HBV Gene Expression Research Articles

Long-term HBV infection of engineered cultures of induced pluripotent stem cell-derived hepatocytes.

HBV infects ~257 million people and can cause hepatocellular carcinoma. Since current drugs are not curative, novel therapies are needed. HBV infects chimpanzee and human livers. However, chimpanzee studies are severely restricted and cost-prohibitive, while transgenic/chimeric mouse models that circumvent the species barrier lack natural HBV infection and disease progression. Thus, in vitro human models of HBV infection are useful in addressing the above limitations. Induced pluripotent stem cell-derived hepatocyte-like cells mitigate the supply limitations of primary human hepatocytes and the abnormal proliferation/functions of hepatoma cell lines. However, variable infection across donors, deficient drug metabolism capacity, and/or low throughput limit iHep utility for drug development. We developed an optimal pipeline using combinations of small molecules, Janus kinase inhibitor, and 3',5'-cAMP to infect iHep-containing micropatterned co-cultures (iMPCC) with stromal fibroblasts within 96-well plates with serum-derived HBV and cell culture-derived HBV (cHBV). Polyethylene glycol was necessary for cell-derived HBV but not for serum-derived HBV infection. Unlike iHep monocultures, iMPCCs created from 3 iHep donors could sustain HBV infection for 2+ weeks. Infected iMPCCs maintained high levels of differentiated functions, including drug metabolism capacity. HBV antigen secretion and gene expression patterns in infected iMPCCs in pathways such as fatty acid metabolism and cholesterol biosynthesis were comparable to primary human hepatocyte-MPCCs. Furthermore, iMPCCs could help elucidate the effects of interferons and direct-acting antiviral drugs on the HBV lifecycle and any hepatotoxicity; iMPCC response to compounds was similar to primary human hepatocyte-MPCCs. The iMPCC platform can enable the development of safe and efficacious drugs against HBV and ultimately help elucidate genotype-phenotype relationships in HBV pathogenesis.

Osthole in Bushen Formula May Block HBV Gene Expression throughNotch and NF-κB Pathways

Osthole is a natural coumarin in the Bushen formulation. Previous studies reported that it has the potential todownregulate Notch and NFκB, which are important signaling pathways in cells. Therefore, we predicted that ostiolemight affect HBV gene expression by down-regulating these two signaling pathways. This study provides experimentalprotocols to apply osthole to mouse hepatocytes and detect the strength of Notch and NFκB signaling pathways inthe cells and HBV gene expression levels. In this work, all possible results were analyzed. Notch signal intensity wasmeasured by assaying NICD, IκB measured NFκB signal intensity, and RT-PCR measured HBV gene expression level.The study of Osthole is meaningful for the research on Chinese medicine. This study will allow a better explanation ofthe mechanism of Bush’s formula and explore the more generalized effects of herbal medicines

Hepatitis B virus promotes hepatocellular carcinoma development by activating GP73 to repress the innate immune response

BackgroundHepatitis B virus (HBV) causes acute and chronic infection in the clinic. Hepatocellular carcinoma (HCC) is closely linked to HBV infection. Serum Golgi protein 73 (GP73) increases during HBV infection. However, the role of GP73 during HBV infection and the occurrence of HBV-related HCC is still poorly understood.MethodsThe underlying role of HBV-induced GP73 in regulating HCC development was investigated in this study. GP73 expression in HBV-related clinical HCC tissues and in HBV-infected hepatoma cells and primary human hepatocytes was evaluated by immunohistochemistry, ELISAs, Western blotting and quantitative real-time PCR (qRT-PCR) analysis. Tumorigenicity of GP73 overexpressed cells was detected by flow cytometry, qRT-PCR, xenograft nude mouse analyses and sphere formation assays. The effects of GP73 and HBV infection on host innate immune responses in hepatocytes were further investigated by Western blotting and qRT-PCR analysis.ResultsInitially, we confirmed that HBV-positive HCC tissues had significantly higher expression of GP73. Ectopic expression of the HBV gene could induce GP73 expression in primary human hepatocytes and hepatoma cells in vitro. In addition, we discovered that GP73 promotes HCC in both normal liver cells and hepatoma cells. We also found that ectopic expression of HBV genes increases GP73 expression, suppressing the host's innate immune responses in hepatocytes.ConclusionsOur results demonstrate that HBV facilitates HCC development by activating GP73 to repress the host's innate immune response. This study adds to our understanding of the pathogenesis of HBV infection-induced HCC. The findings also provide preclinical support for GP73 as a potential HCC prevention or treatment target.

