Limited Effects of Bile Acids and Small Heterodimer Partner onHepatitis B Virus Biosynthesis In Vivo

Vanessa C. Reese,a David D. Moore,b and Alan McLachlana

Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois, USA,a and Department of Molecular and CellularBiology, Baylor College of Medicine, Houston, Texas, USAb

Multiple nuclear receptors, including hepatocyte nuclear factor 4� (HNF4�), retinoid X receptor � (RXR�) plus peroxisomeproliferator-activated receptor � (PPAR�), RXR� plus farnesoid X receptor � (FXR�), liver receptor homolog 1 (LRH1), andestrogen-related receptors (ERRs), have been shown to support efficient viral biosynthesis in nonhepatoma cells in the absenceof additional liver-enriched transcription factors. Although HNF4� has been shown to be critical for the developmental expres-sion of hepatitis B virus (HBV) biosynthesis in the liver, the relative importance of the various nuclear receptors capable of sup-porting viral transcription and replication in the adult in vivo has not been clearly established. To investigate the role of thenuclear receptor FXR and the corepressor small heterodimer partner (SHP) in viral biosynthesis in vivo, SHP-expressing andSHP-null HBV transgenic mice were fed a bile acid-supplemented diet. The increased FXR activity and SHP expression levelsresulting from bile acid treatment did not greatly modulate HBV RNA and DNA synthesis. Therefore, FXR and SHP appear toplay a limited role in modulating HBV biosynthesis, suggesting that alternative nuclear receptors are more critical determinantsof viral transcription in the HBV transgenic mouse model of chronic viral infection. These observations suggest that hepatic bileacid levels or therapeutic agents targeting FXR may not greatly modulate viremia during natural infection.

Hepatitis B virus (HBV) biosynthesis is restricted primarily tothe liver (15). This tropism presumably reflects the limited

tissue distribution of the viral receptor, although the entry mech-anism and proteins involved have not been defined (12). How-ever, the tissue tropism of HBV is also restricted at the level oftranscription (15). Liver-enriched transcription factors, and nu-clear receptors in particular, have been shown to be essential forviral RNA synthesis (19, 44, 47, 52, 53, 61). As HBV replicates bythe reverse transcription (RT) of the pregenomic 3.5-kb RNA syn-thesized from the nucleocapsid or core promoter (58), it is appar-ent that transcription is a major regulatory step governing viralbiosynthesis (53). In the context of viral replication, the nuclearreceptors hepatocyte nuclear factor 4� (HNF4�), retinoid X re-ceptor � (RXR�) plus peroxisome proliferator-activated receptor� (PPAR�), RXR� plus farnesoid X receptor � (FXR�), liver re-ceptor homolog 1 (LRH1), and estrogen-related receptor (ERR)have been shown to be the only transcription factors capable of sup-porting pregenomic RNA synthesis and viral replication in nonhepa-toma cells in the absence of any additional complementing transcrip-tional regulatory machinery (44, 53). These observations suggest thatnuclear receptors have a unique capacity to support HBV transcrip-tion and replication. However, the relative importance of the variousnuclear receptors in governing HBV biosynthesis in vivo has not beenextensively investigated (14, 24). Additionally the coactivator perox-isome proliferator-activated receptor � coactivator 1� (PGC1�) andthe corepressor small heterodimer partner (SHP) differentially mod-ulate nuclear receptor activities and appear to represent importantregulators of HBV biosynthesis (34–36).

The HBV transgenic mouse model of chronic viral infectionhas been used to examine the in vivo role of PPAR� and HNF4� inHBV transcription and replication (14, 24). Under normal phys-iological conditions, PPAR� did not influence HBV biosynthesis,but the activation of PPAR� by synthetic ligands did lead to en-hanced viral biosynthesis (14). These observations demonstratedthat PPAR� can modulate the synthesis of HBV RNA and DNA

under conditions where PPAR� is activated by an appropriatesmall molecule (14, 42). In contrast to PPAR�, HNF4� was shownto be essential for the developmental expression of HBV tran-scripts in the liver and, hence, viral biosynthesis (24, 42). AlthoughHNF4� can support HBV biosynthesis in nonhepatoma cell linesand is essential for viral transcription and replication during liverdevelopment, it is unclear whether this nuclear receptor alonegoverns HBV production in vivo (24, 44, 53). The loss of HNF4�expression during development is associated with the reduced ex-pressions of at least two nuclear receptors, FXR� and LRH1, ca-pable of supporting HBV biosynthesis (20). Consequently, theeffects of the loss of HNF4� on viral RNA and DNA synthesisduring development may be direct or indirect through FXR�,LRH1, or additional transcription factors (20, 24).

In this study, the effect of bile acid treatment on HBV biosyn-thesis was investigated by using the HBV transgenic mouse modelof chronic viral infection (15). Bile acids are the natural ligands forthe nuclear receptor FXR, which regulates endogenous bile acidsynthesis in the liver, in part, through the transcriptional activa-tion of the SHP gene (Fig. 1) (13, 28, 30, 39). SHP is also a memberof the nuclear receptor family of transcription factors, but it lacksa DNA binding domain and generally suppresses gene expressionby binding to various transcription factors, including other nu-clear receptors (Fig. 1) (49). Indeed, SHP decreases the rate-limiting step in bile acid synthesis by inhibiting the liver X recep-tor (LXR)- and LRH1-mediated expression of the cytochromeP450 7A1 (CYP7A1) gene (Fig. 1) (13, 28). Additionally, SHP

Received 4 November 2011 Accepted 7 December 2011

Published ahead of print 14 December 2011

Address correspondence to Alan McLachlan, mclach@uic.edu.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JVI.06742-11

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inhibits its own expression in a negative-feedback loop aimed atmaintaining appropriate bile acid homeostasis within the liver(Fig. 1) (13, 28). Consequently, the effect of bile acid treatment onviral biosynthesis was investigated with SHP-expressing and SHP-null HBV transgenic mice to determine the relative importance ofFXR and SHP for HBV transcription and replication (56). In malemice, a very modest increase in the level of HBV transcription andreplication was observed, which was not apparent in female mice.These observations suggest that neither FXR nor SHP nuclear re-ceptors play a critically important role in the HBV life cycle. Al-though RXR� plus FXR� can support viral biosynthesis in non-hepatoma cells (44), it appears that HNF4� or additional nuclearreceptors are more important for HBV transcription and replica-tion in vivo (24). This suggests that therapeutic modalities limitedto modulating the activities of the nuclear receptors FXR and SHPmay influence HBV biosynthesis to only a limited extent and thatconditions associated with choleostatic liver disease may not di-rectly modulate chronic HBV infection in humans.

