scholarly journals Identification of Aquarius and Senataxin as Restriction Host Factors for Hepatitis B Virus Infection

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 2
Author(s):  
Christina Whitten-Bauer ◽  
Andoni Gómez-Moreno ◽  
Urtzi Garaigorta
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Core Protein ◽  
Sodium Taurocholate ◽  
Hbv Infection ◽  
Loss Of Function ◽  
Glycosylation Pattern ◽  
Hbv Cccdna ◽  
Cellular Factors ◽  
B Virus

Hepatitis B virus (HBV) represents an important human pathogen causing acute and chronic hepatitis. Over 240 million people are chronically infected, many of whom will die due to complications such as liver cirrhosis and hepatocellular carcinoma. Currently approved therapies are very effective in suppressing virus replication and viremia, but they are not curative, because they do not completely eliminate the nuclear episomal DNA form of HBV (cccDNA) that re-establishes infection upon interruption of therapy. Despite our understanding of many aspects of the HBV lifecycle, details of the HBV cccDNA biology remain poorly understood. Our group is pursuing a loss-of-function genetic screening approach, to identify cellular factors regulating HBV infection. A lentivirus-delivered short hairpin RNA (shRNA) library, composed of 384 shRNAs, was used to interrogate the function of 80 DNA damage repair pathway proteins in the establishment of HBV infection. The primary screening identified 10 cellular factors that regulate the HBV infection both positively or negatively. Two of those proteins, aquarius (AQR) and senataxin (SETX), were subsequently validated as factors restricting the HBV infection in independent experiments. Silencing of AQR and SETX led to an increased infection efficiency that was characterized by higher intracellular levels of HBV cccDNA, HBV mRNA, and core protein, and increased HBV e antigen (HBeAg) accumulation in the supernatants of infected cells. The expression level, glycosylation pattern, and localization of the HBV receptor, sodium taurocholate cotransporting polypeptide (NTCP), in AQR- and SETX-downregulated cells was equivalent to that of the control cells. Collectively, our results are compatible with AQR and SETX restricting early steps in the HBV lifecycle and downstream HBV entry, that affect the establishment of the HBV cccDNA pool. Experiments to unravel the function of these proteins in the context of HBV infection are currently underway.

scholarly journals Recent advances in understanding and diagnosing hepatitis B virus infection

F1000Research ◽  
2016 ◽  
Vol 5 ◽  
pp. 2243 ◽  
Author(s):  
Slim Fourati ◽  
Jean-Michel Pawlotsky
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Immune Modulation ◽  
Hepatitis B Surface Antigen ◽  
Sodium Taurocholate ◽  
Hbv Infection ◽  
New Drugs ◽  
Recent Advances ◽  
Hbsag Detection ◽  
B Virus

Hepatitis B virus (HBV) infects approximately 240 million individuals worldwide. Recent advances in the virology, immunopathogenesis, and diagnosis of HBV infection are summarized in this review article. The identification of a hepatocyte-specific cellular receptor for HBV, the sodium taurocholate co-transporting polypeptide (NTCP), made it possible to develop reliable cell culture systems and better understand the early steps of the viral lifecycle. Viral and host factors involved in covalently closed circular DNA synthesis, stability, and transcriptional regulation have also been identified and provide potential targets for new drugs. Based on recent evidence showing trained immunity in immune-tolerant patients, the immune tolerance and immune clearance phases have been renamed the non-inflammatory and inflammatory phases, respectively. New diagnostic and monitoring tools are now available, including rapid diagnostic tests for hepatitis B surface antigen (HBsAg) detection, HBsAg quantification assays, anti-HBc antibody quantification assays, an HBV core-related antigen (HBcrAg) quantification test, new HBV DNA detection and quantification assays, and an HBV RNA quantification test. Their clinical utility is under study. Finally, new antiviral and immune modulation approaches are in the preclinical or early clinical developmental stages, with the goal to achieve functional cure or ideally (if possible) eradication of HBV infection.

scholarly journals Regulation of Molecular Chaperone GRP78 by Hepatitis B Virus: Control of Viral Replication and Cell Survival

2019 ◽  
Vol 40 (3) ◽  
Author(s):  
Wangqin Shu ◽  
Zhiwei Guo ◽  
Lijie Li ◽  
Zhiqi Xiong ◽  
Ziyu Wang ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Er Stress ◽  
Molecular Chaperones ◽  
Inhibitory Effect ◽  
Hbv Infection ◽  
Loss Of Function ◽  
B Virus ◽  
Underlying Mechanisms ◽  
Novel Therapeutic Strategies

