Curcumin inhibited hepatitis B viral entry through NTCP binding

AbstractHepatitis B virus (HBV) has been implicated in hepatitis and hepatocellular carcinoma. Current agents (nucleos(t)ide analogs and interferons) could only attenuate HBV infection. A combination of agents targeting different stages of viral life cycle (e.g., entry, replication, and cccDNA stability) was expected to eradicate the infection. Curcumin (CCM) was investigated for inhibitory action toward HBV attachment and internalization. Immortalized hepatocyte-like cells (imHCs), HepaRG and non-hepatic cells served as host cells for binding study with CCM. CCM decreased viral load, HBeAg, HBcAg (infectivity), intracellular HBV DNA, and cccDNA levels. The CCM-induced suppression of HBV entry was directly correlated with the density of sodium-taurocholate co-transporting polypeptide (NTCP), a known host receptor for HBV entry. The site of action of CCM was confirmed using TCA uptake assay. The affinity between CCM and NTCP was measured using isothermal titration calorimetry (ITC). These results demonstrated that CCM interrupted HBV entry and would therefore suppress HBV re-infection.

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NTCP oligomerization occurs downstream of the NTCP-EGFR interaction during hepatitis B virus internalization

Journal of Virology ◽

10.1128/jvi.00938-21 ◽

2021 ◽

Author(s):

Kento Fukano ◽

Mizuki Oshima ◽

Senko Tsukuda ◽

Hideki Aizaki ◽

Mio Ohki ◽

...

Keyword(s):

Epidermal Growth Factor Receptor ◽

Bile Acid ◽

Hepatitis B Virus ◽

Hepatitis B ◽

Viral Entry ◽

Sodium Taurocholate ◽

Growth Factor Receptor ◽

Host Cells ◽

B Virus ◽

Epidermal Growth

Sodium taurocholate cotransporting polypeptide (NTCP) is a receptor that is essential for hepatitis B virus (HBV) entry into the host cell. A number of HBV entry inhibitors targeting NTCP have been reported to date; these inhibitors have facilitated a mechanistic analysis of the viral entry process. However, the mechanism of HBV internalization into host cells after interaction of virus with NTCP remains largely unknown. Recently, we reported that troglitazone, a thiazolidinedione derivative, specifically inhibits both HBV internalization and NTCP oligomerization, resulting in inhibition of HBV infection. Here, using troglitazone as a chemical probe to investigate entry process, the contribution of NTCP oligomerization to HBV internalization was evaluated. Using surface plasmon resonance and transporter kinetics, we found that troglitazone directly interacts with NTCP and non-competitively interferes with NTCP-mediated bile acid uptake, suggesting that troglitazone allosterically binds to NTCP, rather than to the bile acid-binding pocket. Additionally, alanine scanning mutagenesis showed that a mutation at phenylalanine 274 of NTCP (F274A) caused a loss of HBV susceptibility and disrupted both the oligomerization of NTCP and HBV internalization without affecting viral attachment to the cell surface. An inhibitor of the interaction between NTCP and epidermal growth factor receptor (EGFR), another host cofactor essential for HBV internalization, impeded NTCP oligomerization. Meanwhile, co-immunoprecipitation analysis revealed that neither troglitazone nor the F274A mutation in NTCP affect the NTCP-EGFR interaction. These findings suggest that NTCP oligomerization is initiated downstream of the NTCP-EGFR interaction, and then triggers HBV internalization. This study provides significant insight into the HBV entry mechanisms. Importance Hepatitis B virus (HBV) infection is mediated by a specific interaction with sodium taurocholate cotransporting polypeptide (NTCP), a viral entry receptor. Although the virus-receptor interactions are believed to trigger viral internalization into host cells, the exact molecular mechanisms of HBV internalization are not understood. In this study, we revealed the mode of action whereby troglitazone, a specific inhibitor of HBV internalization, impedes NTCP oligomerization, and identified NTCP phenylalanine 274 as a residue essential for this oligomerization. We further analyzed the association between NTCP oligomerization and HBV internalization, a process that is mediated by epidermal growth factor receptor (EGFR), another essential host cofactor for HBV internalization. Our study provides critical information on the mechanism of HBV entry, and suggests that oligomerization of the viral receptor serves as an attractive target for drug discovery.

