Transcriptional Repression of Human Hepatitis B Virus Genes

天狼星

Journal of Virology, April 1999, p. 3197-3209, Vol. 73, No. 4
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Transcriptional Repression of Human Hepatitis B Virus Genes by a bZIP Family Member, E4BP4
Chao-Kuen Lai and Ling-Pai Ting*
Institute of Microbiology and Immunology, School of Life Science, National Yang-Ming University, Shih-Pai, Taipei 11221, Taiwan, Republic of China


Received 22 September 1998/Accepted 17 December 1998


Box  is an essential element of both the upstream regulatory sequence of the core promoter and the second enhancer, which positively regulate the transcription of human hepatitis B virus (HBV) genes. In this paper, we describe the cloning and characterization of a box  binding protein, E4BP4. E4BP4 is a bZIP type of transcription factor. Overexpression of E4BP4 represses the stimulating activity of box  in the upstream regulatory sequence of the core promoter and the second enhancer in differentiated human hepatoma cell lines. E4BP4 can also suppress the transcription of HBV genes and the production of HBV virions in a transient-transfection system that mimics the viral infection in vivo. Expression of an E4BP4 antisense transcript can, instead, elevate the transcription of the core promoter. A low abundance of E4BP4 protein and mRNA in differentiated human hepatoma cell lines is detected, and E4BP4 is not a major component of box  binding proteins in untransfected differentiated human hepatoma cell lines. C/EBP and C/EBP, in contrast, are major components of the box  binding activity present in nuclear extracts. E4BP4 has a stronger binding affinity towards box  than the endogenous box  binding activity present in nuclear extracts. Structure and function analysis of E4BP4 reveals that DNA binding activity is sufficient to confer the negative regulatory function of E4BP4. These results indicate that binding site occlusion is the mechanism whereby E4BP4 suppresses transcription in HBV.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References  
Hepatitis B virus (HBV) is a small DNA virus with a partially double-stranded 3.2-kb genome. The genome organization of HBV is very compact, with four overlapping open reading frames coding for the surface, core, polymerase, and X proteins. The transcription of these open reading frames is under the control of four promoters: two for surface, one for core and polymerase, and one for X. Two enhancers, enhancer I and enhancer II, play important roles in the transcription regulation of these viral genes. The core promoter is composed of the basal core promoter and its upstream regulatory sequence (70). This promoter produces two 3.5-kb RNAs, i.e., the precore and pregenomic RNAs. Pregenomic RNA has dual functions: (i) it can be packaged into nucleocapsids (core particles) along with viral polymerase and serve as the template for reverse transcription, and (ii) it can serve as mRNA that encodes the core and polymerase proteins. Regulated expression of pregenomic RNA plays a pivotal role in the control of the viral replication cycle. A detailed understanding of the transcription control of viral genes may reveal new targets for therapeutic intervention.

The second enhancer of HBV has a unique bipartite structure in that the cooperation of two noncontiguous elements, box  and box , is required for its enhancer function. It stimulates the transcriptional activity of the simian virus 40 (SV40) early promoter and the HBV surface and X promoters (67, 68). The second enhancer is colocalized with the core upstream regulatory sequence (CURS). Box , for example, is not only an essential component of the second enhancer but also a potent stimulatory element of the CURS. In other words, box  can activate the basal core promoter from an upstream position in differentiated human hepatoma cell lines (HepG2 and HuH-7) (69, 70). A negative regulatory element, designated NRE, which represses the activities of enhancer II and the core promoter, was identified upstream of the CURS (44). The core promoter provides a valuable system to study the positive and negative transcription regulation of eukaryotic promoters.

