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. Author manuscript; available in PMC: 2013 Sep 14.
Published in final edited form as: Int J Biochem Cell Biol. 2009 Nov 24;43(2):230–237. doi: 10.1016/j.biocel.2009.11.016

MULTIPLE NUCLEAR RECEPTORS MAY REGULATE HEPATITIS B VIRUS BIOSYNTHESIS DURING DEVELOPMENT

Vanessa Reese 1, Caitlin Ondracek 1, Christel Rushing 1, Lie Li 1, Claudia E Oropeza 1, Alan McLachlan 1,a
PMCID: PMC3773232  NIHMSID: NIHMS505982  PMID: 19941970

Abstract

Hepatitis B virus (HBV) replicates by the reverse transcription of the viral 3.5kb pregenomic RNA. Therefore the level of expression of this transcript in the liver is a primary determinant of HBV biosynthesis. In vivo neonatal transcription of the HBV 3.5kb pregenomic RNA is developmental regulated by hepatocyte nuclear factor 4α (HNF4α). In addition, viral biosynthesis in non-hepatoma cells can be supported directly by this nuclear receptor. However HBV transcription and replication can be supported by additional nuclear receptors including the retinoid X receptor α/peroxisome proliferator-activated receptor α (RXRα/PPARα), retinoid X receptor α/farnesoid X receptor α (RXRα/FXRα), liver receptor homolog 1 (LRH1) and estrogen-related receptors (ERR) in non-hepatoma cells. Therefore during neonatal liver development, HNF4α may progressively activate viral transcription and replication by binding directly to the proximal HNF4α recognition sequence within the nucleocapsid promoter. Alternatively, HNF4α may support viral biosynthesis in vivo indirectly by activating a network of liver-enriched nuclear receptors that, in combination, direct HBV 3.5kb pregenomic RNA transcription and replication.

Keywords: hepatitis B virus, nuclear receptors, transcription, replication, viral biosynthesis

INTRODUCTION

Hepatitis B virus (HBV) is a major human pathogen that chronically infects approximately 400 million individuals worldwide (Dienstag, 2008). Chronic HBV infections can lead to cirrhosis of the liver and hepatocellular carcinoma which are responsible for about one million deaths annually (Dienstag, 2008). HBV tropism is restricted to the liver of its hosts, man and chimpanzees, presumably because the viral receptor is expressed only on hepatocytes of these species (Raney and McLachlan, 1991). However studies of factors that control the transcription of the HBV genome have indicated that only liver-enriched nuclear receptors are capable of supporting HBV 3.5kb pregenomic RNA synthesis (Tang and McLachlan, 2001). Consequently expression of the viral genome is essentially limited to cells where these transcription factors are abundant, primarily the hepatocytes of the adult liver, adding another level of restriction to the tropism of this virus (Guidotti et al., 1995).

In rodents, the abundance of the liver-enriched nuclear receptors that control HBV transcription is developmentally regulated. The levels of hepatocyte nuclear factor 4α (HNF4α), retinoid X receptor α (RXRα), peroxisome proliferator-activated receptor α (PPARα), farnesoid X receptor α (FXRα) and liver receptor homolog 1 (LRH1) in the liver increase as neonates develop into adults (Kyrmizi et al., 2006; Balasubramaniyan et al., 2005). Concomitantly, the level of HBV transcripts and replication intermediates are very low at birth in HBV transgenic mice and progressively increase throughout subsequent liver development (Guidotti et al., 1995; Li et al., 2009). As the HBV 3.5kb pregenomic RNA encodes the viral nucleocapsid and reverse transcriptase/DNA polymerase, the level of this transcript determines the abundance of the viral replication intermediates as it is also the substrate which is reverse transcribed into viral DNA (Will et al., 1987). The correlation of viral biosynthesis with nuclear receptor levels in the developing liver and the previous observation that HNF4α and RXRα/PPARα can support HBV 3.5kb pregenomic RNA synthesis and replication in non-hepatoma cells raised the central issue of their relative importance for virion production in the liver in vivo (Tang and McLachlan, 2001; Balasubramaniyan et al., 2005; Kyrmizi et al., 2006; Li et al., 2009). To address this question, HBV transgenic mice lacking specific nuclear receptors have been characterized (Guidotti et al., 1999; Li et al., 2009). These studies indicate that PPARα does not affect viral biosynthesis under normal physiological conditions whereas HNF4α is essential for HBV transcription and replication throughout early neonatal development (Guidotti et al., 1999; Li et al., 2009). However the question of the precise mechanism of action of HNF4α throughout development remains to be established.

