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Journal of Virology, November 2008, p. 10832-10840, Vol. 82, No. 21
0022-538X/08/$08.00+0     doi:10.1128/JVI.00883-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Transactivation of the Hepatitis B Virus Core Promoter by the Nuclear Receptor FXR{alpha}{triangledown}

Christophe Ramière,1,2 Caroline Scholtès,1,2 Olivier Diaz,1 Vinca Icard,1,2 Laure Perrin-Cocon,1 Mary-Anne Trabaud,2 Vincent Lotteau,1,2 and Patrice André1,2*

INSERM U851, 21 Avenue Tony Garnier, and Université de Lyon, and Université Lyon 1, and IFR 128, Lyon F-69007, France,1 Hospices Civils de Lyon, Lyon F-69002, France2

Received 26 April 2008/ Accepted 22 August 2008


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ABSTRACT
 
Hepatitis B virus (HBV) core promoter activity is positively and negatively regulated by nuclear receptors, a superfamily of ligand-activated transcription factors, via cis-acting sequences located in the viral genome. In this study, we investigated the role of farnesoid X receptor alpha (FXR{alpha}) in modulating transcription from the HBV core promoter. FXR{alpha} is a liver-enriched nuclear receptor activated by bile acids recognizing hormone response elements by forming heterodimers with retinoid X receptor alpha (RXR{alpha}). Electrophoretic mobility shift assays demonstrated that FXR{alpha}-RXR{alpha} heterodimers can bind two motifs on the HBV enhancer II and core promoter regions, presenting high homology to the consensus (AGGTCA) inverted repeat FXR{alpha} response elements. In transient transfection of the human hepatoma cell line Huh-7, bile acids enhanced the activity of a luciferase reporter containing the HBV enhancer II and core promoter sequences through FXR{alpha}. Moreover, using a greater-than-genome-length HBV construct, we showed that FXR{alpha} also increased synthesis of the viral pregenomic RNA and DNA replication intermediates. The data strongly suggest that FXR{alpha} is another member of the nuclear receptor superfamily implicated in the regulation of HBV core promoter activity and that bile acids could play an important role in the natural history of HBV infection.


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INTRODUCTION
 
Hepatitis B virus (HBV) infection is limited to hepatocytes of humans and primates and is associated with chronic hepatitis, cirrhosis, and hepatocellular carcinoma (10, 19). HBV is an enveloped virus containing a 3.2-kb partially double-stranded DNA genome with four open reading frames. These open reading frames encode the reverse transcriptase, precore, and core proteins; three surface antigen proteins (pre-S1, pre-S2, and S); and the X protein. Regulation of HBV transcription is under the control of four promoters (the core, pre-S1, pre-S2/S, and X promoters) and two enhancer regions (EN1 and EN2). For some authors, the core promoter corresponds to two distinct promoters, namely the precore and pregenomic promoters (44, 46). The core promoter activity, modulated by the EN2 region, plays a critical role in the virus life cycle. It initiates the synthesis of the precore and pregenomic 3.5-kb RNAs. The precore RNA encodes the precore protein, also called HBe antigen. The pregenomic RNA encodes both the polymerase and the core protein and serves as the template for viral DNA synthesis (24, 34).

Multiple cellular transcription factor binding sites have been identified on these regulatory sequences (26, 29, 31, 40, 48, 51), in particular for several nuclear receptors (NR) belonging to the superfamily of ligand-activated transcription factors (1, 11, 13, 14, 22, 23). Two important NR response elements (NRRE), situated in the EN2 (NRREenhII) and the core promoter (NRREpre-C) regions, are recognized by hepatocyte nuclear factor 4 alpha (HNF4{alpha}) and RXR{alpha}-peroxisome proliferator-activated receptor alpha (PPAR{alpha}) heterodimers (13, 30, 44). In hepatoma cell lines, HNF4{alpha} and PPAR{alpha}-RXR{alpha} upregulate synthesis of pregenomic RNA and viral DNA (43). Moreover, in nonhepatoma cells which do not support HBV replication, exogenous expression of these NR is sufficient for synthesis of the 3.5-kb RNA and viral replication intermediates (40). This observation demonstrated that liver-enriched NR play an essential role in the HBV life cycle and are likely to contribute to the restricted tropism of this virus.

In this study, the influence of farnesoid X receptor alpha (FXR{alpha}) on the transcriptional activity from the HBV core promoter was examined. FXR{alpha} is an NR activated by bile acids (25, 28) which is primarily expressed in the liver, intestine, and kidney. FXR{alpha} transactivates several genes implicated in the metabolism of bile acids and lipids, binding preferentially as a heterodimer with RXR{alpha} to regulatory sequences called FXR{alpha} response elements (FXREs). Typical FXREs are inverted repeats of two NR half-site sequences (5'-AGGTCA-3') separated by 1 bp (IR-1 sequences) (7, 16). We identified two putative FXREs in the EN2 region and the core promoter. Using a reporter gene system, we demonstrated that FXR{alpha} transactivated the core promoter through both of these FXREs. Additionally, we observed that, in the context of a whole HBV genome, the global effect of FXR{alpha} was an increase in synthesis of the viral pregenomic RNA and DNA replication intermediates. Our results suggest that FXR{alpha} is another liver-enriched transcription factor implicated in the regulation of core promoter activity and level of viral replication.