MATERIALS AND METHODSTransgenic mice. The production and characterization of the HBV trans-genic mouse lineage 1.3.32 were described previously (15). These HBVtransgenic mice contain a single copy of the terminally redundant, 1.3-genome-length copy of the HBVayw genome integrated into mouse chro-mosomal DNA. High levels of HBV replication occur in the livers of thesemice. The mice used for the breeding experiments were homozygous forthe HBV transgene and were maintained on the SV129 genetic back-ground (22).

The production and characterization of SHP-null mice were describedpreviously (55, 56). These mice do not express SHP, which contributes tobile acid and cholesterol homeostasis (55, 56). The mice used for thebreeding experiments were homozygous null for SHP and were main-tained on the C57B1/129SV hybrid genetic background (55, 56).

SHP-null (�/�) HBV transgenic mice were generated by mating theHBV transgenic mice with the SHP-null mice. The resulting SHP heterozy-gous (�/�) HBV transgenic F1 mice were subsequently mated with the SHP-null mice, and the F2 mice were screened for the HBV transgene and SHP-nullallele by PCR analysis of tail DNA. Tail DNA was prepared by incubating 1 cmof tail in 500 �l of a solution containing 100 mM Tris hydrochloride (pH 8.0),

200 mM NaCl, 5 mM EDTA, and 0.2% (wt/vol) SDS containing 100 �g/mlproteinase K for 16 to 20 h at 55°C. Samples were centrifuged at 14,000 rpm inan Eppendorf 5417C microcentrifuge for 10 min, and the supernatant wasprecipitated with 500 �l of isopropanol. DNA was pelleted by centrifugationat 14,000 rpm in an Eppendorf 5417C microcentrifuge for 10 min and sub-sequently dissolved in 100 �l of a solution containing 5 mM Tris hydrochlo-ride (pH 8.0) and 1 mM EDTA. The HBV transgene was identified by PCRanalysis using the oligonucleotides 5=-TCGATACCTGAACCTTTACCCCGTTGCCCG-3= (oligonucleotide XpHNF4-1; HBV positions 1133 to1159) and 5=-TCGAATTGCTGAGAGTCCAAGAGTCCTCTT-3= (oligo-nucleotide CpHNF4-2; HBV positions 1683 to 1658) and 1 �l of tail DNA.A PCR product of 551 bp indicated the presence of the HBV transgene.The SHP-expressing and -null alleles were identified by PCR analysisusing the oligonucleotides 5=-CTCTGCAGGTCGTCCGACTATTCTG-3= (Exon-1F) and 5=-CCTCGAAGGTCACAGCATCCTG-3= (Exon-1B), located in the deleted first exon of the SHP gene coding region, and5=-CTAGCTAGAGGATCCCCGGGTACC-3= (Gal-PCR 5=) and 5=-AATTCGCGTCTGGCCTTCCTGTAG-3= (Gal-PCR 3=), located in the �-ga-lactosidase (�-gal) cassette, respectively (56), and 1 �l of tail DNA. A PCRproduct of 296 bp indicated the SHP-expressing allele, whereas a PCRproduct of 500 bp indicated the SHP-null allele. The samples were sub-jected to 42 amplification cycles involving denaturation at 94°C for 1 min,annealing at 55°C for 1 min, and extension from the primers at 72°C for 2min. The 20-�l reaction mixtures used were made according to instruc-tions provided by the manufacturer (Genscript) and contained 2.5 unitsof Taq DNA polymerase.

HBV transgenic mice were fed normal rodent chow (control) or ro-dent chow containing 1% (wt/wt) cholic acid (CA) for 7 days (56). Waterwas available ad libitum. Mice were sacrificed, and liver tissue was frozenin liquid nitrogen and stored at �70°C prior to DNA and RNA extraction.

HBV DNA and RNA analysis. Total DNA and RNA were isolatedfrom livers of HBV transgenic mice as described previously (8, 46).DNA (Southern) filter hybridization analyses were performed by using20 �g of HindIII-digested DNA (46). Filters were probed with 32P-labeled HBVayw genomic DNA (10) to detect HBV sequences. RNA(Northern) filter hybridization analyses were performed by using 10�g of total cellular RNA as described previously (46). Filters wereprobed with 32P-labeled HBVayw genomic DNA to detect HBV se-quences and mouse glyceraldehyde-3-phosphate dehydrogenase(GAPDH) cDNA to detect the GAPDH transcript, which was used asan internal control (45).

