critical roles of nuclear receptor response elements in...

JOURNAL OF VIROLOGY,0022-538X/01/$04.00�0 DOI: 10.1128/JVI.75.23.11354–11364.2001

Dec. 2001, p. 11354–11364 Vol. 75, No. 23

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Critical Roles of Nuclear Receptor Response Elements inReplication of Hepatitis B Virus

XIANMING YU AND JANET E. MERTZ*

McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, Wisconsin 53706-1599

Received 29 May 2001/Accepted 22 August 2001

Functional analysis of the roles of the nuclear receptor response elements (NRREs) in the transcription andreplication of hepatitis B virus (HBV) in the context of its whole genome has been hampered by the extensiveoverlapping of the NRREs with the regions encoding viral proteins. We introduced point mutations thatinactivate the NRREs individually without altering the open reading frames of viral proteins. These mutationsin the context of a plasmid containing 1.2 copies of the HBV genome were transiently transfected into thehuman hepatoma cell line Huh7. Inactivation of the NRRE in either the preC promoter (NRREpreC) orenhancer I (NRREenhI) led to moderate reductions in synthesis of viral RNAs. Concurrent inactivation of bothNRREs led to 7- to 8-fold reductions in synthesis of the preC, pregenomic, and preS RNAs and a 15-foldreduction in synthesis of the S RNA. The accumulation of viral DNA in the cytoplasmic nucleocapsids andvirion particles in the culture medium was also reduced seven- to eightfold. These results suggest that theseNRREs are critical for the efficient propagation of HBV in hepatocytes. In cotransfection experiments we alsofound that overexpression of PPAR�-RXR� in the presence of their respective ligands led to a fourfold increasein pregenomic RNA synthesis and a four- to fivefold increase in viral DNA synthesis, while it had little or noeffect on synthesis of the other viral RNAs. Similar effects were observed with overexpression of PPAR�-RXR�in the presence of their respective ligands. This activation was dependent on NRREpreC, because the increasein synthesis of viral RNA and DNA was not observed when this site was mutated. Likewise, no activation ofsynthesis of pregenomic RNA and viral DNA by PPAR�-RXR� was observed in a naturally occurring NR-REpreC

� mutant of HBV. Our results suggest that interactions between nuclear receptors and NRREs presentin the HBV genome may play critical roles in regulating its transcription and replication during HBV infectionof hepatocytes.

Human hepatitis B virus (HBV) is the pathogen for viralhepatitis B. Chronic infection with HBV is associated with livercirrhosis and primary hepatocellular carcinoma. Its 3.2-kb ge-nome contains four overlapping open reading frames encodingthe surface antigens (preS1, preS2, and S proteins), core anti-gens (preC and C proteins), reverse transcriptase (P protein),and transactivator (X protein). These genes are under thecontrol of the preS, S, preC, pregenomic, and X promoters.Transcription from these promoters is regulated by two en-hancer regions named enhancer I and enhancer II. Synthesis ofHBV DNA takes place within the nucleocapsid in the cyto-plasm of infected hepatocytes. The pregenomic RNA playspivotal roles in the viral life cycle, serving both as the templatefor viral DNA synthesis and as the mRNA encoding the C andP proteins, components of the nucleocapsid (16, 17).

Nuclear receptors (NRs) are a superfamily of transcriptionfactors which share domains of similar functionality and se-quence. NRs bind to their respective response elements(NRREs) in a sequence-specific manner, leading to alteredregulation of transcription from nearby promoters. Many NRsalso bind ligands which affect their functional activities (54).NRs play important roles in regulating embryogenesis, celldifferentiation, and a variety of cellular functions (30, 52).

The peroxisome proliferator-activated receptors (PPARs), a

subfamily of NRs, consist of PPAR�, PPAR�, and PPAR�.They play key roles in lipid metabolism (7). The PPARs func-tion as part of a heterodimeric complex with another subfamilyof NRs, the retinoid X receptors (RXRs) (36). The NRRE forPPAR-RXR is typically a direct repeat of two NR half-sitesequences (5�-AGGTCA-3�) separated by 1 bp (DR1) (39).PPAR� is expressed at high levels in the liver (28). WhilePPAR� is normally expressed at low levels in hepatocytes (53),its expression is induced to high levels in hepatoma cells whende novo cholesterol synthesis is inhibited (13). Although thephysiological ligands for the PPARs remain unknown, manysynthetic peroxisome proliferators such as Wy-14,643 and clo-fibric acid can function as ligands for PPAR� (28). A numberof saturated, polyunsaturated, and branch-chained fatty acidsnaturally present in cells, such as metabolites of prostaglandinJ2, can function as ligands for PPAR� (14, 31). The ligand forthe RXRs has been identified as 9-cis-retinoic acid, a metab-olite of vitamin A (24, 33).

The hepatocyte nuclear factor 4� (HNF4�), a liver-enrichedNR, is a transcription factor which activates transcription fromthe promoters of a variety of the genes essential for liverdevelopment and function (35, 48). Its physiological ligandsare unknown, although fatty acyl-coenzyme A thioesters canfuntion as ligands for it (23). HNF4� forms homodimers andbinds to a DR1 element (15, 29).

NRs have been found to play important roles in the lifecycles of a number of animal tumor viruses (19, 32, 37, 58, 63,64). Tur-Kaspa et al. identified a glucocorticoid response ele-

* Corresponding author. Mailing address: McArdle Laboratory forCancer Research, University of Wisconsin Medical School, Madison,WI 53706-1599. Phone: (608)262-2383. Fax: (608)262-2824. E-mail:[email protected].