Human hepatocyte-enriched miRNA-192-3p promotes HBV replication through inhibiting Akt/mTOR signalling by targeting ZNF143 in hepatic cell lines

ABSTRACT Previous studies have revealed multiple tissue- or cell-specific or enriched miRNA profiles. However, miRNA profiles enriched in hepatic cell types and their effect on HBV replication have not been well elucidated. In this study, primary human hepatocytes (PHHs), Kupffer cells (KCs), liver sinusoidal endothelial cells (LSECs), and hepatic stellate cells (HSCs) were prepared from liver specimens of non-HBV-infected patients. Four hepatic cell type-enriched miRNA profiles were identified from purified liver cells miRNA microarray assay. The results revealed that 12 miRNAs, including miR-122-5p and miR-192-3p were PHH-enriched; 9 miRNAs, including miR-142-5p and miR-155-5p were KC-enriched; 6 miRNAs, including miR-126-3p and miR-222-3p were LSEC-enriched; and 14 miRNAs, including miR-214-3p and miR-199a-3p were HSC-enriched. By testing the effect of 11 PHH-enriched miRNAs on HBV production, we observed that miR-192-3p had the greatest pro-virus effect in hepatic cell lines. Moreover, we further found that miR-192-3p promoted HBV replication and gene expression through inhibiting Akt/mTOR signalling by direct targeting of ZNF143 in HepG2.2.15 cells. Additionally, the serum and hepatic miR-192-3p expression levels were significantly higher in chronic hepatitis B patients than in healthy controls and serum miR-192-3p positively correlated with the serum levels of HBV DNA and HBsAg. Collectively, we identified miRNA profiles enriched in four hepatic cell types and revealed that PHH-enriched miR-192-3p promoted HBV replication through inhibiting Akt/mTOR signalling by direct targeting of ZNF143 in hepatic cell lines. Our study provides a specific perspective for the role of hepatic cell type-enriched miRNA in interaction with viral replication and various liver pathogenesis.

Hepatocyte Growth Factor-Dependent Antiviral Activity of Activated cdc42-Associated Kinase 1 Against Hepatitis B Virus.

Activated cdc42-associated kinase 1 (ACK1) is a well-known non-receptor tyrosine kinase that regulates cell proliferation and growth through activation of cellular signaling pathways, including mitogen-activated protein kinase (MAPK). However, the anti-HBV activity of ACK1 has not been elucidated. This study aimed to investigate the role of ACK1 in the HBV life cycle and the mechanism underlying the anti-HBV activity of ACK1. To examine the antiviral activity of ACK1, we established HepG2-ACK1 cells stably overexpressing ACK1. The HBV life cycle, including HBeAg/HBsAg secretion, HBV DNA/transcription, and enhancer activity, was analyzed in HepG2 and HepG2-ACK1 cells with HBV replication-competent HBV 1.2mer (HBV 1.2). Finally, the anti-HBV activity of ACK1 was examined in an HBV infection system. ACK1 suppressed HBV gene expression and transcription in HepG2 and HepG2-ACK1 cells. Furthermore, ACK1 inhibited HBV replication by decreasing viral enhancer activity. ACK1 exhibited its anti-HBV activity via activation of Erk1/2, which consequently downregulated the expression of HNF4α binding to HBV enhancers. Furthermore, hepatocyte growth factor (HGF) induced ACK1 expression at an early stage. Finally, ACK1 mediated the antiviral effect of HGF in the HBV infection system. These results indicated that ACK1 induced by HGF inhibited HBV replication at the transcriptional level by activating the MAPK-HNF signaling pathway. Our findings suggest that ACK1 is a potentially novel upstream molecule of MAPK-mediated anti-HBV activity.

From the Editor’s Desk...

From the Editor’s Desk...

The Functional and Antiviral Activity of Interferon Alpha-Inducible IFI6 Against Hepatitis B Virus Replication and Gene Expression.