Reverse transcription-quantitative PCR (RT-qPCR) was used to mea-sure the levels of SHP, FXR�, bile salt export pump (BSEP), CYP7A1,PGC1�, tumor necrosis factor alpha (TNF-�), and HBV 3.5-kb tran-scripts in mouse liver RNA. After DNase I treatment, 1 �g of RNA wasused for cDNA synthesis using TaqMan reverse transcription reagents(Applied Biosystems, Foster City, CA), followed by real-time PCR quan-tification using SYBR green and an Applied Biosystems 7300 real-timethermocycler (Applied Biosystems). Thermal cycling consisted of an ini-tial denaturation step for 10 min at 95°C followed by 40 cycles of dena-turation (15 s at 95°C) and annealing/extension (1 min at 60°C). Therelative SHP, FXR�, BSEP, CYP7A1, PGC1�, TNF-�, and HBV 3.5-kbRNA expression levels were estimated by using the ��CT method withnormalization to mouse GAPDH RNA (26). The PCR primers usedwere 5=-CTCTGCAGGTCGTCCGACTATTCTG-3= (mouse SHP senseprimer), 5=-CCTCGAAGGTCACAGCATCCTG-3= (mouse SHP anti-sense primer) (56), 5=-TCCGGACATTCAACCATCAC-3= (mouse FXR�sense primer), 5=-TCACTGCACATCCCAGATCTC-3= (mouse FXR�antisense primer) (33), 5=-AAGCTACATCTGCCTTAGACACAGAA-3=(mouse BSEP sense primer), 5=-CAATACAGGTCCGACCCTCTCT-3=(mouse BSEP antisense primer) (33), 5=-AGCAACTAAACAACCTGCCAGTACTA-3= (mouse CYP7A1 sense primer), 5=-GTCCGGATATTCAAGGATGCA-3= (mouse CYP7A1 antisense primer) (33), 5=-AACAATGAGCCTGCGAACAT-3= (mouse PGC1� sense primer), 5=-AAATGAGGGCAATCCGTCTT-3= (mouse PGC1� antisense primer) (23), 5=-CATCT

FIG 1 Components of the regulatory network governing bile acid synthesis inthe liver and their potential effects on nuclear receptor-mediated HBV biosyn-thesis. Bile acids are the ligands for FXR and increase its transcriptional activity(30, 39). FXR activates SHP gene expression, whereas SHP generally repressesnuclear receptor-mediated transcription, including the activities of HNF4,RXR/FXR, RXR/PPAR, LRH1, and ERR (4, 13, 28). FXR directly regulatesbile salt export pump (BSEP) gene expression (1). Cytochrome P450 7A1(CYP7A1) gene expression is regulated by multiple nuclear receptors and en-codes the enzyme mediating the rate-limiting step in bile acid biosynthesis (7,13, 28). Nuclear receptors activate the synthesis of HBV RNA and DNA repli-cation intermediates (RI) (35, 36, 44).

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TCTCAAAATTCGAGTGACAA-3= (mouse TNF-� sense primer), 5=-TGGGAGTAGACAAGGTACAACCC-3= (mouse TNF-� antisense primer)(38), 5=-GCCCCTATCCTATCAACACTTCCGG-3= (HBV 3.5-kb RNAsense primer; positions 2311 to 2335), 5=-TTCGTCTGCGAGGCGAGGGA-3= (HBV 3.5-kb RNA antisense primer; positions 2401 to 2382), 5=-TCTGGAAAGCTGTGGCGTG-3= (mouse GAPDH sense primer), and 5=-CCAGTGAGCTTCCCGTTCAG-3= (mouse GAPDH antisense primer)(23), respectively.

Serum HBV antigen and alanine aminotransaminase analysis.HBeAg analysis was performed by using 2 �l of mouse serum and an HBeenzyme-linked immunosorbent assay according to the manufacturer’sinstructions (Epitope Diagnostics). The level of antigen was determinedto be in the linear range of the assay. Alanine aminotransferase (ALT)activity was determined by using 10 �l of mouse serum according to themanufacturer’s instructions (Genzyme). The level of ALT was determinedin the linear range of the assay by using an ALT positive control (CaymanChemical).

Plasmid constructions. The steps for the cloning of the plasmid con-structs used in the transfection experiments were performed according tostandard techniques (46). HBV DNA sequences in these constructionswere derived from plasmid pCP10, which contains two copies of the HBVgenome (subtype ayw) cloned into the EcoRI site of pBR322 (9). The HBVDNA (4.1-kbp) construct that contains 1.3 copies of the HBV genomeincludes the viral sequence from nucleotides 1072 to 3182 plus 1 to 1990.This plasmid was constructed by cloning the NsiI/BglII HBV DNA frag-ment (nucleotides 1072 to 1990) into pUC13, generating pHBV(1072-1990). Subsequently, a complete copy of the 3.2-kbp viral genome linear-ized at the NcoI site (nucleotides 1375 to 3182 plus 1 to 1374) was clonedinto the unique NcoI site (HBV nucleotide position 1374) of pHBV(1072-1990), generating the HBV DNA (4.1-kbp) construct.

The pCMVHNF4�, pRS-hRXR�, and pCMV-rFXR� vectors expressHNF4�, RXR�, and FXR� polypeptides from rat HNF4�, human RXR�,and rat FXR� cDNAs, respectively, using the cytomegalovirus (CMV)immediate-early promoter (pCMV) and the Rous sarcoma virus longterminal repeat (LTR) (pRS) (6, 28, 31).

Cells and transfections. The human embryonic kidney 293T cell linewas grown in RPMI 1640 medium and 10% fetal bovine serum at 37°C in5% CO2 in air. Transfections for viral RNA and DNA analysis were per-formed as previously described (32), using 10-cm plates containing ap-proximately 1 � 106 cells. The isolation of DNA and RNA was performedat 3 days posttransfection. The transfected DNA mixture was composed of5 �g of HBV DNA (4.1 kbp) plus 1.5 �g of the nuclear receptor expressionvectors pCMVHNF4�, pRS-hRXR�, and pCMV-rFXR� (6, 28, 31). Con-trols were derived from cells transfected with HBV DNA and the expres-sion vectors lacking a nuclear receptor cDNA insert (42). Chenodeoxy-cholic acid (CDCA) at 100 �M was used to activate the nuclear receptorFXR� (53).