Transcription regulation is governed by a constellation of trans-acting cellular factors that bind to specific cis-acting elements that act in either a positive or negative manner. Transcription initiation by RNA polymerase II involves a stepwise assembly of general transcription factors or a holoenzyme on a promoter template to form a preinitiation complex. Transcription activators may stimulate transcription by increasing the assembly of a preinitiation complex. Several distinct models have been proposed as the mechanism of transcription repression (20, 21, 24, 25, 29, 42, 52). In the competition model, repressors may bind directly at or near a transcription start site and compete with the formation of a preinitiation complex in the promoter. Alternatively, activators and repressors may compete for overlapping or closely linked binding sites. In the activator-sequestering model, repressors stoichiometrically bind to particular activators through protein-protein interactions, leading to the formation of complexes with reduced or no DNA binding activity. In the quenching model, repressors and activators may bind to adjacent, nonoverlapping DNA sequences, but the repressors neutralize the ability of the activators to transmit stimulatory signals to the basal transcription machinery. In the fourth model, direct repression, repressors may bind to any of the basal transcription factors, with RNA polymerase II itself, or with a corepressor that ultimately targets the basal machinery. Such interaction may interfere with the formation or the activity of the basal transcription preinitiation complex.

We have previously shown that C/EBP-like proteins can bind to box . C/EBPs (CCAAT/enhancer binding proteins) are a family of highly conserved, leucine zipper-type (bZIP) DNA binding proteins. Members identified so far are C/EBP, C/EBP (also known as NF-IL6, CRP2, LAP, and AGP/EBP), C/EBP (also known as NF-IL6 and CRP3), C/EBP, CRP1, Ig-C/EBP, and GADD153 (also known as CHOP) (1, 5, 6, 15, 31, 32, 36, 53, 54, 65). Different C/EBP family members are characterized by a high degree of sequence homology in the leucine zipper and basic regions. They have, however, much less conserved N-terminal regulatory and transactivation domains (5, 35). C/EBPs have the potential to form homo- and heterodimers with C/EBP family members or bZIP proteins or to interact with proteins that do not contain leucine zippers. Dimerization of C/EBPs is generally required for their DNA binding and transcription activation function (3, 10, 16, 18, 26, 33, 34, 37-41, 45, 46, 48, 58-65).

In this paper, we describe the cloning and characterization of a box  binding protein, E4BP4. Overexpression of E4BP4 represses the stimulating activity of box  in the CURS and the second enhancer. E4BP4 can also repress the transcription of HBV genes and the production of HBV virions in a transient-transfection system. Overexpression of an E4BP4 antisense transcript, on the other hand, can elevate the transcription of the core promoter. Though present in low abundance, E4BP4 can bind to the box  sequence with higher affinity than the box  binding activity present in nuclear extracts. Evidence that binding site occlusion is most likely the mechanism whereby E4BP4 suppresses transcription in HBV is presented.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References  
Isolation of cDNA clones. A ZAPII cDNA library (prepared from Stratagene''''s ZAP cDNA synthesis kit) of human hepatoma HepG2 cells was screened with concatemerized double-stranded synthetic oligonucleotides of box  by the method of Singh (57). The oligonucleotides contained the box  sequence gatCCAAGGTCTTACATAAGAGGACTCTT and its complement, which corresponded to the box  sequence extending from nucleotide (nt) 1644 to 1669 of HBV plus an MboI 5'''' overhang. The oligonucleotides were concatemerized to ~500 bp in size with T4 polynucleotide kinase and T4 DNA ligase and labeled with [-32P]dCTP by random prime labeling. All positive plaques were picked, replated, and clonally purified through secondary and tertiary screenings. cDNA inserts from positive clones were excised in the form of pBluescript plasmid (pSKP4) by coinfection with helper phage. DNA sequences were determined by dideoxy chain termination methods.

Plasmids. The HBV sequence used in the study is of the adw subtype. Numbering of the HBV sequence begins at the unique EcoRI site, which is nt 1. All reporter plasmids used in transfection experiments contain a head-to-tail trimeric tandem repeat, referred to as A3, of a 237-bp BclI-BamHI fragment from the SV40 polyadenylation signal. A3 is placed 5'''' of promoter sequences of interest and has been shown to stop transcription readthrough from spurious upstream initiation.