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ment situated upstream of the enhancer I region of the HBVgenome (55, 56). In recent years, three more NRREs have beenidentified in enhancer I (NRREenhI) (18, 26, 27), enhancer II(NRREenhII) (22), and the preC promoter (NRREpreC) (42, 61)of the HBV genome. Both NRREpreC and NRREenhI allow forthe binding of PPAR-RXR and a number of orphan NRs suchas HNF4� and chicken ovalbumin upstream promoter transcrip-tion factor 1 (COUP-TF1) (18). On the other hand, NRREenhII

only allows for the binding of HNF4� (22).The HBV NRREs are embedded in enhancer and promoter

elements which overlap extensively with coding regions forviral proteins. Therefore, it is difficult to mutate individualNRREs without altering the amino acid sequences of viralproteins as well. Thus, to date, most of our knowledge regard-ing the biological functions of the HBV NRREs has beenobtained utilizing reporter plasmids containing only a portionof the viral genome.

As part of the effort to understand the biology and pathologyof HBV infection and the molecular mechanisms of chronicand fulminant hepatitis B, a few naturally occurring HBVvariants with mutations in NRREs have been analyzed in thecontext of the whole HBV genome. However, the base pairchanges present in all of these variants also cause alterations insome of the amino acids of viral proteins (2, 4, 9, 34). Thus,while the data from these studies indicated that the NRREsprobably function as cis-acting regulatory elements in the lifecycle of HBV, the individual contribution of each NRRE totranscription and replication could not be determined defini-tively. A transgenic mouse model has also been used to inves-tigate regulation of HBV transcription and replication by NRs.Guidotti et al. (20) reported that treatment of HBV transgenicmice with synthetic compounds which are known ligands forPPAR� results in a slight increase in viral RNA synthesis anda large increase in viral DNA synthesis in the livers of femaletransgenic mice.

In an effort to elucidate the mechanisms by which NRsactivate HBV viral DNA synthesis and to explore the possibleuse of NRs and their ligands for treatment of HBV infection,we mutated NRREs in the HBV genome and examined viralRNA and DNA synthesis in hepatoma cells in the presence ofcoexpressed NRs. Previously, we reported that NRs exert dif-ferential regulation of transcription from the preC and pre-genomic promoters in the context of a subgenomic fragment ofHBV, with the effect varying with the NR (61). We report herethat both NRREpreC and NRREenhI do, indeed, function ascis-acting, positive regulatory elements for viral transcriptionand replication in the context of the whole HBV genome. Wealso report that PPAR-RXR and their ligands efficiently up-regulate synthesis of viral DNA through differential regulationof synthesis of the viral RNAs. These results suggest that NRsmay play important roles in modulating the propagation ofHBV in hepatocytes during different stages and courses of itsinfection.

MATERIALS AND METHODS

Site-directed mutagenesis and plasmid construction. Point mutations wereintroduced into the NRREpreC and NRREenhI of the HBV genome by a two-stepPCR-based mutagenesis method (11). All base pair changes were confirmed byDNA sequence analysis. Replication-competent wild-type and NRRE mutantplasmids were constructed by insertion into plasmid pSP65 of 1.2 copies in

tandem of HBV subtype adr (nucleotides [nt] 1403 to 3215 and 1 to 1991)containing either wild-type or mutant NRREs to minimize the redundancy ofHBV sequences while enabling synthesis of full-length pregenomic RNA (Fig.1A). In NRREpreC

� mutant plasmids, both copies of NRREpreC were mutated.Oligonucleotides and EMSAs. Electrophoretic mobility-shift assays (EMSAs)

were performed as described previously (58, 64). The sources of the plasmids forsynthesis of recombinant NR proteins and for transfection experiments weredescribed previously (61). Recombinant proteins COUP-TF1, PPAR�, RXR�,and HNF4� were synthesized in a coupled transcription-translation rabbit re-ticulocyte lysate system (Promega). The NRREpreC 25-bp synthetic oligonucle-otide probe had the sequence 5�-GAGATTAGGTTAAAGGTCTTTGTAC-3�.The NRREenhI 29-bp synthetic oligonucleotide probe had the sequence 5�-ACAATATCTGAACCTTTACCCCGTTGCCC-3�. The nucleotide sequences ofthe mutant NRRE oligonucleotide probes were the same as the wild-type se-quences except for the changes indicated (Fig. 1B and 2A). Competition EMSAswere performed as described previously (58, 64).

Cell line and transfections. The human hepatoma cell line Huh7 was grown at37°C with 5% CO2 in a 1:1 mixture of Dulbecco’s modified Eagle’s medium andF12 medium supplemented with 10% fetal bovine serum. Transient transfectionswere done according to the calcium phosphate precipitation method (44) usingplasmid pUC18 as the carrier DNA. Each 60-mm tissue culture dish of cells wastransfected with a total of 10 �g of plasmid DNA including 3 �g of wild-type ormutant HBV plasmid DNA. The �-galactosidase (�-Gal) expression plasmidpEQ176 (0.75 �g per 60-mm dish) (46) was included as an internal control ineach transfection mixture. In the cotransfection experiments, 1 �g of a PPAR�or PPAR� expression plasmid and 0.25 �g of an RXR� expression plasmid wereincluded in each dish of cells. Cells were plated in medium supplemented with5% charcoal-treated fetal bovine serum 24 h before transfection. After transfec-tion with the calcium phosphate-DNA precipitates for 8 h, the cells were washedand incubated in medium containing 5% charcoal-treated serum and the indi-cated ligands for PPAR and RXR until they were harvested.

Isolation and analysis of viral RNAs. Forty-eight hours after transfection, totalcellular RNA was isolated using an RNeasy Mini kit (Qiagen). The relativeamounts of the viral RNAs were determined by primer extension analysis asdescribed previously (61). The primer for the preC and pregenomic RNAs wasa 24-mer corresponding to HBV nt 1994 to 2017, the primer for the S RNA wasa 20-mer corresponding to nt 137 to 156, and the primer for the preS RNA wasa 24-mer corresponding to nt 3012 to 3035. The primer for �-Gal RNA was a17-mer (5�-GTTTTCCCAGTCACGAC-3�).