ABSTRACT Chronic hepatitis B (CHB) remains a global health problem, carrying a high risk for progression into cirrhosis and liver failure. Molecular chaperones are involved in diverse pathophysiological processes including viral infection. However, the role of molecular chaperones in hepatitis B virus (HBV) infection and its underlying mechanisms remain unclear. Here, we identified GRP78 as one of the molecular chaperones most strongly induced by HBV in human hepatocytes. Gain- and loss-of-function analyses demonstrated that GRP78 exerted an inhibitory effect on HBV transcription and replication. Further study showed that GRP78 was involved in the activation of AKT/mTOR signaling in hepatocytes, which contributed to GRP78-mediated inhibition of HBV. Of note, HBV-upregulated GRP78 was found to play a crucial role in maintaining the survival of hepatocytes via facilitating a mild endoplasmic reticulum (ER) stress. Together, our findings suggest that HBV may sacrifice part of its replication for establishing a persistent infection through induction of GRP78, a master ER stress regulator. Targeting GRP78 may help develop to design novel therapeutic strategies against chronic HBV infection and the associated hepatocellular carcinoma.

scholarly journals Regulation of HBV Replication by Cyclin Docking Motifs in Core Protein

2021 ◽  
Author(s):  
Haitao Liu ◽  
Ji Xi ◽  
Jianming Hu
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Protein Phosphorylation ◽  
Capsid Protein ◽  
Core Protein ◽  
Hbv Infection ◽  
Capsid Assembly ◽  
Cyclin Dependent Kinase ◽  
Terminal Domain ◽  
B Virus

Hepatitis B virus (HBV) capsid or core protein (HBc) consists of an N-terminal domain (NTD) and C-terminal domain (CTD) connected by a short linker peptide. Dynamic phosphorylation and dephosphorylation of HBc regulate its multiple functions in capsid assembly and viral replication. The cellular cyclin-dependent kinase 2 (CDK2) plays a major role in HBc phosphorylation and furthermore, is incorporated into the viral capsid, accounting for most of the “endogenous kinase” activity associated with the capsid. The packaged CDK2 is thought to play a role in phosphorylating HBc to trigger nucleocapsid disassembly (uncoating), an essential step during viral infection. However, little is currently known on how CDK2 is recruited and packaged into the capsid. We have now identified three RXL motifs, in the HBc NTD, known as cyclin docking motifs (CDMs), which mediates the interactions of various CDK substrates/regulators with CDK/cyclin complexes. Mutations of the CDMs in the HBc NTD reduced CTD phosphorylation and diminished CDK2 packaging into the capsid. Also, the CDM mutations showed little effects on capsid assembly and pregenomic RNA (pgRNA) packaging but impaired the integrity of mature nucleocapsids. Furthermore, the CDM mutations blocked covalently closed circular DNA (CCC DNA) formation during infection while having no effect on or enhancing CCC DNA formation via intracellular amplification. These results indicate that the HBc NTD CDMs play a role in CDK2 recruitment and packaging, which, in turn, is important for productive infection. Author Summary Hepatitis B virus (HBV) is an important global human pathogen and persistently infects hundreds of millions of people, who are at high risk of cirrhosis and liver cancer. HBV capsid packages a host cell protein kinase, the cyclin-dependent kinase 2 (CDK2), which is thought to be required to trigger disassembly of the viral nucleocapsid during infection by phosphorylating the capsid protein, a prerequisite for successful infection. We have identified docking sites on the capsid protein for recruiting CDK2, in complex with its cyclin partner, to facilitate capsid protein phosphorylation and CDK2 packaging. Mutations of these docking sites reduced capsid protein phosphorylation, impaired CDK2 packaging into HBV capsids, and blocked HBV infection. These results provide novel insights regarding CDK2 packaging into HBV capsids and the role of CDK2 in HBV infection and should facilitate the development of antiviral drugs that target the HBV capsid protein.

scholarly journals Anti-Hepatitis B Virus Effect and Possible Mechanism of Action of 3,4-O-Dicaffeoylquinic AcidIn VitroandIn Vivo

2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
Yi-Hang Wu ◽  
Bing-Jie Hao ◽  
Hong-Cui Cao ◽  
Wei Xu ◽  
Yong-Jun Li ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Transgenic Mice ◽  
Core Protein ◽  
Heme Oxygenase 1 ◽  
Test Compound ◽  
Hbv Cccdna ◽  
Dicaffeoylquinic Acid ◽  
B Virus

