxy sequencing.
For the yeast two-hybrid screen, Y153 cells were sequentially transformed with pAS-X using the lithium acetate method, followed by transformation with a human cDNA library constructed in [lambda] ACT using mRNA prepared from Epstein-Barr virus-transformed human peripheral lymphocytes [kindly provided
by S. Elledge at the Baylor College of Medicine (
38
)]. Transformants
were plated on 150 mm dishes containing SC medium lacking tryptophan, leucine
and histidine, but containing 50 mM 3-aminotriazole, and incubated for 3 days. His
+
and Leu
+
prototroph colonies were rescreened for [beta]-gal activity using a filter lift assay (
49
). Colonies corresponding to positive (blue color) in this second screen were
then patched onto a master plate for further characterization. Plasmids were recovered and electroporated into
E.coli
strain JA226 (
50
), grown in minimal media lacking leucine but containing 50 [mu]g/ml ampicillin. Isolated plasmids were rescreened by transforming Y153 alone or
Y153 harboring pAS-X, pAS-Rb2 or pAS-SNF1. Positive clones showing specific interactions with X but
not to others were subcloned into Bluescript (Stratagene) or pGEM7Zf(+), and nucleotide sequences of the subcloned cDNA were obtained using dideoxy sequencing (
51
). The final sequence of the XAP2 cDNA was determined from both DNA strands.
To obtain a full length XAP2 cDNA, a HeLa [lambda]gt10 cDNA library (kindly provided by B. Shan at the University of Texas
Health Science Center) was screened with a
32
P-labeled, random- primed 200 bp probe (
Xho
I-
Msc
I fragment from clone X1-3) using standard protocols (
52
). Inserts from positive clones were subcloned into pGEM7Zf(+) and sequences
were determined as described above.
Filter lift assays were performed essentially as described (
49
). Briefly, transformants were allowed to grow at 30oC for 2-4 days, transferred onto nitrocellulose filters, and freezed under liquid nitrogen. Filters were then placed on Whatman 3MM paper presoaked
with 5-bromo-4-chloro-3-indolyl-[beta]-d-galactopyranoside (X gal) solution,
incubated at 30oC, and checked periodically for production of blue color.
For quantitation of [beta]-gal activity in yeast, liquid culture assays were done using
o
-nitrophenyl-[beta]-D-galactoside (ONPG) as described (
53
).
To generate
35
S-labeled XAP2 and CyPA, pGEM-X1-3 (Fig.
2
), pGEM-1-1FL (Fig.
4
) or pGEM-CyPA were transcribed and translated with T7 RNA polymerase and [
35
S]methionine in a transcription/translation coupled system (Promega).
Bacterially-expressed GST protein and GST-X fusion protein were purified according to Frangioni
et al
. (
54
). Briefly, DH5[alpha] cells harboring either the pGSTag or the pGST-X plasmid were grown to log phase and induced with isopropyl-thio-[beta]-d-galactoside (IPTG) for 4 h. After
sonication in STE buffer [10 mM Tris-HCl (pH 8), 150 mM NaCl, 1 mM EDTA and 5 mM dithiothreitol] containing 1%
sarcosyl (w/v, final concentration), solubilized proteins were recovered by centrifugation and incubated with glutathione-agarose beads in the presence of 3% Triton X-100 (final concentration) for 30 min at 4oC, and washed several times with ice-cold phosphate buffer saline (PBS).
For binding assays, beads were mixed with
in vitro
translated,
35
S-labeled proteins for 1 h at room temperature. Unbounded proteins were
washed extensively with STE buffer containing 0.1% NP-40 and bound proteins were eluted from the beads by boiling in SDS loading buffer [50 mM Tris-HCl (pH 6.8), 100 mM dithiothreitol, 2% SDS, 0.1% bromophenol blue and 10% glycerol]. Final products were analyzed on a 15% SDS-polyacrylamide gel and
detected by fluorography.