The total polyadenylated mRNA from transfected Huh7 cells was isolated witholigo(dT) cellulose (60). Northern blot analysis of the viral mRNAs was per-formed as described previously (6). Briefly, denatured RNA was resolved in a 0.6M formaldehyde–1% agarose gel, transferred to a nylon membrane, and probedwith a radiolabeled HBV probe. The blot was then stripped and reprobed witha radiolabeled �-Gal probe. Probes were prepared using a random-prime label-ing system (Amersham) with the 3.2-kb HBV fragment or a 2.9-kb �-Gal frag-ment as the template.

Isolation and analysis of viral DNA. Forty-eight hours after transfection, theHuh7 cells were harvested. Viral nucleic acid was isolated from cytoplasmicnucleocapsids as described previously (49). The relative amounts of viral DNAwere determined by Southern blot analysis (5). To isolate extracellular viralDNA, the medium in which the Huh7 cells had been growing was harvested 5days after transfection. Virion particles were precipitated with 10% polyethyleneglycol, and the viral DNA was isolated according to a method described bySummers et al. (50). The viral DNA was then subjected to Southern blot analysis.The radiolabeled HBV probe was the same as the one used in Northern blotanalyses. To normalize for the efficiency of transfection, �-Gal assays wereperformed with lysates from the same cells.

RESULTS

Generation of NRREenhI and NRREpreC mutants of HBVdefective in binding NRs. Site-directed mutagenesis was per-formed to introduce nucleotide changes into NRREenhI andNRREpreC. These changes were designed such that the aminoacid-coding capacities of the X and P open reading frameswould not be affected (Fig. 2A). To examine the binding ofNRs to these mutant NRREs, EMSAs were performed withrecombinant NRs and radiolabeled synthetic oligonucleotides.As expected, we found that the mutations in these two NRREsdrastically reduced binding by COUP-TF1, HNF4�, and

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PPAR�-RXR� (Fig. 2B). Competition EMSAs were also per-formed to measure the affinities of these NRs for the mutantNRREs relative to the wild-type NRREs. The mutations inNRREpreC reduced its affinities for COUP-TF1, HNF4�, andPPAR�-RXR� 43-, 9-, and 12-fold, respectively (data notshown). The mutations in NRREenhI reduced its affinities forCOUP-TF1, HNF4�, and PPAR�-RXR� 8-, 5-, and 17-fold,respectively (data not shown). Thus, we conclude that thesepoint mutations significantly reduce binding by recombinantCOUP-TF1, HNF4�, and PPAR�-RXR�.

Effects of mutant NRREenhI and NRREpreC on HBV RNAand DNA synthesis. The above NRRE mutations were intro-duced singly and doubly into a replication-competent plasmidcontaining 1.2 copies of the HBV genome (Fig. 1A) to gener-ate plasmids NRREpreC

�, NRREenhI�, and the double mutant

NRREpreC�

,enhI�. These mutant as well as wild-type plasmids

were introduced in parallel into Huh7 cells by transient trans-fection. After incubation at 37°C for 48 h, the cells were har-vested, and RNA and DNA were isolated. Northern blot anal-ysis showed that these mutations in the NRREs led to reducedsynthesis of all of the viral RNAs relative to that with wild-typeNRREs (Fig. 3A).

To examine in greater detail the effects of the NRRE mu-tations on synthesis of the viral RNAs, the HBV RNAs wereanalyzed by primer extension assays with primers specific for

the preC and pregenomic, S, and preS RNAs (Fig. 3B, C, andD, respectively). The mutations in NRREenhI resulted in two-fold reductions in preC, pregenomic, and preS RNA synthesisand a fourfold reduction in S RNA synthesis (lanes 3 in Fig.3B, C, and D, respectively). The mutations in NRREpreC re-sulted in slight reductions in pregenomic, S, and preS RNAsynthesis and a twofold reduction in preC RNA synthesis (lanes 2in Fig. 3B, C, and D, respectively). However, when both of theNRREs were mutant, synthesis of the preC, pregenomic, andpreS RNAs was reduced 7- to 8-fold and that of S RNA wasreduced 15-fold (Fig. 3B through D, lanes 4). We were unableto detect the RNA species corresponding to the X RNA byprimers designed for its detection (data not shown). However,Northern blot analysis showed that the mutants NRREpreC

�,NRREenhI

�, and NRREpreC�

,enhI� reduced synthesis of X

RNA approximately 2-, 4-, and 10-fold, respectively (Fig. 3A,lanes 2 to 4 versus lane 1).

Accumulation of HBV DNA in cytoplasmic nucleocapsidswas examined by Southern blot analysis. As predicted from theeffects of the NRRE mutations on synthesis of the pregenomicRNA, the mutations in NRREpreC led to a slight reduction inviral DNA synthesis, while the mutations in NRREenhI re-sulted in a threefold reduction in viral DNA synthesis (Fig. 4A,lanes 2 and 3 versus lane 1). When both of the NRREs were

FIG. 1. (A) Schematic diagram of plasmid containing 1.2 copies of the HBV genome. A 3.8-kb, terminally redundant variant of the HBVgenome, indicated by the large open rectangle, was inserted into plasmid pSP65. The locations of the P, S, C, and X open reading frames areindicated by solid lines. The locations of the NRREs in enhancer I, enhancer II, and the preC promoter are indicated by small solid rectangles.Shown at the bottom is the structure of the pregenomic RNA synthesized from this plasmid. (B) Nucleotide sequence of the region of the HBVgenome surrounding the NRREpreC. The half-site sequences in the NRREpreC are boxed. The sequence changes in a naturally occurring variantof HBV (CH variant) are shown below the boxed sequences. Right-pointing arrows indicate locations of transcription initiation sites used in thesynthesis of preC and pregenomic RNA (preg RNA).

