(Received for publication, October 11, 1996, and in revised form, January 3, 1997)
From the Department of Molecular Biology, Cancer Research Institute, Kanazawa University, Takara-machi 13-1, Kanazawa 920, Japan
Hepatitis B virus X protein (HBx) transactivates viral and cellular genes through a wide variety of cis-elements. However, the mechanism is still obscure. Our finding that HBx directly interacts with RNA polymerase II subunit 5 (RPB5), a common subunit of RNA polymerases, implies that HBx directly modulates the function of RNA polymerase (Cheong, J. H., Yi, M., Lin, Y., and Murakami, S. (1995) EMBO J. 14, 142-150). In this context, we examined the possibility that HBx and RPB5 interact with other general transcription factors. HBx and RPB5 specifically bound to transcription factor IIB (TFIIB) in vitro, both of which were detected by either far-Western blotting or the glutathione S-transferase-resin pull-down assay. Delineation of the binding regions of these three proteins revealed that HBx, RPB5, and TFIIB each has two binding regions for the other two proteins. Co-immunoprecipitation using HepG2 cell lysates that express HBx demonstrated trimeric interaction in vivo. Some HBx substitution mutants, which had severely impaired transacting activity, exhibited reduced binding affinity with either TFIIB or RPB5 in a mutually exclusive manner, suggesting that HBx transactivation requires the interactions of both RPB5 and TFIIB. These results indicated that HBx is a novel virus modulator that facilitates transcriptional initiation by stabilizing the association between RNA polymerase and TFIIB through communication with RPB5 and TFIIB.
Human hepatitis B virus (HBV)1 is one
major risk factor associated with primary hepatocellular carcinoma.
HBx, the product of the smallest open reading frame of the HBV genome,
is required for virus multiplication in vivo (1, 2).
Transient transfection has demonstrated that HBx transactivates a wide
variety of viral and cellular promoters. The HBx responsible
cis-elements present in RNA polymerase II (RNAPII) promoters
include AP-1, AP-2, ATF, c/EBP, NF-B sites, and serum-responsive
element (3-11). One of the RNA polymerase III promoters is also
transactivated by HBx (12). Several endogenous genes important for cell
proliferation and acute inflammatory response, such as
c-fos, c-jun, and human interleukin-8, are
activated by HBx (3, 7, 13). This broad gene-regulating function
suggests that HBx not only up-regulates the expression of HBV genes by
transactivating the HBV enhancer but also modifies the environment by
transactivating cellular genes in infected cells to facilitate viral
replication. It also implies that HBx plays a positive role in
hepatocellular carcinogenesis (14, 15).
Since HBx cannot bind DNA directly, protein-protein interaction is crucial for HBx transactivation (16). The reported HBx-binding proteins include transcription factors such as TBP (17), RPB5 (18), CREB/ATF2 (19), and Oct-1 (20), a probable DNA repair enzyme (21), a human homologue of Drosophila 20 S proteasome subunit (22, 23), and the tumor suppressor p53 (24, 25). These findings are so diverse that the mechanism of HBx transactivation remains obscure. It has been reported that HBx mediates transcription activation through the modifying signal transduction pathways in cytoplasm such as the activation of AP-1 via protein kinase C and the signaling pathway of Ras-Raf-mitogen-activated protein kinase (4, 5, 26-29). However, it is difficult to reconcile the signal transduction models with the observations that HBx acts in in vitro transcription (30). Since the subcellular localization of HBx is both cytoplasmic and nuclear (16, 31),2 HBx may have a dual role in transcriptional regulation, such that nuclear HBx functions at the promoter level as cytoplasmic HBx influences the regulation of second messenger systems (31). Another model postulates that HBx directly interacts with the transcription machinery. This model is supported by several lines of evidence. HBx can activate transcription in vitro using whole cell extracts (30). HBx fused to LexA or c/EBP DNA-binding domains can transactivate the reporters harboring the responsive cis-elements (9, 32). HBx co-activates potent activators, including acidic activators such as VP16 and p53 as well as non-acidic activators such as E1a (33). Finally, we showed that HBx can specifically bind to RPB5, a common subunit shared by eukaryotic nuclear RNA polymerase I, II, and III, both in vitro and in vivo. RPB5 overexpression can stimulate the transcription of HBx responsible reporters (18) supporting the notion that HBx transactivation acts through direct interaction with the transcription machinery.
