Ten-eleven translocation (Tet) methylcytosine dioxygenase-dependent viral DNA demethylation mediates in vivo hepatitis B virus (HBV) biosynthesis.

Liver-specific ten-eleven translocation (Tet) methylcytosine dioxygenases 2 and 3 (Tet2 plus Tet3)-deficient hepatitis B virus (HBV) transgenic mice fail to support viral biosynthesis. The levels of viral transcription and replication intermediates are dramatically reduced. Hepatitis B core antigen is only observed in a very limited number of pericentral hepatocytes in a pattern that is similar to glutamate-ammonia ligase (Glul), a ß-catenin target gene. HBV transcript abundance in adult Tet-deficient mice resembles that observed in wild-type neonatal mice. Furthermore, the RNA levels of several ß-catenin target genes including Glul, Lhpp, Notun, Oat, Slc1a2, and Tbx3 in Tet-deficient mice were also similar to that observed in wild-type neonatal mice. As HBV transcription is regulated by ß-catenin, these findings support the suggestion that neonatal Tet deficiency might limit ß-catenin target gene expression, limiting viral biosynthesis. Additionally, HBV transgene DNA displays increased 5-methylcytosine (5mC) frequency at CpG sequences consistent with neonatal Tet deficiency being responsible for decreased developmental viral DNA demethylation mediated by 5mC oxidation to 5-hydroxymethylcytosine, a process that might be responsible for the reduction in cellular ß-catenin target gene expression and viral transcription and replication.IMPORTANCEChronic hepatitis B virus (HBV) infection causes significant worldwide morbidity and mortality. There are no curative therapies available to resolve chronic HBV infections, and the small viral genome limits molecular targets for drug development. An alternative approach to drug development is to target cellular genes essential for HBV biosynthesis. In the liver, ten-eleven translocation (Tet) genes encode cellular enzymes that are not essential for postnatal mouse development but represent essential activities for viral DNA demethylation and transcription. Consequently, Tet inhibitors may potentially be developed into therapeutic agents capable of inducing and/or maintaining HBV covalently closed circular DNA methylation, resulting in transcriptional silencing and the resolution of chronic viral infection.

Distinct phenotypic spectra of hepatocellular carcinoma in liver-specific tumor suppressor-deficient hepatitis B virus transgenic mice.

The hepatitis B virus (HBV) transgenic mouse model was used to interrogate the origins of HCC heterogeneity. HBV biosynthesis was used as a marker of liver tumor heterogeneity. Principal component and correlation analysis of HBV and cellular transcript levels demonstrated major differences within and between the gene expression profiles of Apc-deficient, Apc-deficient Pten-deficient, and Pten-deficient HCC. Hence, both oncogenic stimuli and zonal hepatocyte properties determine heterogeneous HCC phenotypes. Additionally, Apc-deficient HCC display decreased expression of Apob, Otc and Tet2 relative to Pten-deficient HCC and control liver tissue suggesting their gene products may represent markers of Apc-deficient HCC. A subset of human HCC with mutations in the ß-catenin gene (CTNNB1) displayed a gene expression profile similar to that observed in the mouse Apc-deficient HCC indicating this model of liver cancer may be useful for interrogating the molecular properties of these tumors and their potential therapeutic vulnerabilities.

Selective effect of ß-catenin on nuclear receptor-dependent hepatitis B virus transcription and replication.

ß-catenin regulates HBV transcription in cell culture and viral biosynthesis in vivo in the transgenic mouse model of chronic HBV infection. Therefore, it is important to understand which transcription factor activities are coactivated by ß-catenin to enhance HBV biosynthesis. The effect of ß-catenin expression in the context of nuclear receptor-mediated HBV transcription was evaluated initially in the human embryonic kidney cell line, HEK293T. Reporter gene and viral replication assays revealed that ß-catenin can coactivate HBV transcription through some, most predominantly liver receptor homolog 1 (LRH1), but not all nuclear receptors known to activate viral biosynthesis. Similarly, ß-catenin activated nuclear receptor-mediated HBV transcription and replication in the human hepatoma cell line, Huh7, primarily through its effect on the farnesoid X receptor α (FXRα). These data indicate that ß-catenin can enhance nuclear receptor-mediated HBV biosynthesis, but the relative importance of various transcription factors is dependent upon the precise cellular environment.