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mutant, viral DNA synthesis was reduced approximately eight-fold (Fig. 4A, lane 4).

To examine whether the effects of the NRRE mutations onsynthesis of intracellular viral DNA in nucleocapsids were alsoreflected in accumulation of extracellular viral DNA in virionparticles, HBV virion DNA was isolated from the tissue culturemedium of the transfected Huh7 cells and analyzed likewise.The mutations in NRREpreC, NRREenhI, or both NRREs re-sulted in two-, four-, and sevenfold reductions of virion DNAlevels in the medium, respectively (Fig. 4B). Thus, we concludethat both of these NRREs in HBV function as positive regu-latory elements, with mutations in NRREenhI having greaternegative effects on viral RNA and DNA synthesis than muta-tions in NRREpreC and with loss of the function of bothNRREs having at least multiplicative effects.

Effects of PPAR�-RXR� and PPAR�-RXR� on viral RNAand DNA synthesis. NRREpreC and NRREenhI are the onlyNRREs in the HBV genome to which PPAR�-RXR� andPPAR�-RXR� are known to bind, with both PPARs requiringthe presence of RXR to form DNA-protein complexes onthese NRREs (61). Data from competition EMSAs indicatedthat both PPAR�-RXR� and PPAR�-RXR� bind NRREpreC

with a higher affinity than they do NRREenhI (61). Further-more, although PPAR� is enriched in the liver (28) whilePPAR� is not (53), immunoshift assays and Western blot anal-ysis with appropriate antisera failed to detect either PPAR� orPPAR� in nuclear extracts of Huh7 cells (data not shown)(42). Thus, we studied the effects of PPAR-RXR on viral geneexpression and DNA synthesis by cotransfection of Huh7 cellswith PPAR and RXR expression plasmids along with wild-typeor NRRE-mutant HBV-containing plasmids. After incubationat 37°C for 40 h in medium containing or lacking ligands forthese receptors, the cells were harvested and analyzed asabove. We found that PPAR�-RXR� activated synthesis ofpregenomic RNA from the wild-type HBV genome 2 1/2- or4-fold when the ligands for PPAR� and RXR� were absent orpresent in the medium, respectively (Fig. 5A, lanes 2 and 3versus lane 1). PPAR�-RXR� also activated synthesis of SRNA from the wild-type genome almost twofold (data notshown) but had little or no effect on synthesis of preC (Fig. 5A,lanes 1 to 3) and preS (data not shown) RNAs. As expectedfrom increased synthesis of pregenomic RNA, overexpressionof PPAR�-RXR� also resulted in four- to fivefold-increasedsynthesis of intracellular viral DNA (Fig. 5B, lanes 1 to 3).

The effect of PPAR�-RXR� on viral RNA synthesis wasfound to be dependent on a functional NRREpreC, becausesynthesis of preC and pregenomic RNAs and synthesis of SRNA were reduced three- and twofold, respectively, when thissite was mutated (Fig. 5A, lanes 4 to 6; also data not shown).As a result of reduced synthesis of pregenomic RNA, theaccumulation of intracellular viral DNA was also reducedthree- to fourfold (Fig. 5B, lanes 4 to 6). On the other hand,overexpression of PPAR�-RXR� still activated synthesis ofpregenomic RNA three- to fourfold and that of S RNA two-fold from the NRREenhI

� plasmid (Fig. 5A, lanes 7 to 9; alsodata not shown) and increased the accumulation of cytoplas-mic viral DNA four- to fivefold (Fig. 5B, lanes 7 to 9).

To examine the possibility that endogenous PPAR� mayhave interfered somewhat with the outcome of these cotrans-fection experiments, analogous experiments were also per-formed with a PPAR� expression plasmid in the presence orabsence of PPAR�’s ligand, 15-deoxy-12,14-prostaglandin J2.In this case, activation of synthesis of pregenomic RNA wasfound to be even more dependent on the presence of theligand, with the increase in pregenomic RNA synthesis fromthe wild-type HBV template being less than twofold in char-coal-treated serum, yet greater than fivefold when the ligandwas added to the medium (Fig. 6A, lane 2 versus lane 3). Asexpected, the accumulation of intracellular viral DNA in-creased concomitantly with pregenomic RNA synthesis (Fig.6B, lanes 1 to 3). On the other hand, PPAR�-RXR� in thepresence of their respective ligands increased S RNA synthesisonly twofold and had little or no effect on preS RNA synthesis(data not shown). The effects of PPAR�-RXR� were alsofound to be largely dependent on the presence of a functional

FIG. 2. Inactivation of the NRREs in the preC promoter andenhancer I. (A) Sequences of the point mutations introduced intoNRREpreC and NRREenhI such that translation of the open readingframes of the X and P genes remains unaffected. The imperfect directrepeats of the NR half-site sequence are boxed. The base pair changesintroduced by mutagenesis are shown as underlined lowercase letters.(B) EMSAs used to determine the binding of NRs to wild-type (WT)and mutant NRREs. Radiolabeled double-stranded oligonucleotidescontaining the wild-type and mutant NRRE sequences were used asprobes. DNA-protein complexes are indicated by the bracket; freeprobes are indicated by the arrow.



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NRREpreC. Overexpression of PPAR�-RXR� led to a three-fold decrease in preC and pregenomic RNA synthesis in theNRREpreC

� mutant (Fig. 6A, lane 4 versus lane 5). This un-expected repression was observed only when ligands were notpresent in the medium (Fig. 6A, lane 5 versus lane 6). Onthe other hand, synthesis of pregenomic RNA still increasedsixfold when NRREenhI was mutated (Fig. 6A, lanes 7 to9), consistent with PPAR-RXR acting primarily throughNRREpreC.