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MATERIALS AND METHODS
 
Materials. Chenodeoxycholic acid (CDCA), all-trans-retinoic acid (RA), and guggulsterone (GGS) were purchased from Sigma (Saint-Quentin Fallavier, France). Stock solutions were prepared with ethanol, chloroform, and dimethyl sulfoxide, respectively, diluted extemporaneously in complete Dulbecco's modified Eagle's medium, and added to the cell cultures at the indicated concentrations.

Plasmids and oligonucleotides. Cloning of the plasmid pGL2-EN2/CP-wt was performed by standard techniques (32). A fragment of the HBV genome (GenBank accession number U95551) corresponding to nucleotides (nt) 1400 to 1902 was amplified from supernatants of HepG2.2.15 cells (35), using the following oligonucleotides: 5'-ATCGGAGCTCTGGATCCTGCGCGGGAC-3' and 5'-ATCGAAGCTTGCCCCAAAGCCACCCAAG-3'. This fragment, which contained the EN2 and core promoter regions, was then inserted between the SacI and HindIII restriction sites into the pGL2 plasmid (Promega, Charbonnières, France) so that the expression of the firefly luciferase reporter gene was governed by the core promoter. Plasmids pGL2-EN2/CP-Em, pGL2-EN2/CP-Cm, and pGL2-EN2/CP-EmCm were created by PCR-based site-directed mutagenesis as previously described (50). The Em mutation converted the HBV sequence located between nt coordinates 1682 and 1694 from 5'-ATGTCAACGACCG-3' to 5'-AtaTCgACcACt-3' (mutations are indicated in lowercase letters). The Cm mutation converted the sequence located between nt coordinates 1764 and 1776 from 5'-AGGTCTTTGTACT-3' to 5'-cGcTaTTTaTACT-3'. All constructs were validated by sequencing (Cogenics Genome Express, Grenoble, France). The HBVx1.3 construct, which contains an overlength HBV genome of 4,195 bp ligated into plasmid pGEM-3Z, was a gift from Gilad Doitsh (Weizmann Institute of Science, Rehovot, Israel) and has previously been described (6). pSG5-FXR{alpha} and pSG5-RXR{alpha}, expression vectors for FXR{alpha} and RXR{alpha}, were gifts from Bart Staels (INSERM U545, Lille, France).

Cells and transfections. The human hepatoma cell line Huh7 was grown at 37°C with 5% CO2 in Dulbecco's modified Eagle's medium containing 100 U/ml of penicillin and 100 µg/ml of streptomycin, supplemented with 10% fetal bovine serum. Transfections for luciferase assays were performed with 24-well plates containing 1 x 105 cells per well with Lipofectamine 2000 (Invitrogen, Cergy-Pontoise, France) according to the manufacturer's recommendations. The transfecting DNA mixture in each well comprised 200 ng of pGL2-EN2/CP-wt plasmid or its mutant derivatives, with 40 ng of pRL-SV40 plasmid serving as an internal control for transfection efficiency. pRL-SV40 (Promega) directs the expression of the Renilla luciferase gene under the control of the simian virus 40 (SV40) early enhancer/promoter. When appropriate, the DNA mixture included 200 ng of pSG5-FXR{alpha} and 100 ng of pSG5-RXR{alpha}. CDCA was used to activate FXR{alpha}. GGS, a natural FXR{alpha} antagonist, was used at 10 µM. At 3 days posttransfection, cells were lysed and assayed for luciferase activity by using a dual-luciferase reporter assay system (Promega). Values shown are averages of results for triplicate cultures.

Transfections for viral RNA and DNA analysis were performed in 10-cm plates containing 106 cells per well. Each well was transfected with 8 µg of the HBVx1.3 construct, with 200 ng of the pRL-SV40 plasmid as an internal control for transfection efficiency. In the cotransfection experiments, 2 µg of pSG5-FXR{alpha} and 1 µg of pSG5-RXR{alpha} were included. To keep the total DNA amount constant, the appropriate amount of pSG5 expression vector lacking the cDNA insert was added. CDCA and RA at 50 µM and 1 µM, respectively, were used to activate the NR FXR{alpha} and RXR{alpha}. GGS was used at 10 µM.