To elucidate the role of HBx-RPB5 interaction in transcription modification, we investigated whether or not HBx and RPB5 interact with other transcription factors. Here we report that both HBx and RPB5 interact with TFIIB through different sites and that they may form a ternary complex that facilitates transcription.
The plasmid pSG5UTPL is a mammalian expression vector derived from pSG5 (Stratagene) (7, 28). The plasmid pGENK1 was used as an Escherichia coli expression vector for GST-fused proteins. The GST-fused proteins have a phosphorylation site for cAMP-dependent protein kinase (28). The full-length and truncated HBx-5D1, -5D2, -5D4, -3D5, and -3D39, and human RPB5-d1, -d2, -d4, and -d13 expression plasmids have been described (18, 28). The truncated HBx-D16 and -D26 mutants and RPB5-d27, -d83, and -d47 mutants were constructed by means of polymerase chain reaction (PCR) cloning. Substitution HBx mutants were constructed by PCR splicing, resulting in three amino acid residues (leucine, proline, and lysine at positions 89-91 of Xm89 and tryptophan, glutamic acid, and glutamic acid at 120-122 positions of Xm120, respectively) being substituted by three alanines or four amino acid residues (alanine, arginine, arginine and methionine at positions 76-79 of Xm76 and leucine, histidine, lysine, and arginine at positions 93-96 of Xm93, respectively) being substituted by glycine, alanine, glycine, and alanine. Plasmid pGENK1-CTD was constructed by inserting the annealed complementary oligonucleotides (TCTCCTAGCTACACCCCAACCTCTCCAAGCTACTCACCAACA) encoding two repeats of the heptapeptide of the CTD into the EcoRI and BamHI sites of pGENK1. TFIIB and TBP cDNAs were derived from B13 and H10, provided by Dr. M. Hirokoshi (34, 35). The inserts were amplified by PCR and inserted into the EcoRI and BamHI sites of pSG5UTPL and pGENK1. The resulting plasmid pSG5UTPL-TFIIB was used as the template with which to construct truncated versions of TFIIB by PCR cloning. TFIIB-d6, -d10, and -d21 encode an initiation codon followed by amino acids 281-316, 124-295, and 21-316. The internal truncated TFIIB-I37 and -I61 mutants encode 17-20 and 37-316 and 45-49 and 61-316, respectively. TFIIB-d1, -d3, and -d9 encode 1-60, 1-124, and 1-295 amino acids of human TFIIB, respectively. All the constructs were sequenced using Taq sequencing kits and a DNA sequencer (370A, Applied Biosystems).
Preparation of Recombinant ProteinsGST-fused proteins were
expressed in E. coli by induction for 3 h at 30 °C
with 0.4 mM
isopropyl--D-thiogalactopyranoside. Cells were harvested
and sonicated in PBST buffer (phosphate-buffered saline containing 1%
Triton X-100). After centrifugation, the extracts (supernatants) were
collected and stored at
80 °C. For purification, the extracts were
incubated with glutathione-Sepharose 4B resin (Pharmacia Biotech Inc.)
for 1 h at room temperature. The beads were precipitated and
washed four times with a 50-fold volume of PBST buffer followed by
elution with 10 mM reduced glutathione in 50 mM
Tris-HCl (pH 8.0). The eluted proteins were divided into aliquots and
stored at
80 °C.
Plasmids pET11d-TFIIB and -TBP (a gift of Dr. R. G. Roeder) were
expressed in BL21 (DE3 pLys) by 0.4 mM
isopropyl--D-thiogalactopyranoside induction at
30 °C. Cells were harvested 3 h postinduction and sonicated in
native binding buffer (20 mM sodium phosphate, 500 mM NaCl, pH 7.8). Histidine-tagged proteins were purified
by incubating the sonication supernatant with nickel resin followed by
extensive washing and elution with imidazole elution buffer (300 mM imidazole, 20 mM sodium phosphate, 500 mM NaCl, pH 6.3).