The anti-hepatitis B activity of 3,4-O-dicaffeoylquinic acid isolated fromLaggera alatawas studied using theD-galactosamine- (D-GalN-) induced hepatocyte damage model, HepG2.2.15 cells, and with HBV transgenic mice.In vitroresults showed that 3,4-O-dicaffeoylquinic acid improved HL-7702 hepatocyte viability and markedly inhibited the production of HBsAg and HBeAg. At a concentration of 100 μg/mL, its inhibitory rates on the expression levels of HBsAg and HBeAg were 89.96% and 81.01%, respectively. The content of hepatitis B virus covalently closed circular DNA (HBV cccDNA) in HepG2.2.15 cells was significantly decreased after the cells were treated with the test compound. In addition, 3,4-O-dicaffeoylquinic acid significantly increased the expression of heme oxygenase-1 (HO-1) in HepG2.2.15 cells.In vivoresults indicated that the test compound at concentrations of 100 μg/mL significantly inhibited HBsAg production and increased HO-1 expression in HBV transgenic mice. In conclusion, this study verifies the anti-hepatitis B activity of 3,4-O-dicaffeoylquinic acid. The upregulation of HO-1 may contribute to the anti-HBV effect of this compound by reducing the stability of the HBV core protein, which blocks the refill of nuclear HBV cccDNA. Furthermore, the hepatoprotective effect of this compound may be mediated through its antioxidative/anti-inflammatory properties and by the induction of HO-1 expression.

scholarly journals A New Role for Capsid Assembly Modulators To Target Mature Hepatitis B Virus Capsids and Prevent Virus Infection

2019 ◽  
Vol 64 (1) ◽  
Author(s):  
Chunkyu Ko ◽  
Romina Bester ◽  
Xue Zhou ◽  
Zhiheng Xu ◽  
Christoph Blossey ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
De Novo ◽  
Core Protein ◽  
Antiviral Effect ◽  
Significant Inhibition ◽  
Hbv Infection ◽  
Capsid Assembly ◽  
Virus Particles ◽  
B Virus

ABSTRACT Hepatitis B virus (HBV) is a major human pathogen, killing an estimated 887,000 people per year. Therefore, potentially curative therapies are of high importance. Following infection, HBV deposits a covalently closed circular DNA (cccDNA) in the nucleus of infected cells that serves as a transcription template and is not affected by current therapies. HBV core protein allosteric modulators (CpAMs) prevent correct capsid assembly but may also affect early stages of HBV infection. In this study, we aimed to determine the antiviral efficacy of a novel, structurally distinct heteroaryldihydropyrimidine (HAP)-type CpAM, HAP_R01, and investigated whether and how HAP_R01 prevents the establishment of HBV infection. HAP_R01 shows a significant inhibition of cccDNA formation when applied during the first 48 h of HBV infection. Inhibiting cccDNA formation, however, requires >1-log10-higher concentrations than inhibition of the assembly of newly forming capsids (half-maximal effective concentration [EC50], 345 to 918 nM versus 26.8 to 43.5 nM, respectively). Biophysical studies using a new method to detect the incoming capsid in de novo infection revealed that HAP_R01 can physically change mature capsids of incoming virus particles and affect particle integrity. Treating purified HBV virions with HAP_R01 reduced their infectivity, highlighting the unique antiviral activity of CpAMs to target the capsid within mature HBV particles. Accordingly, HAP_R01 shows an additive antiviral effect in limiting de novo infection when combined with viral entry inhibitors. In summary, HAP_R01 perturbs capsid integrity of incoming virus particles and reduces their infectivity and thus inhibits cccDNA formation in addition to preventing HBV capsid assembly.

scholarly journals Hepatitis B Virus Infection of a Mouse Hepatic Cell Line Reconstituted with Human Sodium Taurocholate Cotransporting Polypeptide

2016 ◽  
Vol 90 (9) ◽  
pp. 4827-4831 ◽  
Author(s):  
Florian A. Lempp ◽  
Bingqian Qu ◽  
Yong-Xiang Wang ◽  
Stephan Urban
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Cell Line ◽  
Sodium Taurocholate ◽  
Hbv Infection ◽  
Hepatitis B Virus Infection ◽  
Hepatic Cells ◽  
Mouse Liver Cell ◽  
B Virus ◽  
Mouse Hepatocytes