Hepatitis B virus is an enveloped DNA virus, that infects more than three hundred and sixty million people worldwide and leads to severe chronic liver diseases. Interferon-alpha inducible protein 6 (IFI6) is an IFN-stimulated gene (ISG) whose expression is highly regulated by the stimulation of type I IFN-alpha that restricts various kinds of virus infections by targeting different stages of the viral life cycle. This study aims to investigate the antiviral activity of IFI6 against HBV replication and gene expression. The IFI6 was highly induced by the stimulation of IFN-α in hepatoma cells. The overexpression of IFI6 inhibited while knockdown of IFI6 elevated replication and gene expression of HBV in HepG2 cells. Further study determined that IFI6 inhibited HBV replication by reducing EnhII/Cp of the HBV without affecting liver enriched transcription factors that have significant importance in regulating HBV enhancer activity. Furthermore, deletion mutation of EnhII/Cp and CHIP analysis revealed 100 bps (1715-1815 nt) putative sites involved in IFI6 mediated inhibition of HBV. Detailed analysis with EMSA demonstrated that 1715-1770 nt of EnhII/Cp was specifically involved in binding with IFI6 and restricted EnhII/Cp promoter activity. Moreover, IFI6 was localized mainly inside the nucleus to involve in the anti-HBV activity of IFI6. In vivo analysis based on the hydrodynamic injection of IFI6 expression plasmid along with HBV revealed significant inhibition of HBV DNA replication and gene expression. Overall, our results suggested a novel mechanism of IFI6 mediated HBV regulation that could develop potential therapeutics for efficient HBV infection treatment.

Translatomic profiling reveals novel self-restricting virus-host interactions during HBV infection

HBV remains a global threat to human health. It remains incompletely understood how HBV self-restricts in the host during most adult infections. Thus, we performed multi-omics analyses to systematically interrogate HBV-host interactions and the life cycle of HBV. RNA-sequencing and ribosome profiling were conducted with cell-based models for HBV replication and gene expression. The novel translational events or products hereby detected were then characterized, and functionally assessed in both cell and mouse models. Moreover, quasi-species analyses of HBV subpopulations were conducted with patients at immune tolerance or activation phases, using next- or third-generation sequencing. We identified EnhI-SL (Enhancer I-stem loop) as a new cis element in the HBV genome; mutations disrupting EnhI-SL were found to elevate viral polymerase expression. Furthermore, while re-discovering HpZ/P', a previously under-explored isoform of HBV polymerase, we also identified HBxZ, a novel short isoform of HBX. Having confirmed their existence, we functionally characterized them as potent suppressors of HBV gene expression and genome replication. Mechanistically, HpZ/P' was found to repress HBV gene expression partially by interacting with, and sequestering SUPV3L1. Activation of the host immune system seemed to reduce the abundance of HBV mutants deficient in HpZ/P' or with disruptions in EnhI-SL. Finally, SRSF2, a host RNA spliceosome protein that is downregulated by HBV, was found to promote the splicing of viral pre-genomic RNA and HpZ/P' biogenesis. This study has identified multiple self-restricting HBV-host interactions. In particular, SRSF2-HpZ/P' appeared to constitute another negative feedback mechanism in the HBV life cycle. Targeting host splicing machinery might thus represent a strategy to intervene in HBV-host interactions. There remain many unknowns about the natural history of HBV infection in adults. Herein, we identified new HBV-host mechanisms which could be responsible for self-restricting infections. Targeting these mechanisms could be a promising strategy for the treatment of HBV infections.

Antiviral activity of interferon-stimulated gene 20, as a putative repressor binding to hepatitis B virus enhancer II and core promoter.

Background and AimInterferon‐stimulated gene 20 (ISG20) is an interferon‐inducible exonuclease that inhibits the replication of several RNA viruses. In patients with chronic hepatitis B, ISG20 expression is related to the interferon‐α treatment response. However, the molecular mechanism of ISG20‐mediated anti‐hepatitis B virus (HBV) activity is unclear.MethodsWe have investigated the effect of ISG20 on antiviral activity to address that. The life cycle of HBV was analyzed by the ectopic expression of ISG20 in HepG2 and HepG2‐NTCP cells. Finally, to provide physiological relevance of our study, the expression of ISG20 from chronic hepatitis B patients was examined.ResultsInterferon‐stimulated gene 20 was mainly induced by interferon‐β and dramatically inhibited HBV replication. In addition, ISG20 decreased HBV gene expression and transcription. Although ISG20 inhibited HBV replication by reducing viral enhancer activity, the expression of transcription factors that bind the HBV enhancer was not affected. Particularly, ISG20 suppressed HBV enhancer activity by binding to the enhancer II and core promoter (EnhII/Cp) region.ConclusionOur findings suggest that ISG20 exerts the anti‐HBV activity by acting as a putative repressor binding to the HBV EnhII/Cp region.