Characterization of HBV transcripts and viral replication interme-diates. Transfected cells from a single plate were divided equally and usedfor the preparation of total cellular RNA and viral DNA replication inter-mediates as described previously (50), with minor modifications. ForRNA isolation (8), the cells were lysed in a solution containing 1.8 ml of 25mM sodium citrate (pH 7.0), 4 M guanidinium isothiocyanate, 0.5% (vol/vol) sarcosyl, and 0.1 M 2-mercaptoethanol. After the addition of 0.18 mlof 2 M sodium acetate (pH 4.0), the lysate was extracted with 1.8 ml ofwater-saturated phenol plus 0.36 ml of chloroform-isoamyl alcohol (49:1). After centrifugation for 30 min at 3,000 rpm in a Sorvall RT6000centrifuge, the aqueous layer was precipitated with 1.8 ml of isopropanol.The precipitate was resuspended in a solution containing 0.3 ml of 25 mMsodium citrate (pH 7.0), 4 M guanidinium isothiocyanate, 0.5% (vol/vol)sarcosyl, and 0.1 M 2-mercaptoethanol and precipitated with 0.6 ml ofethanol. After centrifugation for 20 min at 14,000 rpm in an Eppendorf5417C microcentrifuge, the precipitate was resuspended in a solutioncontaining 0.3 ml of 10 mM Tris hydrochloride (pH 8.0), 5 mM EDTA,

and 0.1% (wt/vol) sodium lauryl sulfate and precipitated with 45 �l of 2 Msodium acetate plus 0.7 ml of ethanol.

For the isolation of viral DNA replication intermediates, the cells werelysed in 0.4 ml of 100 mM Tris hydrochloride (pH 8.0)– 0.2% (vol/vol)NP-40. The lysate was centrifuged for 1 min at 14,000 rpm in an Eppen-dorf 5417C microcentrifuge to pellet the nuclei. The supernatant wasadjusted to 6.75 mM magnesium acetate plus 200 �g/ml DNase I andincubated for 1 h at 37°C to remove the transfected plasmid DNA. Thesupernatant was readjusted to 100 mM NaCl, 10 mM EDTA, 0.8% (wt/vol) sodium lauryl sulfate, and 1.6 mg/ml pronase and incubated for anadditional 1 h at 37°C. The supernatant was extracted twice with phenol,precipitated with two volumes of ethanol, and resuspended in 100 �l of 10mM Tris hydrochloride (pH 8.0)–1 mM EDTA. RNA (Northern) andDNA (Southern) filter hybridization analyses were performed by using 10�g of total cellular RNA and 30 �l of viral DNA replication intermediates,respectively, as described previously (46). Filter hybridization analyseswere quantified by phosphorimaging using a Packard Cyclone StoragePhosphor system.

RESULTS

A number of nuclear receptors have been reported to regulateHBV transcription and replication in cell culture (Fig. 1) (11, 14,16–18, 25, 35, 36, 40, 42, 44,53, 62–64). However, only PPAR� andHNF4� have been examined for their role in HBV biosynthesis invivo (14, 24). PPAR� was shown previously to have little effect onHBV transcription and replication in the liver of adult HBV trans-genic mice unless activated by a synthetic ligand (14). In contrast,HNF4� was shown to be essential for the developmental expres-sion of HBV transcripts and, hence, HBV replication in vivo (24).RXR� plus FXR� were shown to enhance HBV transcription andreplication in the Huh7 human hepatoma cell line (40) and tosupport viral biosynthesis in nonhepatoma cells (35, 44). Thisfinding suggests that bile acids, the natural FXR� ligands (30, 39),may modulate HBV biosynthesis in vivo (Fig. 1). To examine thispossibility, the effects of bile acid feeding on viral biosynthesis inSHP-expressing and SHP-null HBV transgenic mice were exam-ined. As SHP expression is activated by FXR, and it is a negativeregulator of nuclear receptor-mediated transcription (13, 28), itcould potentially be an in vivo inhibitor of bile acid- and nuclearreceptor-mediated HBV transcription and replication (Fig. 1)(35–37).

Effect of bile acid feeding on FXR- and SHP-regulated geneexpression in SHP-expressing and SHP-null HBV transgenicmice. HBV transgenic mice were bred with SHP-null mice, andHBV transgenic mice hemizygous for the HBV transgene andheterozygous (�/�) for the wild-type SHP-expressing allele orhomozygous (�/�) for the SHP-null allele were identified in theF2 generation. In these studies, both SHP-expressing and SHP-null HBV transgenic mice were fed a diet supplemented with 1%(wt/wt) cholic acid for 7 days (56). Male and female mice of eachgenotype were assayed for levels of liver SHP, FXR�, BSEP,CYP7A1, and PGC1� RNAs after bile acid feeding and comparedwith animals fed a control diet (Fig. 2). As expected, miceheterozygous for the SHP-expressing allele displayed an approxi-mately 2-fold induction of SHP RNA after bile acid feeding (Fig.2A) (56). SHP-null HBV transgenic mice did not express SHPRNA (Fig. 2A). In both male and female HBV transgenic mice, theabundance of the FXR� RNA appeared to be very modestly in-creased due to the absence of SHP (Fig. 2B). Importantly, theinclusion of cholic acid in the diet was associated with a decrease inFXR� transcript levels, as previously noted (56). Additionally,

Reese et al.

