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Chronic hepatitis B infection is caused by hepatitis B virus (HBV) and a total cure is yet to be achieved. The viral covalently closed circular DNA (cccDNA) is the key to establish a persistent infection within hepatocytes. Current antiviral strategies have no effect on the pre-existing cccDNA reservoir. Therefore, the study of the molecular mechanism of cccDNA formation is becoming a major focus of HBV research. This review summarizes the current advances in cccDNA molecular biology and the latest studies on the elimination or inactivation of cccDNA, including three major areas: (1) epigenetic regulation of cccDNA by HBV X protein, (2) immune-mediated degradation, and (3) genome-editing nucleases. All these aspects provide clues on how to finally attain a cure for chronic hepatitis B infection.

During replication of the hepatitis B virus (HBV) in liver cells, the reverse transcription of pre-genomic RNA (pgRNA) is initiated by protein priming at an RNA packaging signal ε located near the 5' end of pgRNA. Heat-shock proteins (Hsps) such as Hsc70, Hsp40, and Hsp90 have been reported to be involved in the reconstitution of HBV polymerase (P protein) and E. The P - E complex initiates the reverse transcription and assembly of nucleocapsids. Hence, blockade of P - ε interactions is an attractive target for drug intervention. We explored the influence of the Hsp inhibitor KNK437 on replication and transcription of the HBV. Three working models were applied: HepG2. 2. 15 cell line; Huh7 cells transfected transiently with the 1. 05 X HBV (pCH9-3091) plasmid; Huh7 cells transfected transiently with the 1. 3 X HBV (pGEM-1. 3 X HBV) plasmid. Cytotoxic effects of KNK437 were detected by the CCK-8 method. Levels of hepatitis B surface antigen (HBsAg) and hepatitis B viral protein (HBeAg) in the media secreted from cells were measured using an ELISA. Intracellular HBV DNAs within nucleocapsids were measured by quantitative polymerase chain reaction (qPCR), and intracellular HBV RNAs by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Transcription of Hsps in cells was determined by qRT-PCR. Data suggested that KNK437 reduced the extracellular secretion of HBsAg and HBeAg in most cases; it downregulated expression of intracellular HBV DNAs within nucleocapsids and RNA transcripts. The lowest rate of viral DNAs in KNK437-treated hepatocytes for all experimental groups was ~1. 5%o (control, 100%), whereas that for RNAs was ~30%. Western blotting revealed KNK437 to inhibit intracellular core expression in HepG2. 2. 15. As a general inhibitor, KNK437 suppressed transcription of hsp70, hsp90b, and hsp4o. These data suggest that KNK437 may be a potent anti-HBV inhibitor for future therapy against chronic hepatitis.

The ultimate goal of chronic hepatitis B virus(HBV)therapy is full eradication of the virus from the liver. However, this is rarely achieved with the clinically available first-line agents (entecavir and tenofovir disoproxil fumarate) due to the inability to eliminate covalently closed circular DNA(cccDNA), which persists in the nucleus of infected hepatocyte cells,and failure of the host to induce an adequate specific immune response to control the infection. Currently, the clinical treatment for chronic HBV infection mainly includes nucleos(t)ide analogues (NAs), non-NAs and immune modulatory agents; however, each agent has individual advantages and drawbacks. It is, therefore, extremely urgent to identify novel targets involved in viral replication and develop novel anti-HBV drugs. In light of the breakthroughs in cccDNA research and host immune treatments, this review aims to summarize the state of the recent HBV drug research and development to highlight future therapeutic strategies to target the virus and host immune response.