Plasmids pSV2CAT, p/BCP-CAT, pCURS/BCP-CAT, p(1613-1851)CAT, p(1687-1851)CAT, pSVpCAT/ENII, pSVpCAT/, and pHBV3.6 were described previously (44, 67, 68, 70).

The recombinant E4BP4 expression plasmid pXa-2-P4 was generated by cloning of the BamHI-KpnI fragment containing the E4BP4 open reading frame into the BamHI and KpnI sites of the PinPoint Xa-2 vector (Promega).

The plasmid pCMVP4 was generated by moving the cDNA inserts of pSKP4 into the BamHI and KpnI sites downstream of the cytomegalovirus (CMV) immediate-early promoter (CMVIE) in pCMVIE. To generate the FLAG-tagged E4BP4 construct, the BamHI-KpnI fragment containing the cDNA insert of pCMVP4 was cloned into the BglII and KpnI sites downstream of the CMVIE in the pFLAG-CMV2 expression vector (Kodak, New Haven, Conn.). The resulting plasmid, pf:E4BP4, was digested with ApaI and SalI and then recircularized to generate pf:E4BP4Apa and pf:E4BP4Sal, respectively. The plasmid pf:E4BP4Pvu was generated by cloning the BamHI-PvuII fragment from pf:E4BP4 into BglII- and SmaI-digested pFLAG-CMV2. To remove the repression domain of E4BP4, pCMVP4 was first digested with BstBI to derive a 5.6-kb BstBI fragment, which was subsequently digested with BamHI, followed by filling in of all 3''''-recessed ends with the Klenow fragment of Escherichia coli DNA polymerase. Two BamHI-BstBI fragments of 4,513 and 1087 bp, were generated. An 885-bp BamHI-HaeIII fragment was derived from the digestion of the 1,087-bp BamHI-BstBI fragment with HaeIII. The 4,513-bp BamHI-BstBI and 885-bp BamHI-HaeIII fragments were ligated together to generate pCMVP4Hae/BstB. The BamHI-KpnI fragment of pCMVP4Hae/BstB was cloned into the BglII and KpnI sites of the pFLAG-CMV2 expression vector to generate the plasmid pf:E4BP4Hae/BstB. All of these constructs were confirmed by DNA sequencing.

The E4BP4 antisense plasmid pCMV4Ns/s was generated by cloning of the 378-bp SalI-SspI fragment, which corresponds to the 5''''-end region of E4BP4 from nt 117 to 494, into the SalI-SmaI sites of the pCMVIE expression vector.

Bacterial fusion proteins. The E4BP4 expression construct pXa-2-P4 was used to express biotinylated fusion proteins in strain JM109. JM109 cells harboring E4BP4 vectors were grown to log phase and induced with 100 µM isopropyl--D-thiogalactopyranoside (IPTG) (Sigma). Six hours following induction, the bacteria were centrifuged and lysed in 1 mg of lysozyme per ml-0.1% Triton X-100-200 U of DNase. The lysates were then clarified by centrifugation at 10,000 × g for 15 min at 4°C and mixed with avidin resin (Promega). Following a 6-h incubation, the resin was washed three times in cold buffer (50 mM Tris-HCl, 4 mM dithiothreitol [DTT], 2 mM EDTA, 10% glycerol). If the fusion protein was to be eluted, the pelleted resin was washed with buffer containing 5 mM biotin. The eluent was aliquoted, quickly frozen under liquid nitrogen, and kept frozen at 70°C.

Preparation of anti-E4BP4 polyclonal antibody. Rabbits were immunized with a 16-amino-acid peptide corresponding to amino acids 446 to 461 of E4BP4. After three booster injections with conjugated peptide (the carrier protein was keyhole limpet hemocyanin or bovine serum albumin), rabbit sera were tested for reactivity with E4BP4 by immunoprecipitation and Western blotting.

