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Huang HE, Colasanti O, Li TF, Lohmann V. Limited impact of hepatitis A virus 3C protease-mediated cleavage on the functions of NEMO in human hepatocytes. J Virol 2025; 99:e0226424. [PMID: 39853114 PMCID: PMC11852894 DOI: 10.1128/jvi.02264-24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2024] [Accepted: 01/06/2025] [Indexed: 01/26/2025] Open
Abstract
NF-κB essential modulator (NEMO) is critically involved in the induction of interferons (IFNs) and pro-inflammatory cytokines. Hepatitis A virus (HAV) 3C protease was recently identified to cleave NEMO in non-hepatic cells. This study aimed at understanding efficiency and function of HAV 3C-mediated NEMO cleavage in hepatocytes. HAV 3C protease and its precursor 3CD strongly affected NEMO abundance in ectopic expression models, which was not observed in HAV replicon cells and upon HAV infection. Using a cleavage-resistant NEMO mutant, we found that specific cleavage by 3C only marginally contributed to NEMO degradation, whereas the magnitude of the effect was due to cytotoxic effects induced by 3C activity. Cleavage efficiency generally did not suffice to disrupt the type I IFN or NF-κB signaling pathways. Knockout of NEMO indeed abrogated both pathways, whereas efficient knockdown had limited the impact on NEMO-mediated signaling, suggesting that low levels of NEMO are sufficient to maintain antiviral responses in hepatocytes. NEMO cleavage was barely detectable in a cell line harboring a persistent HAV replicon or in HAV-infected cells. HAV infection induced a robust innate immune response, which was not affected by efficient knockdown of NEMO, arguing for a limited potential contribution of NEMO cleavage to innate immune counteraction. Overall, our data suggest that HAV 3C is capable of partially cleaving NEMO as reported. However, since minute expression levels of NEMO were sufficient for induction of innate immunity, inefficient NEMO cleavage by HAV is unlikely to contribute to dampening of innate immune responses in hepatocytes.IMPORTANCEHepatitis A virus (HAV) establishes acute infections of the liver, which are always cleared, while a number of mechanisms have been identified contributing to immune escape. Among those, proteolytic cleavage of NF-κB essential modulator (NEMO) by HAV has been suggested to counteract innate immune responses. This study demonstrates that the HAV 3C protease cleaves NEMO inefficiently and does not result in substantial disruption of antiviral signaling. Importantly, NEMO remains capable of inducing an effective immune response in hepatocytes even at low expression levels. Our findings suggest a limited role for NEMO cleavage in HAV's interaction with host immunity and call for a revision of our understanding of HAV counteraction mechanisms.
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Affiliation(s)
- Hao-En Huang
- Department of Infectious Diseases, Molecular Virology, Section Virus-Host Interactions, Heidelberg University, Medical Faculty Heidelberg, Heidelberg, Germany
| | - Ombretta Colasanti
- Department of Infectious Diseases, Molecular Virology, Section Virus-Host Interactions, Heidelberg University, Medical Faculty Heidelberg, Heidelberg, Germany
| | - Teng-Feng Li
- Department of Infectious Diseases, Molecular Virology, Section Virus-Host Interactions, Heidelberg University, Medical Faculty Heidelberg, Heidelberg, Germany
| | - Volker Lohmann
- Department of Infectious Diseases, Molecular Virology, Section Virus-Host Interactions, Heidelberg University, Medical Faculty Heidelberg, Heidelberg, Germany
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Zhou H, Zhu Y, Liu N, Zhang W, Han J. Effect of iron saturation of bovine lactoferrin on the inhibition of hepatitis B virus in vitro. PeerJ 2024; 12:e17302. [PMID: 38737747 PMCID: PMC11086297 DOI: 10.7717/peerj.17302] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2022] [Accepted: 04/04/2024] [Indexed: 05/14/2024] Open
Abstract
Background Hepatitis B virus (HBV) infection poses a major public health problem worldwide. Bovine lactoferrin (bLf) is a natural product that can inhibit HBV, but the effect of iron saturation on its resistance to HBV is unknown. Aims The purpose of this study is to investigate the impact of iron saturation of bLf against HBV. Methods HepG2 cells were cultured in DMEM high glucose containing 10% inactivated fetal calf serum, at 37 °C, in 5% CO2. MTT method was used to detect the cytotoxicity of bLf to HepG2 cells. Apo-bLf and holo-bLf were prepared from bLf. Iron saturation of these proteins was determined by atomic absorption spectrophotometry. Non-cytotoxic concentrations of candidate proteins were used in anti-HBV tests. Fluorescent quantitative polymerase chain reaction was used to detect HBV-DNA. Results The TC50 and TC0of bLf were 54.570 mg/ml and 1.997 mg/ml, respectively. The iron saturation of bLf, apo-bLf and holo-bLf were 10.29%, 8.42% and 85.32%, respectively. In this study, four non-cytotoxic concentrations of candidate proteins (1.5, 1.0, 0.5, and 0.1 mg/ml, respectively) were used to inhibit HBV in HepG2 cells. The results showed that 1.5 mg/ml bLf and 0.1 mg/ml holo-bLf effectively impaired the HBV-DNA amplification in HBV-infected HepG2 cells (P < 0.05). However, apo-bLf, and Fe3+ did not show the anti-HBV effects. Conclusion A total of 1.5 mg/ml bLf and 0.1 mg/ml holo-bLf could inhibit HBV-DNA in HepG2 cells. Complete bLf structure, appropriate concentration and iron saturation of bLf are necessary conditions for anti-HBV effects.
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Affiliation(s)
- Haibo Zhou
- College of Food Science, Northeast Agricultural University, Harbin, Heilongjiang Province, China
| | - Yiwei Zhu
- Chongqing Food Industry Research Institute Co., Ltd., Chongqing, China
| | - Ning Liu
- College of Food Science, Northeast Agricultural University, Harbin, Heilongjiang Province, China
| | - Wencui Zhang
- Institute of Endemic Diseases, Harbin Center for Disease Control and Prevention, Harbin, Heilongjiang Province, China
| | - Jianchun Han
- College of Food Science, Northeast Agricultural University, Harbin, Heilongjiang Province, China
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3
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LYTACs that engage the asialoglycoprotein receptor for targeted protein degradation. Nat Chem Biol 2021; 17:937-946. [PMID: 33767387 PMCID: PMC8387313 DOI: 10.1038/s41589-021-00770-1] [Citation(s) in RCA: 285] [Impact Index Per Article: 71.3] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2020] [Accepted: 02/10/2021] [Indexed: 02/06/2023]
Abstract
Selective protein degradation platforms have afforded new development opportunities for therapeutics and tools for biological inquiry. The first lysosome targeting chimeras (LYTACs) targeted extracellular and membrane proteins for degradation by bridging a target protein to the cation-independent mannose-6-phosphate receptor (CI-M6PR). Here, we developed LYTACs that engage the asialoglycoprotein receptor (ASGPR), a liver-specific lysosomal targeting receptor, to degrade extracellular proteins in a cell type-specific manner. We conjugated binders to a tri-GalNAc motif that engages ASGPR to drive downregulation of proteins. Degradation of EGFR by GalNAc-LYTAC attenuated EGFR signaling compared to inhibition with an antibody. Furthermore, we demonstrated that a LYTAC comprising a 3.4 kDa peptide binder linked to a tri-GalNAc ligand degrades integrins and reduces cancer cell proliferation. Degradation with a single tri-GalNAc ligand prompted site-specific conjugation on antibody scaffolds, which improved the pharmacokinetic profile of GalNAc-LYTACs in vivo. GalNAc-LYTACs thus represent an avenue for cell-type restricted protein degradation.
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Yang Q, Wang P, Wang S, Wang Y, Feng S, Zhang S, Li H. The hepatic lectin of zebrafish binds a wide range of bacteria and participates in immune defense. FISH & SHELLFISH IMMUNOLOGY 2018; 82:267-278. [PMID: 30120977 DOI: 10.1016/j.fsi.2018.08.012] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/14/2018] [Revised: 07/18/2018] [Accepted: 08/07/2018] [Indexed: 06/08/2023]
Abstract
C-type lectins (CTLs) have a diverse range of functions including cell-cell adhesion, immune response to pathogens and apoptosis. Asialoglycoprotein receptor (ASGPR), also known as hepatic lectin, a member of CTLs, was the first animal lectin identified, yet information regarding it remains rather limited in teleost. In this study, we identified a putative protein in zebrafish, named as the zebrafish hepatic lectin (Zhl). The zhl encoded a typical Ca2+-dependent carbohydrate-binding protein, and was mainly expressed in the liver in a tissue specific fashion. Challenge with LPS and LTA resulted in significant up-regulation of zhl expression, suggesting involvement in immune response. Actually, recombinant C-type lectin domain (rCTLD) of Zhl was found to be capable of agglutinating and binding to both Gram-negative and Gram-positive bacteria and enhancing the phagocytosis of the bacteria by macrophages. Moreover, rCTLD specifically bound to insoluble lipopolysaccharide (LPS), lipoteichoic acid (LTA) and peptidoglycan (PGN), which were inhibited by galactose. Interestingly, Zhl was located in the membrane, and its overexpression could inhibit the production of pre-inflammatory cytokines. Taken together, these results indicate that Zhl has immune activity capable of defending invading pathogens, enriching our understanding of the function of ASGPR/hepatic lectin.
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Affiliation(s)
- Qingyun Yang
- Laboratory for Evolution & Development, Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao 266003, China; Department of Marine Biology, Ocean University of China, Qingdao 266003, China
| | - Peng Wang
- Laboratory for Evolution & Development, Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao 266003, China; Department of Marine Biology, Ocean University of China, Qingdao 266003, China
| | - Su Wang
- Laboratory for Evolution & Development, Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao 266003, China; Department of Marine Biology, Ocean University of China, Qingdao 266003, China
| | - Yashuo Wang
- Laboratory for Evolution & Development, Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao 266003, China; Department of Marine Biology, Ocean University of China, Qingdao 266003, China
| | - Shuoqi Feng
- Laboratory for Evolution & Development, Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao 266003, China; Department of Marine Biology, Ocean University of China, Qingdao 266003, China
| | - Shicui Zhang
- Laboratory for Evolution & Development, Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao 266003, China; Department of Marine Biology, Ocean University of China, Qingdao 266003, China
| | - Hongyan Li
- Laboratory for Evolution & Development, Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao 266003, China; Department of Marine Biology, Ocean University of China, Qingdao 266003, China.