In this study, we investigate the role of several additional nuclear receptors in the control of HBV transcription and replication in non-hepatoma cells. Additional liver-enriched nuclear receptors have been shown to bind to and modulate nucleocapsid promoter activity (Li et al., 1998; Ishida et al., 2000; Ramiere et al., 2008). Therefore the effect of RXRα/FXRα and LRH1 plus the related nuclear receptor, estrogen-related receptors (ERR), on HBV 3.5kb pregenomic RNA synthesis and replication in non-hepatoma cells was examined and they were shown individually to have the capacity to support HBV biosynthesis. These observations, in combination with the expression pattern of these nuclear receptors in the developing liver, suggest that HBV has adapted to its host such that viral transcripts, replication intermediates and proteins are synthesized at progressively increasing levels after birth. This pattern of synthesis might induce immunological tolerance, supporting persistent infection, as observed in the majority of human neonatal infections (Dienstag, 2008).

2. Materials and methods

2.1. Plasmid constructions

The steps in the cloning of the plasmid constructs used in the transfection experiments were performed by standard techniques (Sambrook et al., 1989). HBV DNA sequences in these constructions were derived from the plasmid pCP10, which contains two copies of the HBV genome (subtype ayw) cloned into the EcoRI site of pBR322 (Dubois et al., 1980). The HBV DNA (4.1kbp) construct that contains 1.3 copies of the HBV genome includes the viral sequence from nucleotide coordinates 1072 to 3182 plus 1 to 1990 (Fig. 1A). This plasmid was constructed by cloning the NsiI/BglII HBV DNA fragment (nucleotide coordinates 1072 to 1990) into pUC13, generating pHBV(1072–1990). Subsequently, a complete copy of the 3.2kbp viral genome linearized at the NcoI site (nucleotide coordinates 1375 to 3182 plus 1 to 1374) was cloned into the unique NcoI site (HBV nucleotide coordinate 1374) of pHBV(1072–1990), generating the HBV DNA (4.1kbp) construct.

Figure 1.

Figure 1

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Multiple nuclear receptors activate HBV transcription and replication in the human embryonic kidney 293T cell line. (A) Structure of the HBV DNA (4.1kbp) construct used in transient transfection analysis. The 4.1-kbp greater-than-genome length HBV DNA sequence in this construct spans coordinates 1072-3182/1-1990 of the HBV genome (subtype ayw). The locations of the HBV 3.5-kb, 2.4-kb, 2.1-kb and 0.7-kb transcripts are indicated. EnhI/Xp, enhancer I/X-gene promoter region; Cp, nucleocapsid or core promoter; pA, polyadenylation site; PS1p, presurface antigen promoter; Sp, major surface antigen promoter; X, X-gene; S, surface antigen gene; C, core gene; P, polymerase gene; ORF, open reading frame. (B) RNA (Northern) filter hybridization analysis of HBV transcripts. The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) transcript was used as an internal control for RNA loading per lane. (C) DNA (Southern) filter hybridization analysis of HBV replication intermediates. HBV RC DNA, HBV relaxed circular DNA; HBV SS DNA, HBV single stranded DNA. All-trans retinoic acid, clofibric acid and chenodeoxycholic acid at 1µM, 1mM, and 100µM, respectively, were used to activate the nuclear receptors, RXRα, PPARα and FXRα. Cells were transiently transfected with the HBV DNA (4.1kbp) construct and the indicated nuclear receptor expression vectors. Lane 1, Control; lane 2, HNF4α; lane 3, RXRα plus PPARα; lane 4, RXRα plus FXRα; lane 5, LRH1; lane 6, ERRα; lane 6, ERRβ; lane 8, ERRγ.