In conclusion, PPAR�-RXR� and PPAR�-RXR� activatesynthesis of pregenomic RNA and viral DNA in transientlytransfected Huh7 cells. This activation is largely dependent ontheir interaction with NRREpreC; NRREenhI plays only a mi-nor role. The presence of ligands to the receptors greatlystimulates this activation of viral RNA and DNA synthesis,especially by PPAR�-RXR�.

Effects of PPAR�-RXR� on viral RNA and DNA synthesisin a naturally occurring HBV variant. A variant of HBV isfrequently found in chronic and, in certain areas of Asia, ful-minant hepatitis B patients (8, 40). This variant (CH variant)contains changes in two of the conserved base pairs in theHBV NRREpreC: A to T at position 1764 and G to A atposition 1766 (Fig. 1B). It has been reported previously thatthese two point mutations abolish binding of COUP-TF1 toNRREpreC (9, 10). To determine whether the base pairchanges also affect the binding of HNF4� and PPAR�-RXR�

to this site, EMSAs were performed as described above, butwith a 25-bp radiolabeled oligonucleotide corresponding to theNRREpreC sequence in the CH variant as a probe. The two basepair changes in this variant abolished binding to NRREpreC byCOUP-TF1, as expected, and by PPAR�-RXR� (Fig. 7). How-ever, they had little effect on the binding of HNF4� (Fig. 7, lane7 versus lane 3). Competition EMSAs with radiolabeled wild-typeNRREpreC as the probe and unlabeled wild-type versus CHvariant NRREpreC oligonucleotides as competitors showed thatthe mutations in the CH variant reduced its affinities for COUP-TF1 and PPAR�-RXR� 27- and 7-fold, respectively, but had noeffect on the binding of HNF4� to NRREpreC (data not shown).

To study the effects of the mutations in the CH variant onexpression of HBV, these mutations were introduced into theplasmid containing 1.2 copies of the wild-type HBV genome.Transfection experiments were performed with coexpressedPPAR�-RXR� as described above. We found that mutationsin the CH variant led to a two- to threefold increase in syn-thesis of pregenomic RNA but had very little effect on synthe-sis of preC RNA (Fig. 8A, lane 1 versus lane 4). CoexpressedPPAR�-RXR� reduced synthesis of both preC and pre-genomic RNAs from the CH variant genome approximatelytwofold, while it increased synthesis of pregenomic RNA fromthe wild-type genome three- to fourfold (Fig. 8A). Coex-pressed PPAR�-RXR� had little or no effect on the accumu-lation of intracellular viral DNA from the CH variant in the

FIG. 3. Reduced viral RNA synthesis in NRRE mutants of HBV. (A) Autoradiogram of Northern blot analysis of the viral RNAs accumulatedin Huh7 cells transfected with wild-type (WT) and NRRE mutant plasmids. One-half of the poly(A)-selected RNA from a 60-mm dish of cells wasloaded in each lane and analyzed as described in Materials and Methods. (B through D) Autoradiograms of 8 M urea–8% polyacrylamide gelsshowing the results of primer extension assays used to quantify the preC and pregenomic RNAs (B), S RNAs (C), and preS RNA (D). Thepositions of the viral and �-Gal RNAs are indicated by arrows. preg, pregenomic. One-sixth of the RNA from a 60-mm dish of cells was used ineach reaction along with radiolabeled primers specific for the viral and �-Gal RNAs. Numbers at the bottom give the amount of viral RNA in eachlane relative to that synthesized from the wild-type plasmid. These numbers were determined with a PhosphorImager, normalized to �-Gal, andrepresent the means standard errors of the data obtained from three experiments similar to the one for which results are shown. The amountof preC RNA can be calculated by subtracting the pregenomic RNA from the preC-plus-pregenomic RNA in each lane.



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presence or absence of their ligands (Fig. 8B). Thus, we con-clude that PPAR�-RXR� and their ligands fail to significantlyaffect the expression of the HBV genome in the CH variant.

DISCUSSION

Using mutants in the NRREs that abrogate NR bindingwithout disrupting the overlapping coding regions for viralproteins (Fig. 2A), we have examined here the biological func-tions of NRREs in the context of a replication-competentHBV genome. We found that both NRREpreC and NRREenhI

function as cis-acting, positive regulatory elements. Mutagen-esis of either of these NRREs led to decreased synthesis ofboth viral RNA and DNA, with the effects being greatest whenboth sites were mutated (Fig. 3 and 4). Overexpression ofPPAR-RXR led to increased synthesis of both viral pre-genomic RNA and viral DNA (Fig. 5 and 6). Addition ofligands for the PPARs and RXR� to the medium furtherincreased synthesis of pregenomic RNA. This activation wasfound to be dependent only on a functional NRREpreC,although both NRREs can bind PPAR-RXR (Fig. 5 and 6).Lastly, a naturally occurring variant of HBV was shown to be

defective both in the binding of PPAR�-RXR� to its NRREpreC

(Fig. 7) and in activation of synthesis of pregenomic RNA andviral DNA by PPAR�–RXR� and their ligands (Fig. 8). Thus,we conclude that PPAR-RXR and their ligands likely playimportant roles in the replication of HBV, acting primarily viabinding of NRREpreC.

NRREpreC and NRREenhI are positive regulatory elementsessential for efficient viral gene expression and DNA synthesis.NRREpreC was identified in the regulatory region of the preCand pregenomic promoters of HBV. EMSAs and DNase Ifootprinting analyses showed that NRs can bind to this site (42,61). Since NRREpreC is embedded in the preC promoter anddistal to other HBV promoters, it has been hypothesized toregulate primarily synthesis of preC and pregenomic RNAs.However, its biological functions have not been characterizedpreviously in the context of a replication-competent HBV ge-nome in hepatoma cell lines. We have found in this study thatmutations in NRREpreC result in reduced synthesis of preCand pregenomic RNAs (Fig. 3B) and have little or no effect onsynthesis of S and preS RNAs (Fig. 3C and D).