EMSA. Electrophoretic mobility shift assays (EMSAs) were performed as described previously (18), with some modifications. Plasmids pSG5-FXR{alpha} and pSG5-RXR{alpha} were simultaneously in vitro transcribed and translated using a rabbit reticulocyte lysate according to the manufacturer's instructions (Promega). Synthetic oligonucleotides (Invitrogen) were annealed before being end labeled with [{gamma}32P]dATP (Perkin-Elmer, Courtaboeuf, France) or used as unlabeled competitors. The oligonucleotide pairs used to generate the FXR-CONS, FXR-EN2, and FXR-Cp double-stranded probes were 5'-GATCTCAAGAGGTCATTGACCTTTTTG-3' and 5'-GATCCAAAAAGGTCAATGACCTCTTGA-3' (FXR-CONS, which contains a consensus FXRE), 5'-GATCTCTGCAATGTCAACGACCGACCTTGA-3' and 5'-GATCTCAAGGTCGGTCGTTGACATTCAGA-3' (FXR-EN2, HBV coordinates 1682 to 1694), and 5'-GATCGATTAGATTAAAGGTCTTTGTACTAGGA-3' and 5'-GATCTCCTAGACAAAGACCTTTAATCTAATC-3' (FXR-Cp, HBV coordinates 1764 to 1776). 32P-labeled double-stranded oligonucleotides (1 ng) were incubated at room temperature for 20 min with 2 µl of reticulocytes lysates containing or not containing the translated protein factors, 5 µl of binding buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 15% glycerol), 2 µg of poly(dI-dC)-poly(dI-dC), and water to obtain a final volume of 25 µl. The resulting complexes were then separated on a native 4% polyacrylamide gel in 0.5x Tris-glycine-EDTA at 50 V. After electrophoresis, the EMSA complex was visualized by autoradiography. When competition analysis was performed, 200 ng of a cold double-stranded competitor oligonucleotide or 1 µl of antibody was added to the reticulocyte lysate 15 min prior to the addition of the 32P-labeled double-stranded oligonucleotide. FXR{alpha} and RXR{alpha} antibodies (SC-13063 and SC-553, respectively) were purchased from Santa Cruz Biotechnology. The irrelevant antibody used was mouse anti-hepatitis C virus core antigen antibody C-830. The oligonucleotide pair used to generate the double-stranded irrelevant DNA probe was 5'-GATCCTTCTGGGAATTCCTAGATC-3' and 5'-GATCTAGGAATTCCCAGAAGGATC-3'.

Analysis of viral RNA and DNA. At 3 days posttransfection, cells were divided equally and harvested for viral RNA and DNA analysis. Total cellular RNA was isolated using an RNeasy kit (Qiagen) and quantified by determining the absorbance at 260 nm. Northern and Southern blot analyses were performed as described previously (32). For Northern blot analysis, 2 µg of RNA per sample was separated on a 1% agarose-formaldehyde gel and blotted to a nylon membrane (Roche). For Southern blot analysis, viral DNA replication intermediates were isolated as previously described (40), subjected to electrophoresis on a 1% agarose-Tris-borate-EDTA gel, and transferred to a nylon membrane. The membranes were then hybridized with a radiolabeled HBV probe. For viral RNA analysis, the blot was then stripped and reprobed with a Renilla luciferase probe. The HBV radioactive probe was prepared by using the DNA of the X and core gene regions. For the Renilla luciferase DNA probe, a 1-kb fragment of plasmid pRL-SV40 was used. Probes were labeled using a random-prime labeling system (Promega) with [{alpha}-32P]dATP (3,000 Ci/mmol; Perkin Elmer). About 106 cpm labeled probes were used per 1 ml of hybridization buffer. Densitometry was performed with a PhosphorImager.

Primer extension analysis was performed with 5 µg of total cellular RNA per sample by using a primer extension system (Promega) according to the manufacturer's recommendations. The primer used to determine the relative amounts of the precore and pregenomic RNAs was 5'-GAGAGTAACTCCACAGTAT-3' (46). The primer used to analyze the Renilla luciferase transcript was 5-GAAGAGGCCGCGTTACCATG-3'.


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RESULTS
 
Identification of FXR{alpha} binding sites on the core promoter and enhancer II regions. The consensus FXRE was described as an IR-1 sequence, 5'-AGGTCANTGACCT-3' (17). We observed that the HBV EN2 and core promoter regions contained two sequence elements with high homology to this consensus FXRE, corresponding to HBV nt coordinates 1682 to 1694 (in the EN2 region) and HBV nt coordinates 1764 to 1776 (in the core promoter). It is noteworthy that the sequence element identified in the core promoter overlaps NRREpre-C, a binding site for the NR PPAR{alpha}-RXR{alpha}, HNF4{alpha}, and chicken ovalbumin upstream promoter-transcription factor 1 (COUP-TF1) (Fig. 1).