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Abstract
With high morbidity and mortality worldwide, there is great interest in effective therapies for chronic hepatitis B (CHB) virus. There are currently several dozen investigational agents being developed for treatment of CHB. They can be broadly divided into two categories: (1) direct-acting antivirals (DAAs) that interfere with a specific step in viral replication; and (2) host-targeting agents that inhibit viral replication by modifying host cell function, with the latter group further divided into the subcategories of immune modulators and agents that target other host functions. Included among the DAAs being developed are RNA interference therapies, covalently closed circular DNA (cccDNA) formation and transcription inhibitors, core/capsid inhibitors, reverse transcriptase inhibitors, hepatitis B surface antigen (HBsAg) release inhibitors, antisense oligonucleotides, and helioxanthin analogues. Included among the host-targeting agents are entry inhibitors, cyclophilin inhibitors, and multiple immunomodulatory agents, including Toll-like receptor agonists, immune checkpoint inhibitors, therapeutic vaccines, engineered T cells, and several cytokine agents, including recombinant human interleukin-7 (CYT107) and SB 9200, a novel therapy that is believed to both have direct antiviral properties and to induce endogenous interferon. In this review we discuss agents that are currently in the clinical stage of development for CHB treatment as well as strategies and agents currently at the evaluation and discovery phase and potential future targets. Effective approaches to CHB may require suppression of viral replication combined with one or more host-targeting agents. Some of the recent research advances have led to the hope that with such a combined approach we may have a functional cure for CHB in the not distant future.
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Affiliation(s)
- Altaf Dawood
- Department of Internal Medicine, Section of Gastroenterology, University of Nevada School of Medicine, Las Vegas, NV, USA
| | - Syed Abdul Basit
- Department of Internal Medicine, Section of Gastroenterology, University of Nevada School of Medicine, Las Vegas, NV, USA
| | - Mahendran Jayaraj
- Department of Internal Medicine, Section of Gastroenterology, University of Nevada School of Medicine, Las Vegas, NV, USA
| | - Robert G Gish
- Department of Internal Medicine, Section of Gastroenterology, University of Nevada School of Medicine, Las Vegas, NV, USA.
- Division of Gastroenterology and Hepatology, Department of Medicine, Stanford University Medical Center, Stanford, CA, USA.
- Hepatitis B Foundation, Doylestown, PA, USA.
- Asian Pacific Health Foundation, San Diego, CA, USA.
- National Viral Hepatitis Roundtable, Washington, DC, USA.
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Huang X, Leroux JC, Castagner B. Well-Defined Multivalent Ligands for Hepatocytes Targeting via Asialoglycoprotein Receptor. Bioconjug Chem 2016; 28:283-295. [DOI: 10.1021/acs.bioconjchem.6b00651] [Citation(s) in RCA: 56] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Affiliation(s)
- Xiangang Huang
- Institute
of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich, Vladimir-Prelog-Weg 1-5/10, 8093 Zurich, Switzerland
| | - Jean-Christophe Leroux
- Institute
of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich, Vladimir-Prelog-Weg 1-5/10, 8093 Zurich, Switzerland
| | - Bastien Castagner
- Department
of Pharmacology and Therapeutics, McGill University, 3655 Prom. Sir-William-Osler, Montréal, Québec H3G 1Y6, Canada
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Rehman Z, Sadia H, Fahim A, Niazi UHK, Azam MZ. Mutational analysis and interactions of HBV preS1 with asialoglycoprotein receptor. Future Virol 2016. [DOI: 10.2217/fvl-2016-0083] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Background: The mutations in preS1 of a large envelop protein of HBV may have profound implications in HBV receptor binding to hepatocytes and subsequent entry of the virus into host cells. Aims: This study aimed to identify the mutations in preS1 region and the receptor binding interactions of preS1 with hepatocytes. Methods: The mutations were searched through direct sequencing of the preS1 region. Sequence analysis was done through ClustalX and Jalview. Ab initio modeling of preS1 was done through Rosetta and QUARK followed by glycosylation of best model of preS1. Finally the interactions of preS1 with ASGPR was studied using PatchDock and analysis was done using MOE and pyMol. Results: Sequence comparison revealed changes in the preS1 region. Ab initio modeling results showed that preS1 is an overall unstructured protein with the presence of three structural motifs. Docking of preS1 with asialoglycoprotein receptor showed mostly hydrophobic interactions. Conclusion: In conclusion, preS1 sequences from Pakistani isolates were found to be 90% conserved and the predicted structure of preS1 was near to native structure.
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Affiliation(s)
- Zaira Rehman
- Healthcare Biotechnology Department, Atta-ur-Rahman School of Applied Biosciences (ASAB), National University of Sciences & Technology, Islamabad, Pakistan
| | - Hajra Sadia
- Healthcare Biotechnology Department, Atta-ur-Rahman School of Applied Biosciences (ASAB), National University of Sciences & Technology, Islamabad, Pakistan
| | - Ammad Fahim
- Healthcare Biotechnology Department, Atta-ur-Rahman School of Applied Biosciences (ASAB), National University of Sciences & Technology, Islamabad, Pakistan
| | - Umer HK Niazi
- IBERS, Aberystwyth University, Edward Llwyd Building, Penglais Campus, Aberystwyth, Ceredigion, Wales SY23 3FG, UK
| | - Muhammad Z Azam
- National Institute of Liver & Gastrointestinal Diseases, Dow University of Health Sciences, Karachi, Pakistan
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Zhang L, Tian Y, Wen Z, Zhang F, Qi Y, Huang W, Zhang H, Wang Y. Asialoglycoprotein receptor facilitates infection of PLC/PRF/5 cells by HEV through interaction with ORF2. J Med Virol 2016; 88:2186-2195. [DOI: 10.1002/jmv.24570] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 08/30/2023]
Abstract
Although the biological and epidemiological features of hepatitis E virus (HEV) have been studied extensively in recent years, the mechanism by which HEV infects cells is still poorly understood. In this study, coimmunoprecipitation, pull‐down, and ELISA were used to show that the HEV ORF2 protein interacts directly with the ectodomain of both ASGR1 and ASGR2. Susceptibility to HEV correlated positively with the expression level of surface asialoglycoprotein receptor (ASGPR) in cell lines. ASGPR‐directed small interfering RNA (siRNA) in HEV‐infected PLC/PRF/5 cells had no significant effect on HEV release, suggesting that ASGPR mainly regulates the viral binding and entry steps. Both the purified ASGPR ectodomain and anti‐ASGPR antibodies disturbed the binding of HEV to PLC/PRF/5 cells. The classic ASGPR ligands asialofetuin, asialoganglioside, and fibronectin competitively inhibited the binding of HEV to hepatocytes in the presence of calcium. HeLa cell lines stably expressing ASGPR displayed increased HEV‐binding capacity, whereas ASGPR‐knockout PLC/PRF/5 cell lines had lower HEV‐binding capacity. Thus, our study demonstrates that ASGPR is involved in and facilitates HEV infection by binding to ORF2. J. Med. Virol. 88:2186–2195, 2016. © 2016 Wiley Periodicals, Inc.
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Affiliation(s)
- Li Zhang
- Division of HIV/AIDS and Sexually‐Transmitted Virus Vaccines National Institutes for Food and Drug Control Beijing China
| | - Yabin Tian
- Division of HIV/AIDS and Sexually‐Transmitted Virus Vaccines National Institutes for Food and Drug Control Beijing China
| | - Zhiheng Wen
- Division of HIV/AIDS and Sexually‐Transmitted Virus Vaccines National Institutes for Food and Drug Control Beijing China
| | - Feng Zhang
- Division of Monoclonal Antibody Products National Institutes for Food and Drug Control Beijing China
| | - Ying Qi
- Division of HIV/AIDS and Sexually‐Transmitted Virus Vaccines National Institutes for Food and Drug Control Beijing China
| | - Weijin Huang
- Division of HIV/AIDS and Sexually‐Transmitted Virus Vaccines National Institutes for Food and Drug Control Beijing China
| | - Heqiu Zhang
- Department of Bio‐Diagnosis Beijing Institute of Basic Medical Sciences Beijing China
| | - Youchun Wang
- Division of HIV/AIDS and Sexually‐Transmitted Virus Vaccines National Institutes for Food and Drug Control Beijing China
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Jing YY, Liu WT, Guo SW, Ye F, Fan QM, Yu GF, Yu DD, Gao L, Sun K, Han ZP, Li R, Yang Y, Zhao QD, Wu MC, Wang HY, Wei LX. Hepatitis B virus (HBV) receptors: Deficiency in tumor results in scant HBV infection and overexpression in peritumor leads to higher recurrence risk. Oncotarget 2016; 6:42952-62. [PMID: 26515593 PMCID: PMC4767483 DOI: 10.18632/oncotarget.5518] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2015] [Accepted: 10/08/2015] [Indexed: 02/07/2023] Open
Abstract
Hepatitis B virus (HBV) infection is a risk factor for hepatocarcinogenesis and recurrence. Here, we sought to characterize intratumoral and peritumoral expression of HBsAg and its specific receptors in HBsAg-positive hepatocellular carcinoma (HCC) patients and further examined their correlation with the recurrence-free survival (RFS). HCC tissue and adjacent normal tissue specimens were acquired from HBsAg-positive patients. The presence of HBsAg and receptors, as well as hepatic progenitor cells (HPCs) were detected by tissue microassay and immunohistochemistry. Necroinflammatory activity was evaluated by HE staining. The mean IOD of HBsAg and HBV DNA in the intratumoral tissues was markedly lower than that in the peritumoral tissues (P < 0.001). Pearson correlation analysis further showed a significant correlation between the expression of HBsAg and NTCP (r = 0.461, P < 0.001) or ASGPR (r = 0.506, P < 0.001) in peritumoral tissues. And the peritumoral HBsAg and receptors presented a positive association with necroinflammatory activity (P < 0.05). Inflammation induced by HBV infection presented a positive association with HPCs activation (P < 0.05). Additionally, due to lack of HBV receptors, HPCs was not preferentially infected with HBV, but activated HPCs had a significant correlation with HBsAg expression in peritumoral tissues, and the peritumoral HPCs activation was associated with RFS of HCC patients, therefore, the overexpression of HBsAg and receptors in peritumor were also with higher recurrence risk (P < 0.05). In conclusion, lack of HBV receptors resulted in scant HBV infection in tumor cells, and overexpression of HBsAg and receptors in peritumor was strongly associated with higher recurrence risk in HCC patients.