Polyclonal anti-X antibody prepared in rabbits againist HBV X protein expressed in insect
cells was kindly provided by T. H. Lee and J. Butel (Baylor College of
Medicine). Polyclonal antibodies to Gal4DBD and YY1 were obtained from Santa Cruz Biotechnology. Polyclonal antibody to GST-XAP2 fusion protein was raised in Balb-C mice as described by Oettinger
et al
. (
55
). Briefly, 100 [mu]g of GST-XAP2 fusion protein bound to glutathione-agarose beads were injected into five animals intraperitoneally. Booster injections containing 50 [mu]g of proteins were given at 15 day intervals. Forty-five days post-priming, mice were subjected to ocular-vein bleeding and sera were prepared. Antisera
were evaluated for reactivity to bacterially-expressed XAP2 protein in immunoblot analysis using standard protocols (
56
).
Standard protocols were followed (
56
). Briefly, 3 * 10
6
HeLa cells (Fig.
4
) were lysed by boiling for 5 min in sample buffer [62.5 mM Tris-HCl (pH 6.8), 1% SDS, 10% glycerol and 5% [beta]-mercaptoethanol]. Fifty [mu]l of the resulting extract were separated on a 15%
SDS-polyacrylamide gel and transferred onto polyvinylidene difluoride membrane.
After blocking with nonfat dried milk, the membrane was treated with 1:1000
diluted XAP2 polyclonal antiserum followed by 1:7500 diluted alkaline
phosphatase-conjugated rabbit anti-mouse IgG. Subsequently, the blot was developed by 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium.
For each sample in Figure
6
B, yeast transformants were grown at 30oC in 1 ml of selective SC medium containing 2% dextrose to an OD
600
of 1-2. Cells were collected by centrifugation and lysates were prepared
according to a standard protocol (
52
). One-tenth of each sample was resolved on a 12.5% SDS-polyacrylamide gel and
subjected to Western blot analysis with 1:500 diluted anti-Gal4DBD antibody.
HeLa cells were grown on acid-etched cover slips inside a 100 mm tissue culture plate for ~24 h and transfected with 25 [mu]g of pECE-X plasmids as described below. Two days later, cells were washed with ice-cold PBS, and fixed with a 50:50 acetone:methanol mixture for 2 min and then treated with anti-XAP2 (1:600 dilution) and anti-X (1:500 dilution) antibodies. Cells were
then incubated for 30 min at room temperature, followed by washing with PBS and
further incubated with 1:200 diluted sheep anti-mouse IgG coupled with FITC (Sigma) and goat anti-rabbit IgG coupled with Texas-Red (Southern Biotechnology Associates). Subsequently, cells
were subjected to extensive washings with PBS and cover slips were applied with
40% glycerol before analyzing under a Carl Zeiss confocal microscope.
Briefly, cells were collected by scraping into PBS, centrifuged, and resuspended
in 1 ml of ice cold RSB buffer [10 mM HEPES (pH 6.2), 10 mM NaCl, 1.5 mM MgCl
2
and
0.5 mM phenylmethylsulfonyl fluoride] with a Kontes Dounce homogenizer (10 strokes with a type A
pestle). The resulting suspension was centrifuged for 10 min at 700
g
at 4oC and the pellet was resuspended again in 1 ml of RSB buffer. After
homogenization as before, the suspension was combined with the supernatant
obtained from the previous step and centrifuged further for 10 min at 700
g
. The final pellet (nuclear fraction) was washed with RSB buffer and resuspended in 2 ml of
SDS sample buffer and the supernatant (cytoplasmic fraction) was combined with
2 ml of 2* SDS sample buffer. A small aliquot of each fraction was subjected to
electrophoresis and Western blot analysis.
HepG2 or CV1 cells were grown in minimal essential medium supplemented with 10%
fetal bovine serum. Transfections were done using the calcium phosphate method
(
57
) which included a 1 min glycerol shock 4 h after the addition of DNA
precipitate. Forty-eight hours after transfection, cells were harvested and CAT activity was determined (
45
) in 1 h reactions. All transfections were normalized to equal amounts of DNA
with pGEM7Zf(+) or pcDNAI/Amp plasmids.