Figure 1
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  		FIG. 1. Nucleotide sequences of the HBV enhancer II and core promoter regions. The nucleotide coordinates are derived from the GenBank database (HBV genome, subtype ayw; GenBank accession number U95551). The underlined sequences correspond to the previously described response elements NRREenhII (a binding site for HNF4{alpha}) and NRREpre-C (a binding site for PPAR{alpha}/RXR{alpha}, HNF4{alpha}, COUP-TF1, and the testicular receptor TR4) and two binding sites for the HBV enhancer II B1 binding factor hB1F. The arrows indicate the initiation sites of synthesis of the precore and pregenomic RNAs. The two putative FXREs are represented by boxes. For each sequence, the homology with the consensus FXRE is indicated.

EMSAs were performed with two radiolabeled double-stranded oligonucleotides, FXR-EN2 and FXR-Cp, corresponding to the putative FXREs identified in the EN2 and the core promoter regions, respectively. The binding of FXR{alpha} and RXR{alpha} recombinant polypeptides to FXR-EN2 and FXR-Cp sequences was compared to that observed for a perfect inverted repeat sequence (FXR-CONS). The RXR{alpha} polypeptide did not bind the FXR-CONS sequence, whereas two complexes were formed with the FXR{alpha} polypeptide, representing FXR{alpha} binding either as a monomer or as homodimers (Fig. 2A). In the presence of both polypeptides, a unique band was observed, representing binding of FXR{alpha}-RXR{alpha} heterodimers to the FXR-CONS sequence, as expected (4). With FXR{alpha} or RXR{alpha} polypeptides alone, a complex was formed with either the FXR-EN2 or the FXR-Cp sequence (Fig. 2B and C). This complex, which was still observed in the presence of both NR, probably represented binding of FXR{alpha} and RXR{alpha} monomers to FXR-EN2 and FXR-Cp sequences. Similar binding of NR monomers to specific sequences of the HBV genome has previously been described, in particular binding of PPAR{alpha} monomers to NRREpre-C in the presence or absence of RXR{alpha} (30). In the presence of both FXR{alpha} and RXR{alpha} polypeptides, a slower-migrating complex was observed. This complex was not observed when competition analyses were performed with either the double-stranded oligonucleotide probe containing the consensus FXRE (FXR-CONS) or antibodies directed against the DNA-binding site of FXR{alpha} or RXR{alpha}. These results indicate that the slower-migrating complex represents binding of FXR{alpha}-RXR{alpha} heterodimers to FXR-EN2 and FXR-Cp sequences. The affinity of these heterodimeric complexes seems to be higher for the FXR-EN2 sequence than for the FXR-Cp sequence.


Figure 2
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  		FIG. 2. Identification of two FXREs in the HBV enhancer II and core promoter regions. EMSAs were performed with in vitro-expressed FXR{alpha} and RXR{alpha} polypeptides. Binding of the recombinant NR was analyzed with 32P-labeled double-stranded oligonucleotides FXR-CONS, which contains a consensus FXRE (A); FXR-EN2, which contains the putative FXRE located in the EN2 region of HBV (B); and FXR-Cp, which contains the putative FXRE located in the HBV core promoter (C). The specificity of the binding to the probes was verified using unlabeled double-stranded oligonucleotides as competitors or antibodies directed against FXR{alpha} and RXR{alpha}. The arrows indicate the positions of the DNA-protein complexes.

FXR{alpha} activates transcription from the HBV core promoter. To examine the functional properties of the FXR{alpha} binding sites identified in the core promoter and the EN2 region, we performed cotransfections of an expression vector for FXR{alpha} together with the plasmid pGL2-EN2/PC-wt, a luciferase reporter construct containing HBV nt 1400 to 1902, in the hepatoma cell line Huh-7 (Fig. 3A). CDCA, a potent FXR{alpha} activator bile acid, was added at concentrations ranging from 10 µM (which corresponds to normal seric concentrations) to 100 µM (which is observed in cholestatic hepatitis) (8). We first verified that FXR{alpha} and bile acids did not influence transcription of the Renilla luciferase gene from the plasmid pRL-SV40, which was used to normalize the results for transfection efficiency (data not shown). Expression of FXR{alpha} alone did not significantly modify core promoter activity (Fig. 3B). Addition of 50 µM CDCA in the absence of exogenous FXR{alpha} slightly increased the level of transcription of the core promoter, which likely reflects the endogenous expression of FXR{alpha} in Huh-7 cells (data not shown). Expression of FXR{alpha} in the presence of various concentrations of CDCA increased the level of transcription from the core promoter from three- to fivefold, depending on the concentration of CDCA. These results indicate that FXR{alpha} activates transcription from the core promoter in a ligand-dependent manner.