Heterogeneous phenotypes of Pten-null hepatocellular carcinoma in hepatitis B virus transgenic mice parallels liver lobule zonal gene expression patterns.

Chronic HBV infection is a major cause of hepatocellular carcinoma (HCC) worldwide. The phenotypes of HCC are diverse, in part, due to mutations in distinct oncogenes and/or tumor suppressor genes. These genetic drivers of HCC development have generally been considered as major mediators of tumor heterogeneity. Using the liver-specific Pten-null HBV transgenic mouse model of chronic viral infection, a critical role for liver lobule zone-specific gene expression patterns in determining HCC phenotype and ß-catenin-dependent HBV biosynthesis is demonstrated. These observations suggest that the position of the hepatocyte within the liver lobule, and hence its intrinsic gene expression pattern at the time of cellular transformation, make critical contributions to the properties of the resulting liver tumor. These results may explain why therapies targeting pathways modulated by specific identified tumor driver genes display variable treatment efficacy.

ß-Catenin Signaling Regulates the In Vivo Distribution of Hepatitis B Virus Biosynthesis across the Liver Lobule.

ß-Catenin (Ctnnb1) supports high levels of liver gene expression in hepatocytes in proximity to the central vein functionally defining zone 3 of the liver lobule. This region of the liver lobule supports the highest levels of viral biosynthesis in wild-type hepatitis B virus (HBV) transgenic mice. Liver-specific ß-catenin-null HBV transgenic mice exhibit a stark loss of high levels of pericentral viral biosynthesis. Additionally, viral replication that does not depend directly on ß-catenin activity appears to expand to include hepatocytes of zone 1 of the liver lobule in proximity to the portal vein, a region of the liver that typically lacks significant HBV biosynthesis in wild-type HBV transgenic mice. While the average amount of viral RNA transcripts does not change, viral DNA replication is reduced approximately 3-fold. Together, these observations demonstrate that ß-catenin signaling represents a major determinant of HBV biosynthesis governing the magnitude and distribution of viral replication across the liver lobule in vivo. Additionally, these findings reveal a novel mechanism for the regulation of HBV biosynthesis that is potentially relevant to the expression of additional liver-specific genes. IMPORTANCE Viral biosynthesis is highest around the central vein in the hepatitis B virus (HBV) transgenic mouse model of chronic infection. The associated HBV biosynthetic gradient across the liver lobule is primarily dependent upon ß-catenin. In the absence of ß-catenin, the gradient of viral gene expression spanning the liver lobule is absent, and HBV replication is reduced. Therefore, therapeutically manipulating ß-catenin activity in the livers of chronic HBV carriers may reduce circulating infectious virions without greatly modulating viral protein production. Together, these changes in viral biosynthesis might limit infection of additional hepatocytes while permitting immunological clearance of previously infected cells, potentially limiting disease persistence.

Relative DNA Methylation and Demethylation Efficiencies during Postnatal Liver Development Regulate Hepatitis B Virus Biosynthesis.