Recombinant HBx was prepared by digesting the GST-HBx-fused protein
with thrombin as described (36). The HBx protein was purified by
separation in and elution from SDS-PAGE with acetic buffer (50 mM NaOAc, pH 5.2, 0.1% -mercaptoethanol), then stored at
80 °C.
Far-Western blotting proceeded as
reported with some modification. Purified GST-fused proteins were
labeled in vitro as probes with -32P, using
the catalytic subunit of cAMP-dependent protein kinase (Sigma). The target proteins (200 ng each) were
fractionated in SDS-PAGE gels and then electrotransferred onto
nitrocellulose membranes, which were denatured, renatured, blocked with
5% skim milk in modified GBT buffer (10% glycerol, 50 mM
Hepes-NaOH, pH 7.5, 170 mM KCl, 7.5 mM
MgCl2, 0.1 mM EDTA, 0.1 mM
dithiotreitol, 1% Triton X-100) and far-Western blotted (18). The
binding reactions proceeded in modified GBT buffer containing
32P-labeled probe (40-100 ng/ml protein, 2 × 106 cpm/µg of protein), 1% bovine serum albumin, 2 mM unlabeled ATP, and the sonication supernatant of
E. coli JM109 transformed with pGENK1 containing a final
protein concentration of 1 mg/ml GST. The membranes were rotated in the
reaction mixture for 1 h at room temperature, washed five times
with modified GBT buffer, then exposed to x-ray films (X-Omat AR,
Eastman Kodak Co.) for 1-3 days or to imaging plates (Fuji)
overnight.
Equal amounts (approximately 1 µg) of GST, GST-TBP, GST-RPB5, and GST-HBx immobilized on 10 µl of glutathione resin were incubated with 100-ng target proteins in modified GBT buffer containing 1% bovine serum albumin for 2 h at 4 °C. After 4 washes with modified GBT buffer, the bound proteins were eluted, then Western blotted.
Immunoprecipitation and Western BlottingAntibodies against
HBx, RPB5, and TFIIB were generated by immunizing rabbits with purified
GST-fused proteins. IgG fractions were purified by affinity
chromatography using protein A followed by GST (18). HepG2 cells were
transfected under the same conditions as described for CAT assays
except that they were incubated in 100-mm culture dishes (18). The
cells were sonicated in LAC buffer (10% glycerol, 20 mM
HEPES, pH 7.9, 50 mM KCl, 0.4 mM NaCl, 10 mM MgCl2, 0.1 mM dithiothreitol,
0.1 mM EDTA, 9 mM CHAPS, 0.5 mM
phenylmethylsulfonyl fluoride, and 10 µg/ml aprotinin and leupeptin) followed by centrifugation. The supernatants were collected and stored
at 80 °C. The total cell lysate in 500 µl of TBST buffer (50 mM Tris-HCl, pH 7.5, 0.2 M NaCl, 0.05% Tween
20) was clarified by an incubation with 20 µl of swollen protein
A-Sepharose resin (Pharmacia) for 30 min followed by centrifugation.
The supernatants were then immunoprecipitated. After reaction with the
first antibody (3 µg of IgG) for 1 h at 4 °C, 10 µl of 50%
swollen protein A-Sepharose resin was added. Samples were rotated for
another hour followed by 3 washes with washing buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% SDS, 1 mM EDTA). The bound proteins were eluted with elution
buffer (10 mM Tris-HCl, pH 7.4, 2% SDS, 0.3 M
NaCl, and 1 mM EDTA), separated by SDS-PAGE, then
transferred onto nitrocellulose membranes, and Western blotted using
second antibodies to detect HBx, TFIIB, or RPB5. The proteins were
visualized by enhanced chemiluminescence (ECL) according to the
manufacturer's (Amersham) instructions (18).