Using the micro-fast track mRNA isolation kit (Invitrogen), Poly(A)
+
RNA was purified from normal frozen human liver tissues (obtained from the
Liver Tissue Procurement and Distribution System at the University of
Minnesota) and from different cell lines. Two [mu]g of each RNA sample were then separated on a 1% agarose/formaldehyde gel
and transferred onto a Hybond-N
+
nylon membrane using standard protocols (
52
). XAP2 cDNA probe was prepared with [[alpha]-
32
P]dCTP using a random prime kit (Stratagene). Pre-hybridization, hybridization and washing were performed under high
stringency conditions before exposure to X-ray films (
52
). Multiple human tissue Northern blots (Clontech) were treated identically. To control for the relative amount
of RNA in each lane, after hybridization with XAP2, the blots were stripped by
incubation in 0.5% SDS at 95oC, and reprobed with the human [beta]-actin cDNA.
The nucleotide sequence data reported in this paper will appear in GenBank, EMBL
and DDBJ Nucleotide Sequence Databases under the accession number U31913.
To identify human cDNAs encoding proteins that interact with X, we used the
in vivo
two-hybrid system developed by Fields and colleagues (
58
,
59
) as modified by Durfee
et al
. (
38
). A gene encoding the X protein was fused to the DBD of Gal4 that did not
activate transcription by itself in yeast. The Gal4DBD-X fusion gene was introduced into Y153 cells together with a human
lymphoma cDNA library fused to the activation domain of Gal4.
HIS
3 expression was directly selected by plating transformants on media lacking
histidine. To eliminate false positive clones, a second screen was performed
with the
Lac
Z reporter gene, for which
Lac
Z transcription was monitored by growing cells in the presence of X-gal. Further, all positive clones were tested against two unrelated
proteins [the retinoblastoma protein (Rb) and the yeast SNF1 protein] fused to
the DBD to eliminate those that did not show specificity for the X protein.
Of 5.6 * 10
6
colonies screened, 51 grew in the absence of His. Out of these 51 His
+
prototrophs, 23 scored positive for [beta]-gal activity. Though none of the plasmids recovered from the
positive clones produced [beta]-gal activity when singly transformed into yeast, 11 of the clones
were active when cotransformed with plasmids encoding Gal4DBD fused either to
Rb or the yeast SNF1 protein. This suggested that these 11 clones did not
depend on the presence of X for activity. The remaining 12 clones were active
only when cotransformed with plasmid encoding Gal4DBD fused to X, but not to Rb
or SNF1.
Each of the 12 potential X-binding protein cDNAs were subcloned into a Bluescript or pGEM7Zf(+) plasmid and sequenced by the dideoxy
method. Six clones, X1-3, X1-3A, X2, X3, X6 and X10, are overlapping clones of cDNA encoding the
same gene [we will refer to this gene as X-Associate Protein-2 (XAP2)]. Sequence comparison of XAP2 with GenBank and EMBL
databases did not reveal any homologous protein, although a small part of the
cDNA resembles several partial human cDNA fragments deposited in GenBank.
To confirm the specificity of X and XAP2 interaction, clone X1-3 which expresses the Gal4 activation domain (Gal4AD) fused to XAP2, was
retransformed into Y153 cells with plasmids expressing the Gal4DBD, Gal4DBD-X or with Gal4DBD fused to five different unrelated proteins. [beta]-Gal activity (blue color) was observed by the colony filter
lift method only in the presence of a Gal4DBD-X fusion protein but not with other unrelated fusions. Also, the [beta]-gal activity produced in the presence of Gal4DBD-X, as measured by an ONPG assay was almost 10-fold higher compared with Gal4DBD alone or
compared with Gal4DBD fused to other unrelated proteins (Fig.
1
).
The six cDNA clones (X1-3, X1-3A, X2, X3, X6 and X10) isolated from the two-hybrid screen were divided into two independent isolates
according to DNA sequences of the recombinant junctions between the cDNA and
the vector (illustrated in Fig.
3
A). The predicted amino acid sequence was determined by theoretical translation
of the cDNA clone open reading frame (Fig.