The pCMVHNF4α, pRS-hRXR, pCMVPPAR-G, pCMV-rFXRα, pCMX-mLRH1, pSG5-mERRα, pcDNA3–2xFLAG-mERRβ and pSV5-mERRγ vectors express HNF4α, RXR, PPAR-G, FXRα, LRH1, ERRα, ERRβ and ERRγ polypeptides from the rat HNF4α, human RXR, mouse PPARα-G, rat FXRα, mouse LRH1, mouse ERRα, mouse ERRβ and mouse ERRγ cDNAs, respectively, using the CMV immediate-early promoter (pCMV, pCMX and pcDNA3), the Rous sarcoma virus LTR (pRS) or the simian virus 40 early promoter (pSG5 and pSV) (Mangelsdorf et al., 1990; Muerhoff et al., 1992; Chen et al., 1994a; Bonnelye et al., 1997; Knutti et al., 2000; Lu et al., 2000). The PPAR-G polypeptide contains a mutation in the PPAR cDNA changing Glu282 to Gly that may decrease the affinity of the receptor for the endogenous ligand. Consequently, this mutation increases the peroxisome proliferator-dependent (i.e. clofibric acid-dependent) activation of transcription from a peroxisome proliferator response element (PPRE) containing promoter (Muerhoff et al., 1992).

2.2 Cells and transfections

The human embryonic kidney 293T cell line was grown in RPMI-1640 medium and 10% fetal bovine serum at 37C in 5% CO2/air. Transfections for viral RNA and DNA analysis were performed as previously described (McLachlan et al., 1987) using 10 cm plates, containing approximately 1 × 106 cells. DNA and RNA isolation was performed 3 days post transfection. The transfected DNA mixture was composed of 5 µg of HBV DNA (4.1kbp) plus 1.5 µg of the nuclear receptor expression vectors, pCMVHNF4α, pRS-hRXR, pCMVPPAR-G, pCMV-rFXRα, pCMX-mLRH1, pSG5-ERRα, pcDNA3-2xFLAG-mERRβ and pSV5-mERRγ vectors (Mangelsdorf et al., 1990; Muerhoff et al., 1992; Chen et al., 1994a; Bonnelye et al., 1997; Knutti et al., 2000; Lu et al., 2000). Controls were derived from cells transfected with HBV DNA and the expression vectors lacking a nuclear receptor cDNA insert (Raney et al., 1997). All-trans retinoic acid, clofibric acid and chenodeoxycholic acid at 1µM, 1mM and 100µM, respectively, were used to activate the nuclear receptors, RXRα, PPARα and FXRα (Tang and McLachlan, 2001).

2.3 Characterization of HBV transcripts and viral replication intermediates

Transfected cells from a single plate were divided equally and used for the preparation of total cellular RNA and viral DNA replication intermediates as described previously (Summers et al., 1991) with minor modifications. For RNA isolation (Chomczynski and Sacchi, 1987) the cells were lysed in 1.8 ml of 25 mM sodium citrate, pH 7.0, 4 M guanidinium isothiocyanate, 0.5% (v/v) sarcosyl, 0.1 M 2-mercaptoethanol. After addition of 0.18 ml of 2M sodium acetate, pH 4.0, the lysate was extracted with 1.8 ml of water-saturated phenol plus 0.36 ml of chloroform-isoamyl alcohol (49:1). After centrifugation for 30 min. at 3,000 rpm in a Sorval RT6000, the aqueous layer was precipitated with 1.8 ml of isopropanol. The precipitate was resuspended in 0.3 ml of 25 mM sodium citrate, pH 7.0, 4 M guanidinium isothiocyanate, 0.5% (v/v) sarcosyl, 0.1 M 2-mercaptoethanol and precipitated with 0.6 ml of ethanol. After centrifugation for 20 min. at 14,000 rpm in an Eppendorf 5417C microcentifuge, the precipitate was resuspended in 0.3 ml of 10 mM Tris hydrochloride, pH 8.0, 5 mM EDTA, 0.1% (w/v) sodium lauryl sulfate and precipitated with 45 µl of 2 M sodium acetate plus 0.7 ml of ethanol.

For the isolation of viral DNA replication intermediates, the cells were lysed in 0.4 ml of 100 mM Tris hydrochloride, pH 8.0, 0.2% (v/v) NP40. The lysate was centrifuged for 1 min. at 14,000 rpm in an Eppendorf 5417C microcentrifuge to pellet the nuclei. The supernatant was adjusted to 6.75 mM magnesium acetate plus 200 ug/ml DNase I and incubated for 1 hr at 37°C to remove the transfected plasmid DNA. The supernatant was readjusted to 100 mM NaCl, 10 mM EDTA, 0.8% (w/v) sodium lauryl sulfate, 1.6 mg/ml pronase and incubated for an additional 1 hr at 37°C. The supernatant was extracted twice with phenol, precipitated with two volumes of ethanol and resuspended in 100 µl of 10 mM Tris hydrochloride, pH8.0, 1 mM EDTA. RNA (Northern) and DNA (Southern) filter hybridization analysis were performed using 10 µg of total cellular RNA and 30 µl of viral DNA replication intermediates, respectively, as described (Sambrook et al., 1989). Filter hybridization analyses were quantified by phosphorimaging using a Packard Cyclone Storage Phosphor System.