Cell lines, transfection, and CAT assay. The culture and transfection of human hepatoma cell lines HepG2 and HuH-7 were performed as previously described (7). All plasmids used in one set of experiments were simultaneously prepared, checked for supercoiled forms, aliquoted in small amounts, and stored in 70% ethanol. Each set of experiments was performed with two different preparations of plasmids and repeated two to three times for each preparation. The chloramphenicol acetyltransferase (CAT) activity was normalized against the CAT activity exhibited by a control plasmid, pSV2CAT, which was taken as 100%. In pSV2CAT, the expression of the CAT gene is driven by the SV40 early promoter and 72-bp enhancer. When the CAT activity was high, assays were performed on serially diluted cell lysates to ensure that CAT activity fell in a linear range for all assays.

Preparation and heparin-Sepharose fractionation of nuclear extracts. Fractionated nuclear extracts from differentiated human hepatoma cell lines HepG2 and HuH-7 were prepared as previously described (8, 68). The crude and fractionated nuclear extracts were aliquoted, quickly frozen under liquid nitrogen, and kept frozen at 70°C.

Preparation of mini-nuclear extracts from transfected cells. Mini-nuclear extracts were prepared by the method of Schreiber et al. (56). HuH-7 cells were transiently transfected with pFLAG-CMV2, pf:E4BP4, and expression plasmids containing deletion mutants of f:E4BP4 by the calcium phosphate precipitation method. Forty-eight hours later, transfected HuH-7 cells were collected, washed with Tris-buffered saline (TBS) (10 mM Tris-HCl [pH 7.45] and 150 mM NaCl), and pelleted by centrifugation at 1,500 × g for 5 min. The cell pellet was resuspended in TBS, transferred into an Eppendorf tube, and pelleted again by being spun for 20 s in a microcentrifuge. TBS was removed, and the cell pellet was resuspended in cold buffer A (10 mM HEPES [pH 7.9], 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, and 0.5 mM phenylmethylsulfonyl fluoride) by gentle pipetting. The cells were allowed to swell on ice for 15 min, after which a 10% solution of Nonidet P-40 was added and the tube was vigorously vortexed for 10 s. The homogenate was centrifuged for 30 s in a microcentrifuge. The nuclear pellet was resuspended in ice-cold buffer C (20 mM HEPES [pH 7.9], 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM phenylmethylsulfonyl fluoride), and the tube was vigorously rocked at 4°C for 15 min on a shaking plate. The nuclear extract was centrifuged for 5 min in a microcentrifuge at 4°C, and the supernatant was aliquoted, quickly frozen under liquid nitrogen, and kept frozen at 70°C.

Gel shift analysis. The probe was prepared with annealed double-stranded oligonucleotide (100 ng) corresponding to the box  sequence of HBV (Fig. 1A) and end labeled with [-32P]ATP and T4 polynucleotide kinase. Gel shifting and competition experiments were done as previously described (68) except that 5 instead of 10 µg of nuclear extract was used (68). Supershifts were generated with anti-E4BP4 antiserum, anti-FLAG M2 monoclonal antibody (Kodak), or anti-C/EBP polyclonal antibody for C/EBP, C/EBP, or C/EBP (Santa Cruz Biotechnology, Santa Cruz, Calif.).