Hepatitis B virus (HBV) transcription and replication increase progressively throughout postnatal liver development with maximal viral biosynthesis occurring at around 4 weeks of age in the HBV transgenic mouse model of chronic infection. Increasing viral biosynthesis is associated with a corresponding progressive loss of DNA methylation. The loss of DNA methylation is associated with increasing levels of 5-hydroxymethylcytosine (5hmC) residues which correlate with increased liver-enriched pioneer transcription factor Forkhead box protein A (FoxA) RNA levels, a rapid decline in postnatal liver DNA methyltransferase (Dnmt) transcripts, and a very modest reduction in ten-eleven translocation (Tet) methylcytosine dioxygenase expression. These observations are consistent with the suggestion that the balance between active HBV DNA methylation and demethylation is regulated by FoxA recruitment of Tet in the presence of declining Dnmt activity. These changes lead to demethylation of the viral genome during hepatocyte maturation with associated increases in viral biosynthesis. Consequently, manipulation of the relative activities of these two counterbalancing processes might permit the specific silencing of HBV gene expression with the loss of viral biosynthesis and the resolution of chronic HBV infections.IMPORTANCE HBV biosynthesis begins at birth and increases during early postnatal liver development in the HBV transgenic mouse model of chronic infection. The levels of viral RNA and DNA synthesis correlate with pioneer transcription factor FoxA transcript plus Tet methylcytosine dioxygenase-generated 5hmC abundance but inversely with Dnmt transcript levels and HBV DNA methylation. Together, these findings suggest that HBV DNA methylation during neonatal liver development is actively modulated by the relative contributions of FoxA-recruited Tet-mediated DNA demethylation and Dnmt-mediated DNA methylation activities. This mode of gene regulation, mediated by the loss of DNA methylation at hepatocyte-specific viral and cellular promoters, likely contributes to hepatocyte maturation during liver development in addition to the postnatal activation of HBV transcription and replication.

Modulation of hepatitis B virus pregenomic RNA stability and splicing by histone deacetylase 5 enhances viral biosynthesis.

Hepatitis B virus (HBV) is a worldwide health problem without curative treatments. Investigation of the regulation of HBV biosynthesis by class I and II histone deacetylases (HDACs) demonstrated that catalytically active HDAC5 upregulates HBV biosynthesis. HDAC5 expression increased both the stability and splicing of the HBV 3.5 kb RNA without altering the translational efficiency of the viral pregenomic or spliced 2.2 kb RNAs. Together, these observations point to a broader role of HDAC5 in regulating RNA splicing and transcript stability while specifically identifying a potentially novel approach toward antiviral HBV therapeutic development.

The Regulation of HBV Transcription and Replication.

Hepatitis B virus (HBV) is a major human pathogen lacking a reliable curative therapy. Current therapeutics target the viral reverse transcriptase/DNA polymerase to inhibit viral replication but generally fail to resolve chronic HBV infections. Due to the limited coding potential of the HBV genome, alternative approaches for the treatment of chronic infections are desperately needed. An alternative approach to the development of antiviral therapeutics is to target cellular gene products that are critical to the viral life cycle. As transcription of the viral genome is an essential step in the viral life cycle, the selective inhibition of viral RNA synthesis is a possible approach for the development of additional therapeutic modalities that might be used in combination with currently available therapies. To address this possibility, a molecular understanding of the relationship between viral transcription and replication is required. The first step is to identify the transcription factors that are the most critical in controlling the levels of HBV RNA synthesis and to determine their in vivo role in viral biosynthesis. Mapping studies in cell culture utilizing reporter gene constructs permitted the identification of both ubiquitous and liver-enriched transcription factors capable of modulating transcription from the four HBV promoters. However, it was challenging to determine their relative importance for viral biosynthesis in the available human hepatoma replication systems. This technical limitation was addressed, in part, by the development of non-hepatoma HBV replication systems where viral biosynthesis was dependent on complementation with exogenously expressed transcription factors. These systems revealed the importance of specific nuclear receptors and hepatocyte nuclear factor 3 (HNF3)/forkhead box A (FoxA) transcription factors for HBV biosynthesis. Furthermore, using the HBV transgenic mouse model of chronic viral infection, the importance of various nuclear receptors and FoxA isoforms could be established in vivo. The availability of this combination of systems now permits a rational approach toward the development of selective host transcription factor inhibitors. This might permit the development of a new class of therapeutics to aid in the treatment and resolution of chronic HBV infections, which currently affects approximately 1 in 30 individuals worldwide and kills up to a million people annually.

Structurally Diverse Histone Deacetylase Photoreactive Probes: Design, Synthesis, and Photolabeling Studies in Live Cells and Tissue.