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Affiliation(s)
- Ying-Ying Jing
- Tumor Immunology and Gene Therapy Center, Eastern Hepatobiliary Surgery Hospital, The Second Military Medical University, Shanghai, China
| | - Wen-Ting Liu
- Tumor Immunology and Gene Therapy Center, Eastern Hepatobiliary Surgery Hospital, The Second Military Medical University, Shanghai, China
| | - Shi-Wei Guo
- Tumor Immunology and Gene Therapy Center, Eastern Hepatobiliary Surgery Hospital, The Second Military Medical University, Shanghai, China
| | - Fei Ye
- Tumor Immunology and Gene Therapy Center, Eastern Hepatobiliary Surgery Hospital, The Second Military Medical University, Shanghai, China
| | - Qing-Min Fan
- Tumor Immunology and Gene Therapy Center, Eastern Hepatobiliary Surgery Hospital, The Second Military Medical University, Shanghai, China
| | - Guo-Feng Yu
- Tumor Immunology and Gene Therapy Center, Eastern Hepatobiliary Surgery Hospital, The Second Military Medical University, Shanghai, China
| | - Dan-Dan Yu
- Tumor Immunology and Gene Therapy Center, Eastern Hepatobiliary Surgery Hospital, The Second Military Medical University, Shanghai, China
| | - Lu Gao
- Tumor Immunology and Gene Therapy Center, Eastern Hepatobiliary Surgery Hospital, The Second Military Medical University, Shanghai, China
| | - Kai Sun
- Tumor Immunology and Gene Therapy Center, Eastern Hepatobiliary Surgery Hospital, The Second Military Medical University, Shanghai, China.,Central Laboratory, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
| | - Zhi-Peng Han
- Tumor Immunology and Gene Therapy Center, Eastern Hepatobiliary Surgery Hospital, The Second Military Medical University, Shanghai, China
| | - Rong Li
- Tumor Immunology and Gene Therapy Center, Eastern Hepatobiliary Surgery Hospital, The Second Military Medical University, Shanghai, China
| | - Yang Yang
- Tumor Immunology and Gene Therapy Center, Eastern Hepatobiliary Surgery Hospital, The Second Military Medical University, Shanghai, China
| | - Qiu-Dong Zhao
- Tumor Immunology and Gene Therapy Center, Eastern Hepatobiliary Surgery Hospital, The Second Military Medical University, Shanghai, China
| | - Meng-Chao Wu
- Tumor Immunology and Gene Therapy Center, Eastern Hepatobiliary Surgery Hospital, The Second Military Medical University, Shanghai, China
| | - Hong-Yang Wang
- International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Institute/Hospital, The Second Military Medical University, Shanghai, China
| | - Li-Xin Wei
- Tumor Immunology and Gene Therapy Center, Eastern Hepatobiliary Surgery Hospital, The Second Military Medical University, Shanghai, China
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The asialoglycoprotein receptor suppresses the metastasis of hepatocellular carcinoma via LASS2-mediated inhibition of V-ATPase activity. Cancer Lett 2016; 379:107-16. [PMID: 27241665 DOI: 10.1016/j.canlet.2016.05.030] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2016] [Revised: 05/25/2016] [Accepted: 05/25/2016] [Indexed: 12/28/2022]
Abstract
The asialoglycoprotein receptor (ASGR), which is expressed mainly in hepatocytes, is downregulated in poorly differentiated hepatocellular carcinoma (HCC). Here we investigated the role of ASGR1 in HCC metastasis as well as the possible underlying molecular mechanisms. We found that ASGR1 was downregulated in HCC tissue compared with adjacent non-tumorous liver tissue and that lower ASGR1 expression was associated with higher TNM stage and poorer prognosis in HCC patients. ASGR1 overexpression inhibited hepatoma cell migration and invasion in vitro and in vivo, while ASGR1 knockdown had the opposite effects. Furthermore, ASGR1 interacted directly with human longevity assurance homolog 2 of yeast LAG1 (LASS2). Knockdown of LASS2 attenuated the inhibitory effects of ASGR1 on hepatoma cell migration and invasion in vitro. ASGR1 decreased V-ATPase activity in hepatoma cells, and this was reversed by LASS2 knockdown. Finally, HCC patients with low LASS2 levels had poor prognosis, while those with high ASGR1 and LASS2 levels had better prognosis. Thus, ASGR1 may act as a potential metastasis suppressor in HCC, and the combination of ASGR1 and LASS2 may help predict the prognosis of HCC patients.
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11
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Schroeder B, McNiven MA. Importance of endocytic pathways in liver function and disease. Compr Physiol 2015; 4:1403-17. [PMID: 25428849 DOI: 10.1002/cphy.c140001] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Hepatocellular endocytosis is a highly dynamic process responsible for the internalization of a variety of different receptor ligand complexes, trophic factors, lipids, and, unfortunately, many different pathogens. The uptake of these external agents has profound effects on seminal cellular processes including signaling cascades, migration, growth, and proliferation. The hepatocyte, like other well-polarized epithelial cells, possesses a host of different endocytic mechanisms and entry routes to ensure the selective internalization of cargo molecules. These pathways include receptor-mediated endocytosis, lipid raft associated endocytosis, caveolae, or fluid-phase uptake, although there are likely many others. Understanding and defining the regulatory mechanisms underlying these distinct entry routes, sorting and vesicle formation, as well as the postendocytic trafficking pathways is of high importance especially in the liver, as their mis-regulation can contribute to aberrant liver pathology and liver diseases. Further, these processes can be "hijacked" by a variety of different infectious agents and viruses. This review provides an overview of common components of the endocytic and postendocytic trafficking pathways utilized by hepatocytes. It will also discuss in more detail how these general themes apply to liver-specific processes including iron homeostasis, HBV infection, and even hepatic steatosis.
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Affiliation(s)
- Barbara Schroeder
- Department of Biochemistry and Molecular Biology, Center for Basic Research in Digestive Diseases, Mayo Clinic and Foundation, Rochester, Minnesota
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12
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Rehman Z, Fahim A, Sadia H. Deciphering the mystery of hepatitis B virus receptors: A historical perspective. Virusdisease 2015; 26:97-104. [PMID: 26396975 DOI: 10.1007/s13337-015-0260-1] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2015] [Accepted: 06/25/2015] [Indexed: 12/22/2022] Open
Abstract
Hepatitis B virus is one of the major reasons of viral hepatitis with an estimated 350 million infected patients worldwide. Although, the virus was discovered and cloned more than three decades ago, its entry mechanism has still been in investigation. Numerous potential candidates have been proposed and investigated rigorously to reveal HBV entry mechanism and to unveil the first door of viral entry to hepatocytes. This review provides a short account of role of receptors for entry of HBV into hepatocytes. The viral preS1 region of large surface protein is involved in the attachment of HBV to hepatocytes. The putative attachment site of HBV is located at amino acids 21-47 of preS1. So far, several proteins have been proposed to interact with these different regions of the preS1 domain which includes human immunoglobulin A receptor, glyceraldehyde-3-phosphate dehydrogenase, interleukin-6, a 31-kDa protein, HBV binding factor, asialoglycoprotein receptor, nascent polypeptide-associated complex α polypeptide, lipoprotein lipase, hepatocyte-associated heparan sulfate proteoglycans, glucose-regulated protein 75. However, none of them have appeared to be generally accepted as a true receptor for the virus until recently when sodium taurocholate cotransporting polypeptide identified as HBV entry receptor. Current review provides scientific historical perspective of various candidates known to be interacting with preS1 of HBV for their possible role in viral entry.
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Affiliation(s)
- Zaira Rehman
- Healthcare Biotechnology Department, Atta-ur-Rahman School of Applied Biosciences (ASAB), National University of Sciences & Technology (NUST), Sector H-12, Islamabad, Pakistan
| | - Ammad Fahim
- Healthcare Biotechnology Department, Atta-ur-Rahman School of Applied Biosciences (ASAB), National University of Sciences & Technology (NUST), Sector H-12, Islamabad, Pakistan
| | - Hajra Sadia
- Healthcare Biotechnology Department, Atta-ur-Rahman School of Applied Biosciences (ASAB), National University of Sciences & Technology (NUST), Sector H-12, Islamabad, Pakistan
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13
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Pharmacokinetic characteristics, pharmacodynamic effect and in vivo antiviral efficacy of liver-targeted interferon alpha. PLoS One 2015; 10:e0117847. [PMID: 25689509 PMCID: PMC4331089 DOI: 10.1371/journal.pone.0117847] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2014] [Accepted: 01/02/2015] [Indexed: 01/08/2023] Open
Abstract
Interferon alpha (IFNα) is used for the treatment of hepatitis B virus infection, and whilst efficacious, it is associated with multiple adverse events caused by systemic exposure to interferon. We therefore hypothesise that targeting IFN directly to the intended site of action in the liver would reduce exposure in blood and peripheral tissue and hence improve the safety and tolerability of IFNα therapy. Furthermore we investigated whether directing IFN to the reservoir of infection in the liver may improve antiviral efficacy by increasing local concentration in target organs and tissues. Our previous results show that the mIFNα2 fused to an ASGPR specific liver targeting antibody, DOM26h-196-61, results in a fusion protein which retains the activity of both fusion partners when measured in vitro. In vivo targeting of the liver by mIFNα2-DOM26h-196-61, hereafter referred to as targeted mIFNα2, was observed in microSPECT imaging studies in mice. In this study we show by pharmacokinetic analysis that antibody mediated liver-targeting results in increased uptake and exposure of targeted mIFNα2 in target tissues, and correspondingly reduced uptake and exposure in systemic circulation, clearance organs and non-target tissues. We also show that cytokine activity and antiviral activity of liver-targeted IFN is observed in vivo, but that, contrary to expectations, liver-targeting of mIFNα2 using ASGPR specific dAbs actually leads to a reduced pharmacodynamic effect in target organs and lower antiviral activity in vivo when compared to non-targeted mIFNα2-dAb fusions.
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14
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Delgado CL, Núñez E, Yélamos B, Gómez-Gutiérrez J, Peterson DL, Gavilanes F. Study of the putative fusion regions of the preS domain of hepatitis B virus. BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 2014; 1848:895-906. [PMID: 25554595 DOI: 10.1016/j.bbamem.2014.12.020] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/15/2014] [Revised: 12/01/2014] [Accepted: 12/22/2014] [Indexed: 02/09/2023]
Abstract
In a previous study, it was shown that purified preS domains of hepatitis B virus (HBV) could interact with acidic phospholipid vesicles and induce aggregation, lipid mixing and leakage of internal contents which could be indicative of their involvement in the fusion of the viral and cellular membranes (Núñez, E. et al. 2009. Interaction of preS domains of hepatitis B virus with phospholipid vesicles. Biochim. Biophys. Acta 17884:417-424). In order to locate the region responsible for the fusogenic properties of preS, five mutant proteins have been obtained from the preS1 domain of HBV, in which 40 amino acids have been deleted from the sequence, with the starting point of each deletion moving 20 residues along the sequence. These proteins have been characterized by fluorescence and circular dichroism spectroscopy, establishing that, in all cases, they retain their mostly non-ordered conformation with a high percentage of β structure typical of the full-length protein. All the mutants can insert into the lipid matrix of dimyristoylphosphatidylglycerol vesicles. Moreover, we have studied the interaction of the proteins with acidic phospholipid vesicles and each one produces, to a greater or lesser extent, the effects of destabilizing vesicles observed with the full-length preS domain. The ability of all mutants, which cover the complete sequence of preS1, to destabilize the phospholipid bilayers points to a three-dimensional structure and/or distribution of amino acids rather than to a particular amino acid sequence as being responsible for the membrane fusion process.
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Affiliation(s)
- Carmen L Delgado
- Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense, 28040 Madrid, Spain
| | - Elena Núñez
- Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense, 28040 Madrid, Spain
| | - Belén Yélamos
- Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense, 28040 Madrid, Spain
| | - Julián Gómez-Gutiérrez
- Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense, 28040 Madrid, Spain
| | - Darrell L Peterson
- Department of Biochemistry and Molecular Biology, Medical College of Virginia, Virginia Commonwealth University, Richmond, 23298 VA, USA
| | - Francisco Gavilanes
- Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense, 28040 Madrid, Spain.