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Establishment of a novel hepatitis B virus culture system using immortalized human hepatocytes

Scientific Reports ◽

10.1038/s41598-020-78655-x ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Yuichi Akahori ◽

Hiroki Kato ◽

Takashi Fujita ◽

Kohji Moriishi ◽

Yasuhito Tanaka ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Cell Lines ◽

Culture System ◽

Sodium Taurocholate ◽

Three Dimensional ◽

Human Hepatocytes ◽

Host Cells ◽

B Virus ◽

Almost All

AbstractRecent development of hepatitis B virus (HBV) culture systems has made it possible to analyze the almost all steps of the viral life cycle. However, the reproducibility of interaction between HBV and host cells seemed inaccurate in those systems because of utilization of cancer cell lines with a difference from hepatocytes in the majority of cases. In this study, in order to resolve this point, a novel HBV culture system using non-cancer-derived immortalized human hepatocytes derived cell lines, producing exogenous human sodium taurocholate cotransporting polypeptide, was developed. One of the cell clones, E/NtG8 cells, was permissive to both blood-borne HBV (HBVbb) and culture-derived recombinant HBV when cultured in the three-dimensional condition. Furthermore, the production of infectious HBV particles, which showed the similar physicochemical properties to HBVbb, was observed for about a month after HBVbb infection in this system, suggesting that it may reproduce whole steps of the HBV lifecycle under the condition analogous to human liver cells infected with HBV. This system seemed to contribute not only to find novel interactions between HBV and host cells but also to understand mechanism of HBV pathogenesis.

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

Journal of Virology ◽

10.1128/jvi.02832-15 ◽

2016 ◽

Vol 90 (9) ◽

pp. 4827-4831 ◽

Cited By ~ 18

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.

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Evidence for Dual Effects of DNA-Reactive Bile Acid Derivatives (Bamets) on Hepatitis B Virus Life Cycle in an In Vitro Replicative System

Antiviral Chemistry and Chemotherapy ◽

10.1177/095632020201300605 ◽

2002 ◽

Vol 13 (6) ◽

pp. 371-380 ◽

Cited By ~ 8

Author(s):

Marta R Romero ◽

Maria C Martinez-Diez ◽

Monica G Larena ◽

Rocio IR MacIas ◽

Mercedes Dominguez ◽

...

Keyword(s):

Life Cycle ◽

Bile Acid ◽

Hepatitis B Virus ◽

Hepatitis B ◽

Hbv Dna ◽

Drug Uptake ◽

Host Cells ◽

B Virus ◽

Bile Acid Derivatives ◽

Hepg2 2.2.15

A liver targeting strategy to direct antiviral drugs toward hepatitis B virus (HBV) was investigated. As model drugs we used cisplatin-bile acid derivatives (Bamets) to determine the production of virions by HBV-transfected hepatoblastoma cells (HepG2 2.2.15). Drug uptake was determined using flameless atomic absorption spectrometry to measure platinum cell contents. Cytotoxic effect was determined by formazan formation and neutral red uptake tests. The release of viral surface protein was evaluated by ELISA. The abundance of HBV-DNA in the medium was determined by quantitative real-time PCR and its structure by Southern blot analysis. The uptake of Bamets by HepG2 2.2.15 cells was higher than that of cisplatin. At concentrations lower than 10 μM, distinct Bamets have no toxic effect on host cells, whereas cisplatin dramatically reduced cell viability at concentrations higher than 1 μM. All the drugs tested inhibited the release of viral proteins to the medium, but induced a marked and progressive dose-dependent increase in the amount of viral DNA in the medium. This was mainly due to the release of short fragments of HBV-DNA in the case of cisplatin. On the contrary, Bamets induced an enhanced release of circular forms of HBV-DNA. These findings suggest the existence of a dual effect of Bamets on HBV life-cycle by enhancing the production of DNA replicative intermediates but reducing the secretion of complete virions. Altogether these characteristics recommend consideration of these compounds as a useful experimental tool in the investigation of novel liver targeted therapeutic agents based on bile acid derivatives for the treatment of HBV infections, or to carry out further studies on the HBV life cycle.