Figure 3
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  		FIG. 3. FXR{alpha} activates the transcription from the HBV core promoter in Huh-7 cells. (A) Schematic representation of the firefly luciferase reporter plasmid pGL2-EN2/PC-wt. The fragment of the HBV genome comprised between nt 1400 and 1902, which contains the enhancer II and core promoter regions, was cloned between the SacI and HindIII restriction sites of pGL2. Huh-7 cells were cotransfected with pGL2-EN2/PC-wt; pRL-SV40, which contains a Renilla luciferase gene; and expression vectors of FXR{alpha} alone (B) or FXR{alpha} and RXR{alpha} (C). CDCA and GGS, the FXR{alpha} ligands, were added at the indicated concentrations. At 3 days posttransfection, cells were harvested to determine firefly luciferase activity. The results were normalized for transfection efficiency with Renilla luciferase activity and expressed as differences (n-fold) from the control level. The standard deviations of the means are indicated by error bars. Asterisks indicate statistically significant increases in relative activity, as determined by Student's t test (P < 0.05).

We next examined the effect of GGS on core promoter activity. GGS is a natural FXR{alpha} antagonist extracted from the plant Commiphora mukul. GGS inhibits FXR{alpha} activation by bile acids at the micromolar range (42). However, in our experiments, a concentration of 10 µM GGS did not inhibit the CDCA-induced core promoter activation with or without overexpression of FXR{alpha}.

FXR{alpha} binds preferentially to its response elements as a heterodimer with RXR{alpha} (16). Thus, we examined the influence of both FXR{alpha} and RXR{alpha} on core promoter activity. Expression of RXR{alpha} alone slightly increased transcription from the core promoter, whereas a threefold increase was observed with exogenous expression of FXR{alpha} and RXR{alpha} in the absence of CDCA (Fig. 3C). Maximal induction of the transcriptional activity of the core promoter was observed when both NR were expressed in the presence of bile acids. A >10-fold increase of core promoter activity was measured with 50 µM CDCA in the presence of FXR{alpha} and RXR{alpha}. Therefore, it appears that FXR{alpha} and RXR{alpha} synergistically activate the core promoter.

FXR{alpha} transactivates the HBV core promoter through both FXR{alpha} binding site 1 and FXR{alpha} binding site 2. To determine whether FXR{alpha} activates the core promoter by directly interacting with the HBV genome, we performed mutational analysis of the two FXREs identified in the EN2 and the core promoter regions. Several point mutations were introduced into FXR{alpha} binding site 1, located in the EN2 region (plasmid pGL2-EN2/PC-Em); FXR{alpha} binding site 2, located in the core promoter (plasmid pGL2-EN2/PC-Cm); or both sites (plasmid pGL2-EN2/PC-EmCm) (Fig. 4A). We next examined the effects of these mutations on the FXR{alpha}-mediated activation of the core promoter in transient-transfection experiments with Huh-7 cells. When FXR{alpha} binding site 2 was mutated, the basal activity of the core promoter was severely decreased (Fig. 4B). This result was not surprising, as this sequence overlaps NRREpre-C, an essential determinant of core promoter activity. When FXR{alpha} binding site 1 was mutated, transcription from the core promoter was also significantly impaired, suggesting that this sequence is also essential for the proper functioning of the core promoter. However, in each case, FXR{alpha} was still able to enhance transcription from the core promoter, but with a much lesser efficacy than the wild-type sequence. Thus, overexpression of FXR{alpha} plus CDCA led to a 3-fold increase in the presence of a mutant of FXR{alpha} binding site 1 and a 4.2-fold increase in the presence of a mutant of FXR{alpha} binding site 2, compared to a >10-fold increase observed with the wild-type sequence. When both site 1 and site 2 were mutated, the FXR{alpha}-mediated activation of the core promoter was nearly completely abolished. Together, these results indicate that FXR{alpha} transactivates the HBV core promoter through both FXR{alpha} binding site 1 and FXR{alpha} binding site 2, located in the EN2 and the core promoter regions.


Figure 4
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  		FIG. 4. Mutational analysis of the FXREs located in the EN2 and core promoter regions of HBV. (A) Partial sequence of the construct pGL2-EN2/PC-wt showing the wild-type EN2 and core promoter regions of HBV from nt 1678 to nt 1782. FXR{alpha} binding sites are underlined. For each mutant construct (pGL2-EN2/PC-Em, Cm, and EmCm), mutations introduced into FXR{alpha} binding sites 1 and 2 are indicated in lowercase letters, whereas unchanged nucleotides are indicated in capital letters. (B) Relative activities of the wild-type and mutant constructs in Huh-7 cells, with or without exogenously expressed FXR{alpha} in the presence or absence of CDCA. Transfection was performed as described in the legend to Fig. 3. CDCA was added at 100 µM. The transcriptional activities are reported relative to the activity of the pGL2-EN2/PC-wt construct in the absence of exogenously expressed FXR{alpha} and CDCA. The results were normalized for transfection efficiency with Renilla luciferase activity. The standard deviations of the means are indicated in parentheses.

