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Li W, Yang S, Xu P, Zhang D, Tong Y, Chen L, Jia B, Li A, Lian C, Ru D, Zhang B, Liu M, Chen C, Fu W, Yuan S, Gu C, Wang L, Li W, Liang Y, Yang Z, Ren X, Wang S, Zhang X, Song Y, Xie Y, Lu H, Xu J, Wang H, Yu W. SARS-CoV-2 RNA elements share human sequence identity and upregulate hyaluronan via NamiRNA-enhancer network. EBioMedicine 2022; 76:103861. [PMID: 35124429 PMCID: PMC8811534 DOI: 10.1016/j.ebiom.2022.103861] [Citation(s) in RCA: 27] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2021] [Revised: 12/22/2021] [Accepted: 01/18/2022] [Indexed: 01/08/2023] Open
Abstract
BACKGROUND Since late 2019, SARS-CoV-2 infection has resulted in COVID-19 accompanied by diverse clinical manifestations. However, the underlying mechanism of how SARS-CoV-2 interacts with host and develops multiple symptoms is largely unexplored. METHODS Bioinformatics analysis determined the sequence similarity between SARS-CoV-2 and human genomes. Diverse fragments of SARS-CoV-2 genome containing Human Identical Sequences (HIS) were cloned into the lentiviral vector. HEK293T, MRC5 and HUVEC were infected with laboratory-packaged lentivirus or transfected with plasmids or antagomirs for HIS. Quantitative RT-PCR and chromatin immunoprecipitation assay detected gene expression and H3K27ac enrichment, respectively. UV-Vis spectroscopy assessed the interaction between HIS and their target locus. Enzyme-linked immunosorbent assay evaluated the hyaluronan (HA) levels of culture supernatant and plasma of COVID-19 patients. FINDINGS Five short sequences (24-27 nt length) sharing identity between SARS-CoV-2 and human genome were identified. These RNA elements were highly conserved in primates. The genomic fragments containing HIS were predicted to form hairpin structures in silico similar to miRNA precursors. HIS may function through direct genomic interaction leading to activation of host enhancers, and upregulation of adjacent and distant genes, including cytokine genes and hyaluronan synthase 2 (HAS2). HIS antagomirs and Cas13d-mediated HIS degradation reduced HAS2 expression. Severe COVID-19 patients displayed decreased lymphocytes and elevated D-dimer, and C-reactive proteins, as well as increased plasma hyaluronan. Hymecromone inhibited hyaluronan production in vitro, and thus could be further investigated as a therapeutic option for preventing severe outcome in COVID-19 patients. INTERPRETATION HIS of SARS-CoV-2 could promote COVID-19 progression by upregulating hyaluronan, providing novel targets for treatment. FUNDING The National Key R&D Program of China (2018YFC1005004), Major Special Projects of Basic Research of Shanghai Science and Technology Commission (18JC1411101), and the National Natural Science Foundation of China (31872814, 32000505).
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Affiliation(s)
- Wei Li
- Laboratory of RNA Epigenetics, Institutes of Biomedical Sciences & Shanghai Public Health Clinical Center & Department of General Surgery, Huashan Hospital, Cancer Metastasis Institute, Shanghai Medical College, Fudan University, Shanghai 200032, China; Shanghai Key Laboratory of Medical Epigenetics, Shanghai 200032, China
| | - Shuai Yang
- Laboratory of RNA Epigenetics, Institutes of Biomedical Sciences & Shanghai Public Health Clinical Center & Department of General Surgery, Huashan Hospital, Cancer Metastasis Institute, Shanghai Medical College, Fudan University, Shanghai 200032, China; Shanghai Key Laboratory of Medical Epigenetics, Shanghai 200032, China
| | - Peng Xu
- Laboratory of RNA Epigenetics, Institutes of Biomedical Sciences & Shanghai Public Health Clinical Center & Department of General Surgery, Huashan Hospital, Cancer Metastasis Institute, Shanghai Medical College, Fudan University, Shanghai 200032, China; Shanghai Key Laboratory of Medical Epigenetics, Shanghai 200032, China
| | - Dapeng Zhang
- State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China
| | - Ying Tong
- Laboratory of RNA Epigenetics, Institutes of Biomedical Sciences & Shanghai Public Health Clinical Center & Department of General Surgery, Huashan Hospital, Cancer Metastasis Institute, Shanghai Medical College, Fudan University, Shanghai 200032, China; Shanghai Key Laboratory of Medical Epigenetics, Shanghai 200032, China
| | - Lu Chen
- Laboratory of RNA Epigenetics, Institutes of Biomedical Sciences & Shanghai Public Health Clinical Center & Department of General Surgery, Huashan Hospital, Cancer Metastasis Institute, Shanghai Medical College, Fudan University, Shanghai 200032, China; Shanghai Key Laboratory of Medical Epigenetics, Shanghai 200032, China
| | - Ben Jia
- Shanghai Epiprobe Biotechnology Co., Ltd, Shanghai 200233, China
| | - Ang Li
- Institute of Clinical Science & Shanghai Key Laboratory of Organ Transplantation, Zhongshan Hospital, Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China; Shanghai Public Health Clinical Center, Fudan University, Shanghai 201508, China
| | - Cheng Lian
- Laboratory of RNA Epigenetics, Institutes of Biomedical Sciences & Shanghai Public Health Clinical Center & Department of General Surgery, Huashan Hospital, Cancer Metastasis Institute, Shanghai Medical College, Fudan University, Shanghai 200032, China; Shanghai Key Laboratory of Medical Epigenetics, Shanghai 200032, China
| | - Daoping Ru
- Laboratory of RNA Epigenetics, Institutes of Biomedical Sciences & Shanghai Public Health Clinical Center & Department of General Surgery, Huashan Hospital, Cancer Metastasis Institute, Shanghai Medical College, Fudan University, Shanghai 200032, China; Shanghai Key Laboratory of Medical Epigenetics, Shanghai 200032, China
| | - Baolong Zhang
- Laboratory of RNA Epigenetics, Institutes of Biomedical Sciences & Shanghai Public Health Clinical Center & Department of General Surgery, Huashan Hospital, Cancer Metastasis Institute, Shanghai Medical College, Fudan University, Shanghai 200032, China; Shanghai Key Laboratory of Medical Epigenetics, Shanghai 200032, China
| | - Mengxing Liu
- Laboratory of RNA Epigenetics, Institutes of Biomedical Sciences & Shanghai Public Health Clinical Center & Department of General Surgery, Huashan Hospital, Cancer Metastasis Institute, Shanghai Medical College, Fudan University, Shanghai 200032, China; Shanghai Key Laboratory of Medical Epigenetics, Shanghai 200032, China
| | - Cancan Chen
- Laboratory of RNA Epigenetics, Institutes of Biomedical Sciences & Shanghai Public Health Clinical Center & Department of General Surgery, Huashan Hospital, Cancer Metastasis Institute, Shanghai Medical College, Fudan University, Shanghai 200032, China; Shanghai Key Laboratory of Medical Epigenetics, Shanghai 200032, China
| | - Weihui Fu
- Institute of Clinical Science & Shanghai Key Laboratory of Organ Transplantation, Zhongshan Hospital, Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China; Shanghai Public Health Clinical Center, Fudan University, Shanghai 201508, China
| | - Songhua Yuan
- Institute of Clinical Science & Shanghai Key Laboratory of Organ Transplantation, Zhongshan Hospital, Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China; Shanghai Public Health Clinical Center, Fudan University, Shanghai 201508, China
| | - Chenjian Gu
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), Department of Medical Microbiology and Parasitology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China
| | - Lu Wang
- Department of Pulmonary and Critical Care Medicine, Zhongshan Hospital, Fudan University, Shanghai 200032, China
| | - Wenxuan Li
- Laboratory of RNA Epigenetics, Institutes of Biomedical Sciences & Shanghai Public Health Clinical Center & Department of General Surgery, Huashan Hospital, Cancer Metastasis Institute, Shanghai Medical College, Fudan University, Shanghai 200032, China; Shanghai Key Laboratory of Medical Epigenetics, Shanghai 200032, China
| | - Ying Liang
- Laboratory of RNA Epigenetics, Institutes of Biomedical Sciences & Shanghai Public Health Clinical Center & Department of General Surgery, Huashan Hospital, Cancer Metastasis Institute, Shanghai Medical College, Fudan University, Shanghai 200032, China; Shanghai Key Laboratory of Medical Epigenetics, Shanghai 200032, China
| | - Zhicong Yang
- Laboratory of RNA Epigenetics, Institutes of Biomedical Sciences & Shanghai Public Health Clinical Center & Department of General Surgery, Huashan Hospital, Cancer Metastasis Institute, Shanghai Medical College, Fudan University, Shanghai 200032, China; Shanghai Key Laboratory of Medical Epigenetics, Shanghai 200032, China
| | - Xiaoguang Ren
- Laboratory of RNA Epigenetics, Institutes of Biomedical Sciences & Shanghai Public Health Clinical Center & Department of General Surgery, Huashan Hospital, Cancer Metastasis Institute, Shanghai Medical College, Fudan University, Shanghai 200032, China; Shanghai Key Laboratory of Medical Epigenetics, Shanghai 200032, China
| | - Shaoxuan Wang
- Laboratory of RNA Epigenetics, Institutes of Biomedical Sciences & Shanghai Public Health Clinical Center & Department of General Surgery, Huashan Hospital, Cancer Metastasis Institute, Shanghai Medical College, Fudan University, Shanghai 200032, China; Shanghai Key Laboratory of Medical Epigenetics, Shanghai 200032, China
| | - Xiaoyan Zhang
- Institute of Clinical Science & Shanghai Key Laboratory of Organ Transplantation, Zhongshan Hospital, Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China; Shanghai Public Health Clinical Center, Fudan University, Shanghai 201508, China
| | - Yuanlin Song
- Department of Pulmonary and Critical Care Medicine, Zhongshan Hospital, Fudan University, Shanghai 200032, China
| | - Youhua Xie
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), Department of Medical Microbiology and Parasitology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China
| | - Hongzhou Lu
- Shanghai Public Health Clinical Center, Fudan University, Shanghai 201508, China
| | - Jianqing Xu
- Institute of Clinical Science & Shanghai Key Laboratory of Organ Transplantation, Zhongshan Hospital, Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China; Shanghai Public Health Clinical Center, Fudan University, Shanghai 201508, China.