Hepatitis B virus (HBV) enters hepatocytes via its receptor, human sodium taurocholate cotransporting polypeptide (hNTCP). So far, HBV infection has been achieved only in human hepatic cells reconstituted with hNTCP and not in cells of mouse origin. Here, the first mouse liver cell line (AML12) which gains susceptibility to HBV upon hNTCP expression is described. Thus, HBV infection of receptor-expressing mouse hepatocytes does not principally require a human cofactor but can be triggered by endogenous murine determinants.

scholarly journals A Novel Tricyclic Polyketide, Vanitaracin A, Specifically Inhibits the Entry of Hepatitis B and D Viruses by Targeting Sodium Taurocholate Cotransporting Polypeptide

2015 ◽  
Vol 89 (23) ◽  
pp. 11945-11953 ◽  
Author(s):  
Manabu Kaneko ◽  
Koichi Watashi ◽  
Shinji Kamisuki ◽  
Hiroki Matsunaga ◽  
Masashi Iwamoto ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Sodium Taurocholate ◽  
Hbv Infection ◽  
Chemical Library ◽  
Hepatitis D ◽  
Active Inhibitor ◽  
Hepatitis D Virus ◽  
Hbv Genotypes ◽  
B Virus

ABSTRACTAnti-hepatitis B virus (HBV) drugs are currently limited to nucleos(t)ide analogs (NAs) and interferons. A challenge of drug development is the identification of small molecules that suppress HBV infection from new chemical sources. Here, from a fungus-derived secondary metabolite library, we identify a structurally novel tricyclic polyketide, named vanitaracin A, which specifically inhibits HBV infection. Vanitaracin A inhibited the viral entry process with a submicromolar 50% inhibitory concentration (IC50) (IC50= 0.61 ± 0.23 μM), without evident cytotoxicity (50% cytotoxic concentration of >256 μM; selectivity index value of >419) in primary human hepatocytes. Vanitaracin A did not affect the HBV replication process. This compound was found to directly interact with the HBV entry receptor sodium taurocholate cotransporting polypeptide (NTCP) and impaired its bile acid transport activity. Consistent with this NTCP targeting, antiviral activity of vanitaracin A was observed with hepatitis D virus (HDV) but not hepatitis C virus. Importantly, vanitaracin A inhibited infection by all HBV genotypes tested (genotypes A to D) and clinically relevant NA-resistant HBV isolate. Thus, we identified a fungal metabolite, vanitaracin A, which was a potent, well-tolerated, and broadly active inhibitor of HBV and HDV entry. This compound, or its related analogs, could be part of an antiviral strategy for preventing reinfection with HBV, including clinically relevant nucleos(t)ide analog-resistant virus.IMPORTANCEFor achieving better treatment and prevention of hepatitis B virus (HBV) infection, anti-HBV agents targeting a new molecule are in great demand. Although sodium taurocholate cotransporting polypeptide (NTCP) has recently been reported to be an essential host factor for HBV entry, there is a limited number of reports that identify new compounds targeting NTCP and inhibiting HBV entry. Here, from an uncharacterized chemical library, we isolated a structurally new compound, named vanitaracin A, which inhibited the process of entry of HBV and hepatitis D virus (HDV). This compound was suggested to directly interact with NTCP and inhibit its transporter activity. Importantly, vanitaracin A inhibited the entry of all HBV genotypes examined and of a clinically relevant nucleos(t)ide analog-resistant HBV isolate.

scholarly journals In Vitro Infection with Hepatitis B Virus Using Differentiated Human Serum Culture of Huh7.5-NTCP Cells without Requiring Dimethyl Sulfoxide

Viruses ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 97
Author(s):  
Connie Le ◽  
Reshma Sirajee ◽  
Rineke Steenbergen ◽  
Michael A. Joyce ◽  
William R. Addison ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Human Serum ◽  
Dimethyl Sulfoxide ◽  
Hepatoma Cell ◽  
Sodium Taurocholate ◽  
Hbv Infection ◽  
Differentiation Markers ◽  
B Virus

An estimated two billion people worldwide have been infected with hepatitis B virus (HBV). Despite the high infectivity of HBV in vivo, a lack of easily infectable in vitro culture systems hinders studies of HBV. Overexpression of the sodium taurocholate co-transporting polypeptide (NTCP) bile acid transporter in hepatoma cells improved infection efficiency. We report here a hepatoma cell culture system that does not require dimethyl sulfoxide (DMSO) for HBV infection. We overexpressed NTCP in Huh7.5 cells and allowed these cells to differentiate in a medium supplemented with human serum (HS) instead of fetal bovine serum (FBS). We show that human serum culture enhanced HBV infection in Huh7.5-NTCP cells, e.g., in HS cultures, HBV pgRNA levels were increased by as much as 200-fold in comparison with FBS cultures and 19-fold in comparison with FBS+DMSO cultures. Human serum culture increased levels of hepatocyte differentiation markers, such as albumin secretion, in Huh7.5-NTCP cells to similar levels found in primary human hepatocytes. N-glycosylation of NTCP induced by culture in human serum may contribute to viral entry. Our study demonstrates an in vitro HBV infection of Huh7.5-NTCP cells without the use of potentially toxic DMSO.