Regulation of HBV replication and gene expression by miR-501-3p via targeting ZEB2 in hepatocellular carcinoma.

This article has been withdrawn at the request of the author(s) and/or editor. The Publisher apologizes for any inconvenience this may cause.

ERK1/2-HNF4α axis is involved in epigallocatechin-3-gallate inhibition of HBV replication.

Epigallocatechin gallate (EGCG), a major polyphenol in green tea, exhibits diverse biological activities. Previous studies show that EGCG could effectively suppress HBV gene expression and replication, but the role of EGCG in HBV replication and its underlying mechanisms, especially the signaling pathways involved, remain unclear. In this study we investigated the mechanisms underlying EGCG inhibition on HBV replication with a focus on the signaling pathways. We showed that EGCG (12.5−50 μM) dose-dependently inhibited HBV gene expression and replication in HepG2.2.15 cells. Similar results were observed in HBV mice receiving EGCG (25 mg· kg−1· d−1, ip) for 5 days. In HepG2.2.15 cells, we showed that EGCG (12.5−50 μM) significantly activate ERK1/2 MAPK signaling, slightly activate p38 MAPK and JAK2/STAT3 signaling, while had no significant effect on the activation of JNK MAPK, PI3K/AKT/mTOR and NF-κB signaling. By using specific inhibitors of these signaling pathways, we demonstrated that ERK1/2 signaling pathway, but not other signaling pathways, was involved in EGCG-mediated inhibition of HBV transcription and replication. Furthermore, we showed that EGCG treatment dose-dependently decreased the expression of hepatocyte nuclear factor 4α (HNF4α) both at the mRNA and protein levels, which could be reversed by pretreatment with the ERK1/2 inhibitor PD98059 (20 μM). Moreover, we revealed that EGCG treatment dose-dependently inhibited the activity of HBV core promoter and the following HBV replication. In summary, our results demonstrate that EGCG inhibits HBV gene expression and replication, which involves ERK1/2-mediated downregulation of HNF4α.These data reveal a novel mechanism for EGCG to inhibit HBV gene expression and replication.

Occult Hepatitis B Virus Infection: A Review Update

Occult HBV infection (OBI) is defined as HBV DNA detection in serum or in the liver by sensitive diagnostic tests in HbsAg negative individuals with or without serologic markers of previous viral exposure. Since OBI was first described in the late 1970s, there has been increasing concern in this topic. OBI can be both a source of virus contamination in blood and organ donations and the reservoir for full blown hepatitis after reactivation. HBV reactivation depends on viral and host factors but these associations have not been analyzed thoroughly. Although the exact mechanism of OBI yet not proved, intrahepatic persistence of viral covalently closed circular DNA under the host’s strong immune suppression of HBV replication and gene expression seems to be a cause. Current evidence suggests that OBI can favour the progression of fibrosis, cirrhosis, hepatocellular carcinoma and post transfusion hepatitis (PTH). Epidemiological data regarding the global prevalence of OBI vary due to the use of detection methods of different sensitivity and specificity. Appropriate diagnostic techniques must be adopted. Sensitive HBV DNA amplification assay is the gold standard assay for detection of OBI.Bangladesh Journal of Infectious Diseases 2018;5(1):32-38

MiR-200c targets nuclear factor IA to suppress HBV replication and gene expression via repressing HBV Enhancer I activity