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15
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Ebrahim Attia AB, Oh P, Yang C, Tan JPK, Rao N, Hedrick JL, Yang YY, Ge R. Insights into EPR effect versus lectin-mediated targeted delivery: biodegradable polycarbonate micellar nanoparticles with and without galactose surface decoration. SMALL (WEINHEIM AN DER BERGSTRASSE, GERMANY) 2014; 10:4281-4286. [PMID: 25091699 DOI: 10.1002/smll.201401295] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/11/2014] [Revised: 05/30/2014] [Indexed: 06/03/2023]
Abstract
Polymeric micelles with and without galactose are synthesized to study liver targeting ability in an orthotopic HCC rat model. Micelles with galactose accumulate more in the healthy liver tissue instead of HCC, while micelles without galactose amass in HCC by the EPR effect. These micelles show great potential as drug delivery carriers to target either the liver or HCC.
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16
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Adokoh CK, Quan S, Hitt M, Darkwa J, Kumar P, Narain R. Synthesis and Evaluation of Glycopolymeric Decorated Gold Nanoparticles Functionalized with Gold-Triphenyl Phosphine as Anti-Cancer Agents. Biomacromolecules 2014; 15:3802-10. [DOI: 10.1021/bm5010977] [Citation(s) in RCA: 44] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Affiliation(s)
- Christian K. Adokoh
- Department
of Chemistry, University of Johannesburg, P.O. Box 524, Auckland Park 2006, South Africa
| | | | | | - James Darkwa
- Department
of Chemistry, University of Johannesburg, P.O. Box 524, Auckland Park 2006, South Africa
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17
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Abstract
Hepatocytes, like other epithelia, are situated at the interface between the organism's exterior and the underlying internal milieu and organize the vectorial exchange of macromolecules between these two spaces. To mediate this function, epithelial cells, including hepatocytes, are polarized with distinct luminal domains that are separated by tight junctions from lateral domains engaged in cell-cell adhesion and from basal domains that interact with the underlying extracellular matrix. Despite these universal principles, hepatocytes distinguish themselves from other nonstriated epithelia by their multipolar organization. Each hepatocyte participates in multiple, narrow lumina, the bile canaliculi, and has multiple basal surfaces that face the endothelial lining. Hepatocytes also differ in the mechanism of luminal protein trafficking from other epithelia studied. They lack polarized protein secretion to the luminal domain and target single-spanning and glycosylphosphatidylinositol-anchored bile canalicular membrane proteins via transcytosis from the basolateral domain. We compare this unique hepatic polarity phenotype with that of the more common columnar epithelial organization and review our current knowledge of the signaling mechanisms and the organization of polarized protein trafficking that govern the establishment and maintenance of hepatic polarity. The serine/threonine kinase LKB1, which is activated by the bile acid taurocholate and, in turn, activates adenosine monophosphate kinase-related kinases including AMPK1/2 and Par1 paralogues has emerged as a key determinant of hepatic polarity. We propose that the absence of a hepatocyte basal lamina and differences in cell-cell adhesion signaling that determine the positioning of tight junctions are two crucial determinants for the distinct hepatic and columnar polarity phenotypes.
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Affiliation(s)
- Aleksandr Treyer
- Albert Einstein College of Medicine, Department of Developmental and Molecular Biology, Bronx, New York, USA
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18
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Sun P, Zheng J, She G, Wei X, Zhang X, Shi H, Zhou X. Expression pattern of asialoglycoprotein receptor in human testis. Cell Tissue Res 2013; 352:761-8. [PMID: 23604802 DOI: 10.1007/s00441-013-1616-8] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2012] [Accepted: 03/11/2013] [Indexed: 02/05/2023]
Abstract
During acute or chronic hepatitis B virus (HBV) infection, the virus can invade the male reproductive system, pass through the blood-testis barrier and integrate into the germ line, resulting in abnormal spermatozoa. However, the pathway remains unclear. The asialoglycoprotein receptor (ASGR), a potential receptor for HBV, is mainly distributed in hepatocytes. We have examined the distribution of ASGR in human testis and found it in the seminiferous tubules and interstitial region but its enrichment in human testis is much lower than that in liver. By multiple immunoenzyme histochemistry staining, ASGR was precisely co-localized with vimentin (Sertoli cell marker) but not proliferating cell nuclear antigen (spermatogonial cell marker) in testis tissue. ASGR was expressed in human Leydig cells, stromal cells in the seminiferous tubules and Sertoli cells but seldom in spermatogonial cells. Therefore, ASGR could provide HBV with access to the luminal compartment of human testis. The mechanism by which HBV invades germ cells remains unknown.
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Affiliation(s)
- Pingnan Sun
- Department of Pathology, Shantou University Medical College, Shantou, People's Republic of China
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19
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Florian PE, Macovei A, Lazar C, Milac AL, Sokolowska I, Darie CC, Evans RW, Roseanu A, Branza-Nichita N. Characterization of the anti-HBV activity of HLP1-23, a human lactoferrin-derived peptide. J Med Virol 2013; 85:780-8. [DOI: 10.1002/jmv.23549] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 01/07/2013] [Indexed: 11/07/2022]
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20
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Rigopoulou EI, Roggenbuck D, Smyk DS, Liaskos C, Mytilinaiou MG, Feist E, Conrad K, Bogdanos DP. Asialoglycoprotein receptor (ASGPR) as target autoantigen in liver autoimmunity: lost and found. Autoimmun Rev 2012; 12:260-269. [PMID: 22571878 DOI: 10.1016/j.autrev.2012.04.005] [Citation(s) in RCA: 77] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2012] [Accepted: 04/23/2012] [Indexed: 12/11/2022]
Abstract
Asialoglycoprotein receptor (ASGPR) has attracted the attention of liver immunologists for many years. This liver-specific lectin was found to be a major B and T cell autoantigenic target in patients with autoimmune liver diseases, and in particular in autoimmune hepatitis (AIH). This review discusses the biological significance of ASGPR and its relevance to the pathogenesis of autoimmune and virus-triggered liver diseases. We also discuss emerging data on the diagnostic and clinical relevance of anti-ASGPR antibodies in light of recent reports based on commercially available anti-ASGPR enzyme-linked immunosorbent assays. Finally, we critically revisit the data reporting on disease-specific cellular immune responses against ASGPR and their relevance in relation to the pathogenesis of AIH.
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Affiliation(s)
- Eirini I Rigopoulou
- Department of Medicine, University Hospital of Larissa, University of Thessaly Medical School, Viopolis 41110, Larissa, Greece.
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Roggenbuck D, Mytilinaiou MG, Lapin SV, Reinhold D, Conrad K. Asialoglycoprotein receptor (ASGPR): a peculiar target of liver-specific autoimmunity. AUTO- IMMUNITY HIGHLIGHTS 2012; 3:119-25. [PMID: 26000135 PMCID: PMC4389076 DOI: 10.1007/s13317-012-0041-4] [Citation(s) in RCA: 53] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/03/2012] [Accepted: 10/11/2012] [Indexed: 12/11/2022]
Abstract
Asialoglycoprotein receptor (ASGPR) autoantibodies have been considered specific markers of autoimmune hepatitis (AIH). The exact mechanisms responsible for the development of these autoantibodies and leading to autoimmunity to this peculiar liver receptor remain elusive. Furthermore, loss of T cell tolerance to ASGPR has been demonstrated in patients with AIH, but it is poorly understood whether such liver-specific T cell responses bear a pathogenic potential and/or participate in the precipitation of AIH. Newly developed enzyme-linked immunosorbent assays have led to the investigation of the sensitivity and specificity of anti-ASGPR antibodies for AIH. The present review provides an overview of the diagnostic and clinical relevance of anti-ASGPR antibodies. A thorough investigation of the autoreactivity against ASGPR may assist efforts to understand liver autoimmunity in susceptible individuals.
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Affiliation(s)
- Dirk Roggenbuck
- Faculty of Natural Sciences, University of Applied Sciences, Großenhainer Str. 57, 01968 Senftenberg, Germany
- GA Generic Assays GmbH, 15827 Dahlewitz/Berlin, Germany
| | - Maria G. Mytilinaiou
- Division of Transplantation Immunology and Mucosal Biology, Institute of Liver Studies, King’s College London School of Medicine at King’s College Hospital, London, UK
| | - Sergey V. Lapin
- Laboratory of Autoimmune Diagnostics, St. Petersburg Pavlov State Medical University, St.Petersburg, Russia
| | - Dirk Reinhold
- Institute of Molecular and Clinical Immunology, Otto-von-Guericke University, Magdeburg, Germany
| | - Karsten Conrad
- Institute of Immunology, Technical University Dresden, Dresden, Germany
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Hao Z, Zheng L, Kluwe L, Huang W. Ferritin light chain and squamous cell carcinoma antigen 1 are coreceptors for cellular attachment and entry of hepatitis B virus. Int J Nanomedicine 2012; 7:827-34. [PMID: 22359459 PMCID: PMC3284225 DOI: 10.2147/ijn.s27803] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Overexpression of squamous cell carcinoma antigen 1 (SCCA1) in hepatitis G2 (HepG2) and Chinese hamster ovary cells can increase hepatitis B virus (HBV) binding capacity by interacting with the preS1 domain of the HBV surface antigen. However, the magnitude of increase in binding capacity was higher by several orders in the former, indicating the existence of additional factor(s) produced by HepG2 cells, which facilitates HBV attachment. Ferritin light chain (FTL) was identified as the sole high hit candidate by screening human liver cDNA library using a bacterial two-hybrid system with either preS or SCCA1 as the bait. Subsequent in vitro protein–protein interaction assays confirmed the binding activity of FTL to both preS and SCCA1, as well as the formation of triple complex preS-FTL-SCCA1, and narrowed down the binding sites on FTL. In vitro overexpression of FTL could further enhance HBV attachment in both HepG2 and Chinese hamster ovary cells, which were already overexpressing SCCA1. Importantly, in vivo co-expression of human FTL and SCCA1 in mouse liver by means of tailvein hydrodynamic injection increased serum levels of HBV surface antigen transiently 24 hours post challenge with HBV-positive human sera, and a large amount of HBV core antigen-positive hepatocytes around blood vessels could be identified by immunohistochemical staining 48 hours post challenge. The data strongly suggest that FTL and SCCA1 may serve as coreceptors in HBV cellular attachment and virus entry into hepatocytes.
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Affiliation(s)
- Zhaojing Hao
- Department of Biochemistry, School of Life Sciences, Fudan University, Shanghai, People's Republic of China
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24
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Abstract
Influenza A viruses are spherical particles that attach to cells through bonds between hemagglutinin and specific cellular receptors. Numerous studies performed have recently revealed that Sialic acid (SA) is a crucial component of influenza A virus receptors. This brief review summarizes recent advances in our understanding of influenza A virus receptors. The introduction describes the classification of influenza A virus receptors and the review continues with a survey of the distribution of SA in different tissue and host. This is followed by research applications of influenza A virus receptors, and explanation of why receptor studies are so important on a world-wide scale.