RESULTS

3.1. Multiple nuclear receptors can support HBV biosynthesis in nonhepatoma cells

A HBV DNA (4.1kbp) construct that can encode the four HBV transcripts and supports viral replication in hepatoma cells and the hepatocytes of transgenic mice was examined for its ability to support viral transcription and replication in the human embryonic kidney 293T cell line (Fig. 1) (Guidotti et al., 1995; Tang and McLachlan, 2001; Oropeza et al., 2008). In the absence of nuclear receptor expression vectors, the HBV 3.5kb pregenomic RNA is not expressed and viral replication is not apparent in transient transfection analysis (Fig. 1B and C, lane 1). HBV RNAs larger than 3.5kb of unknown origin are apparent and may represent greater-than-genome length transcripts originating from the X-gene promoter (Fig. 1B) (Doitsh and Shaul, 2003). The HBV 2.1kb RNA is expressed in the absence of nuclear receptors presumably because primary control of the major surface antigen promoter activity in transient transfection analysis is mediated by ubiquitous transcription factors (Raney et al., 1992; Raney and McLachlan, 1997; Tang and McLachlan, 2001). In contrast, HNF4α, RXRα/PPARα, RXRα/FXRα, LRH1, ERRβ and ERRγ, stimulate transcription of the HBV 3.5kb RNA and this is associated with the synthesis of encapsidated viral replication intermediates (Fig. 1B and C). ERRα failed to stimulate transcription of the HBV 3.5kb RNA and did not support viral replication under these conditions in the human embryonic kidney 293T cell line (Fig. 1B and C, lane 6). These observations indicate that a number of different nuclear receptors can control the transcription of pregenomic RNA and therefore the ability of HBV to replicate in specific cell types.

To investigate further the mechanism of modulation of viral replication by the different nuclear receptors, a HBV DNA (4.1kbp) construct (Fig. 1A) with a 4-nucleotide mutation in the nucleocapsid promoter proximal HNF4 binding site (HNF4mut) was examined for its ability to support viral transcription and replication in the human embryonic kidney 293T cell line (Figs. 2 and 3A) (Tang and McLachlan, 2001). The mutation blocks nuclear receptor binding to the nucleocapsid promoter proximal HNF4 binding site and inhibits HNF4- and RXRα/PPARα-mediated HBV 3.5kb pregenomic RNA synthesis and viral replication (Fig. 2, lanes 3–6) (Tang and McLachlan, 2001). The nuclear hormone receptor-dependent level of HBV 3.5kb RNA expressed from the HNF4mut construct was two- to four-fold lower than that observed from the wild type construct in the presence of expressed HNF4, RXRα/PPARα and RXRα/FXRα (Fig. 2A, lanes 3–8) but changed only to a very limited extent with the expression of LRH1, ERRβ and ERRγ (Fig. 2A, lanes 9–10 and 13–16). As observed in mouse fibroblasts, HNF4α- and RXRα/PPARα-dependent viral replication derived from the HNF4 mutant construct was greatly reduced compared to the wild type construct indicating the importance of the proximal HNF4 binding site in the nucleocapsid promoter for the control of viral replication by these two nuclear receptors (Fig. 2B, lanes 3–6) (Tang and McLachlan, 2001). RXRα/FXRα-dependent viral replication derived from the HNF4 mutant construct was reduced approximately six-fold compared to the wild type construct indicating the importance of the proximal HNF4 binding site in the nucleocapsid promoter for the control of viral replication by this nuclear receptor although to a somewhat lesser extent than observed with HNF4α and RXRα/PPARα (Fig. 2B, lanes 3–8). In contrast, LRH1-, ERRβ- and ERRγ-dependent viral replication derived from the HNF4 mutant construct was reduced only about two-fold compared to the wild type construct indicating the proximal HNF4 binding site in the nucleocapsid promoter has only a limited role in controlling the level of viral replication by these transcription factors (Fig. 2B, lanes 9–10 and 13–16). These findings indicate that the nucleocapsid promoter proximal HNF4 binding site is essential for high levels of HBV 3.5kb pregenomic RNA synthesis by the dimeric nuclear receptors, HNF4α, RXRα/PPARα and RXRα/FXRα, but is considerably less important for viral transcription and replication mediated by the monomeric nuclear receptors, LRH1, ERRβ and ERRγ. These findings imply that the activities of the various nuclear receptors differ, in part, due to their distinct abilities to promote transcription of the HBV 3.5kb pregenomic RNA from viral regulatory elements other than the nucleocapsid promoter proximal HNF4 binding site. Consequently, the level of viral replication is dependent on both the integrity of the nucleocapsid promoter and the nuclear receptors controlling viral gene expression. As noted previously, the effect of mutation of the nucleocapsid promoter proximal HNF4 binding site on replication is greater than its effect on transcription because it preferentially reduces the level of the HBV 3.5kb pregenomic RNA compared with the HBV 3.5kb precore RNA (Tang and McLachlan, 2001).