BackgroundHepatitis B virus (HBV) chronic infection is a health problem in the worldwide, with a underlying higher risk of liver cirrhosis and hepaticocellular carcinoma. A number of studies indicate that microRNAs (miRNAs) play vital roles in HBV replication. This study was designed to explore the potential molecular mechanism of miR-200c in HBV replication. MethodsThe expression of miR-200c, nuclear factor IA (NFIA) mRNA, HBV DNA, and HBV RNA (pregenomic RNA (pgRNA), and total RNA) were measured by qRCR. The levels of HBsAg and HBeAg were detected by ELISA. NFIA expression at protein level was measured by western blot. The direct interaction between miR-200c and NFIA were identified by Targetscan software and Dual-Luciferase reporter analysis. Enhance I activity were detected by Dual-Luciferase reporter assay. ResultsmiR-200c expression was prominently reduced in pHBV1.3-tranfected Huh7 and in stable HBV-producing cell line (HepG2.2.15). The enforced expression of miR-200c significantly suppressed HBV replication, as demonstrated by the reduced levels of HBV protein (HBsAg and HBeAg) and, DNA and RNA (pgRNA and total RNA) levels. NFIA was proved to be a target of miR-200c and NFIA overexpression notably stimulated HBV replication. In addition, the inhibitory effect of miR-200c on HBV Enhance I activity was abolished following restoration of NFIA. ConclusionsmiR-200c repressed HBV replication by directly targeting NFIA, which might provide a novel therapeutic target for HBV infection.

Inhibition of HBV replication by delivering the dual-gene expression vector pHsa-miR16-siRNA in HepG2.2.15 cells.

This study aimed to construct the dual-gene expression vector pHsa-miR16-siRNA which can express human miR-16 and HBV X siRNA, and examine its regulatory effect on HBV gene expression in the HepG2.2.15 cell line. The expression vectors siR-1583 and pHsa-miR16-siRNA were designed and constructed. HepG2.2.15 cells were transfected with the empty vector, siR-1583, pmiR-16 and pHsa-miR16-siRNA, respectively. ELISA was performed to measure the expression of HBsAg and HBeAg in the culture supernatant 48 and72 h post transfection. Fluorescence quantitative PCR was used to measure the HBV mRNA degradation efficiency and HBV DNA copy number. The results showed that the expression of HBV genes was significantly inhibited in HepG2.2.15 cells transfected with siR-1583, pmiR-16 and pHsa-miR16-siRNA, respectively, when compared with that in cells transfected with the empty vectors, with the inhibitory effect of pHsa-miR16-siRNA being the most significant. ELISA showed that the inhibitory rates of HBsAg and HBeAg in pHsa-miR16-siRNA transfected cells were correspondingly 87.3% and 85.0% at 48 h, and 88.6% and 86.5% at 72 h post transfection (P<0.01 vs. control group). RT-PCR showed that the level of HBV mRNA decreased by 80.2% (t=-99.22, P<0.01), the genomic HBV DNA by 92.8% (t=-73.06, P<0.01), and the supernatant of HBV DNA copy number by 89.8% (t=-47.13, P<0.01) in pHsa-miR16-siRNA transfected group. It was suggested that the dual-gene expression vector pHsa-miR16-siRNA can inhibit the replication of HBV more efficiently than a single-gene expression vector.

A novel orally available small molecule that inhibits hepatitis B virus expression

The hallmarks of chronic HBV infection are a high viral load (HBV DNA) and even higher levels (>100-fold in excess of virions) of non-infectious membranous particles containing the tolerogenic viral S antigen (HBsAg). Currently, standard treatment effectively reduces viremia but only rarely results in a functional cure (defined as sustained HBsAg loss). There is an urgent need to identify novel therapies that reduce HBsAg levels and restore virus-specific immune responsiveness in patients. We report the discovery of a novel, potent and orally bioavailable small molecule inhibitor of HBV gene expression (RG7834). RG7834 antiviral characteristics and selectivity against HBV were evaluated in HBV natural infection assays and in a urokinase-type plasminogen activator/severe combined immunodeficiency humanized mouse model of HBV infection, either alone or in combination with entecavir. Unlike nucleos(t)ide therapies, which reduce viremia but do not lead to an effective reduction in HBV antigen expression, RG7834 significantly reduced the levels of viral proteins (including HBsAg), as well as lowering viremia. Consistent with its proposed mechanism of action, time course RNA-seq analysis revealed a fast and selective reduction in HBV mRNAs in response to RG7834 treatment. Furthermore, oral treatment of HBV-infected humanized mice with RG7834 led to a mean HBsAg reduction of 1.09 log10 compared to entecavir, which had no significant effect on HBsAg levels. Combination of RG7834, entecavir and pegylated interferon α-2a led to significant reductions of both HBV DNA and HBsAg levels in humanized mice. We have identified a novel oral HBV viral gene expression inhibitor that blocks viral antigen and virion production, that is highly selective for HBV, and has a unique antiviral profile that is clearly differentiated from nucleos(t)ide analogues. We discovered a novel small molecule viral expression inhibitor that is highly selective for HBV and unlike current therapy inhibits the expression of viral proteins by specifically reducing HBV mRNAs. RG7834 can therefore potentially provide anti-HBV benefits and increase HBV cure rates, by direct reduction of viral agents needed to complete the viral life cycle, as well as a reduction of viral agents involved in evasion of the host immune responses.