3
B). A consensus sequence for initiation of translation (
60
)
was not found in the 5' end of the two longest cDNAs (X1-3 and X1-3A), and the reading frame remains open at the 5' side, suggesting that these six clones represent only
a partial XAP2 coding sequence.
Although we have shown that X and XAP2 interact both in yeast cells and
in vitro
, we reasoned that if the consequence of X and XAP2 interaction is biologically
significant the two proteins should also colocalize in mammalian cells. The
subcellular location of the X protein is controversial, ranging from
cytoplasmic (
16
,
17
,
62
,
63
), nuclear (
6
,
64
), to nuclear periphery (
12
,
65
-
67
), or more generalized nuclear and cytoplasmic (
12
,
66
,
68
,
69
). To independently assess the subcellular location of X, HeLa cells were
transiently transfected with a plasmid expressing the X protein, fixed with
methanol/acetone, and immunostained with a polyclonal anti-X antibody. As shown in Figure
5
A, images obtained with a confocal laser scanning system indicated that, under
the present conditions, X is localized almost exclusively in the cytoplasm.
Like the X protein, XAP2 was regionally dispersed throughout the cytoplasm
(Fig.
5
B). Interestingly, there were some distinct regions where the distribution of X
and XAP2 overlapped (Fig.
5
C), consistent with the observation that the two proteins physically interacted
in vivo
and
in vitro.
The X protein sequences required for binding XAP2 were examined to determine
whether they coincided with previously defined activation or regulatory
domains. A series of C-terminal X deletion mutants, as well as internal deletion mutants, were
generated and subcloned into pAS1 and tested for their abilities to interact
with XAP2 in the two-hybrid system. As shown in Figure
6
A and Table
0
, deletion of the X C-terminal from amino acids 27 to 154 had no effect on [beta]-gal activities, as measured by filter lift and liquid assays.
However, deletions of amino acids 13-154 or 10-27 (pXD7 and pXI5) eliminated [beta]-gal activities. Three internal deletions outside of
amino acids 13-27 (pXI2, pXI3 and pXI4) had no effect on [beta]-gal activities. Western blot analysis indicated that the two
mutants that did not bind XAP2 were expressed in yeast cells (Fig.
6
B, lanes 2 and 3). Taken together, our data suggest that amino acids 13-26 of X are important for binding XAP2. Interestingly, these 14 amino acids lie within a
previously identified regulatory domain that represses transactivation of the X protein (
70
).
Table 1
To determine the functional significance of X and XAP2 interaction, we tested the ability of XAP2 to influence transcription directed by an X-responsive promoter in the presence of the X protein. We constructed a
plasmid that expresses XAP2 under the control of the human cytomegalovirus (CMV) immediate early promoter, cotransfected it into CV1 (Fig.
7
A) or HepG2 (Fig.
7
B) cells together with a plasmid that expresses the X protein and a reporter.
Consistent with earlier studies (
19
,
28
,
30
,
31
,
71
-
75
), the X protein activated the human immunodeficiency virus type 1 (HIV1), simian virus 40 (SV40) and Rous sarcoma virus (RSV) promoters in CV1 (Fig.
7
A, lanes 2, 6 and 10) and HepG2 cells (Fig.
7
B, lanes 2 and 6). However, when XAP2 expression plasmid was added to the
transfections, transcriptional activation by X was repressed (Fig.
7
A, lanes 3, 7 and 11; Fig.
7
B, lanes 3 and 7).
Using Northern blot analysis, we explored the tissue expression pattern of XAP2
in hope of further understanding the biological significance of X and XAP2 interaction. Interestingly, we found that a message of ~1.25 kb was present in every tissue we examined, with the exception of the
liver (Fig.
8
A; Fig.
8
B, lanes 1 and 2). In addition, XAP2 was found to be abundantly expressed in
three different cell lines including one derived from human hepatocellular carcinoma.
It has been postulated that a key mechanism by which many viral activators
regulate transcription is by forming complexes with cellular factors. The HBV X protein is a promiscuous transcription activator. In order to understand its mechanism we used the HBV X gene as the
bait in a yeast two-hybrid screen to identify proteins that are capable of forming heterologous complexes with X. Six clones
isolated in this screen encode an identical protein that we named XAP2.