Figure 2.

Figure 2

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The nucleocapsid promoter proximal HNF4 binding site is an important determinant of nuclear receptor mediated HBV replication in the human embryonic kidney 293T cell line. Cells were transiently transfected with wild type (wt) or mutant (mt) HBV DNA (4.1kbp) constructs and nuclear receptors. Lanes 1–2, Control; lanes 3–4, HNF4α; lane 5–6, RXRα plus PPARα; lanes 7–8, RXRα plus FXRα; lanes 9–10, LRH1; lanes 11–12, ERRα; lanes 13–14, ERRβ; lanes 15–16, ERRγ. The HBV HNF4mut DNA (4.1kbp) construct contains the 4-nucleotide mutation (Fig. 3A) in the proximal HNF4 binding site of the nucleocapsid promoter that inhibits the binding of nuclear receptors to this recognition sequence (Tang and McLachlan, 2001). The nucleotide substitutions do not alter the X-gene polypeptide sequence. Both nucleocapsid promoter proximal regions in this terminally redundant HBV construct (Fig. 1A) were mutated for this analysis. (B) RNA (Northern) filter hybridization analysis of HBV transcripts. (C) DNA (Southern) filter hybridization analysis of HBV replication intermediates.

Figure 3.

Figure 3

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(A) Sequence of the HBV nucleocapsid promoter region. The 4-nucleotide HNF4 site mutation indicated above the wild type sequence in the green boxes inhibits the binding of nuclear receptors to the proximal HNF4 binding site (Tang and McLachlan, 2001). The nucleotide substitutions do not alter the X-gene polypeptide sequence. The RFX1, Sp1, C/EBP, HNF3 and TBP recognition sequences are underlined (Tang et al., 2001). The nine direct repeat sequence elements related to the hexameric nuclear receptor recognition sequence, AGGTCA, within the nucleocapsid promoter are highlighted in yellow. The identified or presumptive recognition sequences for the nuclear receptors, HNF4α (Raney et al., 1997), RXRα/PPARα (Raney et al., 1997), RXRα/FXRα (Ramiere et al., 2008), LRH1 (Li et al., 1998; Ishida et al., 2000) and ERR, are indicated in blue. The initiation sites for the HBV precore (PC) and pregenomic (or core (C)) 3.5kb RNAs are also indicated. (B) Gene regulatory network interactions among nuclear receptors within the liver that regulate HBV biosynthesis. HNF4α, LRH1, PPARα and ERRα can auto-regulate their own expression (Bailly et al., 2001; Paré et al., 2001; Pineda Torra et al., 2002; Liu et al., 2005; Zhang and Teng, 2007). HNF4α regulates the expression of LRH1, PPARα and FXRα (Paré et al., 2001; Pineda Torra et al., 2002; Zhang et al., 2004). LRH1 can activate HNF4α transcription (Paré et al., 2001). PPARα reciprocally regulates both FXRα and ERRα expression (Pineda Torra et al., 2003; Huss et al., 2004; Zhang et al., 2004; Liu et al., 2005). ERRγ regulates ERRα expression (Liu et al., 2005). Additionally interactions within this regulatory network controlling HBV transcription probably also exit. In particular, it is likely that LRH1 and ERR can reciprocally regulate each others expression given the close similarity in their recognition sequences. For similar reasons, ERR may contribute to HNF4α expression. However, early in neonatal development HNF4α is essential, either directly or indirectly, for HBV biosynthesis (Kyrmizi et al., 2006; Li et al., 2009).