Effects of FXR{alpha} and RXR{alpha} on viral RNA synthesis. Transcription of the 3.5-kb HBV RNA from the core promoter is modulated by the EN2 region. However, in the context of a whole HBV genome, the EN2 region is not autonomous but dependent on an active EN1 region (6). Moreover, it has previously been shown that transcription factor HNF3 increased transcription from the core promoter by using reporter gene constructs (15, 20) but inhibited pregenomic RNA synthesis in the context of a greater-than-genome-length HBV construct (39, 40). Thus, we next determined if the FXR{alpha}-mediated activation of the core promoter that we observed with a luciferase reporter construct lacking the EN1 region correlated with an increase of the viral 3.5-kb RNA synthesis. The influence of FXR{alpha} and RXR{alpha} on viral RNA synthesis was evaluated by transient transfection of Huh-7 cells with a 1.3xHBV DNA construct encoding all viral transcripts (6). Exogenous expression of FXR{alpha} alone did not influence the synthesis of the 3.5-kb HBV RNA (Fig. 5), but a twofold increase was observed in the presence of 50 µM CDCA. The same twofold increase was also observed when the respective ligands of FXR{alpha} and RXR{alpha}, i.e., CDCA and RA, were added without exogenous expression of the NR. As RXR{alpha} and FXR{alpha} are expressed in Huh-7 cells (data not shown), activation of endogenous RXR{alpha} and FXR{alpha} may explain this result. Maximal induction of the 3.5-kb transcript synthesis was observed with exogenous expression of both FXR{alpha} and RXR{alpha} in the presence of CDCA and RA, which induced a nearly threefold increase in the 3.5-kb RNA synthesis. GGS did not significantly modify synthesis of the 3.5-kb RNA.


Figure 5
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  		FIG. 5. FXR{alpha} enhances synthesis of the HBV 3.5-kb RNA. Huh-7 cells were transiently transfected with a 4.2-kb HBV DNA construct together with FXR{alpha} and RXR{alpha} expression vectors. CDCA, GGS, and RA, the FXR{alpha} and RXR{alpha} ligands, were added at the indicated concentrations. Cells were harvested at 3 days posttransfection for total RNA analysis. (A) Northern blot analysis of HBV transcripts was performed with an HBV probe corresponding to the X and core regions. Arrows indicate the locations of the 3.5-, 2.1-, and 0.7-kb transcripts. Hybridization of the Renilla-LUC transcript was used as an internal control for transfection efficiency. (B) Quantitative analysis of the 3.5-kb, 2.1-kb, and 0.7-kb HBV RNAs determined with a PhosphorImager. The mean RNA levels plus standard deviations from two independent experiments are represented. Results for densitometry are expressed relative to the amounts of the RNAs under control conditions (lane 1).

Two other HBV transcripts were also visualized. Synthesis of the 0.7- and 2.1-kb RNAs is regulated by the X promoter and the pre-S2/S promoter, respectively. FXR{alpha} did not modulate synthesis of the 0.7-kb RNA, whereas a moderate increase in the transcription of the 2.1-kb RNA was observed with overexpression of both FXR{alpha} and RXR{alpha} and addition of their ligands. The minor 2.4-kb viral transcript, whose synthesis is regulated by the pre-S1 promoter, was not visualized in our experiments.

FXR{alpha} increases synthesis of the pregenomic RNA and viral DNA. It has previously been demonstrated that transcription of the precore and pregenomic RNAs can be differentially regulated by NR and that the HBV core promoter may actually correspond to two distinct overlapping promoters (41, 43-46). The precore and the pregenomic RNAs exert opposite effects on viral replication. The pregenomic RNA serves as the template for viral DNA synthesis, whereas the precore RNA encodes the HBe antigen, which downregulates the viral replication. Thus, we next analyzed by primer extension assays whether FXR{alpha} modified the relative amounts of these transcripts. Synthesis of the pregenomic RNA was increased 2.6-fold when FXR{alpha} was exogenously expressed in the presence of CDCA and 3.6-fold when RXR{alpha} was also overexpressed (Fig. 6A and C). Synthesis of the precore RNA was enhanced to a lesser extent by FXR{alpha}, with a 1.7-fold increase in the presence of FXR{alpha} plus CDCA and a 2.4-fold increase with both FXR{alpha} and RXR{alpha} overexpressed in the presence of their ligands.