Transient transfection and CAT assays proceeded as described (7, 28). HepG2 cells were cultured in 60-mm plates and transfected. Total cell lysates were prepared from the cells 48 h after transfection. The CAT assay reactions proceeded for 60 min at 37 °C using 20 µg of protein from the transfected cell lysates. The fractionated TLC plates were exposed to imaging plates, and CAT activities were measured as the percentage of conversion to acetylated forms of [14C]chloramphenicol (% acetylation, Amersham) using a bioimage analyzer (BAS1000, Fuji). Transfection and CAT assays were performed at least three times with each combination of transactivator and CAT reporter constructs. Representative data are shown.
Specific HBx-RPB5 interaction is involved in HBx
transactivation, suggesting that RPB5, as a communicating subunit of
RNA polymerase, interacts with general transcription factors (18). We
investigated whether or not RPB5 interacts with other general transcription factors by means of far-Western blotting using
bacterially expressed GST fusion forms of TBP, TFIIB, and CTD (Fig.
1A). Comparable amounts of proteins were
applied and fractionated by SDS-PAGE as shown by Coomassie Blue
staining (lanes 25-30). The RPB5 probe bound to TFIIB with
high affinity (lane 2) as well as to HBx and to itself as
reported (lanes 4 and 6) (18). Similarly, the
TFIIB probe also associated with RPB5 (lane 10), TBP
(lane 7), and with itself. The self-binding of TFIIB agrees
with the reported findings (38). TFIIB and HBx associated with each
other (lanes 12 and 14). None of the probes bound
CTD or GST. The TBP and GST probes did not bind either HBx or RPB5
(lanes 19-24 and data not shown), indicating that the
binding among HBx, RPB5, and TFIIB is specific. These results suggested
that TFIIB is another interaction target of HBx. To confirm the results
of the far-Western blotting, we performed in vitro GST-resin
pull-down assays. As shown in panel a of Fig. 1B,
TFIIB-His6 was efficiently retained by GST-TBP, GST-RPB5, and GST-TFIIB
(lanes 3-5) but barely recovered by GST (lane
2). The in vitro GST-resin pull-down assay was
specific, since TBP-His6 was only retained by GST-TFIIB but not GST-HBx and GST-RPB5 (Fig. 1B, panel b). These results
provided further evidence of the specific binding of TFIIB to RPB5 and
HBx.
Next we performed immunoprecipitation and Western blotting studies to
examine whether these interactions proceed in vivo. HepG2
cell lysates transiently co-transfected with HBx, and RPB5 and TFIIB
were precipitated with anti-RPB5 and anti-TFIIB. Antibodies against
TBP, tubulin, and normal rabbit serum were used as controls. The
antibody-bound proteins were precipitated with Sepharose-bound protein
A, then Western blotted with anti-HBx antibody. HBx was co-precipitated
with RPB5 and TFIIB (Fig. 2A, lanes
3 and 4). Although only a small amount of HBx was
co-precipitated, this phenomenon might be specific since HBx was not
detectably precipitated by the control antibodies (lanes 2,
5, and 6). These results indicate that HBx
associates with RPB5 and TFIIB in vivo when RPB5 and TFIIB
are overexpressed. To determine whether these interactions occur when
RPB5 and TFIIB are expressed at physiological levels, HepG2 cell lysate
transiently transfected with HBx was precipitated with anti-TFIIB. The
antibody-bound proteins were Western blotted for HBx, RPB5, and TFIIB.
HBx and endogenous RPB5 were co-precipitated with TFIIB by anti-TFIIB
(Fig. 2B, lane 6) whereas none of these proteins
were recovered by normal rabbit antibody (lane 5).
Similarly, HBx and endogenous TFIIB were also recovered together with
RPB5 in the anti-RPB5 precipitates (data not shown). When
non-transfected HepG2 cell lysate was precipitated with anti-TFIIB,
RPB5 was co-precipitated whereas no HBx band was detected (Fig.
2B, lane 3). These results indicate that HBx,
RPB5, and TFIIB associate with each other under physiological
conditions. The concurrent precipitation of HBx, RPB5, and TFIIB
implies trimeric interaction or tripartite complex formation (see
below).