DISCUSSION

4.1 Nuclear receptor binding sites in the HBV nucleocapsid promoter

The transcription factors that bind to the nucleocapsid promoter and regulate the level of expression of the HBV 3.5kb RNA have been extensively characterized (Fig. 3) (Tang et al., 2001). A variety of ubiquitous and liver-enriched transcription factors have been identified that contribute to nucleocapsid promoter activity in reporter gene assays but the nuclear receptors, HNF4α and RXRα/PPARα, are the only transcription factors that have been reported previously to support efficient HBV 3.5kb pregenomic RNA synthesis and viral replication in non-hepatoma cells (Tang and McLachlan, 2001). However, additional nuclear receptors have been identified that bind to and regulate nucleocapsid promoter activity in hepatoma cells. Notably, FXRα and LRH1 were found to modulate HBV nucleocapsid promoter activity (Li et al., 1998; Ishida et al., 2000; Ramiere et al., 2008). As the recognition sequence of ERR is very similar to LRH1 (Tsukiyama and Niwa, 1992; Johnston et al., 1997), we surmised that this nuclear receptor might also bind to the nucleocapsid promoter. Therefore, the ability of RXRα/FXRα, LRH1 and ERR to support HBV 3.5kb pregenomic RNA synthesis and viral replication in non-hepatoma cells was determined (Fig. 1). From this analysis, it was apparent that RXRα/FXRα, LRH1 and ERR, in addition to HNF4α and RXRα/PPARα, can direct the efficient expression of the HBV 3.5kb pregenomic RNA and support viral replication in non-hepatoma cells. These findings add an additional level of complexity to the transcriptional regulation of HBV biosynthesis.

4.2 Location of nuclear receptor binding sites within the HBV nucleocapsid promoter

The observation that multiple nuclear receptors can support transcription from the nucleocapsid promoter raised the issue of the importance of the proximal HNF4α recognition site for their activities. Previously it had been shown that the majority of the HNF4α- and RXRα/PPARα-dependent replication in mouse NIH 3T3 fibroblasts was mediated through the proximal HNF4α recognition site within the nucleocapsid promoter (Tang and McLachlan, 2001). Similar findings were observed using the human embryonic kidney 293T cell line and the dimeric nuclear receptors, HNF4α, RXRα/PPARα and RXRα/FXRα (Fig. 2). In contrast, only about a two-fold reduction in viral replication was observed in the human embryonic kidney 293T cell line when the monomeric nuclear receptors, LRH1, ERRβ and ERRγ directed the expression of the HBV 3.5kb pregenomic RNA (Fig. 2). These observations indicate that the monomeric nuclear receptors must interact with the proximal HNF4α recognition site within the nucleocapsid promoter to mediate part of their effect on viral biosynthesis. In addition, there must be other nuclear receptor binding sites within the viral genome for these transcription factors that contribute approximately equally to mediating these effects on HBV transcription and replication (Fig. 3).

Two RXRα/FXRα binding sites have been identified within the nucleocapsid promoter (Fig. 3A) (Ramiere et al., 2008). Therefore it appears that binding to the proximal RXRα/FXRα recognition site, which overlaps with the proximal HNF4α recognition site, within the nucleocapsid promoter contributes the majority of the transcriptional activity whereas binding to the distal site contributes the remaining RXRα/FXRα-mediated promoter activity. These observations indicate that the dimeric nuclear receptors, HNF4α, RXRα/PPARα and RXRα/FXRα, efficiently support viral biosynthesis when they are bound to the proximal nuclear receptor binding sites within the nucleocapsid promoter and only poorly support HBV transcription and replication when bound at distal recognition sites (Figs. 2 and 3A).

Two LRH1 binding sites have been previously identified in the distal nucleocapsid promoter sequences (Li et al., 1998; Ishida et al., 2000). As mutation of the proximal HNF4α recognition site within the nucleocapsid promoter reduces LRH1-mediated HBV biosynthesis approximately in half, it appears that an additional recognition sequence for this nuclear receptor must exist within the proximal HNF4α recognition site. This recognition sequence probably includes the second direct repeat of the HNF4α binding site as the TAAAGGTCT sequence most closely matches the LRH1 consensus sequence, YCAAGGYCR (Tsukiyama and Niwa, 1992). Therefore the remaining promoter activity must be governed by the two distal LRH1 binding sites within the nucleocapsid promoter (Fig. 3B) (Li et al., 1998; Ishida et al., 2000).