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cholic acid appeared to induce the expression of the bile salt ex-port pump (BSEP) transcript in SHP-expressing HBV transgenicmice, and this induction was not apparent in the SHP-null HBVtransgenic mice (Fig. 2C) (56). Most notably, feeding mice a dietwhich included cholic acid resulted in a dramatic inhibition ofCYP7A1 expression that was relieved to a limited extent in theSHP-null mice (Fig. 2D) (56). These observations clearly indicatethat the cholic acid diet was effectively modulating FXR and SHPactivities in the liver in an attempt to maintain normal bile acidhomeostasis, as previously noted (Fig. 1) (28, 56). As PGC1� is acoactivator that can counteract the effect of SHP on HBV biosyn-thesis in cell culture (35, 36), and its expression was previouslyshown to be downregulated in vivo by bile acids (29), the effect ofthe feeding of cholic acid on this transcript in HBV transgenicmice was examined (Fig. 2E). Contrary to previously reportedobservations, cholic acid treatment did not display any consistenteffects on PGC1� RNA levels in these mice, suggesting that thiscoactivator probably does not play a role in modulating the level ofHBV biosynthesis in these particular physiological circumstances.The reason why PGC1� RNA levels were unaffected in the HBVtransgenic mice is unclear but may reflect differences in the ge-netic backgrounds of the mice used in the various studies or themethods used to measure the levels of the PGC1� transcript.

Effects of bile acid feeding on serum HBeAg, liver damage,and inflammation in HBV transgenic mice. HBV transgenicmice displayed a statistically significant increase in the level ofserum HBeAg of approximately 2-fold after bile acid feeding com-pared to the level of serum HBeAg immediately prior to the addi-tion of cholic acid to the control diet (Fig. 3A). All 22 individualmice examined demonstrated an increase in serum HBeAg levelsas a consequence of bile acid feeding, varying from a 10% to a300% elevation in the serum HBeAg level. The increase in serumHBeAg levels appeared to be more pronounced in the SHP-nullHBV transgenic mice than in the SHP-expressing HBV transgenicmice, although the difference in the levels of serum HBeAg in thebile acid-treated SHP-expressing and SHP-null HBV transgenicmice were statistically significant only for the male mice (Fig. 3A).As HBeAg is translated from the HBV 3.5-kb precore RNA (57),these observations suggest that the diet supplemented with cholicacid may have a modest effect on the synthesis of the HBV 3.5-kbprecore RNA in HBV transgenic mice.

As bile acid feeding can be associated with hepatic cytotoxicity(55), the levels of serum alanine aminotransaminase (ALT) wereexamined (Fig. 3B). Indeed, the mice fed bile acid displayedmarked increases in serum ALT levels, which appeared more pro-nounced in male SHP-null HBV transgenic mice. The peak serumALT level was observed between days 4 and 6, and levels declinedup to 2-fold by day 7, suggesting that there was a protective ho-meostatic adjustment to the increased bile acid intake occurring inthe liver (28, 56). The observed elevations in serum ALT levelsraised the possibility that the increases in serum HBeAg levelsobserved for the cholic acid-treated HBV transgenic mice mightbe due to the destruction of hepatocytes replicating the virus, al-though a histological examination of these livers showed that the

FIG 2 Analysis of SHP, FXR�, BSEP, CYP7A1, and PGC1� transcripts in thelivers of HBV transgenic mice. Mice were fed a control or a 1% (wt/wt) cholicacid (CA) diet for 7 days. Shown are data from a quantitative analysis of theSHP (A), FXR� (B), BSEP (C), CYP7A1 (D), and PGC1� (E) transcripts inHBV transgenic mice by RT-qPCR. The GAPDH transcript was used as aninternal control for the quantitation of the SHP, FXR�, BSEP, CYP7A1, andPGC1� RNAs. The mean relative SHP, FXR�, BSEP, CYP7A1, and PGC1�transcript levels plus standard deviations derived from male and female SHP-expressing (�/�) and SHP-null (�/�) HBV transgenic mice are shown. The

levels of transcripts in the cholic acid-fed HBV transgenic mice that are statis-tically significantly different from their levels in the corresponding SHP-expressing or SHP-null HBV transgenic mice (P � 0.05, determined by aStudent t test) are indicated with asterisks.

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livers appeared normal. Indeed, chemically induced liver damagein HBV transgenic mice is associated with increases in both serumALT and HBeAg levels (V. C. Reese and A. McLachlan, unpub-lished data). In addition, it was possible that hepatic cell deathmight be associated with increased inflammation, although leu-kocyte infiltrates were not observed in these livers. To examine thepossibility that hepatic Kupffer cells were becoming activated as aresult of hepatic damage, the levels of TNF-� RNA in the livers of

these mice were measured (Fig. 3C). No significant elevation inTNF-� gene expression levels was observed, indicating the ab-sence of any inflammatory response in the liver in response to thecholic acid diet.

Effect of bile acid feeding on viral transcription in HBVtransgenic mice. HBV transgenic mice that were heterozygous orhomozygous for the SHP-null allele were examined for theirsteady-state levels of HBV transcripts by analysis of the total liverRNA (Fig. 4). The steady-state levels of the HBV 3.5- and 2.1-kbtranscripts in the livers of the HBV transgenic mice with and with-out SHP were not greatly influenced by the cholic acid diet (Fig. 4).Measurements of the levels of the HBV 3.5-kb transcripts indi-cated that they were increased about 1.9-fold by the cholic aciddiet in male SHP-expressing HBV transgenic mice based on RNAfilter hybridization (Fig. 4B) and RT-qPCR (Fig. 4C) analyses.These observations are consistent with the suggestion that bileacids activate FXR, leading to increased transcription from theHBV nucleocapsid promoter (Fig. 1) (35, 40, 44). For the maleSHP-null HBV transgenic mice, the levels of the HBV 3.5-kb tran-scripts were 2.8- and 1.3-fold higher than those observed for theSHP-expressing male HBV transgenic mice based on RNA filterhybridization (Fig. 4B) and RT-qPCR (Fig. 4C) analyses. Theseobservations are consistent with the suggestion that under normalphysiological conditions, SHP can modestly suppress nuclearreceptor-mediated transcription from the HBV nucleocapsid pro-moter (Fig. 1) (35–37). Male SHP-null HBV transgenic mice fedthe cholic acid diet did not display a greater increase in the levels ofthe HBV 3.5-kb transcripts than the SHP-expressing HBV trans-genic mice fed the cholic acid diet or the SHP-null HBV transgenicmice fed the control diet, suggesting that the activation of FXR bybile acids in the absence of the nuclear receptor corepressor SHPdid not lead to an enhanced effect on HBV transcription, as mighthave been predicted (Fig. 1). For female HBV transgenic mice, itwas interesting that neither the cholic acid diet nor the absence ofSHP increased the level of the HBV 3.5-kb transcripts, suggestingthat this transcriptional response to bile acids may be sexuallydimorphic in nature in the liver (Fig. 4).