Seemingly contrary to the present findings, we previouslyreported that mutations in NRREpreC lead to increased syn-thesis of preC RNA (61). However, this study was performedwith expression plasmids containing only a 588-bp subregion ofthe HBV genome (nt 1403 to 1990; see Fig. 1). Therefore,proper functioning of NRREpreC as a positive regulatory ele-ment may require it to be situated at its normal location withinthe context of the whole HBV genome so that appropriateprotein-protein interactions with numerous other regulatoryfactors can occur within their proper contexts.

Increased synthesis of preC RNA by NRREpreC� mutants in

the context of HBV subgenomic fragments was also observedin cell-free transcription assays with nuclear extracts preparedfrom HepG2 and HeLa cells (61). However, COUP-TF1 ismuch more abundant than HNF4� and PPAR� in HepG2 andHeLa nuclear extracts (unpublished data) and likely outcom-petes these other NRs for binding, functioning as a repressorof RNA synthesis.

The HBV enhancer I is a composite regulatory region thatconsists of several motifs to which liver-enriched and ubiqui-tous protein factors are known to bind (3, 12, 41, 43, 47). It hasbeen shown to upregulate all viral promoters in the HBVgenome (1, 25). However, most of our knowledge concerningtranscriptional regulation by enhancer I has been obtained usingreporter plasmids. Garcia et al. (18) reported that NRREenhI

and an adjacent element, EF-C, function interdependently toconfer enhancer and liver-specific activity on enhancer I. Weshowed here that mutations in NRREenhI reduce synthesis ofpreC, pregenomic, and preS RNAs twofold and reduce syn-thesis of S RNA fourfold (Fig. 3). These differential effects ofNRREenhI on the various viral promoters may be due to thedifferent natures or extents of its interactions with the viralpromoters, presumably via protein-protein contacts. Theseproteins may include NRs, their coactivators and corepressors,other regulatory factors, and components of the basal tran-scription machinery.

We reported previously that a number of NRs, includingHNF4�, COUP-TF1, and PPAR�-RXR�, have higher affini-ties for NRREpreC than for NRREenhI (61). Yet our analysisof NRRE mutants here suggests that NRREenhI plays a

FIG. 4. Reduced viral DNA synthesis in NRRE mutants of HBV.(A) Autoradiogram of Southern blot analysis of viral DNA isolatedfrom nucleocapsids present in the cytoplasm of transfected Huh7 cells.Cells were harvested 48 h posttransfection, and nucleocapsid viralDNA was prepared. One-third of the DNA from a 60-mm dish of cellswas loaded in each lane. The total viral DNA from relaxed circular(RC) to single-stranded (SS) DNA in each lane was quantitated witha PhosphorImager. Positions of viral DNAs are indicated by arrows.WT, wild type; DL DNA, duplex linear DNA. The smears between thespecific indicated DNA structures are the result of HBV DNAs withincomplete strands. Numbers at the bottom are means standarderrors of data relative to the wild type obtained in three experimentssimilar to the one for which results are shown. (B) Autoradiogram ofSouthern blot analysis of extracellular viral DNA. Culture medium washarvested for HBV virions 5 days posttransfection. One-half of theviral DNA from the medium of a 60-mm dish of cells was loaded ineach lane. Lanes 1 and 6, molecular markers for SS and DL DNAs,respectively. The DNA band below the DL DNA in lanes 2 through 4is likely DL or RC DNA with an incomplete plus strand. Data werequantified as for panel A.



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global role in regulating viral transcription and replication,while NRREpreC only moderately activates synthesis of pre-genomic RNA and viral DNA (Fig. 3 and 4). One possibleexplanation to reconcile these findings is that the function ofNRREenhI is activated synergistically in vivo by other elementssuch as EF-C that are also present in enhancer I.

In the NRREenhI�

,preC� double mutant, synthesis of preC,

pregenomic, and preS RNAs was down 7- to 8-fold and syn-thesis of S RNA was down 15-fold (Fig. 3), at least a multipli-cative effect relative to the two individually mutated NRREs.One hypothesis consistent with this finding is that the presenceof NRREpreC may compensate in part for loss of NRREenhI toenable NRRE-promoter interactions essential for efficient vi-ral RNA synthesis. Thus, viral gene expression occurs at abasal level only when both NRREs are mutated. The very lowlevel of viral RNA synthesis observed with the double mutantalso suggests that the function of NRREenhI cannot be com-pensated for by other elements in enhancer I.

We did not include here HBV plasmids in which NRREenhII

was also mutated. Nevertheless, the very fact that theNRREpreC NRREenhI double mutant exhibited very low levelsof RNA synthesis indicates that NRREenhII alone is not suffi-cient to sustain synthesis of viral RNA and DNA at wild-typelevels. It has been reported that enhancer II plays a key role asa liver-specific activator of the preC and pregenomic promot-ers (57, 59, 62). We found that a variant of a previously re-ported subgenomic plasmid in which the entire enhancer IIregion (i.e., nt 1403 to 1733) is deleted synthesized 1/10 asmuch preC and pregenomic RNA as the wild type (X. Yu,

unpublished data). On the other hand, an NRREenhII pointmutant defective in binding HNF4� synthesized approximatelyone-half of the wild-type level of preC and pregenomic RNAsin Huh7 cells within the context of the same subgenomic plas-mid (61; X. Yu, unpublished data). Therefore, while enhancerII as a whole is important for synthesis of the preC and prege-nomic RNAs from that HBV subgenomic plasmid, NRREenhII

probably does not contribute significantly to the function ofenhancer II. A detailed analysis of the regulation of synthesisof HBV RNA and DNA by HNF4� will be reported elsewhere(X. Yu and J. E. Mertz, unpublished data).