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Affiliation(s)
- Shengqiang Ge
- Key Laboratory of Animal Infectious Diseases of Ministry of Agriculture, School of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China
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25
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Miki T, Ring A, Gerlach J. Hepatic differentiation of human embryonic stem cells is promoted by three-dimensional dynamic perfusion culture conditions. Tissue Eng Part C Methods 2011; 17:557-68. [PMID: 21210720 DOI: 10.1089/ten.tec.2010.0437] [Citation(s) in RCA: 78] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023] Open
Abstract
The developmental potential of human embryonic stem cells (hESCs) holds great promise to provide a source of human hepatocytes for use in drug discovery, toxicology, hepatitis research, and extracorporeal bioartificial liver support. There are, however, limitations to induce fully functional hepatocytes on conventional two-dimensional (2D) static culture. It had been shown that dynamic three-dimensional (3D) perfusion culture is superior to induce maturation in fetal hepatocytes and prolong hepatic functions of primary adult hepatocytes. We investigated the potential of using a four-compartment 3D perfusion culture to induce hepatic differentiation in hESC. Undifferentiated hESC were inoculated into hollow fiber-based 3D perfusion bioreactors with integral oxygenation. Hepatic differentiation was induced with a multistep growth factor cocktail protocol. Parallel controls were operated under equal perfusion conditions without the growth factor supplementations to allow for spontaneous differentiation, as well as in conventional 2D static conditions using growth factors. Metabolism, hepatocyte-specific gene expression, protein expression, and hepatic function were evaluated after 20 days. Significantly upregulated hepatic gene expression was observed in the hepatic differentiation 3D culture group. Ammonia metabolism activity and albumin production was observed in the 3D directed differentiation culture. Drug-induced cytochrome P450 gene expression was increased with rifampicin induction. Using flow cytometry analysis the mature hepatocyte marker asialoglycoprotein receptor was found on up to 30% of the cells in the 3D system with directed hepatic differentiation. Histological and immunohistochemical analysis revealed structural formation of hepatic and biliary marker-positive cells. In contrast to 2D culture, the 3D perfusion culture induced more functional maturation in hESC-derived hepatic cells. 3D perfusion bioreactor technologies may be useful for further studies on generating hESC-derived hepatic cells.
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Affiliation(s)
- Toshio Miki
- Department of Surgery, McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
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26
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Zhang X, Lin SM, Chen TY, Liu M, Ye F, Chen YR, Shi L, He YL, Wu LX, Zheng SQ, Zhao YR, Zhang SL. Asialoglycoprotein receptor interacts with the preS1 domain of hepatitis B virus in vivo and in vitro. Arch Virol 2011; 156:637-45. [PMID: 21207081 DOI: 10.1007/s00705-010-0903-x] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2010] [Accepted: 12/20/2010] [Indexed: 01/04/2023]
Abstract
BACKGROUND The preS1 domain of the large envelope protein has been identified as an essential viral structure involved in hepatitis B virus (HBV) attachment. However, the cellular receptor(s) for HBV has not yet been identified. AIMS To identify a cell-surface receptor for HBV, which could elucidate the molecular mechanism of HBV infection. METHODS A novel yeast two-hybrid system was used to screen proteins interacting with the preS1 region of HBV. Their interaction was verified by yeast cotransformation, coimmunoprecipitation and mammalian two-hybrid assay, while their intracellular and tissue localization was analyzed by confocal microscopy and immunohistochemistry, respectively. RESULTS Asialoglycoprotein receptor (ASGPR) interacted specifically and directly with the preS1 domain of HBV in vivo and in vitro. The levels of expression of preS1 and ASGPR in the liver were similar and correlated with each other. CONCLUSIONS ASGPR is a candidate receptor for HBV that mediates further steps of HBV entry.
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Affiliation(s)
- Xi Zhang
- Department of Infectious Diseases, The First Affiliated Hospital of Medical College of Xi'an Jiaotong University, No. 277 Yanta West Road, Xi'an, Shaanxi Province, China
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Liu J, Hu B, Yang Y, Ma Z, Yu Y, Liu S, Wang B, Zhao X, Lu M, Yang D. A new splice variant of the major subunit of human asialoglycoprotein receptor encodes a secreted form in hepatocytes. PLoS One 2010; 5:e12934. [PMID: 20886072 PMCID: PMC2944864 DOI: 10.1371/journal.pone.0012934] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2010] [Accepted: 08/29/2010] [Indexed: 11/18/2022] Open
Abstract
BACKGROUND The human asialoglycoprotein receptor (ASGPR) is composed of two polypeptides, designated H1 and H2. While variants of H2 have been known for decades, the existence of H1 variants has never been reported. PRINCIPAL FINDINGS We identified two splice variants of ASGPR H1 transcripts, designated H1a and H1b, in human liver tissues and hepatoma cells. Molecular cloning of ASGPR H1 variants revealed that they differ by a 117 nucleotide segment corresponding to exon 2 in the ASGPR genomic sequence. Thus, ASGPR variant H1b transcript encodes a protein lacking the transmembrane domain. Using an H1b-specific antibody, H1b protein and a functional soluble ASGPR (sASGPR) composed of H1b and H2 in human sera and in hepatoma cell culture supernatant were identified. The expression of ASGPR H1a and H1b in Hela cells demonstrated the different cellular loctions of H1a and H1b proteins at cellular membranes and in intracellular compartments, respectively. In vitro binding assays using fluorescence-labeled sASGPR or the substract ASOR revealed that the presence of sASGPR reduced the binding of ASOR to cells. However, ASOR itself was able to enhance the binding of sASGPR to cells expressing membrane-bound ASGPR. Further, H1b expression is reduced in liver tissues from patients with viral hepatitis. CONCLUSIONS We conclude that two naturally occurring ASGPR H1 splice variants are produced in human hepatocytes. A hetero-oligomeric complex sASGPR consists of the secreted form of H1 and H2 and may bind to free substrates in circulation and carry them to liver tissue for uptake by ASGPR-expressing hepatocytes.
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Affiliation(s)
- Jia Liu
- Division of Clinical Immunology, Tongji Medical College, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, Hubei Province, China
| | - Bin Hu
- Division of Clinical Immunology, Tongji Medical College, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, Hubei Province, China
| | - Yan Yang
- Experimental Medicine Center, Tongji Medical College, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, Hubei Province, China
| | - Zhiyong Ma
- Division of Clinical Immunology, Tongji Medical College, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, Hubei Province, China
| | - Yuan Yu
- Experimental Medicine Center, Tongji Medical College, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, Hubei Province, China
| | - Shenpei Liu
- Experimental Medicine Center, Tongji Medical College, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, Hubei Province, China
| | - Baoju Wang
- Division of Clinical Immunology, Tongji Medical College, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, Hubei Province, China
| | - Xiping Zhao
- Division of Clinical Immunology, Tongji Medical College, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, Hubei Province, China
| | - Mengji Lu
- Department of Microbiology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, China
- Institute of Virology, Medical School, Duisburg-Essen University, Essen, Germany
- * E-mail: (ML); (DY)
| | - Dongliang Yang
- Division of Clinical Immunology, Tongji Medical College, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, Hubei Province, China
- Experimental Medicine Center, Tongji Medical College, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, Hubei Province, China
- * E-mail: (ML); (DY)
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Fibronectin and asialoglyprotein receptor mediate hepatitis B surface antigen binding to the cell surface. Arch Virol 2010; 155:881-8. [PMID: 20364278 DOI: 10.1007/s00705-010-0657-5] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2009] [Accepted: 03/03/2010] [Indexed: 12/15/2022]
Abstract
Both fibronectin and the asialoglycoprotein receptor (ASGPR) have been identified by some investigators as partners for hepatitis B virus (HBV) envelope proteins. Because fibronectin is a natural ligand for ASGPR, we speculated that HBV might attach to ASGPR expressed on the hepatocyte surface via fibronectin. To test this hypothesis, we first confirmed by co-immunoprecipitation that ASGPR, fibronectin and HBsAg bind to each other in HepG2.2.15 cells, and possible binding domains were identified by GST pull-down. In addition, by measuring binding of HBsAg to cells, we found that ASGPR and fibronectin enhanced the binding capability of HBsAg to HepG2 cells, and even to 293T and CHO cells, which normally do not bind HBV. In conclusion, our findings suggest that both fibronectin and ASGPR mediate HBsAg binding to the cell surface, which provides further evidence for the potential roles of these two proteins in mediating HBV binding to liver cells.
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29
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HBV life cycle: entry and morphogenesis. Viruses 2009; 1:185-209. [PMID: 21994545 PMCID: PMC3185491 DOI: 10.3390/v1020185] [Citation(s) in RCA: 84] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2009] [Revised: 07/31/2009] [Accepted: 08/13/2009] [Indexed: 02/07/2023] Open
Abstract
Hepatitis B virus (HBV) is a major cause of liver disease. HBV primarily infects hepatocytes by a still poorly understood mechanism. After an endocytotic process, the nucleocapsids are released into the cytoplasm and the relaxed circular rcDNA genome is transported towards the nucleus where it is converted into covalently closed circular cccDNA. Replication of the viral genome occurs via an RNA pregenome (pgRNA) that binds to HBV polymerase (P). P initiates pgRNA encapsidation and reverse transcription inside the capsid. Matured, rcDNA containing nucleocapsids can re-deliver the RC-DNA to the nucleus, or be secreted via interaction with the envelope proteins as progeny virions.
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Zhou XL, Sun PN, Huang TH, Xie QD, Kang XJ, Liu LM. Effects of hepatitis B virus S protein on human sperm function. Hum Reprod 2009; 24:1575-83. [PMID: 19279032 DOI: 10.1093/humrep/dep050] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
BACKGROUND Hepatitis B virus (HBV) has been determined to exist in semen and male germ cells from patients with chronic HBV infection, but no data are yet available on the impact of HBV S protein (HBs), the main component of HBV envelop protein, on the human reproductive system. The purpose of this article was to investigate the effect of HBs on human sperm function. METHODS Sperm motility analyses, sperm penetration assays, mitochondrial membrane potential assays, immunolocalizations with confocal microscopy and flow cytometry analyses were performed. RESULTS HBs reduced sperm motility in a dose- and time-dependent manner and caused the loss of sperm mitochondrial membrane potential. HBs-HBs monoclonal antibody (MAb) complex apparently aggravated such impairments. After 4 h incubation with HBs at concentrations of 25, 50, 100 microg/ml, the percentages of sperm motility a+b significantly decreased compared with the control (P < 0.01). The fertilization rate and the fertilizing index in HBs-treated group were 40% and 0.57, respectively, which were significantly lower than 90% and 1.6, respectively, in the control (P < 0.01). The asialoglycoprotein receptor (ASGP-R) and HBs were found to localize mainly on the postacrosomal region. Both ASGP-R MAb and asialofoetuin, a high-affinity ligand of ASGP-R, inhibited the HBs-caused loss of sperm motility and mitochondrial membrane potential. CONCLUSIONS HBs had adverse effects on human sperm function, and ASGP-R may play a role in the uptake of HBs into sperm cells, as demonstrated by the competitive inhibition of ASGP-R MAb or asialofoetuin, resulting in diminished impairment caused by HBs.