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FIG. 1.   Binding specificity of recombinant E4BP4 protein. (A) Summary of the oligonucleotide sequences of wild-type box  nt 1636 to 1668 [WT(36)] and 1646 to 1668 [WT(46)] and mutants AB, CD, EF, GH, IJ, and YZ and their binding activities toward recombinant E4BP4. In the WT(36) oligonucleotide, the lowercase letters represent the sequence which is different from the HBV sequence. In mutants, the lowercase letters represent the mutated nucleotides. (B) Competition of binding of recombinant E4BP4 protein to wild-type box  sequence [WT(36)] by wild-type box  sequence [either WT(36) or WT(46)] or six box  mutants in gel shift assays. Recombinant E4BP4 protein was first incubated with competing cold oligonucleotides in molar excess and then tested for its binding to labeled WT36 probe. Lane 1, labeled WT(36) probe with no protein; lane 2: labeled WT(36) probe with recombinant E4BP4 protein but no competitor; lanes 3 to 20, labeled WT(36) probe with recombinant E4BP4 protein and different unlabeled competitors at various molar excesses as indicated. HNF1 is a nonspecific competitor.




For Scatchard plot analysis, 7.5 µg of HuH-7 nuclear extract (0.5 M NaCl fraction) and 7 µl of recombinant E4BP4 protein were incubated with different amounts of 32P-end-labeled box  oligonucleotides ranging from 0.032 to 1.411 ng. The resulting protein-DNA complexes were separated in a 4% polyacrylamide gel, and the bands representing free and bound ligands were identified; this was followed by drying and quantification with a Molecular Dynamics PhosphorImager. Standard Scatchard plot analysis allowed determination of the appropriate Kd values (4, 55).

Northern blotting. Total cellular RNA was prepared from HepG2 cells, HuH-7 cells, or transfected HuH-7 cells with the RNAzol B kit (Cinna/Tiotecx Laboratories, Inc., Houston, Tex.). Twenty or 40 µg of total cellular RNA was electrophoretically separated on a 1% formaldehyde-agarose gel and transferred to a Hybond nylon membrane (Amersham). In addition, nitrocellulose filters containing approximately 2 µg of poly(A)+ RNAs from 16 different adult human tissues (Clontech, Palo Alto, Calif.) were used for Northern analysis. These membranes were hybridized with a 32P-random-prime-labeled 554-bp EcoRI fragment of the E4BP4 cDNA probe or 1,960-bp PstI fragment of the HBV DNA probe and washed at a high stringency under standard conditions. These blots were exposed to Fuji X-ray film at 70°C with an intensifying screen. The signals were normalized by hybridization with a probe for the -actin or glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene followed by quantitation with a Molecular Dynamics PhosphorImager.

Assay for endogenous DNA polymerase activity. To assay for endogenous DNA polymerase activity, the culture supernatant was collected 3 days after transient transfection, treated with 1% Nonidet P-40 for 4 h at room temperature, and centrifuged at 17,000 × g for 30 min at 4°C. The supernatant was then centrifuged at 227,000 × g for 1 h at 4°C. The pellet from the second centrifugation, which contains HBV viral core particles, was resuspended in TNE buffer (10 mM Tris-HCl [pH 7.5], 50 mM NaCl, and 0.1 mM EDTA) and assayed for endogenous polymerase activity as previously described (70).

Western blotting. HuH-7 cells were transfected with pFLAG-CMV2, pf:E4BP4, and plasmids containing deletion mutants of f:E4BP4. After 48 h, cells were collected and lysed in Laemmli sample buffer at 95°C for 10 min. Proteins were separated by electrophoresis through a 10 or 12.5% polyacrylamide gel, transferred onto a Hybond (Amersham) enhanced chemiluminescence (ECL) nitrocellulose membrane, and probed with 6 µg of monoclonal anti-FLAG antibody (Kodak) per ml or with a 1,000× dilution of polyclonal anti-E4BP4 antibody. Blots were incubated with anti-rabbit or anti-mouse immunoglobulin G (IgG)-horseradish peroxidase conjugate (Promega), and immunoreactive proteins were visualized with 4-chloro-1-naphthol (Sigma) or by using the ECL system (Amersham).