HBx, TFIIB, and RPB5 Have Distinct and Separable Binding Sites for Each of the Other Two Proteins
Having established the
interactions among HBx, RPB5, and TFIIB, we mapped the interaction
regions of these proteins by far-Western blotting. As we reported, the
HBx-binding site is located in the middle of RPB5 (10); HBx probe bound
to the constructs harboring this region (73-120 aa) of RPB5, namely
-d1 (47-210 aa), -d13 (47-120 aa), and -d83 (73-120 aa) (Fig.
3A). The TFIIB probe efficiently associated
with the truncated forms of RPB5 proteins harboring the N-terminal
region, namely -d4 (1-46 aa) and -d27 (21-47 aa), but interacted
weakly or barely with constructs having the middle region (-d13, -d1,
and -d83), indicating that the N-terminal portion is necessary for
TFIIB binding (Fig. 3B). These results indicate that in
RPB5, the binding sites of HBx and TFIIB are discrete and
separable.
TFIIB contains two imperfect direct repeats in the C-terminal
two-thirds and one putative zinc finger in the N-terminal portion (39,
40). Far-Western blotting with the HBx probe showed that the
HBx-binding site is located in the two direct repeats, since the probe
bound to the constructs containing the repeats, including TFIIB-full
(1-316 aa), -d9 (1-285 aa), and -d10 (124-285 aa) (Fig. 4A). In contrast, RPB5 binding does not
require the presence of the direct repeats, since RPB5 did not bind to
-d10 (124-285 aa). The RPB5-binding region is within the N-terminal
one-fifth because the RPB5 probe bound to constructs containing this
region, namely TFIIB-d1 (1-60 aa), -d3 (1-124 aa), -d9 (1-285 aa),
and full-length TFIIB (1-316 aa) (Fig. 4B). Since this
region contains a zinc finger, further delineation was conducted to see
whether this motif contributes to RPB5 binding. The results showed that
the zinc finger motif is needed for the binding, since destruction of
the zinc finger motif in -d21 (21-316 aa) reduced the binding ability
to RPB5 probe. Supporting this conclusion, the smallest construct
TFIIB-d16 (21-60 aa) retained only reduced RPB5 binding ability,
whereas the internal deletion mutant proteins, -I37 (17-20 and 37-316
aa) and -I61 (45-49 and 61-316 aa) could not bind to the RPB5 probe
(Fig. 4B). Therefore, the RPB5-binding region is located
within 1-60 aa of TFIIB, and the zinc finger motif is necessary for
the interaction. These results indicated that the binding regions for
HBx and RPB5 in TFIIB are different.
HBx consists of functional transactivation (51-154 aa) and negative
regulatory domains (1-50 aa) (37). Both RPB5- and TFIIB-binding sites
are located within the transactivation domain of HBx (-5D1, 51-154 aa)
but not in the regulation domain (-3D5, 1-50 aa) (Fig. 5). Further truncation of HBx showed that the RPB5- and
TFIIB-binding sites might be different since the TFIIB probe bound to
the constructs containing 102-136 aa, such as -D26 (102-136 aa), -D16
(51-136 aa), -5D4 (72-154 aa), and -5D1 (51-154 aa) (Fig.
5A). The RPB5 probe bound neither -5D4 nor -D26 (Fig.
5B, lanes 4, 5, and 9) but
bound the larger constructs such as D16 (51-136 aa) (lane 8). These results suggested that RPB5 and TFIIB binding requires different regions within the HBx transactivation domain (see below). The TFIIB-binding region is located in the C-terminal (102-136 aa)
while the RPB5 binding seems to require a large portion of the
transactivation domain (51-136 aa).