Figure 6
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  		FIG. 6. FXR{alpha} enhances synthesis of the pregenomic RNA and viral DNA. Huh-7 cells were transfected as described in the legend to Fig. 5. At 3 days posttransfection, cells were divided equally for extraction of total cellular RNA or viral replication intermediates. For each condition, 5 µg of total RNA was used for primer extension analysis of the relative amounts of precore and pregenomic RNAs (A). Detection of the Renilla luciferase transcript served as an internal control for transfection efficiency. 32P-labeled DNA fragments were used as size markers. The lengths of base pairs for each fragment in the marker lane are indicated next to the corresponding band. The expected sizes for the primer extension products were 175 bp for the precore RNA and 135 bp for the pregenomic RNA. For Southern blot analysis of viral replication intermediates (B), hybridization was performed with the same HBV DNA probe used in the Northern blot. The positions of the viral DNAs are indicated by arrows. A duplex linear HBV DNA (DL DNA) was used as a control and size marker. rcDNA, relaxed circular DNA; ssDNA, single-strand DNA. Quantitative analyses of the precore and pregenomic RNAs (C) and viral DNA (D) were performed with a PhosphorImager. Results for densitometry are expressed relative to the levels for the control conditions (lane 1).

To examine the global effect of FXR{alpha} on viral DNA synthesis, Southern blot analysis of HBV DNA accumulated in the cytoplasm of transfected cells was performed. Exogenous expression of FXR{alpha} in the presence of CDCA resulted in a nearly fourfold increase in viral DNA synthesis (Fig. 6B and D). This result correlated with the FXR{alpha}-induced modification of the ratio between the pregenomic and precore transcripts. Thus, we conclude that FXR{alpha} can significantly enhance the level of HBV replication, likely through a preferential activation of the pregenomic RNA synthesis.


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DISCUSSION
 
In this study, we identified two putative FXREs in the HBV genome with homology to the typical inverted repeat sequence recognized by FXR{alpha}. The first FXRE is located in the EN2 region, between HBV nt 1682 and 1694, and is recognized by FXR{alpha}-RXR{alpha} heterodimers. To our knowledge, this sequence has not previously been described as a response element for other NR. Analysis of different HBV genomes in the GenBank database reveals that this sequence is well conserved among genotypes A, B, C, D, E, and G. Interestingly, the nucleotide sequences are somewhat different in genotypes F (5'-CGGTCAATGACCT-3') and H (5'-CGGTCAACGACCT-3') and present even higher homologies to the consensus FXRE. Differences in transcriptional regulation by COUP-TF1 have recently been reported to occur among HBV genotypes (9). Similarly, the influence of FXR{alpha} on different HBV strains could be further explored. The second FXRE is located in the core promoter, between nt 1764 and 1776, and can also bind FXR{alpha}-RXR{alpha} heterodimers. Interpretation concerning this second putative FXRE is rendered difficult as this sequence overlaps the well-characterized response element NRREpre-C, a direct repeat (DR-1) sequence recognized by PPAR{alpha}/RXR{alpha} and HNF4{alpha}, whose role in the modulation of core promoter activity is critical. Though FXR{alpha} interacts preferentially with IR-1 sequences, the capacity of this NR to bind to DR-1 sequences has also been demonstrated, although with a weaker affinity (17). This suggests that a competition could exist between PPAR{alpha}, RXR{alpha}, HNF4{alpha}, and FXR{alpha} for binding to this cis-acting region of the HBV genome.

The functional significance of these two FXREs was investigated by transient transfection in the hepatoma cell line Huh-7, demonstrating that bile acids, through FXR{alpha}, activate the HBV core promoter. This effect was modulated by exogenous expression of RXR{alpha}, the FXR{alpha} preferential transactivation partner. Mutational analysis of FXR{alpha} binding sites 1 and 2 confirmed that FXR{alpha}-induced activation of the core promoter was mediated through both FXREs. Whereas EMSA experiments suggested that the affinity of FXR{alpha}-RXR{alpha} heterodimers was higher for the FXRE located in the EN2 region, results for the functional analysis did not reveal a significantly predominant role for either site in FXR{alpha}-induced activation of the core promoter. We cannot exclude that other FXR{alpha} binding sites exist in the HBV genome. However, results for the mutational analysis suggest that the two FXREs identified in this study are the principal cis-acting sequences responsible for the FXR{alpha}-induced activation of the HBV core promoter.

A recent study showed that the small heterodimer partner (SHP) whose expression is under the control of FXR{alpha} could inhibit HBV replication in the HepG2 cell line, likely through a direct interaction with HNF4{alpha} (27). However, using a greater-than-genome-length construct, we showed that the global effect of FXR{alpha} activation by bile acids, despite the induction of SHP expression in Huh-7 cells (data not shown), is an increased transcription of the HBV pregenomic RNA and, consequently, an increase in the level of viral replication. This suggests that the effect of FXR{alpha} on HBV transcription primarily depends on its direct binding to the FXREs and that the FXR{alpha}-induced expression of SHP is not sufficient to counterbalance the global activation of the core promoter. Northern blot analysis also revealed that FXR{alpha} does not influence the synthesis of the 0.7-kb RNA but slightly increases the synthesis of the 2.1-kb RNA. These results are in agreement with previous work demonstrating that transcription from the X promoter is essentially under the control of EN1 (6) whereas transcription from the pre-S2/S promoter is also modulated by the EN2 region (47).