Immunofluorescence. HuH-7 cells cultured on Chamber Slides (Nunc) were transfected with pFLAG-CMV2, pf:E4BP4, and plasmids containing deletion mutants of f:E4BP4. Following washing in phosphate-buffered saline (PBS), the cells were fixed with 2% formaldehyde in PBS for 20 min at room temperature, permeabilized by expoure to cold acetone for 3 min, and washed once with PBS. The permeabilized cells were detected with 6 µg of monoclonal anti-FLAG antibody (Kodak) per ml for 1 h and then with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Cappel) and 0.1 µg of Hoechst 33258 per ml in 1% bovine serum albumin-PBS for 1 h at room temperature. Finally, the cells were washed three times with PBS and examined by fluorescence microscopy.


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References  

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E4BP4 as a box  binding protein. To search for the box  binding protein(s), we screened a cDNA library made from a differentiated human hepatoma cell line, HepG2, with labeled concatemers of the box  sequence. A cDNA which was identical to that of a previously described transcription factor, E4BP4 (also named NF-IL3A), was obtained. A member of the bZIP family, E4BP4 has been identified as a binding protein for a variety of promoters, including the ATF site in the E4 promoter of adenovirus, the CRE/ATF-like site of the interleukin-1 (IL-1) promoter, the gamma interferon promoter, and the A site of the IL-3 promoter (11, 12, 27, 71). E4BP4 contains 462 amino acids. The bZIP domain of E4BP4 is located in the N-terminal region of the protein (see Fig. 6A). E4BP4 has been shown to function as a dimer (12).

To examine the binding specificity of E4BP4, recombinant E4BP4 protein was obtained by fusion of the E4BP4 open reading frame with that of the 1.3S subunit of the Proprionibacterium shermanii transcarboxylase. Labeled double-stranded oligonucleotides (nt 1636 to 1668) corresponding to the wild-type box  sequence were incubated with recombinant E4BP4 protein in gel shift assays. Oligonucleotides containing different mutated box  sequences were added in molar excess as competitors in gel shift assays. As shown in Fig. 1B, mutants IJ, GH, EF, and YZ competed efficiently for binding, while mutants AB and CD did not. These results, summarized in Fig. 1A, indicated that the sequence from nt 1650 to 1662 in box  was the binding site for E4BP4. This segment contains the sequence cTTACaTAAg (the lowercase letters represent the sequence which is different from the consensus E4BP4 binding sequence), which resembled the consensus E4BP4 binding sequence (A/G)T(G/T)A(T/C)GTAA(T/C) (12).

We then examined the binding specificity of overexpressed E4BP4 protein in cells. E4BP4 was first epitope tagged with the FLAG sequence at its N terminus. The coding sequence of FLAG-E4BP4 was then cloned downstream of the CMVIE in the expression plasmid pf:E4BP4. An empty vector, pFLAG-CMV2, was included as a negative control. The expression of E4BP4 was detected with both anti-FLAG monoclonal antibody and anti-E4BP4 antiserum. The anti-E4BP4 antiserum was raised against a peptide derived from the C-terminal region of E4BP4 (see Materials and Methods). Antihemagglutinin (anti-HA) monoclonal antibody and/or preimmune serum was used as a negative control.

HuH-7 cells were transfected with pf:E4BP4 or empty vector in transient-transfection assays. Total cell lysates from untransfected and transfected cells were obtained for Western and gel shift experiments. As shown in Fig. 2A, two forms of E4BP4 were detected with anti-E4BP4 antiserum in pf:E4BP4-transfected cells. Only the larger form of E4BP4 was detected with anti-FLAG monoclonal antibody. The small form of E4BP4 is most likely the translation product from the AUG initiation codon of E4BP4 in the FLAG-tagged E4BP4 from a downstream position. The apparent molecular masses of these two forms of E4BP4 are approximately 64 and 61 kDa instead of the estimated 51 kDa. The reason for this discrepancy is not clear, although an earlier report suggests that phosphorylation may play a role (11). No signal was observed in cells transfected with empty vectors or in untransfected cells. No signal was observed with preimmune serum or anti-HA antibody.