| | - Hailin Wang
- State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China.
| | - Wenqiang Yu
- Laboratory of RNA Epigenetics, Institutes of Biomedical Sciences & Shanghai Public Health Clinical Center & Department of General Surgery, Huashan Hospital, Cancer Metastasis Institute, Shanghai Medical College, Fudan University, Shanghai 200032, China; Shanghai Key Laboratory of Medical Epigenetics, Shanghai 200032, China.
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In Vitro Systems for Studying Different Genotypes/Sub-Genotypes of Hepatitis B Virus: Strengths and Limitations. Viruses 2020; 12:v12030353. [PMID: 32210021 PMCID: PMC7150782 DOI: 10.3390/v12030353] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2020] [Revised: 03/04/2020] [Accepted: 03/06/2020] [Indexed: 12/11/2022] Open
Abstract
Hepatitis B virus (HBV) infects the liver resulting in end stage liver disease, cirrhosis, and hepatocellular carcinoma. Despite an effective vaccine, HBV poses a serious health problem globally, accounting for 257 million chronic carriers. Unique features of HBV, including its narrow virus-host range and its hepatocyte tropism, have led to major challenges in the development of suitable in vivo and in vitro model systems to recapitulate the HBV replication cycle and to test various antiviral strategies. Moreover, HBV is classified into at least nine genotypes and 35 sub-genotypes with distinct geographical distributions and prevalence, which have different natural histories of infection, clinical manifestation, and response to current antiviral agents. Here, we review various in vitro systems used to study the molecular biology of the different (sub)genotypes of HBV and their response to antiviral agents, and we discuss their strengths and limitations. Despite the advances made, no system is ideal for pan-genotypic HBV research or drug development and therefore further improvement is required. It is necessary to establish a centralized repository of HBV-related generated materials, which are readily accessible to HBV researchers, with international collaboration toward advancement and development of in vitro model systems for testing new HBV antivirals to ensure their pan-genotypic and/or customized activity.
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Park S, Ha YN, Dezhbord M, Lee AR, Park ES, Park YK, Won J, Kim NY, Choo SY, Shin JJ, Ahn CH, Kim KH. Suppression of Hepatocyte Nuclear Factor 4 α by Long-term Infection of Hepatitis B Virus Contributes to Tumor Cell Proliferation. Int J Mol Sci 2020; 21:ijms21030948. [PMID: 32023898 PMCID: PMC7037729 DOI: 10.3390/ijms21030948] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2019] [Revised: 01/24/2020] [Accepted: 01/29/2020] [Indexed: 12/15/2022] Open
Abstract
Hepatitis B virus (HBV) infection is a major factor in the development of various liver diseases such as hepatocellular carcinoma (HCC). Among HBV encoded proteins, HBV X protein (HBx) is known to play a key role in the development of HCC. Hepatocyte nuclear factor 4α (HNF4α) is a nuclear transcription factor which is critical for hepatocyte differentiation. However, the expression level as well as its regulatory mechanism in HBV infection have yet to be clarified. Here, we observed the suppression of HNF4α in cells which stably express HBV whole genome or HBx protein alone, while transient transfection of HBV replicon or HBx plasmid had no effect on the HNF4α level. Importantly, in the stable HBV- or HBx-expressing hepatocytes, the downregulated level of HNF4α was restored by inhibiting the ERK signaling pathway. Our data show that HNF4α was suppressed during long-term HBV infection in cultured HepG2-NTCP cells as well as in a mouse model following hydrodynamic injection of pAAV-HBV or in mice intravenously infected with rAAV-HBV. Importantly, HNF4α downregulation increased cell proliferation, which contributed to the formation and development of tumor in xenograft nude mice. The data presented here provide proof of the effect of HBV infection in manipulating the HNF4α regulatory pathway in HCC development.
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Affiliation(s)
| | | | | | | | | | | | | | | | | | | | | | - Kyun-Hwan Kim
- Correspondence: ; Tel.: +82-2-2030-7833; Fax: +82-2-2049-6192
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Riazalhosseini B, Mohamed Z, Apalasamy YD, Shafie NS, Mohamed R. Interleukin-6 gene variants are associated with reduced risk of chronicity in hepatitis B virus infection in a Malaysian population. Biomed Rep 2018; 9:213-220. [PMID: 30271596 DOI: 10.3892/br.2018.1126] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2018] [Accepted: 06/29/2018] [Indexed: 02/06/2023] Open
Abstract
Interleukin-6 (IL-6) is a cytokine with a critical role in regulating the immune response to infectious disease. Studies have indicated that polymorphisms in the IL-6 gene may be linked to hepatitis B virus (HBV) infection. The purpose of the present study was to examine the association among IL-6 SNPs and haplotypes with HBV infection risk in a Malaysian population. A total of 1,246 Malaysian subjects with and without chronic hepatitis B were recruited for this study. Three IL-6 polymorphisms (rs2069837, rs1800796 and rs2066992) were genotyped using a Sequenom MassARRAY® platform. The results suggested that GC and CC genotypes of rs1800796 as well as GT and TT genotypes of rs2066992 were associated with protection against HBV infection (P<0.001). Furthermore, haplotypes GG and CT exhibited a significant association with protection against HBV (P=0.003 and =0.005, respectively); and haplotypes GG and CT exhibited a significant association with clearance of HBV infection (P=0.035 and =0.037, respectively). The present study indicates that two IL-6 SNPs (rs1800796 and rs2066992) are associated with clearance of chronic HBV or protection against HBV infection at allelic, genotypic and haplotypic levels.
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Affiliation(s)
- Behnaz Riazalhosseini
- Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur 50603, Malaysia
| | - Zahurin Mohamed
- Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur 50603, Malaysia
| | - Yamunah Devi Apalasamy
- Social Wellbeing Research Centre, Faculty of Economics and Administration, University of Malaya, Kuala Lumpur 50603, Malaysia
| | - Noor Shafila Shafie
- Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur 50603, Malaysia
| | - Rosmawati Mohamed
- Department of Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur 50603, Malaysia
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Lai YH, Sun CP, Huang HC, Chen JC, Liu HK, Huang C. Epigallocatechin gallate inhibits hepatitis B virus infection in human liver chimeric mice. BMC COMPLEMENTARY AND ALTERNATIVE MEDICINE 2018; 18:248. [PMID: 30189898 PMCID: PMC6127945 DOI: 10.1186/s12906-018-2316-4] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/16/2018] [Accepted: 08/28/2018] [Indexed: 12/12/2022]
Abstract
Background Persistent hepatitis B virus (HBV) infection causes liver cirrhosis and hepatocellular carcinoma and constitutes a major worldwide health problem. Currently, anti-HBV drugs are limited to peginterferon and nucleos(t)ide analogs, which are costly and have considerable side effects; the development of novel, effective anti-HBV agents is crucial. Methods Catechins are a major group of compounds found in green tea extract and epigallocatechin gallate (EGCG) has been shown to have antiviral properties, including inhibition of cellular entry by HBV. FRG (Fah−/−/ Rag2−/−/ IL-2Rγ/−) mice were used in this study to generate chimeras carrying human primary hepatocytes, to facilitate investigation of the inhibitory effect of EGCG on HBV infection. Results Here, we show the inhibitory effect of EGCG on HBV infection and replication in HuS-E/2 cells. The inhibitory effect of EGCG on HBV infection in vivo was confirmed by monitoring HBV DNA and HBsAg in serum and immunostaining the liver tissues of the human liver chimeric mice. Conclusions The effects of EGCG suggest a robust strategy for the treatment of HBV infection and EGCG may have therapeutic potential for the treatment of HBV-associated liver diseases.