Sequence comparison of XAP2 with protein sequences in the GenBank and EMBL databases indicate it is a novel protein.
Three lines of evidence confirmed that XAP2 is a genuine X-associated protein. First, we shown that XAP2 interacts only with X but
not a number of other unrelated proteins in the yeast two-hybrid system. Second, using a GST-X fusion protein we obtained biochemical evidence that X and XAP2
interact
in vitro.
Finally, we used immunocytochemical methods to demonstrate similar subcellular
distributions of X and XAP2 in mammalian cells.
Analysis of X mutants indicated that the interaction with XAP2 probably occurred
through X residues 13-26. This N-terminal region is highly conserved among all mammalian
hepadnaviruses (reviewed in ref.
18
). The importance of this region with respect to transactivation is unclear. It
has been reported that deletion of the N-terminal rendered the X protein incapable of transactivation of the HBV enhancer (
76
) but did not affect its ability to transactivate the SV40 early
promoter/enhancer, the RSV long terminal repeat (
30
), or a hybrid SV40/HBV promoter/enhancer (
70
). Similarly, a finer deletion of amino acids 4-20 indicated that this region is not important in the transactivation of
the SV40 early promoter/enhancer (
77
). However, there is evidence that the N-terminal one-third of the X protein can bind the X protein itself and repress the
X transactivation function (
70
,
78
). In addition, this identical region is sensitive to transrepression as well (
70
). Our finding that XAP2 interacts with this particular domain raises the
possibility that the X-X interaction and X transrepression function described previously is
mediated by association of X and XAP2. One can imagine that XAP2 functions as a bridging protein that brings two X molecules together. Alternatively, XAP2 may compete for
binding to X protein, preventing X-X dimer formation, and hence repress X transactivation.
Many cellular proteins have the ability to bind and inhibit the transactivation
function of transcription activators. For examples, the MDM-2 protein can suppress p53 transactivation (
79
,
80
), the Id family proteins bind to and inhibit transcription activation by the family of basic helix-loop-helix transcription factors (
81
-
85
), p107 can suppress c-Myc transactivation (
86
), and a cellular protein BS69 inhibits E1A transactivation (
87
). Perhaps the best characterized case of suppression of transactivation is the
interaction between the Rb protein, transcription factor E2F, and the
adenovirus E1A protein. Studies of transcription control mediated by E1A
identified a cellular DNA-binding protein, termed E2F, that recognizes key regulatory elements in
many promoters (
88
). In the last several years, it became clear that when E2F is complexed with
cellular Rb, its ability to activate transcription is prevented. Several laboratories have demonstrated that E1A can release E2F from the Rb complex, making it available to bind other
proteins and hence activate transcription (
89
-
91
). Although a number of cellular proteins have been shown to bind the X protein,
XAP2 is the first cellular protein identified that appears to directly inhibit
X transactivation. It is possible that XAP2 has a similar function to Rb in
that it forms complexes with certain transcription factors. In this case, X
could then release these transcription factors from XAP2, making them available
to activate transcription. In contrast, when XAP2 is abundant it would prevent
the X protein from releasing transcription factors for activation.
Understanding the normal function of XAP2 and its binding proteins will help
resolve whether this is a novel mechanism for X transactivation.
Another possible, though not mutually exclusive, significance of the X and XAP2
interaction is that XAP2 may function as a cytoplasmically located inhibitor.
Our finding that X and XAP2 colocalize in the cytoplasm of HeLa cells suggests
that by association with X, XAP2 may prevent X from entering the nucleus to
activate transcription. This would be similar to the transcription factor NF-[kappa]B whose ability to activate transcription is regulated by association and dissociation with the cytoplasmically located inhibitor I[kappa]B (
92
,
93
). Experiments involving comparisons of X subcellular localization to its ability to transactivate, similar to those
described by Doria
et al
. (
68
), as well as studies to compare XAP2 expression with X subcellular localization
are now in progress.