Histone deacetylase (HDAC) activity is modulated in vivo by post-translational modifications and formation of multiprotein complexes. Novel chemical tools to study how these factors affect engagement of HDAC isoforms by HDAC inhibitors (HDACi) in cells and tissues are needed. In this study, a synthetic strategy to access chemically diverse photoreactive probes (PRPs) was developed and used to prepare seven novel HDAC PRPs 9-15. The class I HDAC isoform engagement by PRPs was determined in biochemical assays and photolabeling experiments in live SET-2, HepG2, HuH7, and HEK293T cell lines and in mouse liver tissue. Unlike the HDAC protein abundance and biochemical activity against recombinant HDACs, the chemotype of the PRPs and the type of cells were key in defining the engagement of HDAC isoforms in live cells. Our findings suggest that engagement of HDAC isoforms by HDACi in vivo may be substantially modulated in a cell- and tissue-type-dependent manner.

Peroxisome proliferator-activated receptor γ coactivator family members competitively regulate hepatitis b virus biosynthesis.

Transcriptional coactivators represent critical components of the transcriptional pre-initiation complex and are required for efficient gene activation. Members of the peroxisome proliferator-activated receptor gamma coactivator 1 (PGC1) family differentially regulate hepatitis b virus (HBV) biosynthesis. Whereas PGC1α has been shown to be a potent activator of HBV biosynthesis, PGC1ß only very poorly activates HBV RNA and DNA synthesis in human hepatoma (HepG2) and embryonic kidney (HEK293T) cells. Furthermore, PGC1ß inhibits PGC1α-mediated HBV biosynthesis. These observations suggest that a potential competition between human hepatoma (HepG2) and embryonic kidney (HEK293T) cells PGC1α and PGC1ß for common transcription factor target(s) may regulate HBV transcription and replication in a context and signal transduction pathway dependent manner.

PGC1α Transcriptional Adaptor Function Governs Hepatitis B Virus Replication by Controlling HBcAg/p21 Protein-Mediated Capsid Formation.

In the human hepatoma cell line Huh7, the coexpression of the coactivators peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α), cyclic AMP-responsive element binding protein binding protein (CBP), steroid receptor coactivator 1 (SRC1), and protein arginine methyltransferase 1 (PRMT1) only modestly increase hepatitis B virus (HBV) biosynthesis. However, by utilizing the human embryonic kidney cell line HEK293T, it was possible to demonstrate that PGC1α alone can support viral biosynthesis independently of the expression of additional coactivators or transcription factors. In contrast, additional coactivators failed to support robust HBV replication in the absence of PGC1α. These observations indicate that PGC1α represents a novel adaptor molecule capable of recruiting the necessary transcriptional machinery to the HBV nucleocapsid promoter to modestly enhance viral pregenomic 3.5-kb RNA synthesis. Although this change in transcription is associated with a similar modest change in hepatitis B virus core antigen polypeptide (HBcAg/p21) synthesis, it mediates a dramatic increase in viral capsid production and robust viral replication. Therefore, it is apparent that the synthesis of cytoplasmic HBcAg/p21 above a critical threshold level is required for the efficient assembly of HBV replication-competent viral capsids.IMPORTANCE Hepatitis B virus (HBV) is a major human pathogen, and novel targets for the development of additional therapeutic agents are urgently needed. Here we demonstrate that the coactivator peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) serves as a unique adaptor molecule for the recruitment of additional coactivator proteins, which can finely regulate HBV transcription. The consequence of this precise regulation of viral RNA levels by PGC1α is a subtle increase in cytoplasmic HBcAg/p21 polypeptide translation, which shifts the equilibrium from dimer formation dramatically in favor of viral capsid assembly. These findings suggest that both PGC1α and capsid assembly may represent attractive targets for the development of antiviral agents against chronic HBV infection.

Hepatic deficiency of the pioneer transcription factor FoxA restricts hepatitis B virus biosynthesis by the developmental regulation of viral DNA methylation.