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Abstract
We have developed a miniature human liver (liver-sinusoid-on-a-chip) model using a dual microchannel separated by a porous membrane. Primary human hepatocytes and immortalized bovine aortic endothelial cells were co-cultured on opposite sides of a microporous membrane in a dual microchannel with continuous perfusion. Primary human hepatocytes in this system retained their polygonal morphology for up to 26 days, while hepatocytes cultured in the absence of bovine aortic endothelial cells lost their morphology within a week. In order to demonstrate the utility of our human-liver-sinusoid-on-a-chip, human hepatocytes in this system were directly infected by Hepatitis B Virus (HBV). Expression of the HBV core antigen was detected in human hepatocytes in the microchannel system. HBV replication, measured by the presence of cell-secreted HBV DNA, was also detected. Importantly, HBV is hepatotropic, and expression of HBV RNA transcripts is dependent upon expression of hepatocyte-specific factors. Moreover, HBV infection requires expression of the human-hepatocyte-specific HBV cell surface receptor. Therefore, the ability to detect HBV replication and Hepatitis B core Antigen (HBcAg) expression in our microfluidic platform confirmed that hepatocyte differentiation and functions were retained throughout the time course of our studies. We believe that our human-liver-sinusoid-on-a-chip could have many applications in liver-related research and drug development.
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Lan T, Chang L, Wu L, Yuan YF. IL-6 Plays a Crucial Role in HBV Infection. J Clin Transl Hepatol 2015; 3:271-6. [PMID: 26807383 PMCID: PMC4721895 DOI: 10.14218/jcth.2015.00024] [Citation(s) in RCA: 59] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/27/2015] [Revised: 09/17/2015] [Accepted: 10/11/2015] [Indexed: 01/05/2023] Open
Abstract
Interleukin-6 (IL-6), a cytokine mainly produced by activated monocytes, has broad pleiotropic actions that affect the functions of a variety of lymphoid cells. The roles of IL-6 in regulating immunity to infections are currently being defined. Remarkably, IL-6-mediated cellular and humoral immune responses play a crucial role in determining the outcome of viral infection. This article reviews the current knowledge on the critical role of IL-6 in hepatitis B virus (HBV) infection. As a competent intermediary, IL-6 derived from activated monocytes plays an important role in promoting lymphocytes responses that are essential for effective viral control. However, as a mediator of inflammation, IL-6 is also involved in the development of HBV-induced liver cirrhosis and exacerbating liver injury. Overall, the current data point to IL-6 as an immunoregulatory cytokine in HBV infection. Immunotherapeutic strategies aimed at optimizing the beneficial effects of IL-6 in HBV infection may prove to be an ordeal in the future, as they should foster the strengths of IL-6 while circumventing potential drawbacks.
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Affiliation(s)
- Tian Lan
- Zhongnan Hospital of Wuhan University, Department of Hepatobiliary Surgery, Wuhan University, Wuhan, China
| | - Lei Chang
- Zhongnan Hospital of Wuhan University, Department of Hepatobiliary Surgery, Wuhan University, Wuhan, China
| | - Long Wu
- Zhongnan Hospital of Wuhan University, Department of Hepatobiliary Surgery, Wuhan University, Wuhan, China
| | - Yu-Feng Yuan
- Zhongnan Hospital of Wuhan University, Department of Hepatobiliary Surgery, Wuhan University, Wuhan, China
- Correspondence to: Yu-Feng Yuan, Zhongnan Hospital of Wuhan University, Department of Hepatobiliary Surgery, Wuhan University, Wuhan 430071, Hubei, China. Tel: +86-027-67812888, Fax: +86-027-67812892, E-mail:
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Suresh V, Krishnakumar K, Asha V. A new fluorescent based screening system for high throughput screening of drugs targeting HBV-core and HBsAg interaction. Biomed Pharmacother 2015; 70:305-16. [DOI: 10.1016/j.biopha.2015.02.002] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2015] [Accepted: 02/08/2015] [Indexed: 12/28/2022] Open
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Russell MW, Whittum-Hudson J, Fidel PL, Hook EW, Mestecky J. Immunity to Sexually Transmitted Infections. Mucosal Immunol 2015. [DOI: 10.1016/b978-0-12-415847-4.00112-9] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
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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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Suresh V, Sojan J, Krishna Radhika N, Asha VV. Anti-HBV activity of the different extracts from Phyllanthus rheedei Wight in cell culture based assay systems. JOURNAL OF ETHNOPHARMACOLOGY 2014; 156:309-315. [PMID: 25219604 DOI: 10.1016/j.jep.2014.08.028] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/17/2014] [Revised: 07/28/2014] [Accepted: 08/23/2014] [Indexed: 06/03/2023]
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE Phyllanthusrheedei Wight is a plant used by Muthuvan tribes of Kerala for treating liver related diseases. MATERIALS AND METHODS The different extracts of Phyllanthus rheedei were analysed on cell lines were viz, PLC/PRF, Hep3B, FLCII10 and HepG2215 for its anti-HBV property. The analysis was done through ELISA, SQRT-PCR and immuno blotting. The most active extract was then divided in to fractions using HPTLC and the most active fraction was further identified. RESULTS From the screening experiments it was shown that the ethanol extract of this plant has the maximum activity in lowering the viral markers like HBsAg, HBV Core and HBV X protein and whole virions with comparatively lesser cytotoxicity. The dose responses of this particular extract were further established. CONCLUSIONS This study concluded that the ethanol extract of Phyllanthusrheedei is very much effective in preventing the multiplication of HBV at the cellular level. This study scientifically validated the tribal claim of the use of this plant for severe liver disorders.
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Affiliation(s)
- V Suresh
- Plant Based Bioactives and Disease Biology, Rajiv Gandhi Centre for Biotechnology, Trivandrum 695014, Kerala, India
| | - Jose Sojan
- Department of Botany, Govt. Victoria College, Palakkad 678001, Kerala, India
| | - N Krishna Radhika
- Division of Crop Improvement, Central Tuber Crops Research Institute (ICAR), Trivandrum 695017, Kerala, India
| | - V V Asha
- Plant Based Bioactives and Disease Biology, Rajiv Gandhi Centre for Biotechnology, Trivandrum 695014, Kerala, India.
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Hepatitis B virus preS1-derived lipopeptide functionalized liposomes for targeting of hepatic cells. Biomaterials 2014; 35:6130-41. [DOI: 10.1016/j.biomaterials.2014.04.037] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2014] [Accepted: 04/14/2014] [Indexed: 12/21/2022]
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Jeulin H, Velay A, Murray J, Schvoerer E. Clinical impact of hepatitis B and C virus envelope glycoproteins. World J Gastroenterol 2013; 19:654-664. [PMID: 23429668 PMCID: PMC3574591 DOI: 10.3748/wjg.v19.i5.654] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/14/2012] [Accepted: 12/17/2012] [Indexed: 02/06/2023] Open
Abstract
Chronic infection by either hepatitis B virus (HBV) or hepatitis C virus (HCV) share epidemiological characteristics with risks for development of severe complications such as liver cirrhosis and hepatocellular carcinoma. HBV and HCV also share a high genetic variability. Among highly variable regions, viral genes encoding surface proteins (hepatitis B surface antigen, E1/E2 HCV glycoproteins) play key roles in the stimulation of the host-related immune response and viral entry into hepatocytes. Specific segments of HBV envelope proteins (preS1, “a” determinant) are crucial in the entry process into permissive cells. HCV entry is a complex multistep process involving multiple cell cofactors (glycosaminoglycans, low density lipoprotein receptor, SR-B1, CD81, claudin-1, occludin, EGFR, EphA2) in the interaction with HCV E1/E2 envelope glycoproteins. In vitro both viruses can be controlled by antibody-mediated neutralization targeting viral envelope, also essential in preventing HBV infection in vivo as observed through successful vaccination using HBs antigen. But preventive vaccination and/or therapeutic pressure can influence HBV and HCV variability. For HBV, the patterns of antiviral drug resistance in chronic hepatitis are complex and the original pol/S gene overlap has to be taken into account. Treatment-induced HBV mutations in pol could indeed generate S mutants with subsequent modified antigenicity or increased cancer induction. Variability of HBV and HCV envelope proteins combining high exposure to selective pressures and crucial functional roles require investigation in the context of diagnostic, vaccination and treatment tools. In this editorial a synthesis is performed of HBV and HCV envelope properties at the entry step and as antigenic proteins, and the subsequent clinical impact.