Effects of bile acid feeding on viral replication intermediatesin HBV transgenic mice. The alterations in the levels of replica-tion intermediates in the livers of SHP-expressing and SHP-nullHBV transgenic mice on the control or cholic acid diet closelyparalleled the observed changes in HBV 3.5-kb RNA abundances(Fig. 4 and 5). Measurements of the levels of the HBV replicationintermediates indicated that they were increased about 1.3-fold bythe cholic acid diet in male SHP-expressing HBV transgenic micebased on the DNA filter hybridization analysis (Fig. 5B). In themale SHP-null HBV transgenic mice, the levels of the HBV repli-cation intermediates were 2.3-fold higher than those observed forthe SHP-expressing male HBV transgenic mice based on the DNAfilter hybridization analysis (Fig. 5B). These observations are con-sistent with data from the transcriptional analysis (Fig. 4) andsuggest that cholic acid may activate and that SHP may inhibitHBV 3.5-kb RNA synthesis from the HBV nucleocapsid promoterto modulate HBV biosynthesis (Fig. 1) (35–37). As observed forHBV 3.5-kb RNA synthesis, female HBV transgenic mice dis-played very limited, and statistically insignificant, alterations inlevels of viral replication intermediates in response to the cholicacid diet or the loss of SHP (Fig. 5). These observations suggestthat the transcriptional regulatory network involving FXR andSHP, which helps to maintain bile acid homeostasis within the

FIG 3 Effect of bile acid feeding on serum HBeAg levels, ALT levels, andTNF-� transcripts in the livers of HBV transgenic mice. (A) Serum HBeAglevels were measured on day 0 (d0) and day 7 for mice fed the 1% (wt/wt)cholic acid diet (optical density [OD] at 450 nm). The mean HBeAg levels plusstandard deviations derived from male and female SHP-expressing (�/�) andSHP-null (�/�) HBV transgenic mice are shown. The levels of serum HBeAgin the cholic acid-fed HBV transgenic mice from day 0 to day 7 that are statis-tically significantly different by a paired Student t test (P � 0.05) are indicatedwith asterisks. (B) Serum ALT levels were measured on day 0, day 4, day 6, andday 7 of the 1% (wt/wt) cholic acid diet (units per liter). The mean ALT levelsplus standard deviations derived from serum samples of male and female SHP-expressing and SHP-null HBV transgenic mice taken during the initial pre-treatment (day 0) and during peak activity (days 4 to 6) are shown. (C) Quan-titative analysis of TNF-� transcripts by RT-qPCR in HBV transgenic mice.The GAPDH transcript was used as an internal control for the quantitationof TNF-� RNA. The mean relative TNF-� transcript levels plus standard de-viations derived from male and female SHP-expressing and SHP-null HBVtransgenic mice are shown. Mice were fed a control or a 1% (wt/wt) cholic acid(CA) diet for 7 days.

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liver (13, 28), may preferentially modulate HBV transcription andreplication in males, possibly contributing to the greater diseaseburden associated with chronic HBV infection in men than inwomen (21, 27, 60).

Absence of synergistic modulation of HNF4- and FXR�-directed HBV biosynthesis in human embryonic kidney 293Tcells. Bile acid feeding in the presence or absence of SHP has amodest effect on viral transcription and replication in male HBVtransgenic mice and essentially no effect on viral biosynthesis infemale HBV transgenic mice (Fig. 4 and 5). This appears contraryto the effects observed in cell cultures, where FXR and SHP cangreatly modulate HBV biosynthesis (35–37, 44). However, to

FIG 4 RNA (Northern) filter hybridization and RT-qPCR analysis of HBV tran-scripts in the livers of HBV transgenic mice. (A) RNA (Northern) filter hybridiza-tion of groups of two representative mice of each sex and genotype. The probesused were HBVayw genomic DNA plus GAPDH cDNA. SHP-expressing (�/�)HBV transgenic mice heterozygous for the SHP allele and SHP-null (�/�) HBVtransgenic mice are indicated. Mice were fed a control (�) or a 1% (wt/wt) cholicacid (�) diet for 7 days. The glyceraldehyde-3-phosphate dehydrogenase(GAPDH) transcript was used as an internal control for the quantitation of theHBV 3.5-kb RNA. (B) Quantitative analysis of the HBV 3.5-kb transcript level inHBV transgenic mice. The mean HBV 3.5-kb transcript levels plus standard devi-ations derived from 6 control male SHP-expressing HBV transgenic mice, 7 cholicacid-fed male SHP-expressing HBV transgenic mice, 8 control male SHP-nullHBV transgenic mice, 4 cholic acid-fed male SHP-null HBV transgenic mice, 3control female SHP-expressing HBV transgenic mice, 6 cholic acid-fed femaleSHP-expressing HBV transgenic mice, 5 control female SHP-null HBV transgenicmice, and 5 cholic-acid fed female SHP-null HBV transgenic mice are shown. Thelevels of the HBV 3.5-kb transcript in the cholic acid-fed HBV transgenic mice thatare statistically significantly different from their levels in the corresponding SHP-expressing or SHP-null HBV transgenic mice (P � 0.05 by Student’s t test) areindicated with asterisks. The levels of the HBV 3.5-kb RNA detected in the controlmale SHP-expressing HBV transgenic mice were also statistically significantly dif-ferent from the levels in the control male SHP-null HBV transgenic mice by theStudent t test (P � 0.05). (C) Quantitative analysis by RT-qPCR of the HBV 3.5-kbtranscript in the HBV transgenic mice analyzed in panel B. The GAPDH transcriptwas used as an internal control for the quantitation of the HBV 3.5-kb RNAs. Themean relative HBV 3.5-kb transcript levels plus standard deviations are shown.The levels of the transcripts in the cholic acid-fed HBV transgenic mice that arestatistically significantly different from their levels in the corresponding SHP-expressing or SHP-null HBV transgenic mice (P � 0.05, determined by Student’st test) are indicated with asterisks.