Following the demonstration of the essential role of NR in the HBV life cycle, NR agonists and antagonists were envisaged as possible new therapeutic targets against HBV. Bile acids were recently shown to enhance genotype 1 hepatitis C virus replication in a subgenomic system, and GGS, an FXR{alpha} antagonist, significantly inhibited this effect (33). However, in our study, GGS showed little or no effect in inhibiting core promoter activity or viral RNA synthesis. This observation may be explained by the fact that GGS, after being described as a pure FXR{alpha} antagonist, is now envisioned as a gene-selective FXR{alpha} modulator. For instance, GGS has been shown to enhance the expression of the bile salt export pump, one of the target genes for FXR{alpha} (5). As its antagonist effect seems to be dependent, at least partly, on the nucleotide sequences of the FXREs, it will be interesting to examine the influence of GGS on different HBV genotypes or test other synthetic FXR{alpha} modulators.

Several studies have described in detail the essential role of NR PPAR{alpha}-RXR{alpha} and HNF4{alpha} in the HBV life cycle (30, 40, 43-45). The relative influence of FXR{alpha} compared to that of these NR remains to be clarified, but our results strongly suggest that FXR{alpha} may also contribute to the modulation of the HBV core promoter activity in vivo and subsequently to the level of viral replication. Interestingly, in HBV transgenic mice, synthesis of the 3.5-kb RNA greatly predominates in hepatocytes and proximal convoluted renal tubules (12). The liver and kidney express not only NR PPAR{alpha} and HNF4{alpha} but also FXR{alpha} (38). Thus, FXR{alpha} could be another factor implicated in the HBV-restricted tropism at the transcriptional level.

Shlomai and Shaul showed recently that peroxisome proliferator-activated receptor {gamma} coactivator 1{alpha} (PGC-1{alpha}), a coactivator of several transcription factors controlling energy and nutrient homeostasis, coactivates HBV replication after short-term fasting in mice (36). Moreover, this process was at least partly dependent on HNF4{alpha}. Subsequently, they proposed the concept of "metabolovirus" to describe the regulation of HBV replication (37). In the metabolovirus model, the regulation of HBV transcription is modulated by nutritional signals and is similar to what was found for hepatic metabolic genes, such as the PEPCK and G6Pase genes, both implicated in gluconeogenesis. Upon starvation, PGC-1{alpha} is induced and coactivates HNF4{alpha}, resulting in the upregulation of HBV replication. Interestingly, PGC-1{alpha} is also known to coactivate FXR{alpha} and stimulate the expression of FXR{alpha} target genes. During the response to short-term fasting, whereas PPAR{alpha} and HNF4{alpha} stimulate the beta-oxidation and gluconeogenesis pathways, respectively (21), FXR{alpha} is supposed to participate in this metabolic response by decreasing triglyceride production and secretion (49) and also by modulating gluconeogenesis (2). Based on the metabolovirus model, we suggest that bile acids, through FXR{alpha}, are implicated in the metabolic regulation of HBV transcription.

To our knowledge, the only work that examined in vivo the relations between bile acids and viral hepatitis focused on ursodeoxycholic acid (3), which is a very weak FXR{alpha} activator. In our experiments, bile acids increased HBV core promoter activity in a dose-dependent manner. A modest but significant effect was measured with 10 µM CDCA, which corresponds to normal seric levels. Concentrations leading to the maximal induction, i.e., 50 µM and 100 µM CDCA, are observed only in cholestatic hepatitis and cirrhosis (8). Possible correlations in vivo between bile acid concentrations and the natural history of HBV infection, in terms of risk of chronicity, severity of evolution, occurrence of exacerbation episodes, and prognosis, should be further examined to evaluate the potential benefit of therapeutics such as FXR{alpha} modulators or bile acid sequestrants.


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ACKNOWLEDGMENTS
 
We are grateful to Gilad Doitsh and Yosef Shaul (Weizmann Institute of Science, Rehovot, Israel) for the 1.3xHBV DNA construct and to Bart Staels (U545 INSERM, Lille, France) for expression vectors for FXR{alpha} and RXR{alpha}.

This work was supported by a grant from Agence Nationale de Recherche sur le Sida et les Hépatites (ANRS).


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FOOTNOTES
 
* Corresponding author. Mailing address: INSERM U851, 21 avenue Tony Garnier, Lyon F-69007, France. Phone: 33 4 37 28 23 27. Fax: 33 4 37 28 23 41. E-mail: patrice.andre{at}inserm.fr Back

{triangledown} Published ahead of print on 3 September 2008. Back


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Journal of Virology, November 2008, p. 10832-10840, Vol. 82, No. 21
0022-538X/08/$08.00+0     doi:10.1128/JVI.00883-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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