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MESH Headings
- Animals
- Antiviral Agents/therapeutic use
- Drug Resistance, Viral
- Genotype
- Hepacivirus/drug effects
- Hepacivirus/genetics
- Hepacivirus/immunology
- Hepacivirus/metabolism
- Hepacivirus/pathogenicity
- Hepatitis B Vaccines
- Hepatitis B virus/drug effects
- Hepatitis B virus/genetics
- Hepatitis B virus/immunology
- Hepatitis B virus/metabolism
- Hepatitis B virus/pathogenicity
- Hepatitis B, Chronic/diagnosis
- Hepatitis B, Chronic/drug therapy
- Hepatitis B, Chronic/immunology
- Hepatitis B, Chronic/prevention & control
- Hepatitis B, Chronic/virology
- Hepatitis C, Chronic/diagnosis
- Hepatitis C, Chronic/drug therapy
- Hepatitis C, Chronic/immunology
- Hepatitis C, Chronic/prevention & control
- Hepatitis C, Chronic/virology
- Host-Pathogen Interactions
- Humans
- Phenotype
- Prognosis
- Viral Envelope Proteins/genetics
- Viral Envelope Proteins/metabolism
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14
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Sueki R, Maekawa S, Miura M, Kadokura M, Komase K, Shindo H, Kanayama A, Ohmori T, Shindo K, Amemiya F, Nakayama Y, Uetake T, Inoue T, Sakamoto M, Enomoto N. Correlation between pretreatment viral sequences and the emergence of lamivudine resistance in hepatitis B virus infection. J Med Virol 2012; 84:1360-8. [PMID: 22825814 DOI: 10.1002/jmv.23314] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
The emergence of amino acid or nucleotide substitutions leads to lamivudine resistance in hepatitis B virus (HBV) infected patients. The aim of this study was to investigate whether viral sequences help predict the emergence of lamivudine resistance. The study subjects comprised 59 consecutive patients infected with HBV treated with daily therapy of 100 mg lamivudine. Among those, 32 patients with adequate pretreatment serum preservation were investigated for the correlation between viral amino acid substitutions and the appearance of lamivudine resistance with consideration of clinical background by determining dominant HBV full open reading frames. Viral resistance to lamivudine emerged in 28 of 59 patients (47%) in a median period of 2.45 years. Sequence comparisons of HBV genomes between patients who later developed lamivudine resistance and patients who did not revealed the existence of significant differences between the two groups in the pre-S1 84 (P = 0.042), pre-S2 1 (P = 0.017) and 22 (P = 0.015), and polymerase tp 95 (P = 0.046), judged by a log-rank test. Viral sequence analyses revealed the presence of amino acid substitutions in HBV pre-S1 and pre-S2 that may be associated with the emergence of lamivudine resistance during chronic HBV infection.
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Affiliation(s)
- Ryota Sueki
- First Department of Internal Medicine, Faculty of Medicine, University of Yamanashi, Chuo, Yamanashi, Japan
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15
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Rawat S, Clippinger AJ, Bouchard MJ. Modulation of apoptotic signaling by the hepatitis B virus X protein. Viruses 2012; 4:2945-72. [PMID: 23202511 PMCID: PMC3509679 DOI: 10.3390/v4112945] [Citation(s) in RCA: 57] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2012] [Revised: 10/23/2012] [Accepted: 10/31/2012] [Indexed: 12/18/2022] Open
Abstract
Worldwide, an estimated 350 million people are chronically infected with the Hepatitis B Virus (HBV); chronic infection with HBV is associated with the development of severe liver diseases including hepatitis and cirrhosis. Individuals who are chronically infected with HBV also have a significantly higher risk of developing hepatocellular carcinoma (HCC) than uninfected individuals. The HBV X protein (HBx) is a key regulatory HBV protein that is important for HBV replication, and likely plays a cofactor role in the development of HCC in chronically HBV-infected individuals. Although some of the functions of HBx that may contribute to the development of HCC have been characterized, many HBx activities, and their putative roles during the development of HBV-associated HCC, remain incompletely understood. HBx is a multifunctional protein that localizes to the cytoplasm, nucleus, and mitochondria of HBV‑infected hepatocytes. HBx regulates numerous cellular signal transduction pathways and transcription factors as well as cell cycle progression and apoptosis. In this review, we will summarize reports in which the impact of HBx expression on cellular apoptotic pathways has been analyzed. Although various effects of HBx on apoptotic pathways have been observed in different model systems, studies of HBx activities in biologically relevant hepatocyte systems have begun to clarify apoptotic effects of HBx and suggest mechanisms that could link HBx modulation of apoptotic pathways to the development of HBV-associated HCC.
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Affiliation(s)
- Siddhartha Rawat
- Graduate Program in Molecular and Cellular Biology and Genetics, Drexel University College of Medicine, Philadelphia, PA 19102, USA;
| | - Amy J. Clippinger
- Department of Cancer Biology, Abramson Family Cancer Research Institute, School of Medicine, University of Pennsylvania Philadelphia, PA 19104, USA;
| | - Michael J. Bouchard
- Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA 19102, USA
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16
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Hu Z, Li M, Huang B, Liu J, Yu L, Chen G. Detection of hepatitis B virus PreS1 antigen using a time-resolved fluoroimmunoassay. J Immunoassay Immunochem 2012; 33:156-65. [PMID: 22471606 DOI: 10.1080/15321819.2011.609576] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
Abstract
The hepatitis B virus (HBV) PreS1 antigen is expressed at the distal most region of the envelope protein and contains the hepatocyte receptor-binding site. The presence of the HBV PreS1 antigen in serum and liver of HBsAg-positive patients is a new marker used for diagnosing HBV infection, and is indicative of viral replication. Our objective is to establish a method of time-resolved fluoroimmunoassay (TRFIA) with higher sensitivity and broader detection range for detecting serum HBV PreS1 antigen. Eu(3+) labeling of antibodies was performed with respective labeling kits, and Eu(3+) fluorescence intensity was measured with an auto DELFIA1235 TRFIA analyzer. The established method was evaluated for its performance. Serum specimens (574 in total) from Wuxi People's Hospital were analyzed for PreS1 antigen using the TRFIA and ELISA. The precision, specificity, and sensitivity of the TRFIA were clearly better than ELISA. The detection limit was 0.01 ng/mL. The average recovery rate for PreS1 antigens was 103.3%. There was significant correlation between the PreS1 antigen results obtained by TRFIA and ELISA in 374 serum samples with HBV >10(3) IU/mL (χ(2) = 25.04, p < 0.01) and 183 HbeAg-positive serum samples (χ(2) = 12.07, p < 0.01). Normal reference ranges were established at 0-0.32 ng/mL based on the values obtained from 100 healthy controls. TRFIA is a significantly effective method for clinical detection of serum HBV PreS1 antigens.
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Abstract
Hepatitis B is a DNA virus affecting hundreds of millions of individuals worldwide. As the clinical sequelae of cirrhosis and hepatocellular cancer are increasingly recognized to be related to viral levels, the impetus increases to offer treatment to those previously not treated. With the development of more robust antivirals with reasonable safety profiles, long-term treatment is becoming more common. The oral nucleos(t)ide analogs have become the preferred first-line therapies for most genotypes of hepatitis B. Five are now available, all with different potencies and resistance profiles. Long-term data spanning several years are now available for most compounds in this arena. This article focuses on the common natural variants and those secondary to nucleos(t)ide therapy, as well as diagnostic methods to detect resistance.
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Affiliation(s)
- Fred Poordad
- David Geffen School of Medicine at UCLA, Hepatology and Liver Transplantation, Cedars-Sinai Medical Center, Suite 1060, Los Angeles, CA 90048, USA.