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Affiliation(s)
- Xiao-Ling Zhou
- Research Center for Reproductive Medicine, Shantou University Medical College, Shantou 515041, People's Republic of China
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Gao YF, Yu L, Li JB, Wei SF, Li X, Shen JL. Inhibition of hepatitis B virus gene expression and replication by artificial microRNA targeted ASGPR1. Shijie Huaren Xiaohua Zazhi 2009; 17:699-704. [DOI: 10.11569/wcjd.v17.i7.699] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the inhibitory effects on hepatitis B virus (HBV) replication and expression by transfecting artificial microRNA targeted ASGPR1 into HepG2.2.15 cells.
METHODS: Three amiRNA-HBV plasmids were constructed and transfected into HepG2.2.15 cells via LipofectamineTM 2000 reagent. The level of ASGPR1 mRNA was measured by semi-quantitative RT-PCR. The level of ASGPR1 protein was measured by western blot. HBV antigen secretion was detected in the cells with transient and stable transfection by time-resolved fluoroimmunoassays (TRFIA). HBV DNA replication was examined by fluorescence quantitative PCR.
RESULTS: Three amiRNA significantly reduced ASGPR1 mRNA and protein expression, and the greatest reduction was seen in amiRNA-ASGPR1-610 transfected group. Expressions of ASGPR1 mRNA and protein were down-regulated by 57.3% and 49.8% at 72 h(P < 0.01). At the virus level, three amiRNA-ASGPR1 plasmids obviously inhibited the secretion of HBsAg and HBeAg with the greatest reduction seen in amiRNA-ASGPR1-610 transfected group. Expression levels of HBsAg and HBeAg were down-regulated by 31.3% and 33.6% after 72 h (P < 0.01) and HBV DNA level was down-regulated by 29.7% at 72 h (P < 0.01).
CONCLUSION: In HepG2.2.15 cells, HBV replication and expression could be inhibited by artificial microRNA targeted ASGPR1. Artificial microRNA targeted ASGPR1 could be a promising therapeutic approach for chronic HBV infection.
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Núñez E, Yélamos B, Delgado C, Gómez-Gutiérrez J, Peterson DL, Gavilanes F. Interaction of preS domains of hepatitis B virus with phospholipid vesicles. BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 2009; 1788:417-24. [DOI: 10.1016/j.bbamem.2008.10.014] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/25/2008] [Revised: 09/24/2008] [Accepted: 10/22/2008] [Indexed: 12/16/2022]
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Khatri K, Rawat A, Mahor S, Gupta PN, Vyas SP. Hepatitis B surface protein docked vesicular carrier for site specific delivery to liver. J Drug Target 2008; 13:359-66. [PMID: 16278155 DOI: 10.1080/10611860500230294] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/25/2022]
Abstract
The intrinsic liver tropism of liposomes can be augmented by the addition of targeting features such as the incorporation of hepatotropic elements of the hepatitis viruses. Hepatitis B virus is known to infect hepatocytes after viremia by asialoglycoprotein receptor mediated uptake. However, the specificity of hepatitis B virus surface protein (HBsAg) towards hepatocytes has confronting reports. In the present study, we evaluated the functional ability of HBsAg to be employed as a ligand for targeting hepatocytes. We prepared (14)C labeled small unilamellar vesicles (SUVs) composed of egg PC/Cholesterol/N-glutarylphosphatidylethanolamine (NGPE) in a 60:30:10 molar ratio. HBsAg was covalently linked to SUVs using a water-soluble carbodiimide (EDC) mediated conjugation with NGPE. In vitro cell binding and uptake studies revealed that bioprotein docked carrier system was efficiently taken up by HepG2 cells by the receptor mediated endocytosis. The biodistribution behaviour of plain and HBsAg coated liposomes was also examined followed by intravenous injection. The study revealed that almost 75% of the radioactivity was recovered in the liver after 4 h of injection that was nearly three-fold greater in magnitude than the plain liposomes. Further, fractionation of liver into liver parenchymal cells (PC) and non-parenchymal cells confirmed the preferential localization of the HBsAg coated liposomal carrier in the parenchymal cells.
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Affiliation(s)
- Kapil Khatri
- Drug Delivery Research Laboratory, Department of Pharmaceutical Sciences, Dr. Hari Singh Gour Vishwavidyalaya, Sagar, India
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Leistner CM, Gruen-Bernhard S, Glebe D. Role of glycosaminoglycans for binding and infection of hepatitis B virus. Cell Microbiol 2007; 10:122-33. [DOI: 10.1111/j.1462-5822.2007.01023.x] [Citation(s) in RCA: 83] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
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Abstract
Hepadnaviridae is a family of hepatotropic DNA viruses that is divided into the genera orthohepadnavirus of mammals and avihepadnavirus of birds. All members of this family can cause acute and chronic hepatic infection, which in the case of human hepatitis B virus (HBV) constitutes a major global health problem. Although our knowledge about the molecular biology of these highly liver-specific viruses has profoundly increased in the last two decades, the mechanisms of attachment and productive entrance into the differentiated host hepatocytes are still enigmatic. The difficulties in studying hepadnaviral entry were primarily caused by the lack of easily accessible in vitro infection systems. Thus, for more than twenty years, differentiated primary hepatocytes from the respective species were the only in vitro models for both orthohepadnaviruses (e.g. HBV) and avihepadnaviruses (e.g. duck hepatitis B virus [DHBV]). Two important discoveries have been made recently regarding HBV: (1) primary hepatocytes from tree-shrews; i.e., Tupaia belangeri, can be substituted for primary human hepatocytes, and (2) a human hepatoma cell line (HepaRG) was established that gains susceptibility for HBV infection upon induction of differentiation in vitro. A number of potential HBV receptor candidates have been described in the past, but none of them have been confirmed to function as a receptor. For DHBV and probably all other avian hepadnaviruses, carboxypeptidase D (CPD) has been shown to be indispensable for infection, although the exact role of this molecule is still under debate. While still restricted to the use of primary duck hepatocytes (PDH), investigations performed with DHBV provided important general concepts on the first steps of hepadnaviral infection. However, with emerging data obtained from the new HBV infection systems, the hope that DHBV utilizes the same mechanism as HBV only partially held true. Nevertheless, both HBV and DHBV in vitro infection systems will help to: (1) functionally dissect the hepadnaviral entry pathways, (2) perform reverse genetics (e.g. test the fitness of escape mutants), (3) titrate and map neutralizing antibodies, (4) improve current vaccines to combat acute and chronic infections of hepatitis B, and (5) develop entry inhibitors for future clinical applications.
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Affiliation(s)
- Dieter Glebe
- Institute of Medical Virology, Justus-Liebig University of Giessen, Frankfurter Strasse 107, D-35392 Giessen, Germany.
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Park JH, Kim KL, Cho EW. Detection of surface asialoglycoprotein receptor expression in hepatic and extra-hepatic cells using a novel monoclonal antibody. Biotechnol Lett 2006; 28:1061-9. [PMID: 16799763 DOI: 10.1007/s10529-006-9064-0] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2006] [Accepted: 03/28/2006] [Indexed: 11/26/2022]
Abstract
The asialoglycoprotein receptor (ASGPR) is a heterodimeric membrane protein which is involved in the internalization of desialylated glycoproteins and also in the binding and uptake of various pathogenic viruses. To facilitate the analysis of ASGPR expression, we generated a monoclonal antibody, termed ASSA-1, that is specific to the ASGPR H1 subunit based on ELISA and Western blots analysis. ASSA-1 also reacted to surface-displayed ASGPR in live cells thus enabling analysis of ASGPR expression by immunofluorescence flow cytometry, which we used to analyze established human liver cell lines previously confirmed to be positive for ASGPR mRNA expression. In agreement with previous reports, surface ASGPR was also detected in extra-hepatic cells and, surprisingly, even in human T cell lines, which was then further confirmed in activated, but not in resting, primary human peripheral blood lymphocytes. These observations suggest that ASGPR has a broad pattern of expression that even extends into cells from the immune system, which biological meanings still have to be analyzed. We expect that monoclonal antibody ASSA-1 will serve as a new powerful tool in analyzing the biological role of ASGPR in hepatic and extra-hepatic cells.
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Affiliation(s)
- Jung-Hyun Park
- Systemic Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Yusong, Daejon, South Korea
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Yang J, Bo XC, Ding XR, Dai JM, Zhang ML, Wang XH, Wang SQ. Antisense oligonucleotides targeted against asialoglycoprotein receptor 1 block human hepatitis B virus replication. J Viral Hepat 2006; 13:158-65. [PMID: 16475991 DOI: 10.1111/j.1365-2893.2005.00666.x] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Chronic hepatitis B virus (HBV) infection is a major worldwide public health problem. Better therapeutics and treatment strategies are urgently needed because of ineffective clinical treatment. Our previous study showed that asialoglycoprotein receptor 1 (ASGPR1) was upregulated by HBV but downregulated by lamivudine in HepG2.2.15 cells. It has also been reported that ASGPR is a candidate receptor for HBV attachment to hepatocytes. Therefore, as a major subunit of ASGPR, ASGPR1, might be a potential target for anti-HBV drugs. To validate this hypothesis, antisense oligonucleiotides (ASODNs) were used to downregulate ASGPR1 level in HepG2.2.15 cells. By using the MFOLD web server and BLAST searches, five ASODNs theoretically targeting ASGPR1 were selected. After 72 h post-transfection, HBV-DNA level in cell medium were examined by real-time polymerase chain reaction (PCR). Hepatitis B surface antigen (HBsAg) and Hepatitis B e antigen (HBeAg) were detected using enzyme-linked immunosorbent assay (ELISA). ASGPR1 mRNA and protein level were measured by semi-quantitative reverse transcriptase (RT)-PCR and Western blot analysis respectively. The results showed that ASODN2 significantly downregulated ASGPR1 level. It also reduced HBV-DNA, HBsAg and HBeAg level in cell medium as observed with lamivudine. In contrast, the sense sequence and scrambled sequence of ASODN2 had no effect on ASGPR1 and HBV markers in HepG2.2.15 cells. This indicated that ASODN2 could specifically reduce HBV replication in vitro. Additionally, cell proliferation and apoptosis assay suggested that downregulation of ASGPR1 did not affect cell viability. We, therefore, proposed that ASODNs targeted against ASGPR1 could block HBV replication without the influence of other changes, and ASGPR1 could be targeted for anti-HBV drug development.