Hepatitis B virus (HBV) infection is one of the most serious and prevalent health problems worldwide. Current anti-HBV medications have a number of drawbacks, such as adverse effects and drug resistance; thus, novel potential anti-HBV reagents are needed. Selenium (Se) has been shown to be involved in both human immunodeficiency virus and hepatitis C virus infections, but its role in HBV infection remains unclear. To address this, sodium selenite (Na2SeO3 ) was applied to three HBV cell models: HepG2.2.15 cells, and HuH-7 cells transfected with either 1.1 or 1.3× HBV plasmids. Cytotoxicity of Na2SeO3 was examined by Cell Counting Kit-8. Levels of viral antigen expression, transcripts, and encapsidated viral DNA were measured by enzyme-linked immunosorbent assay, northern blot, and Southern blot, respectively. There was no obvious cytotoxicity in either HepG2.2.15 or HuH-7 cells with <2.5 µM Na2SeO3 . Below this concentration, Na2SeO3 suppressed HBsAg and HBeAg production, HBV transcript level, and amount of genomic DNA in all three tested models, and suppression level was enhanced in line with increases in Na2 SeO3 concentration or treatment time. Moreover, the inhibitory effect of Na2SeO3 on HBV replication can be further enhanced by combined treatment with lamivudine, entecavir, or adefovir. Thus, the present study clearly proves that Na2SeO3 suppresses HBV protein expression, transcription, and genome replication in hepatoma cell models in a dose- and time-dependent manner., (© 2015 Wiley Periodicals, Inc.)

Hepatitis B virus (HBV) infection is one of the most serious and prevalent viral diseases in the world. Although several anti-HBV drugs have been used clinically, their side and adverse effects limit treatment efficacy. Therefore, it is necessary to identify novel potential anti-HBV agents. The flavonol quercetin has shown activity against some retroviruses, but its effect on HBV remains unclear. In the present study, quercetin was incubated with HepG2.2.15 cells, as well as HuH-7 cells transfected with an HBV plasmid. Quercetin was shown to significantly reduce Hepatitis B surface antigen (HBsAg) and Hepatitis B e antigen (HBeAg), secretion and HBV genomic DNA levels in both cell lines. In addition, co-incubation with lamivudine (3TC), entecavir (ETV), or adefovir (Ade) further enhanced the quercetin-induced inhibition of HBV replication. This inhibition was partially associated with decreased heat shock proteins and HBV transcription levels. The results indicate that quercetin inhibited HBV antigen secretion and genome replication in human hepatoma cell lines, which suggests that quercetin may be a potentially effective anti-HBV agent.

Primary human hepatocytes are considered the ideal cellular model for in-vitro studies of liver-specific pathology, such as hepatitis B virus (HBV) infection. However, poor accessibility, limited cell numbers, and lot-to-lot variation of primary human hepatocytes limit their broad application. Human fetal hepatocytes were isolated from postmortem embryonic liver tissues by two-step collagenase perfusion and cryopreserved. A monolayer of cryopreserved human fetal hepatocytes was established by optimizing such conditions as cell density and viability and purification of viable cells by Percoll. Finally, revived human fetal hepatocytes were characterized and infected with HBV. A large number of viable human fetal hepatocytes could be isolated and cryopreserved, with seeding density and viability being critical for the establishment of a compact monolayer culture. Using low-viability cryopreserved human fetal hepatocytes, a typical monolayer was established by purification with Percoll. The revived cells were actively proliferative, showed identical morphologic characteristics to non-cryopreserved cells, and had a typical hepatic gene expression profile. Moreover, this optimized model was susceptible to HBV infection and could be used to screen entry inhibitors against HBV infection. In conclusion, these methods can be used on human fetal hepatocytes to provide a cell bank for studies of the early stages of HBV infection., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Hepatitis B virus (HBV) transcription and replication are essentially restricted to hepatocytes. Based on the HBV enhancer and promoter complex that links hepatic glucose metabolism to its transcription and replication, HBV adopts a regulatory system that is unique to the hepatic gluconeogenic genes. CRTC2, the CREB-regulated transcription coactivator 2, is a critical switch modulating the gluconeogenic program in response to both hormonal and intracellular signals. However, the relationship between CRTC2 and HBV transcription and replication remains unclear., Methods: To analyze the influence of CRTC2 on HBV transcription and replication, CRTC2 expression construct or siRNA was cotransfected with plasmids containing enhancer II/core promoter complex-controlled luciferase or 1.3× wtHBV genome in Huh-7 cells. Luciferase activity, HBV core protein expression, HBV transcripts, and DNA replication intermediates were measured by luciferase assays, western blots, real-time polymerase chain reaction (PCR), and Southern blots, respectively. Forskolin (FSK) or phosphorylation-defective CRTC2 mutants were further utilized to elucidate the potential mechanism. siRNA against peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α) was also used to examine the mediator involved in CRTC2-regulated HBV biosynthesis in Huh-7 cells., Results: CRTC2 overexpression increased HBV transcription and replication in Huh-7 cells, including levels of core protein expression, mRNA, and DNA replication intermediates. Correspondingly, CRTC2 knock down by siRNA reduced HBV biosynthesis. FSK treatment strongly enhanced the effect of CRTC2 through triggering the dephosphorylation and nuclear entry of CRTC2. The phosphorylation-defective mutant (S171A/S275A) of CRTC2 localized in the nucleus and was constitutively active, which dramatically promoted HBV transcription and replication similar to FSK-treated wild-type CRTC2. Knock down of PGC1α, whose expression was induced by CRTC2, greatly compromised the enhancing effect of CRTC2 on HBV transcription and replication., Conclusions: Our results clearly indicate that non-phosphorylated CRTC2 strongly enhances HBV biosynthesis through inducing PGC1α expression. Further study of the mechanisms will elucidate the importance of metabolic signals on HBV transcription and replication, and offer insight into potential targets for developing anti-HBV agents.