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18
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Jiang Y, Wang AH, Shao LH, Wang G, Yao YY, Sai LT, Chen FZ, Zheng F, Li Y, Ma LX. A new cell culture system for infection with hepatitis B virus that fuses HepG2 cells with primary human hepatocytes. J Int Med Res 2009; 37:650-61. [PMID: 19589247 DOI: 10.1177/147323000903700307] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022] Open
Abstract
Hepatitis B virus (HBV) infection exhibits a very narrow host range and shows a strong tropism for liver parenchymal cells, however none of the previously established experimental models can reproduce the natural process of HBV infection. In the present study, primary human hepatocytes were fused with HepG2 cells to establish the hybrid HepCHLine-4 cell line with high susceptibility to HBV. The HepCHLine-4 cells expressed HBV-specific antigen when co-incubated with HBV-positive serum from a hepatitis B patient. Post-infection, HBV relaxed circular DNA and covalently closed circular DNA were detected in HepCHLine-4 cells using a nested polymerase chain reaction, and HBV-specific particles were visualized by electron microscopy of the culture media of HepCHLine-4 cells. HepG2 cells were not susceptible to HBV infection under the same conditions. The HepCHLine-4 cells can be sub-cultured for > 12 months while maintaining susceptibility to HBV and may, therefore, be useful for studying HBV infection and the viral life cycle in human hepatocytes.
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Affiliation(s)
- Y Jiang
- Department of Infectious Diseases, Qilu Hospital, Shandong University, Jinan, Shandong Province, China
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19
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Wang Z, Yuan Z, Jin L. Gene delivery into hepatocytes with the preS/liposome/DNA system. Biotechnol J 2009; 3:1286-95. [PMID: 18830969 DOI: 10.1002/biot.200800125] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023]
Abstract
Gene delivery into human hepatocytes remains a critical issue for the development of liver-directed gene therapy. Gene delivery based on non-viral vectors is an attractive approach relative to viral vectors. In this report, novel delivery system of preS/liposome/DNA virus-like particle (VLP) was developed for gene transfection into hepatocytes in vivo and in vitro. Plasmid pCMVbeta, expressing beta-galactosidase, was encapsulated with cationic liposome, and then the histidine-tagged preS domain of hepatitis B virus was coated on the surface of liposome/DNA to form preS/liposome/ DNA VLP. Transfection efficiencies of preS/liposome/DNA, liposome/DNA, naked DNA and preS were analyzed using several different human cell lines. The highest transfection efficiency was found using preS/liposome/DNA VLP as the transfection reagent in human hepatocyte (HH) cell line. Results show that preS domain of hepatitis B virus coated on liposome/DNA can be used for highly efficient gene transfection into human hepatocytes. Moreover, the target characteristic of preS/liposome/DNA was analyzed in vivo. After preS/liposome/DNA VLP was injected into immunocompromised (Nude) mice via the tail vein, most of beta-galactosidase was expressed in the liver; however, no significant target expression was found with the injection of liposome/ DNA or naked DNA. Our results show that preS/liposome/DNA VLP can be used as a novel liver-specific gene delivery system.
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Affiliation(s)
- Zhijun Wang
- CAS-MPG Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, People's Republic of China.
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20
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Hellström UB, Madalinski K, Sylvan SP. PreS1 epitope recognition in newborns after vaccination with the third-generation Sci-B-Vac vaccine and their relation to the antibody response to hepatitis B surface antigen. Virol J 2009; 6:7. [PMID: 19154574 PMCID: PMC2635352 DOI: 10.1186/1743-422x-6-7] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2008] [Accepted: 01/20/2009] [Indexed: 12/12/2022] Open
Abstract
Background Sci-B-Vac™ is a recombinant, hepatitis B vaccine derived from a mammalian cell line and containing hepatitis B surface antigen (HBsAg) as well as preS1 and preS2 antigens. Few studies have been performed on the antibody responses to preS1 in relation to the antibody to hepatitis B surface antigen (anti-HBs) response during immunisation of healthy children with preS-containing vaccines. Results In this study 28 healthy newborns were randomly selected to receive either 2.5 ug or 5.0 ug of the Sci-B-Vac vaccine. Children received three doses of vaccine according to a 0-, 1-, 6-month scheme. Antibodies against the S-protein and three synthetic peptides mimicking three B-cell preS1 epitopes, (21–32 amino acid epitope), (32–47 amino acid epitope) and the C-terminal (amino acid epitope 94–117) were determined at 6 and 9 months. Fourteen (50%) of the 28 newborns had detectable levels of anti-preS1 (21–32) antibodies; 15 (54%) were anti-preS1 (32–47) reactive and 12 (43%) were anti-preS1 (94–117) reactive at 6 or 9 months after initiation of the vaccination. Significantly higher levels of anti-HBs were observed in the sera of patients with detectable anti-preS1 (32–47) reactivity (24 550 ± 7375 IU/L, mean ± SEM) as compared with the non-reactive sera (5991 ± 1530 IU/L, p < 0.05). The anti-HBs levels were significantly lower if none (p < 0.05) or one (p < 0.025) of the preS1 (21–32, 32–47, 94–117) peptides were recognised compared with the anti-HBs levels if two or three peptides were recognised. Conclusion Recognition of several preS1 epitopes, and in particular, the epitope contained within the second half of the hepatocyte binding site localised in the hepatitis B surface protein of the third-generation hepatitis B vaccine is accompanied by a more pronounced antibody response to the S-gene-derived protein in healthy newborns.
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Affiliation(s)
- Ulla B Hellström
- Department of Communicable Disease Control and Prevention, Uppsala County Council, Sweden.
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21
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Chen B, Lian M, Xu S, Luo M, Zheng X. A chemical lipid modification of recombinant preS antigen to study the mechanism of HBV attachment to the host cell. J Biotechnol 2008; 137:8-13. [PMID: 18675858 DOI: 10.1016/j.jbiotec.2008.06.010] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2008] [Revised: 06/12/2008] [Accepted: 06/26/2008] [Indexed: 10/21/2022]
Abstract
Surface antigen preS of Hepatitis B virus plays fundamental roles in mediating receptor recognition and virus internalization. Myristoylation at N-terminal Gly(2) residue of preS is essential for viral attachment and infectivity. A number of myristoylated proteins have been shown to undergo a conformational change (myristoyl switch) that alters their affinity to cell membrane. However, there is little knowledge about what effect this fatty acylation contributes in virus-host cell interaction. Here we demonstrated a new method for lipid modification of recombinant preS protein at N-terminal residue 2 with alkylating chemicals. Modified preS was able to inhibit HBV penetrating into HepG2 cells with an increased efficiency compared to unmodified form. Flow cytometric analysis indicated that lipid modification enhanced the binding affinity of preS to hepatocytes, but not resulting from hydrophobic interaction. CD analysis further revealed a conformational change of modified preS in the presence of membrane mimetics. These findings imply that the conformation transition induced by fatty acylation is important for efficient attachment of virus to cell receptors, and this method of chemical lipid modification provides a basis for designing therapeutic inhibitors to Hepatitis B virus.
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Affiliation(s)
- Bin Chen
- National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871, China
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22
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Lian M, Zhou X, Chen B, Li C, Gu X, Luo M, Zheng X. Identification of the critical regions in hepatitis B virus preS required for its stability. J Pept Sci 2008; 14:307-12. [PMID: 17918766 DOI: 10.1002/psc.929] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
As a hepatitis B virus (HBV) envelope domain, preS plays significant roles in receptor recognition and viral infection. However, the regions critical for maintaining a stable and functional conformation of preS are still unclear and require further investigation. In order to unravel these regions, serially truncated fragments of preS were constructed and expressed in Escherichia coli. Their solubility, stability, secondary structure, and affinity to polyclonal antibodies and hepatocytes were examined. The results showed that amino acids 31-36 were vital for its stable conformation, and the absence of 10-36 amino acids significantly reduced its binding to polyclonal antibodies as well as hepatocytes. The most stable fragment 1-120 (preS1 + N-terminal 12 amino acids of preS2), perhaps the core of preS, was discovered, which bound to HepG2 cells most tightly. Moreover, the availability of large amounts of well-folded and stable preS1-120 enables us to carry out further structural determination and mechanistic study on HBV infection.