Although computer searches of the database failed to detect any proteins with
sequence identity to XAP2, a closer inspection of the XAP2 sequence revealed a
region (amino acids 264-275) that is similar to a region of the X protein (amino acids 107-118). Interestingly, amino acids 105-115 of the X protein, coincide with a site termed X1 that has been shown to interact with
cellular proteins and is necessary for X transactivation (
61
). This observation, together with the finding that excess XAP2 can prevent X
transactivation, suggests that XAP2 may inhibit X transactivation simply by
sequestering cellular proteins important for X transactivation. Elucidation of the exact mechanism by which XAP2 inhibits X activity will provide insight into the molecular basis of X transactivation.
The chief biologic features of HBV infection are species specificity and
relative hepatotropism (reviewed in ref.
94
). In productive infections, viral antigens and DNA are found primarily within liver cells (although viral DNA sequences have been detected in low copy numbers in cells other than hepatocytes). Furthermore, successful infection of permanent cell lines by HBV has not yet been
documented. Propagation of HBV in human hepatoma cell lines has been achieved with modest success only after transfection with large amounts of HBV DNA (
4
,
95
-
97
). Although there are probably multiple factors that contribute to HBVs
hepatotropism, our finding that XAP2 is abundantly expressed in every human
tissue we examined, with the exception of the liver, is intriguing. If XAP2 is
complexed with X and prevents the X protein's normal function; and since XAP2
is not expressed (or expressed in a very low level) in the human liver, this
may partially allow for X to contribute to the virus' hepatotropism. Second,
our finding that XAP2 is present in different cell lines (including one that is
derived from human hepatocellular carcinoma, Hep G2), may also partially
explain why X is not essential for the viral life cycle
in vitro
but is important for viral infection
in vivo
(
1
-
4
), and why attempts to propagate HBV
in vitro
have not been successful.
While most previous studies of the X protein focused on the transcriptional
activation potential of X, clearly X may also play a role in the viral life
cycle (
1
,
2
) and the development of liver cancer (
6
,
7
). Our findings that XAP2 inhibits X transactivation and is expressed in low
level in normal human liver, point to the possibility that XAP2 may also
interfere with HBV replication and transformation
in vivo.
The cloning of XAP2 allows us now to systematically characterize the protein,
and determine its role not only in X-mediated transcription activation, but also in HBV infection and
transformation. Ultimately, it may be possible to target XAP2 to inactivate X
as an antiviral agent for treatment of patients with chronic hepatitis B.
Current sequence comparisons with the BLAST algorithm show that partial DNA sequences for XAP2 have been isolated randomly (accession numbers T17260, R50134, Z41802, F02680, F03063, R46618, T32201,
H17133, Z46173 and R50188). No function for these partial cDNAs has been
described.
We thank Janet Butel, Barbara Christy, Holly Dressler, Steve Elledge, Teh-Hsiu Lee and Bei Shan for generous gifts of antibodies, plasmids and libraries; Jarvis Gao for help with confocal microscopy; Lisa Hoegler for technical support; Dave Bearss for help with
preparation of some figures; Dave Bearss and Paul D. Gardner for discussion and
critical reading of the manuscript. This work was supported by grants from the
National Cancer Institute (R01-CA61257) and the San Antonio Cancer Institute.
*To whom correspondence should be addressed at: Molecular Oncology Program,
Moffit Cancer Center and Research Institute, University of South Florida, 12902
Magnolia Drive, Tampa, FL 33612, USA. Tel: +1 813 979 6754; Fax: +1 813 979
6700; Email: setoe@moffitt.usf.edu
+
Present address: Department of Molecular and Cell Biology, University of
California, Berkeley, CA 94720, USA
Bait/prey
Gal4AD-XAP2
Gal4DBD (pAS1)
1
Gal4DBD-X (pASX)
10.9 +- 1.3
pXD1
21.3 +- 2.5
pXD3
11.3 +- 2.1
pXD5
9.1 +- 1.4
pXD8
13.6 +- 2.9
pXD6
11.9 +- 1.7
pXD7
1.2 +- 0.4
pXI5
3.0 +- 0.8
pXI2
12.4 +- 1.6
pXI3
23.4 +- 2.5
pXI4
12.3 +- 1.5
REFERENCES
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