Within a few days of being isolated, primary human hepatocytes undergo a rapid dedifferentiation process and lose susceptibility to hepatitis B virus (HBV) infection in vitro. This fact has limited their further application. In this study, a convenient and feasible method of preventing this dedifferentiation was established, by co-culturing human fetal hepatocytes with hepatic non-parenchymal cells to maintain the differentiation features of human fetal hepatocytes. Isolated hepatic cells were seeded at a low density, and cultured in dimethyl sulfoxide-free medium for a month to allow rapid proliferation of non-parenchymal cells. Subsequently, 2% dimethyl sulfoxide was added to induce formation of typical hepatic islands, in which hepatocytic features could be further maintained for up to an additional 3 months. These hepatic islands, formed of piled-up hepatocytes, were surrounded and invaded by non-parenchymal cells. Protein expression profiles showed that the human fetal hepatocytes underwent a rapid maturation process, and the hepatocytic features were well preserved. Most importantly, these human fetal hepatocytes still exhibited susceptibility to HBV infection after long-term maintenance, for as long as 10 weeks. This co-culture method has overcome the pre-existing disadvantages of primary human hepatocytes for virological studies, and provides a valuable approach to long-term maintenance of primary human hepatocytes for studies of HBV infection for prolonged periods., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Hepatitis B virus (HBV) biosynthesis is primarily restricted to hepatocytes due to the governing of liver-enriched nuclear receptors (NRs) on viral RNA synthesis. The liver-enriched NR hepatocyte nuclear factor 4α (HNF4α), the key regulator of genes implicated in hepatic glucose metabolism, is also a primary determinant of HBV pregenomic RNA synthesis and HBV replication. Peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α) coactivates and further enhances the effect of HNF4α on HBV biosynthesis. Here, we showed that the acetyltransferase General Control Non-repressed Protein 5 (GCN5) acetylated PGC1α, leading to alteration of PGC1α from a transcriptionally active state into an inactive state. As a result, the coactivation activity of PGC1α on HBV transcription and replication was suppressed. Apparently, an acetylation site mutant of PGC1α (PGC1αR13) still had coactivation activity as GCN5 could not suppress the coactivation activity of the mutant. Moreover, a catalytically inactive acetyltransferase mutant GCN5m, due to the loss of acetylation activity, failed to inhibit the coactivation function of PGC1α in HBV biosynthesis. Our results demonstrate that GCN5, through its acetyltransferase activity, inhibits PGC1α-induced enhancement of HBV transcription and replication both in vitro and in vivo.