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DOCK11 and DENND2A play pivotal roles in the maintenance of hepatitis B virus in host cells

PLoS ONE ◽

10.1371/journal.pone.0246313 ◽

2021 ◽

Vol 16 (2) ◽

pp. e0246313

Author(s):

Shinichi Hashimoto ◽

Takayoshi Shirasaki ◽

Taro Yamashita ◽

Sadahiro Iwabuchi ◽

Yutaka Suzuki ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Limit Of Detection ◽

Low Frequency ◽

Hbv Dna ◽

Small Gtpase ◽

Host Cells ◽

Sequencing Analysis ◽

Circular Dna ◽

B Virus

Human hepatitis B virus (HBV) infection remains a serious health problem worldwide. However, the mechanism for the maintenance of HBV in a latent state within host cells remains unclear. Here, using single-cell RNA sequencing analysis, we identified four genes linked to the maintenance of HBV in a liver cell line expressing HBV RNA at a low frequency. These genes included DOCK11 and DENND2A, which encode small GTPase regulators. In primary human hepatocytes infected with HBV, knockdown of these two genes decreased the amount of both HBV DNA and covalently closed circular DNA to below the limit of detection. Our findings reveal a role for DOCK11 and DENND2A in the maintenance of HBV.

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

10.1101/729822 ◽

2019 ◽

Cited By ~ 1

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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A STUDY OF HEPATITIS B VIRUS GENOTYPES IN CHRONIC HEPATITIS B PATIENTS

Journal of Medicine and Pharmacy ◽

10.34071/jmp.2011.3.4 ◽

2011 ◽

pp. 25-29

Author(s):

Keyword(s):

Chronic Hepatitis ◽

Hepatitis B ◽

Chronic Hepatitis B ◽

Clinical Laboratory ◽

Laboratory Data ◽

Hbv Dna ◽

Hbv Genotypes ◽

B Virus ◽

Virus Genotypes ◽

Genotype C

Aims: To measure the prevalence of HBV genotypes in chronic hepatitis B patients and their relation to HBeAg and HBV DNA level. Methods: 81 patients were enrolled in this study from January 2009 to December 2010. Clinical, laboratory data were collected during the patient’s hospitalization. Sera were quantitatively tested for HBeAg and HBV DNA. HBV genotyping was made by real-time PCR. Results: Among the 81 patients, 60.5% had genotype B, 26.7% had genotype C and 8.6% had mixed genotype B-C. Prevalence of symptoms (fatigue, anorexia, insomnia...) was higher in genotype C than in genotype B. Genotype C patients had positivity higher HBeAg than genotype B patients (56% vs. 38,8%, p <0.05). The rate of HBV DNA > 107 copies/mL was higher in genotype C group than in genotype B group (36% vs. 28,6%, p > 0.05). Conclusions: Most of the patients had genotypes B or C. Patients with genotype C had positive HBeAg and may be related to higher serological HBV DNA level than in genotype B.