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Affiliation(s)
- J Yang
- Beijing Institute of Radiation Medicine, Beijing, China
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Yang J, Bo XC, Yao J, Yang NM, Wang SQ. Differentially expressed cellular genes following HBV: potential targets of anti-HBV drugs? J Viral Hepat 2005; 12:357-63. [PMID: 15985005 DOI: 10.1111/j.1365-2893.2005.00611.x] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Abstract
The aim of the study was to screen for cellular genes that are differentially expressed following hepatitis B virus (HBV) infection, in an attempt to identify potential targets of anti-HBV drugs. An oligonucleotide microarray containing 231 virus-infection-associated genes was prepared. Differential gene expression in HepG2.2.15 cells compared to control with HepG2 cells was analysed by this in-house microarray. The change in gene expression in HepG2.2.15 cells treated by lamivudine on days 4 and 8 after exposure was also studied. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used to comfirm the differentially expressed genes induced by HBV and lamivudine. There were 31 upregulated and four downregulated genes in HepG2.2.15 cells compared with the HepG2 control cells. Eleven genes were consistently altered by lamivudine at both time points. Of the 31 genes that were upregulated in HepG2.2.15 cells, there were seven genes which were downregulated by lamivudine. Of the four downregulated genes, there was one gene which was upregulated by lamivudine. Of the differentially expressed genes induced by HBV and lamivudine, the expression of five genes was confirmed by semi-quantitative RT-PCR. These results shed new light on the effects of HBV and lamivudine on cellular gene expression. Differentially expressed genes induced by HBV and lamivudine could potentially become new anti-HBV drug targets in novel therapies.
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Affiliation(s)
- J Yang
- Beijing Institute of Radiation Medicine, Beijing, China
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39
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Yeh CT, Lai HY, Chu SP, Tseng IC. Anti-sense expression of a metallopeptidase gene enhances nuclear entry of HBV-DNA. Biochem Biophys Res Commun 2004; 323:32-7. [PMID: 15351696 DOI: 10.1016/j.bbrc.2004.08.079] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2004] [Indexed: 01/14/2023]
Abstract
Although several putative hepatitis B virus (HBV) receptors have been identified, none of them is capable of initiating HBV replication in a non-permissive human cell line. Using an Epstein-Barr virus-based extrachromosomal replication system, we have screened through a human liver cDNA library and successfully identified a clone capable of facilitating nuclear transport of HBV-DNA during the early phase of HBV infection. This clone contained a cDNA encoding a metallopeptidase-like protein in anti-sense orientation. Pretreatment of naïve HepG2 cells with 1,10-phenanthroline, an inhibitor for liver metallopeptidases, led to nuclear entry of HBV-DNA after HBV infection. However, cccDNA was still undetectable in the nuclei, indicating other cellular factors required to complete the replication cycle were still missing. Our present data suggest that in the initial stage of HBV infection, liver metallopeptidase constitutes a barrier for effective nuclear entry of HBV genomic DNA. Attenuation of metallopeptidase activity may facilitate HBV infection.
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Affiliation(s)
- Chau-Ting Yeh
- Liver Research Unit, Chang Gung Memorial Hospital and Chang Gung University, College of Medicine, Taipei, Taiwan, ROC.
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40
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Marzi A, Gramberg T, Simmons G, Möller P, Rennekamp AJ, Krumbiegel M, Geier M, Eisemann J, Turza N, Saunier B, Steinkasserer A, Becker S, Bates P, Hofmann H, Pöhlmann S. DC-SIGN and DC-SIGNR interact with the glycoprotein of Marburg virus and the S protein of severe acute respiratory syndrome coronavirus. J Virol 2004; 78:12090-5. [PMID: 15479853 PMCID: PMC523257 DOI: 10.1128/jvi.78.21.12090-12095.2004] [Citation(s) in RCA: 291] [Impact Index Per Article: 13.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
The lectins DC-SIGN and DC-SIGNR can augment viral infection; however, the range of pathogens interacting with these attachment factors is incompletely defined. Here we show that DC-SIGN and DC-SIGNR enhance infection mediated by the glycoprotein (GP) of Marburg virus (MARV) and the S protein of severe acute respiratory syndrome coronavirus and might promote viral dissemination. SIGNR1, a murine DC-SIGN homologue, also enhanced infection driven by MARV and Ebola virus GP and could be targeted to assess the role of attachment factors in filovirus infection in vivo.
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Affiliation(s)
- Andrea Marzi
- Institute for Clinical and Molecular Virology, University Erlangen-Nürnberg, Nikolaus-Fiebiger-Center, Glückstrasse 6, D-91054 Erlangen, Germany
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41
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Lu X, Block T. Study of the early steps of the Hepatitis B Virus life cycle. Int J Med Sci 2004; 1:21-33. [PMID: 15912187 PMCID: PMC1074507 DOI: 10.7150/ijms.1.21] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/02/2004] [Accepted: 03/03/2004] [Indexed: 02/07/2023] Open
Abstract
Hepatitis B virus (HBV) is a human pathogen, causing the serious liver disease. Despite considerable advances in the understanding of the natural history of HBV disease, most of the early steps in the virus life cycle remain unclear. Virus attachment to permissive cells, fusion and penetration through cell membranes and subsequent genome release, are largely a mystery. Current knowledge on the early steps of HBV life cycle has mostly come from molecular cloning, expression of individual genes and studies of the infection of duck hepatitis B virus (DHBV) with duck primary duck hepatocytes. However, considering of the difference of the surface protein of HBV and DHBV both in the composition and sequence, the degree to which information from DHBV applies to human HBV attachment and entry may be limited. A major obstacle to the study HBV infection is the lack of a reliable and sensitive in vitro infection system. We have found that the digestion of HBV and woodchuck hepatitis virus (WHBV) by protease V8 led to the infection of HepG2 cell, a cell line generally is refractory for their infection [Lu et al. J Virol. 1996. 70. 2277-2285 . Lu et al. Virus Research. 2001. 73(1): 27-4].. Further studies showed that a serine protease inhibitor Kazal (SPIK) was over expressed in the HepG2 cells. Therefore, it is possible that to silence the over expressed SPIK and thus to reinstate the activity of indispensable cellular proteases can result in the restoration of the susceptibility of HepG2 cells for HBV infection. The establishing a stable cell line for study of the early steps of HBV life cycle by silencing of SPIK is discussed.
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42
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Lu YY, Chen TY, Cheng J, Liang YD, Wang L, Liu Y, Zhang J, Shao Q, Li K, Zhang LX. Cloning of a gene coding for novel mutant of asialoglycoprotein receptor 2 binding to hepatitis B virus X protein in hepatocytes. Shijie Huaren Xiaohua Zazhi 2003; 11:1126-1130. [DOI: 10.11569/wcjd.v11.i8.1126] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM The pathogenesis of HBV-induced malignant transformation is incompletely understood. The X protein of hepatitis B virus (HBxAg) is a multifunctional protein that can influence a variety of signal transduction pathways within the cell and is essential for establishing natural viral infection, it also has been implicated in the development of liver cancer associated with chronic infection. Further understanding of the interaction between HBxAg and proteins in hepatocytes is of great significance for the prevention of the development of hepatocellular carcinoma (HCC).
METHODS HBxAg bait plasmid was constructed by ligating the HBxAg gene with a yeast expression vector pGBKT7, then transformed into yeast AH109 (a type). The transformed yeast cells were amplified and mated with yeast cells Y187(α type) containing liver cDNA library plasmid pCAT2 in 2×YPDA medium. Diploid yeast cells were plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-α-gal for selection twice. Plasmid of true positive blue colonies was extracted and analysed by DNA sequencing and blast in GenBank. After the complete sequence of the novel mutant of asialoglycoprotein receptor 2 (ASGPR2) was amplified from the mRNA of HepG2 cell by reverse transcription polymerase chain reaction (RT-PCR) and cloned into pGADT7 vector, the recombined plasmid was translated by using reticulocyte lysate and analysed by immunoprecipitation technique in vitro together with HBxAg.
RESULTS Eighteen genes in forty-one positive colonies were obtained, one of them is a novel mutant of ASGPR2, which is 80 % homologous to natural ASGPR2. The complete sequence of the mutant was amplified from the mRNA of HepG2 cell by RT-PCR successfully. The interaction between HBx and ASGPR2 mutant was further confirmed by immunoprecipitation technique.
CONCLUSION Interaction between HBx and ASGPR2 mutant can be observed in both yeast cell and in vitro.
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Affiliation(s)
- Yin-Ying Lu
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuan Zhonglu, Beijing 100039, China
| | - Tian-Yan Chen
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuan Zhonglu, Beijing 100039, China
| | - Jun Cheng
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuan Zhonglu, Beijing 100039, China
| | - Yao-Dong Liang
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuan Zhonglu, Beijing 100039, China
| | - Lin Wang
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuan Zhonglu, Beijing 100039, China
| | - Yan Liu
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuan Zhonglu, Beijing 100039, China
| | - Jian Zhang
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuan Zhonglu, Beijing 100039, China
| | - Qing Shao
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuan Zhonglu, Beijing 100039, China
| | - Ke Li
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuan Zhonglu, Beijing 100039, China
| | - Ling-Xia Zhang
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuan Zhonglu, Beijing 100039, China
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Beckebaum S, Cicinnati VR, Zhang X, Ferencik S, Frilling A, Grosse-Wilde H, Broelsch CE, Gerken G. Hepatitis B virus-induced defect of monocyte-derived dendritic cells leads to impaired T helper type 1 response in vitro: mechanisms for viral immune escape. Immunology 2003; 109:487-95. [PMID: 12871214 PMCID: PMC1783010 DOI: 10.1046/j.1365-2567.2003.01699.x] [Citation(s) in RCA: 113] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2003] [Revised: 04/29/2003] [Accepted: 06/04/2003] [Indexed: 12/15/2022] Open
Abstract
Dendritic cells (DC) are the most potent antigen-presenting cells and play a central role in the induction of antiviral immune responses. Recently, we have shown that monocyte-derived DC (MoDC) from patients with chronic hepatitis B virus (HBV) infection are functionally impaired. In our present study MoDC from healthy subjects were propagated in vitro and inoculated with HBV particles to investigate the precise mechanisms that underly MoDC dysfunction. T-cell proliferation assays revealed an impaired allostimulatory capacity of HBV-inoculated MoDC (HBV-MoDC) as well as a lower potential of stimulating autologous T cells against a recall antigen in comparison to control-MoDC. Interleukin-2, tumour necrosis factor-alpha and interferon-gamma production by T cells in proliferation assays with HBV-MoDC was significantly lower than with control-MoDC and correlated with lower IL-12 production in HBV-MoDC cultures. The presence of the nucleoside analogue lamivudine (3TC), an inhibitor of HBV replication, restored impaired allostimulatory function of HBV-MoDC and up-regulated major histocompatibility complex class II expression. These results show that HBV infection compromises the antigen-presenting function of MoDC with concomitant impairment of T helper cell type 1 responses. This may play an important role for viral immune escape leading to chronic HBV infection. However, 3TC treatment can overcome HBV-MoDC-related T-cell hyporeactivity and this underscores its important role in enhanced immune responses to HBV.