As a highly efficient delivery system, lentiviral vectors (LVs) have become a powerful tool to assess the antiviral efficacy of RNA drugs such as short hairpin RNA (shRNA) and decoys. Furthermore, recent advanced systems allow controlled expression of the effector RNA via coexpression of a tetracycline/doxycycline (DOX) responsive repressor (tTR-KRAB). Herein, this system was utilized to assess the antiviral effects of LV-encoded shRNAs targeting three conserved regions on the pregenomic RNA of hepatitis B virus (HBV), namely the region coding for the reverse transcriptase (RT) domain of the viral polymerase (LV-HBV-shRNA1), the core promoter (CP; LV-HBV-shRNA2), and the direct repeat 1 (DR1; LV-HBV-shRNA3). Transduction of just the LV-HBV-shRNA vectors into the stably HBV expressing HepG2.2.15 cell line showed significant reductions in secreted HBsAg and HBeAg, intracellular HBcAg as well as HBV RNA and DNA replicative intermediates for all vectors, however, most pronouncedly for the DR1-targeting shRNA3. The corresponding vector was therefore applied in the DOX-controlled system. Notably, strong interference with HBV replication was found in the presence of the inducer DOX whereas the antiviral effect was essentially ablated in its absence; hence, the silencing effect of the shRNA and consequently HBV replication could be strictly regulated by DOX. This newly established system may therefore provide a valuable platform to study the antiviral efficacy of RNA drugs against HBV in a regulated manner, and even be applicable in vivo.

Introduction: The Hepatitis B Virus (HBV) genome contains four ORFs, S (surface), P (polymerase), C (core) and X. S is completely overlapped by P and as a consequence the overlapping region is subject to distinctive evolutionary constraints compared to the remainder of the genome. Specifically, a non-synonymous substitution in one coding frame may produce a synonymous substitution in the alternative frame, suggesting a possible conflict between requirements for diversifying and purifying forces. To examine how these contrasting requirements are balanced within this region, we investigated the relationship amongst positive selection sites, conserved regions, epitopes and elements of protein structure to consider how HBV balances the contrasting evolutionary pressures., Methodology/results: 323 HBV genotype D genome sequences were collected and analyzed to identify sites under positive selection and highly conserved regions. Epitopes sequences were retrieved from previously published experimental studies stored in the Immune Epitope Database. Predicted secondary structures were used to investigate the association between structure and conservation. Entropy was used as a measure of conservation and bivariate logistic regression was used to investigate the relationship between positive selection/conserved sites and epitope/secondary structure regions. Our results indicate: (i) conservation in S is primarily dictated by α-helix elements in the protein structure, (ii) variable residues are mainly located in PreS, the major hydrophilic region (MHR) and the C-terminus, (iii) epitopes in S, which are directly targeted by the host immune system, are significantly associated with sites under positive selection., Conclusions: The highly variable spacer domain in P, which corresponds to PreS in S, appears to act as a harbor for the accumulation of mutations that can provide flexibility for conformational changes and responding to immune pressure.

Hepatitis B virus (HBV) is a noncytopathic human hepadnavirus that causes acute, chronic hepatitis and hepatocellular carcinoma (HCC). As the clinical utility of current therapies is limited, new anti-HBV agents and sources for such agents are still highly sought after. Here, we report that Mucroporin-M1, a scorpion venom-derived peptide, reduces the amount of extracellular HBsAg, HBeAg, and HBV DNA productions of HepG2.2.15 cells in a dose-dependent manner and inhibits HBV capsid DNA, HBV intracellular RNA replication intermediates and the HBV Core protein in the cytoplasm of HepG2.2.15 cells. Using a mouse model of HBV infection, we found that HBV replication was significantly inhibited by intravenous injection of the Mucroporin-M1 peptide. This inhibitory activity was due to a reduction in HBV promoter activity caused by a decrease in the binding of HNF4α to the precore/core promoter region. Furthermore, we confirmed that Mucroporin-M1 could selectively activate mitogen-activated protein kinases (MAPKs) and lead to the down-regulation of HNF4α expression, which explains the decreased binding of HNF4α to the HBV promoter. Moreover, when the protein phosphorylation activity of the MAPK pathway was inhibited, both HNF4α expression and HBV replication recovered. Finally, we proved that treatment with the Mucroporin-M1 peptide increased phosphorylation of the MAPK proteins in HBV-harboring mice. These results implicate Mucroporin-M1 peptide can activate the MAPK pathway and then reduce the expression of HNF4α, resulting in the inhibition of HBV replication in vitro and in vivo. Our work also opens new doors to discovering novel anti-HBV agents or sources.