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   FIG. 2.   DNA binding specificity of overexpressed E4BP4. HuH-7 cells at a density of 1.1 × 107 cells per 15-cm-diameter plate were untransfected (un) or transfected with 62.5 µg of pFLAG-CMV2 (flag) or pf:E4BP4 as indicated. Mini-nuclear extracts were prepared as described in Materials and Methods. (A) E4BP4 expression in transfectants determined by Western blot analysis. Mini-nuclear extracts (20 µg per lane) were used for Western blot analysis, with 1,000× dilutions of anti-E4BP4 (lanes 1 to 3) and preimmune serum (lanes 4 to 6), 6 µg of anti-FLAG ( flag) antibody per ml (lanes 7-9), and 10 µg of anti-HA antibody per ml (lanes 10 to 12). Immunoreactive proteins were visualized with 4-chloro-1-naphthol. (B) Binding of E4BP4 to the box  sequence. Five micrograms of mini-nuclear extracts was incubated with 105 cpm of labeled box  probe [WT(36)] in gel shift experiments. The sources of nuclear extracts are indicated. For supershift experiments, 1 µl of preimmune serum (lane 5) or anti-E4BP4 (lane 6) or 3 µg of anti-FLAG antibody (lane 11) was added. (C) DNA binding specificity of E4BP4. Five micrograms of mini-nuclear extracts was incubated with the labeled wild-type box  probe [WT(36)] in the presence of unlabeled WT(36) or different mutant competitors at various molar excesses as indicated.




Nuclear extracts derived from the transfected cells described above were used in gel shift assays. As shown in Fig. 2B, box  binding activity was present in both untransfected cells and cells transfected with pf:E4BP4 or empty vector. In the nuclear extracts obtained from pf:E4BP4 transfectants, a supershift of box  binding activity by anti-FLAG and anti-E4BP4 antibodies was observed. This supershift was not seen with a control preimmune serum. These results indicated that E4BP4 could indeed bind to the box  sequence. They also showed that E4BP4 is not a major component of endogenous box  binding proteins (Fig. 2C). Moreover, inclusion of unlabeled competing oligonucleotides in molar excess showed that wild-type box  and the EF sequences could effectively abolish the box  binding activity that was supershifted with anti-FLAG antibody. FLAG-tagged E4BP4 expressed in transient transfection, therefore, exhibited the same binding specificity as its bacterially expressed counterpart. Identical results were obtained with another E4BP4-transfected differentiated human hepatoma cell line, HepG2 (data not shown).

To determine the intracellular localization of E4BP4, immunofluorescence of E4BP4-transfected and untransfected HuH-7 cells with anti-FLAG antibody was performed. E4BP4 was detected as a nuclear protein. Identical results were obtained with anti-E4BP4 antiserum (data not shown).

Repression of the transcription stimulation effect of box  by E4BP4. We have previously shown that a single copy of the box  sequence in an upstream position stimulates the transcription of the HBV basal core promoter in both HepG2 and HuH-7 cells (68). To examine the effect of E4BP4 on box , the reporter plasmid p/BCP-CAT was cotransfected with pf:E4BP4 or pFLAG-CMV2 vector in HepG2 and HuH-7 cells. p/BCP-CAT contains a CAT reporter gene, driven by the HBV basal core promoter, which is preceded by an upstream box  sequence. Increasing amounts of E4BP4 expression plasmids were cotransfected with the reporter plasmid. The expression level of f:E4BP4 and the transcriptional activity of the basal core promoter as measured by CAT assays were determined (Fig. 3). Coexpression of E4BP4 reduced the CAT activity by 35- and 14-fold in HepG2 and HuH-7 cells, respectively (Fig. 3). Cotransfection with pFLAG-CMV2, which had no insert, had no significant effect. It is worth noting that the suppression by E4BP4 was observed with the expression of E4BP4 at a very low level (data not shown). Since the promoter activity of the basal core promoter (pBCP-CAT) is already very low, it is difficult to examine the effect of E4BP4 on the basal core promoter directly. To circumvent this problem, we placed another positive element from the CURS upstream of the basal core promoter instead of box . This reporter plasmid, p(1687-1851)CAT, contained an extra sequence from nt 1687 to 1743 in addition to the basal core promoter. E4BP4 decreased the activity of p(1687-1851)CAT by threefold (Fig. 4). Weak suppression of the basal promoter by E4BP4 has been previously noted (12). Taken together, these data indicated that E4BP4 suppressed the transcription-stimulatory activity of the basal core promoter mediated by box .