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Affiliation(s)
- Susanne Beckebaum
- Department of Gastroenterology and Hepatology, Department of General Surgery and Transplantation, and Institute of Immunology, University of Essen, Germany
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Abstract
In a natural setting, hepatitis delta virus (HDV) is only found in patients that are also infected with hepatitis B virus (HBV). In hepatocytes infected with these two viruses, HDV RNA genomes are assembled using the envelope proteins of HBV. Since 1986, we have known that HDV has a small single-stranded RNA genome with a unique circular conformation that is replicated using a host RNA polymerase. These and other features make HDV and its replication unique, at least among agents that infect animals. This mini-review focuses on advances gained over the last 2-3 years, together with an evaluation of HDV questions that are either unsolved or not yet solved satisfactorily.
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Affiliation(s)
- John M Taylor
- Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111-2497, USA.
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45
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Hong SR, Lee YM, Akaike T. Evaluation of a galactose-carrying gelatin sponge for hepatocytes culture and transplantation. ACTA ACUST UNITED AC 2003; 67:733-41. [PMID: 14613220 DOI: 10.1002/jbm.a.10138] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
This study proposes a new three-dimensional culture of mouse hepatocytes in a porous galactose-carrying modified gelatin sponge matrix. The modification of gelatin using galactose residues significantly increased the attachment of hepatocytes on the substrate. A modified gelatin sponge with lactobionic acid (MGLA) was prepared to increase the specific interaction between the hepatocytes and the matrix. Hepatocytes cultured in a three-dimensional MGLA sponge released much less lactate dehydrogenase than those cultured on a collagen Type I-coated monolayer. Moreover, the survival rate of hepatocytes cultured on an MGLA sponge was longer than the survival rate of hepatocytes cultured on a collagen Type I-coated monolayer. Hepatic specific metabolic functions, namely, the secretion of serum albumin and the synthesis of urea, were well maintained and promoted by spheroidal hepatocytes formed in the MGLA sponge.
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Affiliation(s)
- Sung Ran Hong
- School of Chemical Engineering, College of Engineering, Hanyang University, Seungdong-ku, Seoul, 133-791, Korea
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46
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Favre D, Berthillon P, Trépo C. Removal of cell-bound lipoproteins: a crucial step for the efficient infection of liver cells with hepatitis C virus in vitro. COMPTES RENDUS DE L'ACADEMIE DES SCIENCES. SERIE III, SCIENCES DE LA VIE 2001; 324:1141-8. [PMID: 11803815 DOI: 10.1016/s0764-4469(01)01397-x] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
Abstract
Hepatitis C virus (HCV) is of major social, medical and economic importance. The prevalence of HCV is approximatively 1% in most developed countries, and much higher in developing countries. HCV infection is the second major cause, after hepatitis B virus infection, for the generation of chronic liver disease and hepatocellular carcinoma. To date, the only reliable model for the study of HCV infection is the chimpanzee. Indeed, there is no robust in vitro infection system, yet. There is thus an urgent need for such an in vitro infection system in order to evaluate therapeutic agents. Here, a process is provided for infecting hepatocyte cell lines with hepatitis C virus in vitro. It is strongly suggested that cell-bound lipoproteins are playing a crucial role during the infection process. In order to obtain a robust infection, the cell-bound lipoproteins have first to be removed from their cellular receptor prior to the addition of viral inocula originating from human sera, the latter being made originally of a virus-lipoprotein complex.
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Affiliation(s)
- D Favre
- Institut national de la santé et de la recherche médicale, virus des hépatites, rétrovirus humains et pathologies associées, Inserm unité 271, 151, cours Albert Thomas, 69424 Lyon, France.
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47
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Hartmann-Stühler C, Prange R. Hepatitis B virus large envelope protein interacts with gamma2-adaptin, a clathrin adaptor-related protein. J Virol 2001; 75:5343-51. [PMID: 11333915 PMCID: PMC114939 DOI: 10.1128/jvi.75.11.5343-5351.2001] [Citation(s) in RCA: 51] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
For the outcome of a hepatitis B virus (HBV) infection, the viral L envelope protein with its pre-S domain performs pivotal functions by mediating attachment of HBV to liver cells, envelopment of viral capsids, release of (sub)viral particles, regulation of supercoiled DNA amplification, and transcriptional transactivation. To assess its multiple functions and host-protein assistance involved, we initiated a two-hybrid screen using the L-specific pre-S1 domain as bait. With this approach, we have identified gamma2-adaptin, a putative member of the clathrin adaptor proteins responsible for protein sorting and trafficking, as a specific binding partner of L protein. Evidence for a physical interaction between L protein and gamma2-adaptin was also demonstrated by affinity chromatography and coimmunoprecipitation, and the binding sites were mapped to the L-specific pre-S1 domain and the gamma2-adaptin-specific ear domain. The specificity of the interaction was further sustained by the failure of gamma1-adaptin, a closely related gamma2-adaptin homologue, to associate with L protein. Analysis of an L mutant protein indicates that the L-gamma2-adaptin interaction strictly depends on the pre-S1 domain of transmembrane L protein oriented to the cytosol and thus appears to occur in the cytosolic environment. Interestingly, coexpression of the two interacting partners in transfected cells resulted in recruitment of gamma2-adaptin by L protein onto cis-Golgi-like structures, strongly indicating that the association is physiologically relevant. Together, the results suggest a role for gamma2-adaptin in L-mediated processes of viral biogenesis and/or pathogenesis, such as facilitating and guiding HBV assembly.
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Affiliation(s)
- C Hartmann-Stühler
- Institute for Medical Microbiology and Hygiene, Johannes Gutenberg-Universität Mainz, D-55101 Mainz, Germany
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Heinz D, Peters M, Prange R, Gerken G, Rose-John S. Possible role of human interleukin-6 and soluble interleukin-6 receptor in hepatitis B virus infection. J Viral Hepat 2001; 8:186-93. [PMID: 11380796 DOI: 10.1046/j.1365-2893.2001.00281.x] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
Human interleukin-6 has been shown to promote hepatitis B virus (HBV) infection. However, it is not clear whether this influence is the result of a direct interaction between interleukin-6 (IL-6) and the HBV envelope proteins or of a rather indirect mechanism. A direct interaction of IL-6 and the preS region of the large envelope protein (L-protein) of HBV has been reported. In this study we assessed the binding of IL-6 and of the IL-6 receptor subunits to the preS region of the L-protein of HBV. Binding of IL-6 and IL-6 receptor subunits sIL-6R and gp130 to preS was assessed by immunoprecipitation with recombinant preS proteins. In patient sera IL-6 and sIL-6R concentrations were analysed with respect to the course of hepatitis B infection during and after interferon-alpha (IFN-alpha) therapy. The IL-6 and IL-6 receptor subunits could not be precipitated with recombinant preS proteins. In sera of patients who responded to IFN-alpha therapy by virus elimination, a significant increase in sIL-6R concentration was measured. No increase in sIL-6R levels was seen in patients who did not respond to IFN-alpha. Hence, IL-6 and IL-6 receptor subunits do not bind to preS directly. A possible role for sIL-6R in the elimination of HBV infection is discussed.
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Affiliation(s)
- D Heinz
- I. Medizinische Klinik, Abteilung Pathophysiologie, Johannes Gutenberg Universität Mainz, Mainz, Germany
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Núñez E, Wei X, Delgado C, Rodríguez-Crespo I, Yélamos B, Gómez-Gutiérrez J, Peterson DL, Gavilanes F. Cloning, expression, and purification of histidine-tagged preS domains of hepatitis B virus. Protein Expr Purif 2001; 21:183-91. [PMID: 11162405 DOI: 10.1006/prep.2000.1368] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
The preS domains of the hepatitis B virus are hydrophilic polypeptides that have been implicated, among other functions, in the binding of the virus to hepatocytes and in the induction of virus-neutralizing antibodies. A method of overproducing the preS domains of two different subtypes, adw and ayw, has been developed by adding a 6x His tag at the carboxy-terminal end of the polypeptides. Codons for the 6x His were added in reverse primers used to amplify the corresponding cDNAs. The polymerase chain reaction products were cloned into the expression vectors pET-3d (subtype ayw) and pT7-7 (subtype adw), under the control of the inducible bacteriophage T7 RNA polymerase promoter. Upon induction with isopropyl-beta-d-thiogalactopyranoside, proteins were overexpressed and purified by affinity chromatography on a Ni-nitrilotriacetic acid agarose column. This method yielded 20-40 mg of highly pure and very stable proteins per liter of cell culture. Circular dichroism and fluorescence spectroscopy of isolated preS-his-ayw and preS-his-adw, as well as their ability to bind polymerized human serum albumin, indicate that the 6x His tag does not modify the native-like conformation and, therefore, they may be considered as useful tools to study the function of these viral polypeptide regions.
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Affiliation(s)
- E Núñez
- Departamento de Bioquímica y Biología Molecular, Universidad Complutense, 28040 Madrid, Spain
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Meier M, Bider MD, Malashkevich VN, Spiess M, Burkhard P. Crystal structure of the carbohydrate recognition domain of the H1 subunit of the asialoglycoprotein receptor. J Mol Biol 2000; 300:857-65. [PMID: 10891274 DOI: 10.1006/jmbi.2000.3853] [Citation(s) in RCA: 140] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The human asialoglycoprotein receptor (ASGPR), also called hepatic lectin, is an integral membrane protein and is responsible for the clearance of desialylated, galactose-terminal glycoproteins from the circulation by receptor-mediated endocytosis. It can be subdivided into four functional domains: the cytosolic domain, the transmembrane domain, the stalk and the carbohydrate recognition domain (CRD). The galactose-binding domains belong to the superfamily of C-type (calcium-dependent) lectins, in particular to the long-form subfamily with three conserved intramolecular disulphide bonds. It is able to bind terminal non-reducing galactose residues and N-acetyl-galactosamine residues of desialated tri or tetra-antennary N-linked glycans. The ASGPR is a potential liver-specific receptor for hepatitis B virus and Marburg virus and has been used to target exogenous molecules specifically to hepatocytes for diagnostic and therapeutic purposes.Here, we present the X-ray crystal structure of the carbohydrate recognition domain of the major subunit H1 at 2.3 A resolution. While the overall fold of this and other known C-type lectin structures are well conserved, the positions of the bound calcium ions are not, indicating that the fold is stabilised by alternative mechanisms in different branches of the C-type lectin family. It is the first CRD structure where three calcium ions form an intergral part of the structure. In addition, the structure provides direct confirmation for the conversion of the ligand-binding site of the mannose-binding protein to an asialoglycoprotein receptor-like specificity suggested by Drickamer and colleagues. In agreement with the prediction that the coiled-coil domain of the ASGPR is separated from the CRD and its N-terminal disulphide bridge by several residues, these residues are indeed not alpha-helical, while in tetranectin they form an alpha-helical coiled-coil.
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Affiliation(s)
- M Meier
- M.E. Müller Institute for Structural Biology, University of Basel, Klingelbergstrasse 70 CH-4056 Basel, Switzerland
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