Chronic hepatitis B virus (HBV) infection affects nearly 300 million people worldwide. Progress in understanding HBV immunopathogenesis and developing curative therapies has been hindered by the lack of suitable small animal models. HBV exhibits strict host and tissue tropism, with productive infection largely restricted to human and chimpanzee hepatocytes. Murine hepatocytes are resistant to HBV infection, even with ectopic expression of the human HBV entry receptor sodium taurocholate cotransporting polypeptide (huNTCP), because they apparently fail to form covalently closed circular DNA (cccDNA), the viral episome required for productive infection and persistence. To investigate the mechanisms restricting HBV infection in murine cells, we developed a piggyBac transposon-based system that efficiently generates inducible stable cell lines supporting HBV replication and cccDNA formation via intracellular amplification-a pathway that shares similar nucleocapsid uncoating and nuclear import of relaxed circular DNA (rcDNA) that occur during de novo infection. Remarkably, all tested murine hepatocyte and hepatoma cell lines, across multiple mouse genetic backgrounds, supported cccDNA formation at levels comparable to human cells, indicating that nucleocapsid uncoating and rcDNA nuclear import are not limited in mice. Given that huNTCP-expressing murine hepatocytes support hepatitis D virus infection, which shares early entry events with HBV, our findings reveal that the dominant restriction to HBV infection in huNTCP-expressing murine hepatocytes lies at a late entry step preceding nucleocapsid uncoating. By defining this mechanistic block, our study advances understanding of HBV host tropism and provides a foundation for facilitating development of fully HBVsusceptible mouse models.

In the hepatis B virus (HBV) transgenic mouse model of chronic infection, the forkhead box protein A/hepatocyte nuclear factor 3 (Foxa/HNF3) family of pioneer transcription factors are required to support postnatal viral demethylation and subsequent HBV transcription and replication. Liver-specific Foxa-deficient mice with hepatic expression of only Foxa3 do not support HBV replication but display biliary epithelial hyperplasia with bridging fibrosis. However, liver-specific Foxa-deficient mice with hepatic expression of only Foxa1 or Foxa2 also successfully restrict viral transcription and replication but display only minimal alterations in liver physiology. These observations suggest that the level of Foxa activity, rather than the combination of specific Foxa genes, is a key determinant of HBV biosynthesis. Together, these findings suggest that targeting Foxa activity could lead to HBV DNA methylation and transcriptional inactivation, resulting in the resolution of chronic HBV infections that are responsible for approximately one million deaths annually worldwide. IMPORTANCE:The current absence of curative therapies capable of resolving chronic hepatis B virus (HBV) infection is a major clinical problem associated with considerable morbidity and mortality. The small viral genome limits molecular targets for drug development, suggesting that the identification of cellular factors essential for HBV biosynthesis may represent alternative targets for therapeutic intervention. Genetic Foxa deficiency in the neonatal liver of HBV transgenic mice leads to the transcriptional silencing of viral DNA by CpG methylation without affecting viability or displaying an obvious phenotype. Therefore, limiting liver Foxa activity therapeutically may lead to the methylation of viral covalently closed circular DNA (cccDNA), resulting in its transcriptional silencing and ultimately the resolution of chronic HBV infection.

Human cytomegalovirus (HCMV) infects a wide range of cell types in the body, including a variety of epithelial cell types. Despite the significance of epithelial cells during infection, HCMV has been difficult to study in epithelial cells. In this study, we examined HCMV infection in mammary and prostate epithelial cell lines, finding that the virus establishes a semi-permissive, biosynthetically abortive state. Building on previous work, we hypothesized that shifting epithelial cells to a mesenchymal cell state would restore HCMV biosynthesis and progeny production. To test this hypothesis, we induced epithelial-to-mesenchymal transition (EMT) using TGF-β and the EMT-transcription factor (EMT-TF) SNAIL. We found that shifting strongly epithelial cell lines to a mesenchymal cell state shifted HCMV infection from a semi-permissive to fully permissive state. This effect appeared to involve two distinct mechanisms: EMT-sensitive enhancement of viral entry and EMT-sensitive enhancement of viral mRNA translation. Although the precise mechanisms remain elusive, our findings identify the epithelial-mesenchymal cell state axis as an important regulator of HCMV infection and provide new insights into how cellular differentiation states influence viral replication. They also raise the possibility that the EMT pathway, a fundamental pathway involved in development and cancer metastasis, could regulate HCMV infection in-vivo, potentially contributing to viral persistence or pathogenesis in epithelial tissues. ### Competing Interest Statement The authors have declared no competing interest.

Chronic 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.

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 β-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.

β-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 β-catenin to enhance HBV biosynthesis. The effect of β-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 β-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, β-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 β-catenin can enhance nuclear receptor-mediated HBV biosynthesis, but the relative importance of various transcription factors is dependent upon the precise cellular environment.

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 β-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.

β-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 β-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 β-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 β-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 β-catenin. In the absence of β-catenin, the gradient of viral gene expression spanning the liver lobule is absent, and HBV replication is reduced. Therefore, therapeutically manipulating β-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.

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.

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.

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.

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.

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, PGC1β only very poorly activates HBV RNA and DNA synthesis in human hepatoma (HepG2) and embryonic kidney (HEK293T) cells. Furthermore, PGC1β inhibits PGC1α-mediated HBV biosynthesis. These observations suggest that a potential competition between human hepatoma (HepG2) and embryonic kidney (HEK293T) cells PGC1α and PGC1β for common transcription factor target(s) may regulate HBV transcription and replication in a context and signal transduction pathway dependent manner.

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.

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.

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 vs. nonpregnant CYP2D6-humanized (tg- CYP2D6) mice was compiled via microarray experiments followed by qRT-PCR verification. As a result, seven transcription factors (ATF5, EGR1, FOXA3, JUNB, KLF9, KLF10, and REV-ERB α ) were found to be upregulated 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 HNF4 α , 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 HNF4 α -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 HNF4 α transactivation of CYP2D6. role of promoter contexts in modulating HNF4 α transactivation of its target genes and the presence of additional regulatory factors potentially involved in CYP2D6 regulation during pregnancy. In this study, we show that KLF9 expression is upregulated during pregnancy in the livers of tg-CYP2D6 mice. The roles of KLF9 in HNF4 α transactivation of the CYP2D6 promoter as well as in CYP2D6 induction during pregnancy were investigated. Our results illustrate the interplay among hepatic transcription factors KLF9, HNF4 α , and SHP contributing to the regulation of CYP2D6 expression during pregnancy. Affymetrix GeneChip list differentially genes at term postpartum, compared control group (virgin). prediction of putative factor binding sites performed using These results may provide a basis to improve drug therapy during pregnancy. Also, considering the roles of CYP2D6 in the pathophysiology of neurological diseases (in addition to drug metabolism), better understanding of the regulation of CYP2D6 expression may allow for the identification of new drug targets. In this regard, whether the regulatory pathways involving KLF9, SHP, and HNF4 α play a role in governing CYP2D6 expression in non-pregnant subjects is currently being investigated.

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.