As the consensus sequence for LRH1 is very similar to the consensus binding site sequence for ERR, TCAAGGTCA (Johnston et al., 1997), it was of interest to determine if ERR might also support HBV biosynthesis in non-hepatoma cells. In the human embryonic kidney 293T cell line, ERRβ and ERRγ support viral biosynthesis whereas ERRα failed to mediate detectable levels of HBV replication (Figs. 1C and 2B). However, complementation with coactivators demonstrated that all the ERR isoforms have the potential to support viral biosynthesis (Rushing and McLachlan, unpublished results). Mutation of the proximal HNF4α recognition site within the nucleocapsid promoter decreases both ERRβ- and ERRγ-dependent HBV replication approximately two-fold indicating that additional ERR recognition sites must exist within the distal promoter sequences. The distal ERR binding sites likely correspond to the LRH1 binding sites (Fig. 3B). Indeed the locations of the ERR binding sites predicted using Consite (http://asp.ii.uib.no:8090/cgi-bin/CONSITE/consite/) software, in combination with the appropriate transcription factor weight matrix data, identified the distal LRH1 binding site at approximately nucleotide coordinate 1650 and the second direct repeat of the proximal HNF4α binding site at approximately nucleotide coordinate 1765 as the most likely ERR recognition sequences within the nucleocapsid promoter (Fig. 3A). Therefore it appears that approximately half the transcriptional activity mediated by ERR is due to binding to distal recognition sites within the nucleocapsid promoter and the remainder is mediated through the proximal binding site which can functionally interact with several nuclear receptor including ERR, LRH1, RXRα/FXRα, RXRα/PPARα and HNF4α (Fig. 3A).

4.3 In vivo role of nuclear receptors in the biosynthesis of HBV during development

Analysis of HBV biosynthesis in non-hepatoma cells has demonstrated that nuclear receptors are the only transcription factors capable of supporting HBV 3.5kb pregenomic RNA synthesis and viral replication (Fig. 1) (Tang and McLachlan, 2001). In fact the nucleocapsid promoter contains nine direct repeat sequence elements related to the hexameric nuclear receptor recognition sequence, AGGTCA, within 250 nucleotides of the initiation sites of the HBV 3.5kb RNAs (Fig. 3A) (Mangelsdorf et al., 1995; Mangelsdorf and Evans, 1995). This observation suggests that the in vivo role of nuclear receptors in HBV biosynthesis is probably complex. This is supported by the finding that multiple nuclear receptors, interacting with several different recognition elements within the nucleocapsid promoter, can support viral replication in non-hepatoma cells (Figs. 1–Fig. 3). As only man and chimpanzee are highly susceptible to HBV infection, the only small animal model of chronic HBV infection readily amenable to studying the in vivo role of nuclear receptors in the viral life cycle is the HBV transgenic mouse (Guidotti et al., 1995). Based on our cell culture studies and using the HBV transgenic mouse model of chronic HBV infection, the role of PPARα in HBV biosynthesis was investigated in vivo (Guidotti et al., 1999). This analysis demonstrated that PPARα did not play a role in determining the level of viral biosynthesis in the liver under normal physiological conditions (Guidotti et al., 1999). However, treatment of HBV transgenic mice with peroxisome proliferators enhanced viral transcription and replication in a PPARα-dependent manner indicating that this nuclear receptor can activate viral biosynthesis under certain circumstances (Guidotti et al., 1999).

The second nuclear receptor examined in the HBV transgenic mouse model of chronic infection was HNF4α (Li et al., 2009). HNF4α is a transcription factor that is essential for the viability of the mouse (Chen et al., 1994b; Hayhurst et al., 2001). However utilizing the conditional tissue-specific deletion of HNF4α in the neonatal liver, it was possible to determine the role of this nuclear receptor in HBV biosynthesis during early postnatal development (Li et al., 2009). Loss of HNF4α expression resulted in the absence of HBV transcription and replication in the livers of the HBV transgenic mice demonstrating that HNF4α is essential for viral biosynthesis in vivo (Li et al., 2009). This finding supports the analysis in non-hepatoma cells where HNF4α binding to the proximal recognition sequence within the nucleocapsid promoter is essential for HBV 3.5kb pregenomic RNA synthesis and viral replication (Figs. 1 and 2) (Tang and McLachlan, 2001).