In vivo reduction of hepatitis B virus antigenemia and viremia by antisense oligonucleotides

Current treatment of chronic hepatitis B virus infection (CHB) includes interferon and nucleos(t)ide analogues, which generally do not reduce HBV surface antigen (HBsAg) production, a constellation that is associated with poor prognosis of CHB. Here we evaluated the efficacy of an antisense approach using antisense oligonucleotide (ASO) technology already in clinical use for liver targeted therapy to specifically inhibit HBsAg production and viremia in a preclinical setting. A lead ASO was identified and characterized in vitro and subsequently tested for efficacy in vivo and in vitro using HBV transgenic and hydrodynamic transfection mouse and a cell culture HBV infection model, respectively. ASO treatment decreased serum HBsAg levels ⩾2 logs in a dose and time-dependent manner; HBsAg decreased 2 logs in a week and returned to baseline 4 weeks after a single ASO injection. ASO treatment effectively reduced HBsAg in combination with entecavir, while the nucleoside analogue alone did not. ASO treatment has pan-genotypic antiviral activity in the hydrodynamic transfection system. Finally, cccDNA-driven HBV gene expression is ASO sensitive in HBV infected cells in vitro. Our results demonstrate in a preclinical setting the efficacy of an antisense approach against HBV by efficiently reducing serum HBsAg (as well as viremia) across different genotypes alone or in combination with standard nucleoside therapy. Since the applied antisense technology is already in clinical use, a lead compound can be rapidly validated in a clinical setting and thus, constitutes a novel therapeutic approach targeting chronic HBV infection.

Abstract 1132: Gluconeogenesis, lipogenesis, and HBV replication are commonly regulated by PGC-1α-dependent pathway

Abstract Peroxisome proliferator-activated receptor-γ coactivator 1 (PGC-1α), a major metabolic regulator of gluconeogenesis and lipogenesis, is known to be strongly induced to coactivate HBV gene expression in the liver of fasting mice. In this study, we found that the combination of 8-Br-cAMP and glucocorticoids showed the synergistic induction of PGC-1α and its downstream gene expression, including the expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase). Moreover, HBV corepromoter activity was synergistically enhanced by the combination of 8-Br-cAMP and glucocorticoid treatment. Graptopetalum paraguayense (GP), a herbal medicine, is commonly used in Taiwan to treat liver disorders. Here, we found that a partially purified fraction of GP (named HH-F3) dose dependently suppressed 8-Br-cAMP/glucocorticoid-induced G6Pase, PEPCKand PGC-1α expression as well as suppressed HBV core promoter activity. Additionally, HH-F3 blocked HBV core promoter activity via the inhibition of PGC-1α expression. The ectopic expression of PGC-1α effectively reversed the HH-F3-mediated inhibition of HBV core promoter activity.HH-F3-reduced HBV surface antigen expression, HBV mRNA production, core protein level, and HBV replication were drastically rescued by ectopically expressed PGC-1α. In addition, HH-F3 could also inhibit fatty acid synthase (FASN) expression as well as decrease lipid accumulation by the down-regulation of PGC-1α in human hepatocarcinoma cell lines. In conclusion, HH-F3 could inhibit HBV replication, gluconeogenesis, and lipogenesis through the down-regulation of PGC-1α. Our study indicates that targeting PGC-1α may be a novel therapeutic strategy for HBV infection. Together, HH-F3mighthave potential use for the treatment of chronic hepatitis B patients with associated metabolic syndrome. Citation Format: Wei-Hsiang Hsu, Hong-Jhih Jhuang, Chi-Ying Huang. Gluconeogenesis, lipogenesis, and HBV replication are commonly regulated by PGC-1α-dependent pathway. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1132. doi:10.1158/1538-7445.AM2015-1132