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Affiliation(s)
- Min Lian
- National Laboratory of Protein Engineering and Plant Genetic Engineering, Peking University, Beijing, 100871, China
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23
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Hellström U, Lindh M, Krogsgaard K, Sylvan S. Demonstration of an association between detection of IgG antibody reactivity towards the C-terminal region of the preS1 protein of hepatitis B virus and the capacity to respond to interferon therapy in chronic hepatitis B. J Gastroenterol Hepatol 2008; 23:804-10. [PMID: 17931371 DOI: 10.1111/j.1440-1746.2007.05174.x] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
BACKGROUND AND AIM The treatment of hepatitis B virus (HBV) remains complex, with somewhat unpredictable responses. The aim of this study was to determine the predictive value of the pretreatment presence of circulatory antibodies towards a synthetic peptide mimicking the amino acids 94-117 of the preS1 protein of HBV and the capacity to respond to alpha-inteferon (IFN-alpha) treatment. METHODS The anti preS1(94-117) antibodies were measured by a peptide-based enzyme-linked immunosorbent assay (ELISA) and the response to INF-alpha therapy was judged by the effect on the viral kinetics as measured by an assay based on quantitative polymerase chain reaction during the treatment and follow up. RESULTS We found a significant (P < 0.001) correlation between the pretreatment presence of anti preS1(94-117) antibodies and a decrease in viral levels on follow up after the end of IFN-alpha therapy. The combined response of HBV DNA suppression (P < 0.001), hepatitis B e antigen (HBeAg) loss (P < 0.0001), anti-HBe seroconversion (P < 0.005) and AST aminotransferase normalization (P < 0.01) was also highly associated with the pretreatment presence of anti preS1(94-117) antibodies. CONCLUSION The positive predictive value (PPV) of anti preS1(94-117) in determining a virological response was 83% and the negative predictive value (NPV) was 100%, indicating that in the absence of pretreatment anti preS1 reactivity virtually no patient has the capacity to respond to IFN-alpha therapy. Our findings may help to improve the efficacy of INF-alpha therapy for chronic hepatitis B (CHB) by guiding the selection of patients for treatment and optimizing the clinical management of the individual patient.
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Affiliation(s)
- Ulla Hellström
- Department of Communicable Disease Control and Prevention, Karolinska Hospital, Stockholm, Sweden
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24
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Abbas N, Ahmad A, Shakoori AR. Overexpression and purification of PreS region of hepatitis B virus antigenic surface protein adr subtype in Escherichia coli. BMB Rep 2008; 40:1002-8. [PMID: 18047797 DOI: 10.5483/bmbrep.2007.40.6.1002] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
PreS domain of Hepatitis B virus (HBV) surface antigen is a good candidate for an effective vaccine as it activates both B and T cells besides binding to hepatocytes. This report deals with overexpression and purification of adr subtype of surface antigen that is more prevalent in Pakistan. PreS region, comprising 119 aa preS1 region plus a 55 aa preS2 region plus 11 aa from the N-terminal S region, was inserted in pET21a+ vector, cloned in E. coli DH5alpha cells and expressed in E. coli BL21 codon+ cells. The conditions for over expression were optimized using different concentrations of IPTG (0.01-5 mM), and incubating the cells at different temperatures (23-41 degrees C) for different durations (0-6 h). The cells were grown under the given optimized conditions (0.5 mM IPTG concentration at 37 degrees C for 4 h), lysed by sonication and the protein was purified by ion exchange chromatography. On the average, 24.5 mg of recombinant protein was purified per liter of culture. The purified protein was later lyophilized and stored at -80 degrees.
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Affiliation(s)
- Naaz Abbas
- School of Biological Sciences, University of the Punjab, New Campus, Lahore 54590, Pakistan
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Chi SW, Kim DH, Lee SH, Chang I, Han KH. Pre-structured motifs in the natively unstructured preS1 surface antigen of hepatitis B virus. Protein Sci 2007; 16:2108-17. [PMID: 17766372 PMCID: PMC2204132 DOI: 10.1110/ps.072983507] [Citation(s) in RCA: 48] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
The preS1 surface antigen of hepatitis B virus (HBV) is known to play an important role in the initial attachment of HBV to hepatocytes. We have characterized structural features of the full-length preS1 using heteronuclear NMR methods and discovered that this 119-residue protein is inherently unstructured without a unique tertiary structure under a nondenaturing condition. Yet, combination of various NMR parameters shows that the preS1 contains "pre-structured" domains broadly covering its functional domains. The most prominent domain is formed by residues 27-45 and overlaps with the putative hepatocyte-binding domain (HBD) encompassing residues 21-47, within which two well-defined pre-structured motifs, formed by Pro(32)-Ala(36) and Pro(41)-Phe(45) are found. Additional, somewhat less prominent, pre-structured motifs are also formed by residues 11-18, 22-25, 37-40, and 46-50. Overall results suggest that the preS1 is a natively unstructured protein (NUP) whose N-terminal 50 residues, populated with multiple pre-structured motifs, contribute critically to hepatocyte binding.
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Affiliation(s)
- Seung-Wook Chi
- Molecular Cancer Research Center, Division of Molecular Therapeutics, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea
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Jung J, Kasuya T, Tanizawa K, Kuroda S. Bio-nanocapsules for In vivo Pinpoint Drug Delivery. YAKUGAKU ZASSHI 2007; 127:797-805. [PMID: 17473521 DOI: 10.1248/yakushi.127.797] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
To maximize the beneficial effects and minimize the side effect of drugs, DDS (drug delivery system) has been attracted many researchers in the recent drug development. Especially, the in vivo pinpoint delivery system for drugs is very important and key technology for developing the next generations of anti-cancer drugs and gene therapies. Bio-nanocapsule (BNC) is recombinant yeast-derived hepatitis B virus surface antigen particle, which has been used as a recombinant hepatitis B vaccine for the last 20 years in the world. BNC can incorporate various materials (chemical compounds, proteins, genes, siRNA, etc) by the fusion with liposome, and deliver them to the organs and tissues in vivo specifically by the action of bio-recognition molecules on the BNC's surface. The transfection efficiency is significantly higher than that of liposome, because BNC harbors the complete set of hepatitis B virus infection machinery. Recently, we succeeded in the in vivo retargeting of BNC by displaying either antibody or homing peptide, less than 10 amino acid residues for in vivo targeting. BNC is a hybrid of liposome and virus, and very flexible system for in vivo retargeting. BNC might be very promising carriers in the next generation of DDS.
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Affiliation(s)
- Joohee Jung
- The Institute of Scientific and Industrial Research, Osaka University, Ibaraki City, Japan
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27
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Desai M, Pal R, Deshmukh R, Banker D. Replication of TT virus in hepatocyte and leucocyte cell lines. J Med Virol 2005; 77:136-43. [PMID: 16032745 DOI: 10.1002/jmv.20426] [Citation(s) in RCA: 38] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
The TT virus (TTV) is a non-enveloped, single-stranded, circular, DNA virus, first isolated from a patient with hepatitis of unexplained etiology. The much deliberated pathological role of the virus continues to be conjectural in the absence of a suitable in vitro replication model. So far, the liver and the bone marrow have been shown to be the main sites of TTV replication. In this study, the human cell lines HepG2 and Chang Liver, the rat hepatoma cell line MH1C1, phytohemagglutinin (PHA)-stimulated TTV-negative peripheral blood mononuclear cell (PBMC) cultures and the B lymphoblast cell line, Raji were investigated as potential in vitro replication systems for TTV. The cell lines were infected with an inoculum prepared by pooling TTV genotype1 DNA positive sera and monitored for virus replication. Of the three hepatocyte cell lines, while the HepG2 and MH1C1 cell lines did not support TTV replication, the Chang Liver cell line showed clear morphological changes as a result of the in vitro infection, which included clumping and granular degeneration of the entire cell sheet over a period of 6 days. The infected cells also showed presence of virus-specific mRNA representative of viral transcription. The consistent presence of infectious viral particles in the supernatant culture fluid at 24-hr fluid replacement intervals indicated limited extra-cellular release of viral particles. The PHA-stimulated TTV-negative PBMC cultures and the Raji cell line were also able to support TTV replication and released significant levels of infectious viral particles into the supernantant culture fluid.
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Affiliation(s)
- Mayura Desai
- Department of Microbiology, Sir Hurkisondas Nurrotumdas Medical Research Society, Mumbai, India
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28
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Deng Q, Zhuang M, Kong YY, Xie YH, Wang Y. Screening for PreS specific binding ligands with a phage displayed peptides library. World J Gastroenterol 2005; 11:4018-23. [PMID: 15996026 PMCID: PMC4502097 DOI: 10.3748/wjg.v11.i26.4018] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To construct a random peptide phage display library and search for peptides that specifically bind to the PreS region of hepatitis B virus (HBV).
METHODS: A phage display vector, pFuse8, based on the gene 8 product (pVIII) of M13 phage was made and used to construct a random peptide library. E.coli derived thioredoxin-PreS was purified with Thio-bond beads, and exploited as the bait protein for library screening. Five rounds of bio-panning were performed. The PreS-binding specificities of enriched phages were characterized with phage ELISA assay.
RESULTS: A phage display vector was successfully constructed as demonstrated to present a pVIII fused HBV PreS1 epitope on the phage surface with a high efficiency. A cysteine confined random peptide library was constructed containing independent clones exceeding 5±108 clone forming unit (CFU). A pool of phages showing a PreS-binding specificity was obtained after the screening against thio-PreS with an enrichment of approximately 400 times. Five phages with high PreS-binding specificities were selected and characterized. Sequences of the peptides displayed on these phages were determined.