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Generation of Full-Length Functional Antibody against PreS2 of Hepatitis B Virus in Hepatic Cells In Vitro from Bicistrons Mediated by Gutless Adenovirus

BioDrugs ◽

10.2165/11316940-000000000-00000 ◽

2009 ◽

Vol 23 (6) ◽

pp. 391-397

Author(s):

Yuexiang Yin ◽

Fang Lin ◽

Qiang Zhuang ◽

Li Liu ◽

Cheng Qian

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Full Length ◽

Hepatic Cells ◽

B Virus

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272 Use of LioCyx-M, autologous hepatitis B virus (HBV)-Specific T cell receptor (TCR) T-cells, in advanced HBV-related hepatocellular carcinoma (HCC)

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0272 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A297-A297

Author(s):

Fu-Sheng Wang ◽

Fanping Meng ◽

Jiehua Jin ◽

Yuanyuan Li ◽

Regina Wanju Wong ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

T Cells ◽

Hepatitis B Virus ◽

T Cell ◽

Hepatitis B ◽

T Cell Receptor ◽

Dose Level ◽

Cell Receptor ◽

Hbv Dna ◽

B Virus

BackgroundWe have demonstrated the ability of Hepatitis-B-virus (HBV)-specific T cell receptor (TCR) bioengineered T cells to recognize and lyse Hepatocellular carcinoma (HCC) cells expressing HBV antigens derived from HBV-DNA integration in patients with liver transplant.1 LioCyx-M is an immunotherapeutic product composing of autologous T cells transiently modified with in-vitro transcribed mRNA encoding HBV-specific TCR. The transient TCR expression makes LioCyx -M amenable to a dose escalating posology.MethodsThe primary endpoint of this phase 1 trial is to assess the safety and tolerability of LioCyx-M in patients with advanced HBV-HCC without curative treatment options. Eligible patients were diagnosed with Barcelona clinic liver cancer stage B or C HCC (Child-Pugh < 7 points), receiving >1 year antiviral treatment prior to enrollment. These patients had matching HLA class I genotypes which present HBV encoded antigen. Peripheral blood was collected from each patient prior to each dose for LioCyx-M manufacturing. Patients received 4 escalating doses of 1×104 cells/kg, 1×105 cells/kg, 1×106 cells/kg, 5×106 cells/kg bodyweight (BW) in the first treatment cycle, each intravenously administered weekly. Patients underwent 1-month safety assessment post the 4th infusion, according to Common Terminology NCI CTCAE Version 4.0.3. If there were no dose associated toxicities, patients were eligible to continue administration of LioCyx-M at dose of 5 × 106 cells/kg BW weekly. Tumor response per RECIST 1.1 criteria and survival time were assessed.ResultsAt data cutoff (30 April 2020), eight patients were enrolled, with a median age of 53 (range: 49 - 67). These patients received a median number of 6 (range: 4 - 12) infusions of LioCyx-M. 1 patient developed Grade 3 elevations in alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT), aspartate aminotransferase (AST) and bilirubin after receiving LioCyx-M at dose level of 1×105 cells/kg BW. Another patient had Grade 1 transient fever after receiving LioCyx-M at dose level 5×106 cells/kg BW in the 4th, 5th and 6th infusions. No treatment-related adverse events (trAEs) such as cytokine release syndrome or neurotoxicity were observed. No fatal trAEs were observed. The median time to progression was 1.9 months (range: 0.2 - 9.5 months). The median overall survival was 34 months (range: 3 - 38.2 months).ConclusionsThe encouraging clinical outcome and tolerable safety highlight the good benefit-risk profile of LioCyx-M. Therefore, further exploration of efficacy of LioCyx-M treatment for advanced HBV-HCC is warranted in a Phase 2 proof-of-concept clinical study.AcknowledgementsFunding: Lion TCR.Trial RegistrationNCT03899415Ethics ApprovalThe study was approved by Fifth Medical Center of Chinese PLA General Hospital’s Ethics Board, approval number R2016185DI010.ReferenceTan AT, Yang N, Lee Krishnamoorthy T, et al. Use of Expression Profiles of HBV-DNA Integrated Into Genomes of Hepatocellular Carcinoma Cells to Select T Cells for Immunotherapy. Gastroenterology 2019;156(6):1862–1876.e9.

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