In the studies presented here, we were careful to ensure thatthe mutations in the NRREs did not affect the sequences of theviral proteins encoded by these regions of the HBV genome(Fig. 2A). Nevertheless, the theoretical possibility exists thatthey may have affected posttranscriptional events such as theformation of secondary structures important for genome rep-lication and/or stability of viral RNAs.

PPAR-RXR activation of synthesis of pregenomic RNA andviral DNA. We showed here that PPAR-RXR and their ligandscan play important roles in the replication of HBV, probablyvia direct binding to NRREpreC. For example, synthesis ofpregenomic RNA increased four- to fivefold when PPAR�-RXR� or PPAR�-RXR� was overexpressed by cotransfectionwith expression plasmids (Fig. 5A and 6A). As expected, syn-thesis of viral DNA increased concomitantly (Fig. 5B and 6B).On the other hand, overexpression of PPAR-RXR had mini-mal effects on synthesis of S, preC, and preS RNAs (Fig. 5Aand 6A; also data not shown). The S protein is already in vast

FIG. 5. PPAR�-RXR� activates synthesis of RNA and DNA of HBV. After incubation with the indicated DNA in a calcium phosphateprecipitate for 8 h, Huh7 cells were washed and incubated in medium containing 1 mM clofibric acid (Sigma), a ligand for PPAR�, and 1 �M9-cis-retinoic acid (Sigma), a ligand for RXR�, for 40 h before they were harvested for RNA and DNA. (A) Autoradiogram showing the primerextension reactions of preC and pregenomic (preg) RNAs. One-sixth of the RNA from a 60-mm dish of cells was used in each primer extensionreaction. WT, wild type. (B) Autoradiogram of Southern blot analysis of cytoplasmic viral DNA. One-third of the DNA from a 60-mm dish of cellswas loaded in each lane. Quantitations were performed as described in the legends to Fig. 3 and 4. Numbers at the bottom are means standarderrors of data obtained from three experiments similar to the one for which results are shown. RC DNA, relaxed circular DNA; DL DNA, duplexlinear DNA; SS DNA, single-stranded DNA.



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excess in HBV-infected hepatocytes, and the preS1 protein isonly a minor component of the viral envelope and toxic tohepatocytes when overexpressed. This differential regulationof transcription from viral genes may avoid wasteful produc-

tion of these two proteins and help to direct the cellular ma-chinery to the synthesis of more C and P proteins, thus allow-ing for efficient virion production in hepatocytes.

After we completed our studies reported here, Tang andMcLachlan published their finding that expression of PPAR�-RXR� and HNF4 enables synthesis of the 3.5-kb HBV RNAand HBV DNA replication in otherwise nonpermissive mouseNIH 3T3 cells (51). These complementary experiments indi-cate clearly that these NRs are major players in controlling thetranscription and replication of HBV. However, whereas theyfailed to detect any 3.5-kb HBV RNA in their nonliver cells inthe absence of exogenously expressed NRs, we still observedsome 3.5-kb HBV RNA with the double mutant in Huh7 cells,albeit at low levels (Fig. 3A). Likely, additional liver-enrichedtranscription factors and cis-acting regulatory elements withinthe HBV genome are responsible for the differences observedbetween Huh7 and NIH 3T3 cells.

The specific activation of the pregenomic promoter byPPAR-RXR was dependent on the presence of a functionalNRREpreC, but not NRREenhI (Fig. 5A and 6A). PPAR-RXRincreased synthesis of pregenomic RNA to the same extentfrom wild-type and NRREenhI

� HBV plasmids (Fig. 5A and6A). One model consistent with this observation is thatNRREpreC may become a stronger activator of the pregenomicpromoter in the presence of overexpressed PPAR-RXR andtheir ligands due to its higher affinity for PPAR-RXR andproximity to the promoter. On the other hand, synthesis ofpreC and pregenomic RNAs was actually slightly repressed byPPAR-RXR in the NRREpreC

� mutant in the absence of li-gand (Fig. 5A and 6A). Likewise, Tang and McLachlan re-ported that their NRREpreC mutant also preferentially re-duced the level of pregenomic RNA and greatly reduced viral

FIG. 6. PPAR�-RXR� activates synthesis of viral RNA and DNA in Huh7 cells. The amounts of RNA and DNA used in each assay were thesame as those stated for Fig. 5. Transfection of Huh7 cells was carried out as described for Fig. 5 except that a PPAR�-specific ligand,15-deoxy-12,14-prostaglandin J2, was used (8 �M) (Cayman Chemical). (A) Autoradiogram showing primer extension reactions used to quantifypreC and pregenomic (preg) RNAs. (B) Autoradiogram of a Southern blot analysis of cytoplasmic viral DNA. Quantitations were performed asdescribed in the legends to Fig. 3 and 4. Numbers at the bottom are means standard errors of data obtained from three experiments similar tothe one for which results are shown. RC DNA, relaxed circular DNA; DL DNA, duplex linear DNA; SS DNA, single-stranded DNA.

FIG. 7. Effects of base pair changes in the NRREpreC of the CHvariant of HBV on its binding to NRs. Shown here are results ofEMSAs performed with the indicated NRs and radiolabeled, double-stranded oligonucleotides containing the wild-type (WT) and CH vari-ant NRREpreC sequences as probes. Bracket, DNA-protein complexes;arrow, free probe.



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replication when PPAR�-RXR� was overexpressed in NIH3T3 cells (51). Remaining unclear is the reason for this re-duced pregenomic RNA synthesis when PPAR-RXR is over-expressed in NRREpreC mutants.