Although HNF4α is essential for viral biosynthesis in early neonatal development in the HBV transgenic mouse, it is still not established if the effects of HNF4α in vivo are direct or indirect. However, it is apparent that the effect of HNF4α occurs at the level of viral transcription as the loss of this nuclear receptor is associated with a corresponding decrease in HBV transcription (Li et al., 2009). If the effect of HNF4α is not directly on viral transcription, it most likely affects this process by altering the activities of additional nuclear receptors that can support HBV biosynthesis (Fig. 1) which, like HNF4α, are involved in the complex transcription factor regulatory network required to produce and maintain the differentiated hepatic phenotype (Kyrmizi et al., 2006). As loss of PPARα does not affect HBV biosynthesis under normal physiological conditions, it seems most likely that LRH1 and FXRα represent the alternative liver-enriched nuclear receptors that might direct HBV transcription and replication. Indeed, HNF4α is known to modulate the levels of expression of LRH1, PPARα and FXRα during early neonatal development (Fig. 3B) (Pineda Torra et al., 2002; Balasubramaniyan et al., 2005; Kyrmizi et al., 2006). Therefore, it is possible that HNF4α controls HBV biosynthesis indirectly by increasing the expression of LRH1, PPARα and FXRα during early neonatal development and thereby indirectly activating HBV replication. To examine this possibility, HBV transgenic mice lacking LRH1 or FXRα in the liver will have to be characterized for their ability to support viral transcription and replication throughout postnatal development.

The potential role of ERR in HBV biosynthesis is less apparent. ERRβ in not expressed in the liver and therefore is unlikely to contribute to viral transcription (Bookout et al., 2006). Likewise, ERRα and ERRγ are expressed in the liver at modest levels but are also expressed in a variety of other tissues that have not been shown to support viral biosynthesis (Bookout et al., 2006). These observations suggest ERR may not contribute directly to HBV biosynthesis although it may enhance the activities of other nuclear receptors either directly by binding to the nucleocapsid promoter or possibly indirectly by modulating PPARα, and possibly HNF4α, activity within the liver (Fig. 3B).

4.4 Adaptation of HBV to host development as an effective strategy for viral persistence

The parallel increase in HNF4α expression and HBV biosynthesis levels observed during postnatal development suggest that neonatal HBV infection in man may be associated with a slow, developmentally regulated increase in viral transcription, replication and antigen expression. This may contribute to host immunological tolerance to viral antigens and subsequent life long chronic infection (Milich et al., 1990; Dienstag, 2008). In addition, the essential nature of the HNF4α transcription factor for host viability (Chen et al., 1994b; Hayhurst et al., 2001; Parviz et al., 2003) probably ensures that HBV transcription and replication cannot be resolved without killing infected hepatocytes further limiting the hosts’ ability to resolve infection and increasing the probability of viral persistence. This adaptation may have a significant selective advantage and help to explain the success of this pathogen that currently chronically infects approximately 400 million individuals worldwide (Dienstag, 2008).

ACKNOWLEDGMENTS

This manuscript is dedicated to my friend and colleague, Dr. Robert H. Costa. You are dearly missed. We are grateful to Dr. Anastasia Kralli (The Scripps Research Institute, La Jolla, CA) for the plasmids pSG5-mERRα, pcDNA3-2xFLAG-mERRβ and pSV5-mERRγ, Dr. Eric F. Johnson (The Scripps Research Institute, La Jolla, CA) for the plasmids pCMVHNF4 and pCMVPPAR-G, Dr. Ronald M. Evans (The Salk Institute, La Jolla, CA) for the plasmids pCMV-rFXR and pRS-hRXR, and Dr. David Mangelsdorf (Southwestern Medical Center, Dallas, TX) for the plasmid pCMX-mLRH1. This work was supported by Public Health Service grant AI30070 from the National Institutes of Health.

Contributor Information

Vanessa Reese, Email: vreese2@uic.edu.

Caitlin Ondracek, Email: condra2@uic.edu.

Christel Rushing, Email: crushi2@uic.edu.

Lie Li, Email: lieli26@uic.edu.

Claudia E. Oropeza, Email: coropeza@uic.edu.

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