Hydrodynamic injection (HI) with a replication competent hepatitis B virus (HBV) genome may lead to transient or prolonged HBV replication in mice. However, the prolonged HBV persistence after HI depends on the specific backbone of the vector carrying HBV genome and the genetic background of the mouse strain. We asked whether a genetically closely related hepadnavirus, woodchuck hepatitis virus (WHV), may maintain the gene expression and replication in the mouse liver after HI. Interestingly, we found that HI of pBS-WHV1.3 containing a 1.3 fold overlength WHV genome in BALB/c mouse led to the long presence of WHV DNA and WHV proteins expression in the mouse liver. Thus, we asked whether WHV genome carrying foreign DNA sequences could maintain the long term gene expression and persistence. For this purpose, the coding region of HBV surface antigen (HBsAg) was inserted into the WHV genome to replace the corresponding region. Three recombinant WHV-HBV genomes were constructed with the replacement with HBsAg a-determinant, major HBsAg, and middle HBsAg. Serum HBsAg, viral DNA, hepatic WHV protein expression, and viral replication intermediates were detected in mice after HI with recombinant genomes. Similarly, the recombinant genomes could persist for a prolonged period of time up to 45 weeks in mice. WHV and recombinant WHV-HBV genomes did not trigger effective antibody and T-cell responses to viral proteins. The ability of recombinant WHV constructs to persist in mice is an interesting aspect for the future investigation and may be explored for in vivo gene transfer. [ABSTRACT FROM AUTHOR]

Replication of hepatitis B virus (HBV) via protein-primed reverse transcription is initiated by binding of the viral P protein to the conserved ε stem-loop on the pregenomic (pg) RNA. This triggers encapsidation of the complex and the ε-templated synthesis of a short P protein-linked DNA oligonucleotide (priming) for subsequent minus-strand DNA extension. ε consists of a lower and upper stem, a bulge containing the priming template, and an apical loop. The nonhelical subelements are considered important for DNA synthesis and pgRNA packaging whereas the role of the upper stem is not well characterized. Priming itself could until recently not be addressed because in vitro generated HBV P - ε complexes showed no activity. Focussing on the four A residues at the base and tip of the upper ε stem and the two U residues in the loop we first investigated the impact of 24 mutations on viral DNA accumulation in transfected cells. While surprisingly many mutations were tolerated, further analyzing the negatively acting mutations, including in a new cell-free priming system, revealed divergent position-related impacts on pgRNA packaging, priming activity and possibly initiation site selection. This genetic separability implies that the ε RNA undergoes multiple distinct interactions with P protein as pgRNA encapsidation and replication initiation progress, and that the strict conservation of ε in nature may reflect its optimal adaptation to comply with all of them. The data further define the most attractive mutants for future studies, including as decoys for interference with HBV replication. [ABSTRACT FROM AUTHOR]

Introduction: The Hepatitis B Virus (HBV) genome contains four ORFs, S (surface), P (polymerase), C (core) and X. S is completely overlapped by P and as a consequence the overlapping region is subject to distinctive evolutionary constraints compared to the remainder of the genome. Specifically, a non-synonymous substitution in one coding frame may produce a synonymous substitution in the alternative frame, suggesting a possible conflict between requirements for diversifying and purifying forces. To examine how these contrasting requirements are balanced within this region, we investigated the relationship amongst positive selection sites, conserved regions, epitopes and elements of protein structure to consider how HBV balances the contrasting evolutionary pressures. Methodology/Results: 323 HBV genotype D genome sequences were collected and analyzed to identify sites under positive selection and highly conserved regions. Epitopes sequences were retrieved from previously published experimental studies stored in the Immune Epitope Database. Predicted secondary structures were used to investigate the association between structure and conservation. Entropy was used as a measure of conservation and bivariate logistic regression was used to investigate the relationship between positive selection/conserved sites and epitope/secondary structure regions. Our results indicate: (i) conservation in S is primarily dictated by α-helix elements in the protein structure, (ii) variable residues are mainly located in PreS, the major hydrophilic region (MHR) and the C-terminus, (iii) epitopes in S, which are directly targeted by the host immune system, are significantly associated with sites under positive selection. Conclusions: The highly variable spacer domain in P, which corresponds to PreS in S, appears to act as a harbor for the accumulation of mutations that can provide flexibility for conformational changes and responding to immune pressure. [ABSTRACT FROM AUTHOR]