The FoxA family of pioneer transcription factors regulates hepatitis B virus (HBV) transcription, and hence viral replication. Hepatocyte-specific FoxA-deficiency in the HBV transgenic mouse model of chronic infection prevents the transcription of the viral DNA genome as a result of the failure of the developmentally controlled conversion of 5-methylcytosine residues to cytosine during postnatal hepatic maturation. These observations suggest that pioneer transcription factors such as FoxA, which mark genes for expression at subsequent developmental steps in the cellular differentiation program, mediate their effects by reversing the DNA methylation status of their target genes to permit their ensuing expression when the appropriate tissue-specific transcription factor combinations arise during development. Furthermore, as the FoxA-deficient HBV transgenic mice are viable, the specific developmental timing, abundance and isoform type of pioneer factor expression must permit all essential liver gene expression to occur at a level sufficient to support adequate liver function. This implies that pioneer transcription factors can recognize and mark their target genes in distinct developmental manners dependent upon, at least in part, the concentration and affinity of FoxA for its binding sites within enhancer and promoter regulatory sequence elements. This selective marking of cellular genes for expression by the FoxA pioneer factor compared to HBV may offer the opportunity for the specific silencing of HBV gene expression and hence the resolution of chronic HBV infections which are responsible for approximately one million deaths worldwide annually due to liver cirrhosis and hepatocellular carcinoma.

Krüppel-like factor 9 promotes hepatic cytochrome P450 2D6 expression during pregnancy in CYP2D6-humanized mice.

Cytochrome P450 2D6 (CYP2D6), a major drug-metabolizing enzyme, is responsible for metabolism of approximately 25% of marketed drugs. Clinical evidence indicates that metabolism of CYP2D6 substrates is increased during pregnancy, but the underlying mechanisms remain unclear. To identify transcription factors potentially responsible for CYP2D6 induction during pregnancy, a panel of genes differentially expressed in the livers of pregnant versus nonpregnant CYP2D6-humanized (tg-CYP2D6) mice was compiled via microarray experiments followed by real-time quantitative reverse-transcription polymerase chain reaction(qRT-PCR) verification. As a result, seven transcription factors-activating transcription factor 5 (ATF5), early growth response 1 (EGR1), forkhead box protein A3 (FOXA3), JUNB, Krüppel-like factor 9 (KLF9), KLF10, and REV-ERBα-were found to be up-regulated in liver during pregnancy. Results from transient transfection and promoter reporter gene assays indicate that KLF9 itself is a weak transactivator of CYP2D6 promoter but significantly enhances CYP2D6 promoter transactivation by hepatocyte nuclear factor 4 (HNF4α), a known transcriptional activator of CYP2D6 expression. The results from deletion and mutation analysis of CYP2D6 promoter activity identified a KLF9 putative binding motif at -22/-14 region to be critical in the potentiation of HNF4α-induced transactivation of CYP2D6. Electrophoretic mobility shift assays revealed a direct binding of KLF9 to the putative KLF binding motif. Results from chromatin immunoprecipitation assay showed increased recruitment of KLF9 to CYP2D6 promoter in the livers of tg-CYP2D6 mice during pregnancy. Taken together, our data suggest that increased KLF9 expression is in part responsible for CYP2D6 induction during pregnancy via the potentiation of HNF4α transactivation of CYP2D6.

Hepatocyte-targeted RNAi therapeutics for the treatment of chronic hepatitis B virus infection.