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   FIG. 3.   Repression of the box  activity by E4BP4. HuH-7 (lanes 1 to 14) and HepG2 (lanes 15 to 28) cells were transfected with 8 µg of pBCP-CAT only (lanes 1 and 15), 8 µg of p/BCP-CAT only (lanes 2 and 16), 8 µg of p/BCP-CAT plus 31.2, 62.5, 125, 250, 500, or 1,000 ng of pf:E4BP4 (lanes 3 to 8 and 17 to 22, respectively), or 8 µg of p/BCP-CAT plus 31.2, 62.5, 125, 250, 500 or 1,000 ng of pFLAG-CMV2 (lanes 9 to 14 and 23 to 28, respectively). The cell densities for HuH-7 and HepG2 cells were 1.5 × 106 and 2.8 × 106 per 5-cm-diameter plate, respectively. The ability of a cotransfected expression vector to modulate the box  activity was determined by CAT assay. (A) Autoradiogram of CAT activities in a representative assay. (B) Diagram showing the suppression of CAT activity produced by p/BCP-CAT in the presence of an increasing amount of cotransfected pf:E4BP4. The diagram shows the CAT activity exhibited by p/BCP-CAT relative to that of pBCP-CAT. Results were quantitated by PhosphorImager counting as described in Materials and Methods.





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   FIG. 4.   Repression of the activities of the CURS and second enhancer by E4BP4. HepG2 cells at a density of 1.4 × 106 per well in a six-well plate were either transfected with 4 µg of pBCP-CAT or cotransfected with p/BCP-CAT, pCURS/BCP-CAT, pNRE-CURS/BCP-CAT, pSVpCAT/ENII, pSVpCAT/, or a p(1687-1851)CAT control plasmid in the presence of 500 ng of either pCMVP4 (CMV-E4BP4) or pCMVIE (CMV). The diagram shows the CAT activity exhibited by p/BCP-CAT relative to that of pBCP-CAT. Results were quantitated with a PhosphorImager as described in Materials and Methods. The data represent the mean results obtained from at least four experiments. Error bars represent the standard errors of the mean values obtained.




Since box  is a functional element of the CURS (nt 1636 to 1741), the effect of E4BP4 on the entire CURS was determined. The positive regulatory activity of the CURS was reduced sevenfold in HepG2 cells (Fig. 4).

Box  is an essential component of enhancer II of HBV. Enhancer II has a unique bipartite structure, and the cooperation of two noncontiguous sequence motifs, box  and box , is required for its function (68). As shown in Fig. 4, E4BP4 could also suppress the stimulating activity of enhancer II. This suppressive effect was seen with the enhancer in its entirety (from nt 1636 to 1741) as well as with its minimal essential elements (box  and box ).

We have previously identified a negative regulatory element designated NRE. The sequence from nt 1613 to 1621 is essential for NRE activity. Located upstream of the CURS, NRE represses the activity of CURS and enhancer II (reference 44 and our unpublished results). We then examined whether E4BP4 could suppress the transcription-stimulatory activity of the CURS in the presence of NRE. A 25-fold reduction in CAT activity was observed in HepG2 cells (Fig. 4). E4BP4 therefore suppressed the stimulating activity of the CURS in the absence or presence of NRE.