CpG islands of hepatitis B virus genome isolated from Chinese patients

There are differences in the distribution and length of HBV CpG islands and the viral mutations contribute greatly to the development of HBV-related diseases. However, little is known regarding the effects of such difference and mutations in HBV genotypes B and C sequences on the regulation of HBV gene expression and their clinical outcomes. To study the distribution, length and genetic trait of CpG islands in normal and mutant sequences of HBV genotypes B and C, 320 HBV isolates from Chinese patients were retrieved from GenBank. Programs CLUSTALX 1.83 and MethPrimer were employed to perform multiple sequence alignments and to predict CpG islands, respectively. 72.0% genotype B isolates contained three conventional CpG islands, and 76.1% genotype C only contained CpG islands II and III. 14.6% genotype B and 7.5% genotype C contained three novel CpG islands. In genotype B, lengths of conventional CpG islands between normal and mutant isolates exhibited substantial variations, but in genotype C, those were relatively stable. CpG island II could be “truncated” or “split”. “Truncated” region mutations were associated with structural and functional abnormalities of HBV genes. Rate of “split” CpG island II in genotype B was much higher than that in genotype C. In the majority of isolates from HCC and HBV–ACLF, genotype C lacked CpG island I and novel islands. Distribution, length and genetic trait of CpG islands in HBV genotypes B and C might affect their methylation status, and further affect regulation of HBV gene expression, leading to different clinical outcomes.

Inhibition of hepatitis B virus gene expression and replication by hepatocyte nuclear factor 6.

Hepatitis B virus (HBV), a small enveloped DNA virus, chronically infects more than 350 million people worldwide and causes liver diseases from hepatitis to cirrhosis and liver cancer. Here, we report that hepatocyte nuclear factor 6 (HNF6), a liver-enriched transcription factor, can inhibit HBV gene expression and DNA replication. Overexpression of HNF6 inhibited, while knockdown of HNF6 expression enhanced, HBV gene expression and replication in hepatoma cells. Mechanistically, the SP2 promoter was inhibited by HNF6, which partly accounts for the inhibition on S mRNA. Detailed analysis showed that a cis element on the HBV genome (nucleotides [nt] 3009 to 3019) was responsible for the inhibition of the SP2 promoter by HNF6. Moreover, further analysis showed that HNF6 reduced viral pregenomic RNA (pgRNA) posttranscriptionally via accelerating the degradation of HBV pgRNA independent of La protein. Furthermore, by using truncated mutation experiments, we demonstrated that the N-terminal region of HNF6 was responsible for its inhibitory effects. Importantly, introduction of an HNF6 expression construct with the HBV genome into the mouse liver using hydrodynamic injection resulted in a significant reduction in viral gene expression and DNA replication. Overall, our data demonstrated that HNF6 is a novel host factor that can restrict HBV replication via both transcriptional and posttranscriptional mechanisms. HBV is a major human pathogen whose replication is regulated by host factors. Liver-enriched transcription factors are critical for many liver functions, including metabolism, development, and cell proliferation, and some of them have been shown to regulate HBV gene expression or replication in different manners. In this study, we showed that HNF6 could inhibit the gene expression and DNA replication of HBV via both transcriptional and posttranscriptional mechanisms. As HNF6 is differentially expressed in men and women, the current results may suggest a role of HNF6 in the gender dimorphism of HBV infection.

Interleukin-21 inhibits HBV replication in vitro.

Cytokines are crucial factors in the non-cytolytic antiviral process to inhibit HBV gene expression and replication. Interleukin (IL)-21 has been suggested to play an important role in HBV infection, but it remains unknown whether IL-21 can inhibit HBV replication or how it inhibits HBV replication. In this study, we investigated the influence of IL-21 on HBV replication based on human hepatoma Huh7.93 cells co-cultured with human peripheral blood mononuclear cells (PBMCs) and the possible correlation among IL-21, interferon-γ, tumour necrosis factor-α and IL-10. We demonstrated that the decrease of IL-21 expression and the increase of IL-10 expression in PBMCs could promote HBV replication in vitro. We further revealed that IL-21 is not only able to effectively suppress HBV replication directly but also reduce HBV replication by inhibition of IL-10 secretion. Our results provide important evidence for the non-cytolytic antiviral mechanism mediated by cytokines and their interactions in chronic hepatitis B.