CONCLUSION: A phage library has been constructed, with random peptides displaying as pVIII-fusion proteins. Specific PreS-binding peptides have been obtained, which may be useful for developing antivirals against HBV infection.
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Affiliation(s)
- Qiang Deng
- Institute of Biochemistry and Cell Biology, 320 Yue-Yang Road, Shanghai 200031, 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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Glebe D, Urban S, Knoop EV, Cag N, Krass P, Grün S, Bulavaite A, Sasnauskas K, Gerlich WH. Mapping of the hepatitis B virus attachment site by use of infection-inhibiting preS1 lipopeptides and tupaia hepatocytes. Gastroenterology 2005; 129:234-45. [PMID: 16012950 DOI: 10.1053/j.gastro.2005.03.090] [Citation(s) in RCA: 207] [Impact Index Per Article: 10.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
BACKGROUND & AIMS Studies on the early steps in the life cycle of hepatitis B virus have been hampered by the lack of readily available target cells. In this study, we mapped a defined virus attachment site to primary hepatocytes that is essential for infection. METHODS We used purified virus particles from human carrier plasma as an inoculum and primary cultures of tupaia hepatocytes as susceptible target cells and studied the inhibitory effect of amino-terminally acylated preS1-derived lipopeptides on infection interference. RESULTS Infectivity of virus could be blocked efficiently in this system by amino-terminally acylated peptides containing amino acids 2-18 from the preS1 domain. The addition of amino acids 28-48 enhanced the inhibitory capacity, whereas amino acids 49-78 did not contribute to inhibition. Myristoylated preS1 peptides 2-48 bound strongly to tupaia hepatocytes but not to nonhepatic cells or rodent hepatocytes and thereby inhibited infection even at concentrations of 1 nmol/L completely. Particles consisting only of the small hepatitis B surface protein-the active component of current hepatitis B vaccines-did not bind at all to tupaia hepatocytes, but the addition of the preS1 domain to the particles allowed binding. CONCLUSIONS The preS1 sequence 2-48 mediates attachment of the virus to its target cells, whereas the small surface protein seems to be involved in other steps. These findings indicate that the current subunit hepatitis B vaccines may be improved by the addition of distinct preS1 epitopes. Moreover, preS1 lipopeptides are promising candidates for specific antiviral therapy against hepatitis B infections.
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Affiliation(s)
- Dieter Glebe
- Institute of Medical Virology, Justus-Liebig University Giessen, Germany.
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31
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Abstract
The mechanisms of the attachment and penetration of hepatitis B virus remain obscure. It has been demonstrated that the preS1 region is essential for viral assembly and infectivity, however, as its cellular receptor has still not been identified unequivocally, we used a yeast two-hybrid system to screen the cellular proteins that can interact with preS1 protein. The protein recovered from a human liver cDNA library was nascent polypeptide-associated complex alpha polypeptide. The interaction between preS1 and nascent polypeptide-associated complex alpha polypeptide was verified by mating experiment and coimmunoprecipitation of COS7 cell lysates expressing both proteins. Based on these results, we speculate that nascent polypeptide-associated complex alpha polypeptide is a functional target of hepatitis B virus preS1 protein in cells.
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Affiliation(s)
- Dan Li
- Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province, China.
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32
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Abstract
AIM: To express the complete PreS region of HBV in E.coli with good solubility and stability, and to establish an effective method for purification of the recombinant PreS protein.
METHODS: The complete PreS region (PreS1 and PreS2) was fused into a series of tags including glutathione S-transferase (GST), dihydrofolate reductase (DHFR), maltose binding protein (MBP), 6×histidine, chitin binding domain (CBD), and thioredoxin, respectively. Expression of recombinant PreS fusion proteins was examined by SDS-PAGE analysis and confirmed by Western blot. Two fusion proteins, thio-PreS, and PreS-CBD, with desirable solubility and stability, were subjected to affinity purification and further characterization.
RESULTS: Recombinant PreS fusion proteins could be synthesized with good yields in E.coli. However, most of these proteins except for thio-PreS and PreS-CBD were vulnerable to degradation or insoluble as revealed by SDS-PAGE and Western blot. Thio-PreS could be purified by affinity chromatography with nickel-chelating sepharose as the matrix. However, some impurities were also co-purified. A simple freeze-thaw treatment yielded most of the thio-PreS proteins in solution while the impurities were in the precipitate. Purified thio-PreS protein was capable of inhibiting the binding of HBV virion to a specific monoclonal antibody against an epitope within the PreS1 domain.
CONCLUSION: Increased solubility and stability of the complete PreS region synthesized in E.coli can be achieved by fusion with the thioredoxin or the CBD tag. A simple yet highly effective method has been established for the purification of the thio-PreS protein. Purified thio-PreS protein likely assumes a native conformation, which makes it an ideal candidate for studying the structure of the PreS region as well as for screening antivirals.
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Affiliation(s)
- Qiang Deng
- State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai 200031, China
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34
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Lee JY, Locarnini S. Hepatitis B virus: pathogenesis, viral intermediates, and viral replication. Clin Liver Dis 2004; 8:301-20. [PMID: 15481342 DOI: 10.1016/j.cld.2004.02.009] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Although HBV has the potential to generate an almost limitless spectrum of quasispecies during chronic infection, the viability of the majority of these quasispecies is almost certainly impaired due to constraints imposed by the remarkably compact organization of the HBV genome. On the other hand, single mutations may affect more than one gene and result in complex and unpredictable effects on viral phenotype. Better understanding of the constraints imposed by gene overlap and of genotype-phenotype relationships should help in the development of improved antiviral strategies and management approaches. Although the probability of developing viral resistance is directly proportional to the intensity of selection pressure and the diversity of quasispecies, potent inhibition of HBV replication should be able to prevent development of drug resistance because mutagenesis is replication dependent. If viral replication can be suppressed for a sufficient length of time, viral load should decline to a point where the continued production of quasispecies with the potential to resist new drug treatments no longer occurs. Clinical application of this concept will require optimization of combination therapies analogous to highly active antiretroviral therapy (HAART) for HIV infection. Total cure of hepatitis B will require elimination of the intranuclear pool of viral minichromosomes, which will probably only be achieved by normal cell turnover, reactivation of host immunity, or elucidation of the antiviral mechanisms operating during cytokine clearance in acute hepatitis B (see Fig. 1).
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Affiliation(s)
- Jia-Yee Lee
- Victorian Infectious Diseases Reference Laboratory, 10 Wreckyn Street, North Melbourne, Victoria 3051, Australia
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35
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Moore PL, Ong S, Harrison TJ. Squamous cell carcinoma antigen 1-mediated binding of hepatitis B virus to hepatocytes does not involve the hepatic serpin clearance system. J Biol Chem 2003; 278:46709-17. [PMID: 12975381 DOI: 10.1074/jbc.m302842200] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023] Open
Abstract
The cellular receptor for hepatitis B virus (HBV) has not yet been identified. A recent candidate is a homologue of squamous cell carcinoma antigen 1 (SCCA1), a serpin. This study confirms that transfection of SCCA1 into mammalian cells (both hepatocyte-derived and of non-hepatocyte origin) results in increased HBV binding. Furthermore, virus bound to transfected cells is protected significantly from degradation by trypsin (75% compared with 30% in untransfected cells). The possibility that HBV enters cells via the hepatic clearance system for serpin-enzyme complexes was investigated by analysis of the reactive site loop of SCCA1. Functional and deletion mutants of SCCA1 were constructed by site-directed mutagenesis and compared with the wild type construct. In no case was virus binding reduced by functional alterations or deletions within the reactive site loop. A possible role for the low density lipoprotein receptor-related protein (LRP) in binding virus was investigated. SCCA1 transfection of Huh7 cells was shown to result in up-regulation of LRP expression, reaching levels observed in total liver. However, the use of receptor-associated protein (RAP), a competitive ligand for LRP, suggests than LRP up-regulation is not responsible for enhanced virus binding to SCCA1-transfected cells.