Garcia et al. (18) have reported that overexpression ofRXR� can activate a heterologous promoter containing mul-tiple copies of NRREenhI 8- to 15-fold. On the other hand, wefailed to observe activation of the HBV promoters within thecontext of a whole wild-type HBV genome when we overex-pressed only RXR� in the presence of its ligand (data notshown). We also failed to observe significant activation orrepression of viral promoters in Huh7 cells when we addedonly ligands for PPARs and RXR� to the culture mediumwithout concomitant overexpression of NRs (data not shown).These findings are consistent with our failure to detect thePPARs in nuclear extracts prepared from Huh7 cells and sug-gest that the natural concentrations of PPAR� and PPAR� inHuh7 cells are probably too low for efficient activation ofreplication of HBV.

Guidotti et al. (20) reported that treatment of HBV trans-genic mice with the peroxisome proliferators Wy-14,643 andclofibric acid results in a �2-fold increase in HBV 3.5-kb RNAlevels and in 2- to 3-fold and 7- to 14-fold increases in viralDNA accumulation in male and female mice, respectively. Themuch larger increase in viral DNA replication than in viraltranscription in female mice treated with ligands was hypoth-esized to be due to the basal level of pregenomic RNA beinglow, and, therefore, the level of synthesis of the C protein intheir livers also being low, with the moderate increase in pre-

genomic RNA after treatment with ligands raising the concen-tration of the C protein in hepatocytes above the thresholdneeded for efficient formation of nucleocapsids and viral DNAsynthesis.

The HBV transgenic mouse model is an excellent system inwhich to study the HBV life cycle in the liver and the hostimmune response and pathogenesis caused by HBV infection.On the other hand, transfection of cultured cells is also avaluable tool in that it allows for ready analysis of phenotypicchanges resulting from mutations in cis-acting regulatory ele-ments and viral genes. As shown here, the use of hepatoma celllines allows for assessment of the contributions of individualNRREs to viral gene expression in the context of functionalHBV enhancers and other cis-acting regulatory elements. Italso enables screening for NRs and ligands which may modu-late viral gene expression and virion production.

The NRREpreC� phenotype of the CH variant of HBV. The

CH variant of HBV contains mutations in one of the twohalf-sites of the NRREpreC (Fig. 1B). When we introducedthese two point mutations into the HBV genome, we foundthat synthesis of pregenomic RNA and viral DNA increasedapproximately twofold while preC RNA accumulation re-mained similar to that in the wild type (Fig. 8). Others havereported either reduced synthesis of preC RNA and secretionof HBV e antigen but no effect on synthesis of pregenomicRNA (9, 21, 45), activation of pregenomic RNA synthesis (38),no effect on viral DNA synthesis (21), or increased viral DNAsynthesis (9, 38, 45). These discrepancies may be attributed tothe use of different subtypes of HBV (ayw, adw2, and adr),

FIG. 8. Sequence changes in the NRREpreC of the CH variant of HBV abolish activation of synthesis of viral RNA and DNA by PPAR�-RXR�. (A) Autoradiogram of primer extension reactions used to quantify preC and pregenomic (preg) RNAs synthesized from the wild-type(WT) and CH variant HBV plasmids. Transient transfections of Huh7 cells and the concentrations of ligands for PPAR� and RXR� added to themedium were as described for Fig. 5. (B) Autoradiogram of Southern blot analysis of cytoplasmic viral DNA present in the same cells. Numbersat the bottom are means standard errors of data quantified from three experiments similar to the one for which results are shown. RC DNA,relaxed circular DNA; DL DNA, duplex linear DNA; SS DNA, single-stranded DNA.



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plasmid constructs (monomer, dimer, and 1.2-mer), and hep-atoma cell lines (HepG2 and Huh7).

We also studied the responses of the CH variant genome tooverexpression of PPAR�-RXR� in the presence of their li-gands. The CH variant was found to exhibit a phenotype sim-ilar to, but less severe than, that of our artificial NRREpreC

mutant, with pregenomic RNA synthesis being reduced ap-proximately twofold and viral DNA synthesis being largelyunaffected (Fig. 8). The less severe phenotype may be attrib-uted to the following facts: (i) the CH variant still retainsbinding by HNF4� in its NRREpreC (Fig. 7), while our NRREpreC

mutations abolish HNF4� binding as well (Fig. 2B); (ii) themutations in the CH variant create a new HNF1 binding site inNRREpreC (34); and (iii) the mutations in the CH variant alsoled to changes in two of the amino acids in the X protein (34).

We have shown here dependence on a functional NRREpreC

in PPAR�-RXR�-mediated activation of viral RNA and DNAsynthesis. Thus, the low level of responsiveness of the CHvariant to PPAR�-RXR� is likely due, at least in part, toinactivation of NRREpreC. Presumably, this nonresponsivenessto PPARs confers some advantage on the CH variant formaintaining its presence in hepatocytes. For example, manyphysiological and environmental changes and exposure to per-oxisome proliferators and other compounds which can func-tion as ligands for PPARs may upregulate the synthesis andactivity of PPARs in hepatocytes. In wild-type HBV infection,this will result in elevated synthesis of viral RNA, protein, andDNA. The increased synthesis of viral C protein and its displayon the cell surface may render the hepatocytes more vulnera-ble to the host immune system. With mutated NRREpreC, theCH variant can avoid this upregulation of its replication andthus retain the chronicity of its infection.

In summary, we conclude that NRREpreC and NRREenhI

are important positive regulatory elements for HBV gene ex-pression and replication. These NRREs differentially regulatetranscription from various viral promoters to facilitate efficientnucleocapsid formation and virion production. PPAR-RXRand their ligands increase synthesis of both the pregenomicRNA and viral DNA. Analysis of artificial NRRE mutants anda naturally occurring NRRE mutant suggests that this activa-tion is largely dependent on the function of NRREpreC.

ACKNOWLEDGMENTS

We thank Shannon Reagan for technical assistance. We also thankDan Loeb, Jeff Habig, Paul Lambert, Richard Kraus, and MichaelFarrell for helpful discussions and comments on the manuscript.

This work was supported by Public Health Service research grantsCA22443 and CA07175 from the National Cancer Institute.

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