scholarly journals Functional Characterization of Naturally Occurring Variants of Human Hepatitis B Virus Containing the Core Internal Deletion Mutation

1998 ◽  
Vol 72 (3) ◽  
pp. 2168-2176 ◽  
Author(s):  
Thomas Ta-Tung Yuan ◽  
Min-Hui Lin ◽  
Sui Min Qiu ◽  
Chiaho Shih
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Core Protein ◽  
Hbv Infection ◽  
Wild Type ◽  
Internal Deletion ◽  
The Core ◽  
Naturally Occurring ◽  
Human Hepatitis ◽  
B Virus

ABSTRACT Naturally occurring variants of human hepatitis B virus (HBV) containing the core internal deletion (CID) mutation have been found frequently in HBV carriers worldwide. Despite numerous sequence analysis reports of CID variants in patients, in the past decade, CID variants have not been characterized functionally, and thus their biological significance to HBV infection remains unclear. We report here two different CID variants identified from two patients that are replication defective, most likely due to the absence of detectable core protein. In addition, we were unable to detect the presence of the precore protein and e antigen from CID variants. However, the production of polymerase appeared to be normal. The replication defect of the CID variants can be rescued in trans by complementation with wild-type core protein. The rescued CID variant particles, which utilize the wild-type core protein, presumably are enveloped properly since they can be secreted into the medium and band at a position similar to that of mature wild-type Dane particles, as determined by gradient centrifugation analysis. Our results also provide an explanation for the association of CID variants with helper or wild-type HBV in nature. The significance of CID variants in HBV infection and pathogenesis is discussed.

scholarly journals E-cadherin binds glycosylated sodium-taurocholate cotransporting polypeptide to facilitate hepatitis B virus entry

2019 ◽  
Author(s):  
Qin Hu ◽  
Fei-Fei Zhang ◽  
Liang Duan ◽  
Bo Wang ◽  
Pu Li ◽  
...  
Keyword(s):  
Public Health ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Sodium Taurocholate ◽  
Hbv Infection ◽  
Host Cells ◽  
Heparg Cells ◽  
B Virus ◽  
Hepatocyte Polarity ◽  
E Cadherin

AbstractHepatitis B virus (HBV) continues to pose a serious public health risk and is one of the major causes of chronic liver disease and hepatocellular carcinoma. Current antiviral therapy does not effectively eradicate HBV and, thus, further investigation into the mechanisms employed by HBV to allow for invasion of host cells, is critical for the development of novel therapeutic agents. Sodium-taurocholate cotransporting polypeptide (NTCP) has been identified as a functional receptor for HBV. However, the specific mechanism by which HBV and NTCP interact remains unclear. Herein we show that the expression of E-cadherin was upregulated in cells expressing HBV, while knockdown of E-cadherin in HepG2-NTCP cells, HepaRG cells and primary human hepatocytes served to significantly inhibit infection by HBV and HBV pseudotyped particles. Alternatively, exogenous E-cadherin expression was found to significantly enhance HBV uptake by HepaRG cells. Further, mechanistic studies identified glycosylated NTCP localized to the cell membrane via E-cadherin binding, which subsequently allowed for more efficient binding between NTCP and the preS1 of the large HBV surface proteins. E-cadherin was also found to play a key role in establishing and maintaining hepatocyte polarity, which is essential for efficient HBV infection. These observations suggest that E-cadherin facilitates HBV entry through regulation of NTCP distribution and hepatocyte polarity.Author SummaryHepatitis B Virus (HBV) still seriously endangers public health. It is very important to understand the mechanism of HBV invading host cells for developing new therapy target. Sodium-taurocholate cotransporting polypeptide (NTCP) is the key receptor mediating HBV invasion, while other molecules also exhibit important roles in ensuring efficient and productive HBV infection. This study reports that E-cadherin facilitates HBV entry by directly interacting with glycosylated NTCP to mediate its distribution on the hepatocyte membrane and also affects the efficacy of HBV invasion by influncing hepatocyte polarity.

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