RPB5 and TFIIB Binding Are Essential for the Transactivation Activity of HBx
HBx, RPB5, and TFIIB associated with each other
through distinct binding sites, which may contribute to the
transactivation function of HBx. To investigate this notion, we
attempted to introduce substitution mutations into the HBx
transactivation domain. Since we could not find a single mutation
within the HBx transactivation domain that markedly affects the
transactivation function as reported by others (41), we constructed
mutants with multiple mutations (three or four substitutions in a row
as described under "Experimental Procedures"). The HBx mutant
proteins were examined for transactivation activities and binding
affinity for RPB5 and TFIIB. All the mutants except Xm76 had severely
impaired transactivation activity (Fig. 6A,
left panel). The difference in the transactivation
activities was not because of a different expression level or stability
of the HBx mutants, since comparable HBx mutant proteins were detected by Western blotting (Fig. 6A, right panel). RPB5
binding of Xm89 and Xm93 was reduced whereas the TFIIB binding was
similar to that of wild HBx (Fig. 6B, compare lanes
2 and 3 with lane 7). In contrast, mutation
Xm120 had reduced binding to TFIIB, but there was no effect upon RPB5
binding (compare lane 4 to lane 7). The double
mutant, Xm93 m120, exhibited reduced binding affinity for TFIIB and
RPB5 (data not shown). The mutant Xm76 with normal transactivation
activity retained binding affinity with both the TFIIB and RPB5
(lane 1). The results showed that RPB5 and TFIIB bind to
different regions in the HBx transactivation domain and suggested that
both the RPB5- and TFIIB-binding regions are essential for HBx
transactivation function.
HBx, RPB5, and TFIIB May Form a Ternary Complex
Since HBx,
RPB5, and TFIIB have two different binding sites each for the other two
partners, these three proteins may form a tripartite complex. We
examined this possibility by means of a GST-resin pull-down assay. The
GST-fusion form of the full size RPB5 protein immobilized on
glutathione-Sepharose resin was incubated in a mixture containing HBx
and TFIIB-His6. After extensive washing, bound proteins were separated
by SDS-PAGE and visualized by Western blotting (Fig.
7A). Both HBx and TFIIB were retained by
GST-RPB5 resin (lane 2) but not by GST resin (lane
3). This result suggests that RPB5 simultaneously binds HBx and
TFIIB. We then examined the possibility that these proteins form a
trimeric complex using RPB5 mutants. The RPB5-full and -d1, harboring
the HBx-binding site, bound HBx independently of the presence of TFIIB
(Fig. 7B, lanes 7-10). However, RPB5-d4, which
has the TFIIB- but not HBx-binding site, efficiently pulled down HBx
only when TFIIB was present in the binding reaction (lane
12). As negative controls, neither GST nor GST-TBP bound HBx
regardless of the presence of TFIIB (lanes 3-6). These
results showed that RPB5-d4 associates with HBx indirectly through the
bridging of TFIIB and that HBx, RPB5, and TFIIB simultaneously interact
with one another to form a trimeric complex in vitro.
We present evidence of specific interactions among TFIIB, RPB5, and HBx, which were detected in vitro by far-Western blotting and the GST-resin pull-down assay as well as in vivo by co-immunoprecipitation. Since each of these three proteins has two distinct and separable binding sites for the other two, they may form a trimeric complex. The simultaneous binding of HBx and TFIIB to RPB5 detected by GST-resin pull-down assay, as well as the finding that the HBx-binding-defective deletion mutant RPB5-d4 (1-47 aa) bound HBx through the bridging of TFIIB, indicated trimeric complex formation at least in vitro. Our results imply that trimeric interaction or complex formation is required for HBx transactivation, since the HBx mutations, which reduced the binding ability either to TFIIB or RPB5, markedly impaired transactivation activity.
TFIIB is conserved from archaebacteria to humans (42, 43). In combination with RNA polymerase II, TFIIB determines the transcription start site (44, 45). The importance of TFIIB in activated transcription has been revealed by its interactions with a variety of gene-specific transcriptional regulator proteins (see Refs. 46 and 47 and references therein), cofactors (48-50), and general transcription factors such as TFIID, TFIIF, and RNA polymerase II (37, 38, 51, 52). The specific interaction of HBx and TFIIB implies that TFIIB serves as a regulation target of HBx. Several virus regulators interact with TFIIB, such as VP16, ICP4 of herpes simplex virus, EBNA-2 of Epstein-Barr virus, IE2 of human cytomegalovirus, E2TA of bovine papillomavirus, and Tat of human T-cell lymphotrophic virus (37, 53). HBx can be categorized as a novel type of transcriptional modulator that communicates with RNA polymerase and the general transcription factor TFIIB, since it is distinct from the precedents that act through binding distal cis-elements and communicating with TFIIB to activate transcription. The HBx-binding region was confined within the two direct repeat regions of TFIIB, which is necessary for the binding of TBP, VP16, and some cofactors (37, 39, 54, 55). HBx may modify TFIIB function by inducing a conformational change in TFIIB through interaction with the two direct repeats, like VP16 (37).