FIG 5 DNA (Southern) filter hybridization analysis of HBV DNA replicationintermediates in the livers of HBV transgenic mice. (A) DNA (Southern) filterhybridization of groups of two representative mice of each sex and each geno-type. The probe used was HBVayw genomic DNA. SHP-expressing (�/�)HBV transgenic mice heterozygous for the SHP allele and SHP-null (�/�)HBV transgenic mice are indicated. Mice were fed a control (�) or a 1%(wt/wt) cholic acid (�) diet for 7 days. The HBV transgene (Tg) was used as aninternal control for the quantitation of the HBV replication intermediates. RC,HBV relaxed circular replication intermediates; SS, HBV single-stranded rep-lication intermediates. (B) Quantitative analysis of HBV DNA replication in-termediate (RI) levels in HBV transgenic mice. The mean HBV DNA replica-tion intermediate levels plus standard deviations derived from 6 control maleSHP-expressing HBV transgenic mice, 7 cholic acid-fed male SHP-expressingHBV transgenic mice, 8 control male SHP-null HBV transgenic mice, 4 cholicacid-fed male SHP-null HBV transgenic mice, 3 control female SHP-expressing HBV transgenic mice, 6 cholic acid-fed female SHP-expressingHBV transgenic mice, 5 control female SHP-null HBV transgenic mice, and 5cholic acid-fed female SHP-null HBV transgenic mice are shown. The levels ofthe replication intermediates in the cholic acid-fed HBV transgenic mice werenot statistically significantly different from their levels in the correspondingSHP-expressing or SHP-null HBV transgenic mice by Student’s t test (P �0.05). The levels of replication intermediates detected in the control maleSHP-expressing HBV transgenic mice were statistically significantly differentfrom the levels in the control male SHP-null HBV transgenic mice by a Studentt test (P � 0.05).

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more closely approximate the conditions observed in vivo, theconsequence of expressing multiple nuclear receptors for HBVbiosynthesis was investigated by using nonhepatoma cells (Fig. 6).The transfection of the replication-competent HBV DNA (4.1-kbp) construct alone into 293T cells left untreated or treated withbile acid failed to support viral biosynthesis (Fig. 6, lanes 1 and 2).The transfection of the HBV DNA (4.1-kbp) construct with anHNF4 expression vector in the absence or presence of bile acidsupported HBV transcription and replication (Fig. 6, lanes 3 and4). The transfection of the HBV DNA (4.1-kbp) construct withRXR� plus FXR� expression vectors in the presence, but not inthe absence, of bile acid supported robust HBV transcription andreplication (Fig. 6, lanes 5 and 6). Critically, the transfection of theHBV DNA (4.1-kbp) construct with HNF4, RXR�, and FXR�expression vectors in the presence of bile acid supported a level ofHBV transcription and replication that was similar to that ob-served with the HNF4 or RXR� plus FXR� expression vectorsalone (Fig. 6, lane 8). This indicates that HNF4 and RXR� plus

FXR� do not synergistically or even additively cooperate to pro-mote HBV transcription. This observation may explain the lim-ited effect of bile acid treatment on HBV biosynthesis in the HBVtransgenic mice. HNF4 and other nuclear receptors, such as LRH1and ERR, which are capable of supporting HBV biosynthesis, areconstitutively expressed in the liver. The activation of FXR by bileacids must compete with these additional nuclear receptors toactivate transcription from the HBV nucleocapsid promoter (Fig.1). Based on the cell culture analysis (Fig. 6), it is possible that invivo, even the activation of FXR by bile acids is not sufficient tofurther enhance HBV transcription and replication, which are be-ing directed by constitutively active nuclear receptors, such asHNF4, that appear to be essential for viral biosynthesis (Fig. 1)(24). Interestingly, the expression of HNF4 and RXR� plus FXR�in the absence of bile acid appears to support less HBV RNA andDNA synthesis than that observed with HNF4 alone (Fig. 6, lanes3, 5, and 7), suggesting that in the absence of a ligand, RXR� plusFXR� may actually inhibit HNF4-mediated viral biosynthesis,presumably by competing for the proximal nuclear receptor bind-ing site in the HBV nucleocapsid promoter (40, 42, 44).

DISCUSSION

The transcriptional regulation of HBV in cell cultures has beenextensively studied. Indeed, transfection analyses with various celllines have led to the identification of cis-acting regulatory se-quence elements within the viral promoters which interact withthe trans-acting factors that regulate HBV transcription (19, 47,52, 61). This type of analysis has identified a variety of ubiquitousand liver-enriched transcription factors that modulate HBV tran-scription in cell culture (19, 47, 52, 61). Such studies have beenextended to examine the roles of specific transcription factors incontrolling viral biosynthesis in cell culture (35, 36, 44, 53). AsHBV transcription and replication occur in the HepG2 and Huh7human hepatoma cell lines when transfected with HBV DNA (5,48, 51, 54, 59), it has not been readily possible to determine whichtranscription factors are essential for HBV biosynthesis using thisapproach (53). However, the complementation of nonhepatomacells with liver-enriched transcription factors has permitted theevaluation of the specific roles of individual factors in HBV tran-scription and replication (35, 36, 44, 53). Using this approach, ithas been possible to demonstrate that the only individual liver-enriched transcription factors capable of supporting HBV biosyn-thesis in nonhepatoma cells belong to the nuclear receptor super-family (44, 53). Interestingly, the HBV nucleocapsid promotercontains several recognition sites for these transcription factors,and multiple members of the nuclear receptor superfamily, in-cluding HNF4, RXR� plus PPAR�, RXR� plus FXR�, LRH1, andERR, have been shown to be capable of supporting HBV biosyn-thesis in nonhepatoma cells (Fig. 1) (44, 53).