RNA interference (RNAi)-based therapeutics have the potential to treat chronic hepatitis B virus (HBV) infection in a fundamentally different manner than current therapies. Using RNAi, it is possible to knock down expression of viral RNAs including the pregenomic RNA from which the replicative intermediates are derived, thus reducing viral load, and the viral proteins that result in disease and impact the immune system's ability to eliminate the virus. We previously described the use of polymer-based Dynamic PolyConjugate (DPC) for the targeted delivery of siRNAs to hepatocytes. Here, we first show in proof-of-concept studies that simple coinjection of a hepatocyte-targeted, N-acetylgalactosamine-conjugated melittin-like peptide (NAG-MLP) with a liver-tropic cholesterol-conjugated siRNA (chol-siRNA) targeting coagulation factor VII (F7) results in efficient F7 knockdown in mice and nonhuman primates without changes in clinical chemistry or induction of cytokines. Using transient and transgenic mouse models of HBV infection, we show that a single coinjection of NAG-MLP with potent chol-siRNAs targeting conserved HBV sequences resulted in multilog repression of viral RNA, proteins, and viral DNA with long duration of effect. These results suggest that coinjection of NAG-MLP and chol-siHBVs holds great promise as a new therapeutic for patients chronically infected with HBV.

Independent activation of hepatitis B virus biosynthesis by retinoids, peroxisome proliferators, and bile acids.

In the human hepatoma cell line HepG2, retinoic acid, clofibric acid, and bile acid treatment can only modestly increase hepatitis B virus (HBV) biosynthesis. Utilizing the human embryonic kidney cell line 293T, it was possible to demonstrate that the retinoid X receptor α (RXRα) plus its ligand can support viral biosynthesis independently of additional nuclear receptors. In addition, RXRα/peroxisome proliferator-activated receptor α (PPARα) and RXRα/farnesoid X receptor α (FXRα) heterodimeric nuclear receptors can also mediate ligand-dependent HBV transcription and replication when activated by clofibric acid and bile acid, respectively, independently of a requirement for the ligand-dependent activation of RXRα. These observations indicate that there are at least three possible modes of ligand-mediated activation of HBV transcription and replication existing within hepatocytes, suggesting that multiple independent mechanisms control viral production in the livers of infected individuals.

Limited effects of bile acids and small heterodimer partner on hepatitis B virus biosynthesis in vivo.

Multiple nuclear receptors, including hepatocyte nuclear factor 4α (HNF4α), retinoid X receptor α (RXRα) plus peroxisome proliferator-activated receptor α (PPARα), RXRα plus farnesoid X receptor α (FXRα), liver receptor homolog 1 (LRH1), and estrogen-related receptors (ERRs), have been shown to support efficient viral biosynthesis in nonhepatoma cells in the absence of additional liver-enriched transcription factors. Although HNF4α has been shown to be critical for the developmental expression of hepatitis B virus (HBV) biosynthesis in the liver, the relative importance of the various nuclear receptors capable of supporting viral transcription and replication in the adult in vivo has not been clearly established. To investigate the role of the nuclear receptor FXR and the corepressor small heterodimer partner (SHP) in viral biosynthesis in vivo, SHP-expressing and SHP-null HBV transgenic mice were fed a bile acid-supplemented diet. The increased FXR activity and SHP expression levels resulting from bile acid treatment did not greatly modulate HBV RNA and DNA synthesis. Therefore, FXR and SHP appear to play a limited role in modulating HBV biosynthesis, suggesting that alternative nuclear receptors are more critical determinants of viral transcription in the HBV transgenic mouse model of chronic viral infection. These observations suggest that hepatic bile acid levels or therapeutic agents targeting FXR may not greatly modulate viremia during natural infection.

Role of peroxisome proliferator-activated receptor gamma coactivator 1alpha in AKT/PKB-mediated inhibition of hepatitis B virus biosynthesis.