The RPB5-binding site was mapped within the N-terminal part of TFIIB (1-60 aa), and we found that the zinc finger is necessary for the binding. The site is mapped within the region (1-106 aa) important for the recruitment of RNA polymerase II to promoters (39, 55, 56), but seems to be distinct from the putative TFIIF-binding region, because the yeast TFIIB mutation E62K, outside of the RPB5-binding region, can be suppressed by the largest subunit of TFIIF (56). Therefore, the N-terminal part of TFIIB may play an important role in transcription initiation through interacting with RPB5, the exposed subunit of RNA polymerase. The N-terminal part of TFIIB including the zinc finger and its adjacent regions is involved in the transcriptional regulation of the glutamine-rich activator, ftz Q (47).
Two models of transcriptional initiation have been proposed. According to the stepwise model, TFIID first binds the promoter through its TBP subunit, perhaps assisted by TFIIA. The TFIID-promoter complex is subsequently recognized by TFIIB, which acts as a bridging factor by recruiting RNAPII/TFIIF (57, 58). Since HBx can simultaneously bind RPB5 and TFIIB, it may facilitate recruiting RNAPII/TFIIF to the promoter and/or stabilize the pre-initiation complex through HBx-RPB5-TFIIB interactions. Another model is that the RNA polymerase holoenzyme, a large complex containing RNAPII and other factors such as TFIIF, TFIIB, TFIIH, and mediators, is directly recruited to the promoter (58-60). Activators may enhance the initiation by interaction with a component of the holoenzyme (58, 59, 61). According to the latter model, HBx may promote holoenzyme assembly or stabilize it through communication with TFIIB and RPB5. Alternatively, HBx may facilitate the recycling of transcription factors, which is thought to be a regulating step during the transcription process (62). HBx expressed by HepG2 has been recovered together with RPB5 in the fast sedimenting fractions (over 1000 kDa) by glycerol gradient centrifugation (18), suggesting that HBx is associated with the holoenzyme and that it modulates the function of the holoenzyme.
Despite the direct interaction of HBx and basal transcription factors,
HBx transactivation requires distinct cis-elements, such as
AP1-, NF-B-, and SRF-binding sites, in a given reporter as reported
by several groups. Therefore an obvious question is how the
HBx-RPB5-TFIIB trimeric interaction enables promoter selectivity in HBx
transactivation. HBx alone does not transactivate the pGal4-CAT reporter but facilitates the activation of Gal4-VP16, suggesting that
HBx acts during activated, but not basal transcription (33, 63).3 In this regard, HBx may directly or
indirectly communicate with some transcriptional activators (19, 20).
Another possibility is the presence of RPB5-binding negative factor(s)
or a co-repressor that can be replaced by HBx. The latter was suggested
from the observation that the overexpression of RPB5 or its HBx-binding region exhibits transacting ability that requires HBx-responsive cis-elements (18). These two possibilities are not mutually exclusive. Isolating and characterizing the host proteins that interact
with RPB5 may clarify the mechanism of HBx transactivation.
Both the RPB5- and TFIIB-binding regions in the transactivation domain of HBx were indispensable for HBx transactivation, suggesting that HBx transactivates through direct interactions with general transcription factors. However, these results do not exclude the possibility that HBx transactivates through other routes such as the Ras signaling pathways in the cytoplasm. If HBx has several independent pathways for transactivation, some mutations of HBx that differentially affect them may be isolated, and host proteins that selectively affect one of these pathways may be identified, as shown for human T-cell lymphotrophic virus Tax-1 (64).
We are grateful to M. Horikoshi for human TFIIB and TBP, to R. G. Roeder for TFIIB-His6 and TBP-His6 plasmids, to K. Matsushima, M. Seiki, and S. Kaneko for critical discussion, and to C. Matsushima and F. Momoshima for technical assistance.