Although these cell culture studies have uncovered a previouslyunappreciated critical role for nuclear receptors in HBV biosyn-thesis, it remains unclear which members of the nuclear receptorsuperfamily are critical for the synthesis of HBV RNA and DNA invivo. The absence of any small-animal models of human HBVinfection seriously limits examinations of the in vivo role of nu-clear receptors in viral transcription and replication. To addressthis problem, the HBV transgenic mouse model of chronic HBVinfection was developed and was shown to recapitulate many ofthe postentry steps of the virus life cycle (15). Consequently, thisanimal model has become the most manipulatable small-animal

FIG 6 HNF4- and FXR�-directed HBV biosynthesis in human embryonickidney 293T cells. (A) RNA (Northern) filter hybridization analysis of HBVtranscripts. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) tran-script was used as an internal control for RNA loading per lane. (B) DNA(Southern) filter hybridization analysis of HBV replication intermediates.HBV RC DNA, HBV relaxed circular DNA; HBV SS DNA, HBV single-stranded DNA. Chenodeoxycholic acid (CDCA) at 100 �M was used to acti-vate the nuclear receptor FXR�. Cells were transiently transfected with theHBV DNA (4.1-kbp) construct and the indicated nuclear receptor expressionvectors. Lane 1, control; lane 2, control plus CDCA; lane 3, HNF4�; lane 4,HNF4� plus CDCA; lane 5, RXR�/FXR�; lane 6, RXR�/FXR� plus CDCA;lane 7, RXR�/FXR� plus HNF4�; lane 8, RXR�/FXR� plus CDCA plusHNF4�.

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model where the in vivo role of transcription factors affecting HBVRNA and DNA synthesis can be evaluated (2, 3, 14, 23, 24, 41, 43).Indeed, the model has been used to demonstrate that HNF4� isessential for the developmental expression of HBV biosynthesisand that PPAR� does not play a role in governing HBV biosyn-thesis under normal physiological conditions but can be activatedby using synthetic ligands to enhance viral transcription and rep-lication (14, 24).

In the current study, the role of bile acids, their nuclear recep-tor FXR, and the corepressor SHP in HBV biosynthesis was inves-tigated in vivo by using the HBV transgenic mouse model system(Fig. 1). In general, activated FXR appears to have a very modesteffect on the synthesis of HBV RNA and DNA, and SHP seems topartially inhibit viral biosynthesis in male HBV transgenic miceunder normal physiological conditions (Fig. 4 and 5). In contrast,HBV biosynthesis was unaffected in the female HBV transgenicmice examined under identical conditions (Fig. 4 and 5). Thissuggests that there are subtle differences in the regulation of HBVbiosynthesis by the bile acid-FXR-SHP regulatory network inmale and female mice (Fig. 1) (13, 28). The reasons for thesedifferences are currently unclear, but it is worth noting that dis-ease progression is generally more severe in males than in females,indicating that there are sexually dimorphic differences present inboth mice and humans (21, 27, 60).

In an attempt to explain the very modest effect of FXR activa-tion on HBV biosynthesis in vivo, the relative importance of HNF4and RXR� plus FXR� was examined by using nonhepatoma cells(Fig. 6). Previously, it was shown that both these nuclear receptorscan direct the expression of the HBV 3.5-kb RNA and, hence, viralreplication (35, 36, 44, 53). Notably, the simultaneous expressionof both these nuclear receptors does not lead to any enhancementof HBV RNA and DNA synthesis over that seen with the transcrip-tion factors alone (Fig. 6). The absence of any additive or syner-gistic effects on viral biosynthesis in cell cultures suggests an ex-planation for the lack of any major effect of the bile acid diet in vivo(Fig. 4 and 5). In the liver, HNF4 is presumably constitutivelyactive. The activation of FXR by the bile acid diet clearly alteredthe expressions of both FXR and SHP target genes, as expected(Fig. 1 and 2) (13, 28, 29). However, activated FXR had only aminimal effect on HBV biosynthesis, presumably because it can-not efficiently enhance HNF4-mediated HBV nucleocapsid pro-moter activity (Fig. 6). The ability of FXR to modestly increaseviral biosynthesis in male mice but not in female mice may reflectsubtle differences in the nature of the transcription factors gov-erning HBV nucleocapsid promoter activity in the different sexes.Regardless of these differences, it is important that the HBV trans-genic mouse model reflects only the changes in expression associ-ated with a single viral replication cycle, and even a small differ-ence in the replication efficiency can lead to a dramaticamplification of viral titers over an infection period that repre-sents many replication cycles. Therefore, it is possible that bile acidmetabolism in the liver might contribute to some of the differ-ences in viral synthesis and disease outcomes associated with HBVinfections in men and women (Fig. 1) (21, 27, 60).

ACKNOWLEDGMENTS

We thank Luca G. Guidotti and Francis V. Chisari (The Scripps ResearchInstitute, La Jolla, CA) for providing the HBV transgenic mice and BrunoSainz, Jr., and Susan L. Uprichard (University of Illinois at Chicago, Chi-cago, IL) for assistance with the RT-qPCR analysis. We are grateful to Eric

F. Johnson (The Scripps Research Institute, La Jolla, CA) for plasmidpCMVHNF4 and Ronald M. Evans (The Salk Institute, La Jolla, CA) forplasmids pRS-hRXR� and pCMV-rFXR�.

This work was supported by Public Health Service grant AI30070 fromthe National Institutes of Health.

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