Hepatitis B virus (HBV) transcription and replication are essentially restricted to hepatocytes because liver-enriched transcription factors govern viral RNA synthesis. The level of transcription from the HBV promoters depends on both the transcription factors binding to these regulatory sequence elements and their ability to recruit coactivators capable of mediating assembly of the transcription preinitiation complex containing RNA polymerase II. Nuclear receptors are a primary determinant of HBV pregenomic RNA synthesis and, hence, viral replication. Peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) enhances the activity of nuclear receptors and, consequently, HBV biosynthesis. PGC1α is also an important target of signal transduction pathways involved in hepatic glucose and lipid homeostasis, suggesting that this coactivator may have an important role in modulating HBV biosynthesis under various physiological conditions. Consistent with this suggestion, v-akt murine thymoma viral oncogene homolog/protein kinase B (AKT/PKB) is shown to modulate PGC1α activity and, hence, HBV transcription and replication in a cell line-specific manner. In addition, AKT can modulate HBV replication in some but not all cell lines at a posttranscriptional step in the viral life cycle. These observations demonstrate that growth and nutritional signals have the capacity to influence viral production, but the magnitude of these effects will depend on the precise cellular context in which they occur.

Multiple nuclear receptors may regulate hepatitis B virus biosynthesis during development.

Hepatitis B virus (HBV) replicates by the reverse transcription of the viral 3.5 kb 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 3.5 kb 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 3.5 kb pregenomic RNA transcription and replication.

Distinct regulation of hepatitis B virus biosynthesis by peroxisome proliferator-activated receptor gamma coactivator 1alpha and small heterodimer partner in human hepatoma cell lines.

The human hepatoma cell lines HepG2 and Huh7 have been used extensively to study hepatitis B virus (HBV) transcription and replication. Both cell lines support transcription of the 3.5-kb viral pregenomic RNA and subsequent viral DNA synthesis by reverse transcription. The effects of the coactivator peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC1alpha) and corepressor small heterodimer partner (SHP) on HBV transcription and replication mediated by nuclear receptors were examined in the context of individual nuclear receptors in nonhepatoma cells and in hepatoma cells in an attempt to determine the relative contribution of the various nuclear receptors to viral biosynthesis in the hepatoma cells. PGC1alpha and SHP modulated viral biosynthesis differently in the human hepatoma cell lines HepG2 and Huh7, indicating distinct modes of transcriptional regulation. Consistent with this suggestion, it appears that retinoid X receptor alpha/farnesoid X receptor alpha and liver receptor homolog 1 or estrogen-related receptor beta (ERRbeta) may contribute to the majority of the viral replication observed in HepG2 cells, whereas ERRalpha and ERRgamma are probably responsible for the majority of viral biosynthesis in Huh7 cells. Therefore, this approach indicates that the transcriptional regulation of HBV biosynthesis in HepG2 and Huh7 cells is primarily controlled by different transcription factors.

Peroxisome proliferator-activated receptor gamma Coactivator 1alpha and small heterodimer partner differentially regulate nuclear receptor-dependent hepatitis B virus biosynthesis.

Hepatitis B virus (HBV) biosynthesis involves the transcription of the 3.5-kb viral pregenomic RNA, followed by its reverse transcription into viral DNA. Consequently, the modulation of viral transcription influences the level of virus production. Nuclear receptors are the only transcription factors known to support viral pregenomic RNA transcription and replication. The coactivator peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC1alpha) and corepressor small heterodimer partner (SHP) have central roles in regulating energy homeostasis in the liver by modulating the transcriptional activities of nuclear receptors. Therefore, the effect of PGC1alpha and SHP on HBV transcription and replication mediated by nuclear receptors was examined in the context of individual nuclear receptors in nonhepatoma cells and in hepatoma cells. This analysis indicated that viral replication mediated by hepatocyte nuclear factor 4alpha, retinoid X receptor alpha (RXRalpha) plus peroxisome proliferator-activated receptor alpha (PPARalpha), and estrogen-related receptor (ERR) displayed differential sensitivity to PGC1alpha activation and SHP inhibition. The effects of PGC1alpha and SHP on viral biosynthesis in the human hepatoma cell line Huh7 were similar to those observed in the nonhepatoma cells expressing ERRalpha and ERRgamma. This suggests that these nuclear receptors, potentially in combination with RXRalpha plus PPARalpha, may have a major role in governing HBV transcription and replication in this cell line. Additionally, this functional approach may help to distinguish the transcription factors in various liver cells governing viral biosynthesis under a variety of physiologically relevant conditions.