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Affiliation(s)
- Penelope L Moore
- Centre for Hepatology, Department of Medicine, Royal Free and University College Medical School, Rowland Hill Street, London NW3 2PF, United Kingdom
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36
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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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N/A. N/A. Shijie Huaren Xiaohua Zazhi 2003; 11:1004-1006. [DOI: 10.11569/wcjd.v11.i7.1004] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
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Abstract
Virus infection is initiated by recognition and attachment of the virus to the cell surface. Despite the fact that this interaction determines the virus-related pathogenesis, its molecular basis remained obscure for HBV. This process is mediated primarily by the viral envelope and the cellular receptors. HBV infection is not exceptional in this regard but its putative receptors have not been identified yet. The recent development of protocols to establish HBV susceptible cell lines and unique tools to measure HBV-cell attachment at a single cell resolution set the stage for the study of HBV-host cell interaction. These studies revealed that the QLDPAF epitope of the HBV surface antigen large protein (LHBsAg) plays a major role in this process. Quantitative measurements suggested the presence of a second player in this process and both act synergistically to improve cell attachment. As the step of virus-cell attachment is potentially susceptible to specific inhibitors, understanding the molecular basis of virus-cell attachment can be expected to have therapeutic impacts.
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Affiliation(s)
- Nir Paran
- Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel
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39
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Gao CX, Honke K, Taniguchi N. Carbohydrate Binding Activity of Annexin V toward a Bisecting N-Acetylglucosamine. Methods Enzymol 2003; 363:34-47. [PMID: 14579566 DOI: 10.1016/s0076-6879(03)01042-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/27/2023]
Affiliation(s)
- Cong-Xiao Gao
- Department of Biochemistry, Osaka University Medical School, Suita, Osaka 565-0871, Japan
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40
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41
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Abstract
Heparin, a sulfated polysaccharide belonging to the family of glycosaminoglycans, has numerous important biological activities, associated with its interaction with diverse proteins. Heparin is widely used as an anticoagulant drug based on its ability to accelerate the rate at which antithrombin inhibits serine proteases in the blood coagulation cascade. Heparin and the structurally related heparan sulfate are complex linear polymers comprised of a mixture of chains of different length, having variable sequences. Heparan sulfate is ubiquitously distributed on the surfaces of animal cells and in the extracellular matrix. It also mediates various physiologic and pathophysiologic processes. Difficulties in evaluating the role of heparin and heparan sulfate in vivo may be partly ascribed to ignorance of the detailed structure and sequence of these polysaccharides. In addition, the understanding of carbohydrate-protein interactions has lagged behind that of the more thoroughly studied protein-protein and protein-nucleic acid interactions. The recent extensive studies on the structural, kinetic, and thermodynamic aspects of the protein binding of heparin and heparan sulfate have led to an improved understanding of heparin-protein interactions. A high degree of specificity could be identified in many of these interactions. An understanding of these interactions at the molecular level is of fundamental importance in the design of new highly specific therapeutic agents. This review focuses on aspects of heparin structure and conformation, which are important for its interactions with proteins. It also describes the interaction of heparin and heparan sulfate with selected families of heparin-binding proteins.
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Affiliation(s)
- Ishan Capila
- S328 College of Pharmacy, University of Iowa, 115 S. Grand Avenue, Iowa City 52242, USA
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42
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43
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44
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Sung VMH, Lai MMC. Murine retroviral pseudotype virus containing hepatitis B virus large and small surface antigens confers specific tropism for primary human hepatocytes: a potential liver-specific targeting system. J Virol 2002; 76:912-7. [PMID: 11752180 PMCID: PMC136820 DOI: 10.1128/jvi.76.2.912-917.2002] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
We have developed a system for producing murine leukemia virus (MLV) pseudotyped with human hepatitis B virus (HBV) large (L) and small (S) surface antigens (HBsAg) for targeting primary human hepatocytes. Using the MLV(HBV) pseudotype virus containing a beta-galactosidase reporter gene, we demonstrated that this pseudotype virus exhibits strict tropism for primary human hepatocytes, similar to the natural target cell specificity of HBV. It does not infect any of the established tissue culture cell lines, including human hepatoma cell lines (HepG2 and Huh-7), or rat primary hepatocytes. The infectivity of MLV(HBV) for human hepatocytes was inhibited by anti-HBs antibody. The L form of HBsAg was both necessary and sufficient for virus infectivity, but the presence of both L and S forms enhanced the surface expression of HBsAg and thus increased virus production. The middle form of HBsAg was not necessary. This pseudotype virus bypasses the requirement for the liver-specific transcription factors for HBV replication, enabling direct study of HBV tissue tropism conferred by the viral envelope proteins. This virus also offers a potential liver-specific targeting system for gene therapy.
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Affiliation(s)
- Vicky M H Sung
- Department of Molecular Microbiology and Immunology, Howard Hughes Medical Institute, Keck School of Medicine, University of Southern California, Los Angeles, California 90033, USA
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45
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Pizarro JC, Vulliez-le Normand B, Riottot MM, Budkowska A, Bentley GA. Structural and functional characterization of a monoclonal antibody specific for the preS1 region of hepatitis B virus. FEBS Lett 2001; 509:463-8. [PMID: 11749974 DOI: 10.1016/s0014-5793(01)03190-8] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
The monoclonal antibody 5a19, raised against the ay serotype of hepatitis B virus, binds to the segment of the preS1 region comprising residues 37-43, which is implicated in attachment of the virus to hepatocytes. The dissociation constant, derived from kinetic studies using surface plasmon resonance techniques, is in the low nanomolar range. The nucleotide sequence of the variable domains has been determined and the corresponding germ-line genes have been identified. The three-dimensional structure of the Fab fragment has been determined by X-ray crystallography to 2.6 A resolution.
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Affiliation(s)
- J C Pizarro
- Unité d' Immunologie Structurale, Institut Pasteur, Paris, France
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46
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Paran N, Geiger B, Shaul Y. HBV infection of cell culture: evidence for multivalent and cooperative attachment. EMBO J 2001; 20:4443-53. [PMID: 11500372 PMCID: PMC125578 DOI: 10.1093/emboj/20.16.4443] [Citation(s) in RCA: 68] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
Hepadnaviruses do not infect cultured cells, therefore our knowledge of the mechanism of the early stages of virus-cell interaction is rather poor. In this study, we show that dimethylsulfoxide (DMSO)-treated HepG2 hepatoblastoma cells are infected efficiently by serum-derived hepatitis B virus (HBV) as monitored by viral gene expression and replication markers. To measure virus attachment, a variety of HBV surface proteins (HBsAgs) were conjugated to polystyrene beads and their capacity to attach cells was visualized and quantified by light microscopy at a single-cell resolution. Remarkably, DMSO increases the attachment efficiency by >200-fold. We further identify the QLDPAF sequence within preS1 as the receptor-binding viral domain epitope. Interestingly, a similar sequence is shared by several cellular, bacterial and viral proteins involved in cell adhesion, attachment and fusion. We also found that the small HBsAg contains a secondary attachment site that recognizes a distinct receptor on the cell membrane. Furthermore, we provide evidence in support of multivalent HBV attachment with synergistic interplay. Our data depict a mechanistic view of virus attachment and ingestion.
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Affiliation(s)
- Nir Paran
- Departments of
Molecular Genetics and Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel Corresponding author e-mail:
| | - Benjamin Geiger
- Departments of
Molecular Genetics and Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel Corresponding author e-mail:
| | - Yosef Shaul
- Departments of
Molecular Genetics and Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel Corresponding author e-mail:
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47
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François G, Kew M, Van Damme P, Mphahlele MJ, Meheus A. Mutant hepatitis B viruses: a matter of academic interest only or a problem with far-reaching implications? Vaccine 2001; 19:3799-815. [PMID: 11427251 DOI: 10.1016/s0264-410x(01)00108-6] [Citation(s) in RCA: 100] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Affiliation(s)
- G François
- WHO Collaborating Centre for Prevention and Control of Viral Hepatitis, Department of Epidemiology and Social Medicine, Universiteit Antwerpen, Universiteitsplein 1, B-2610 Antwerpen, Belgium.
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48
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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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49
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Maeng CY, Oh MS, Park IH, Hong HJ. Purification and structural analysis of the hepatitis B virus preS1 expressed from Escherichia coli. Biochem Biophys Res Commun 2001; 282:787-92. [PMID: 11401532 DOI: 10.1006/bbrc.2001.4641] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023]
Abstract
The preS1 of hepatitis B virus (HBV) is located at the outermost part of the envelope protein and possesses several functionally important regions such as hepatocyte receptor-binding site and virus-neutralizing epitopes. As the first step to understand the structure-function relationship for the preS1 antigen, we have purified the preS1 and performed its structural characterization by circular dichroism (CD) spectroscopy. The preS1 was purified to near homogeneity from bacterially expressed glutathione S-transferase (GST)-preS1 fusion protein by two-step purification, affinity chromatography on glutathione-agarose column, and cation-exchange chromatography on Mono S column. The CD analysis showed that the purified preS1, which was largely unstructured in aqueous solution, acquired a significant (16%) alpha-helical structure when analyzed in 50% trifluoroethanol or 20 mM SDS. The results suggest that the preS1 assumes a mainly unstructured conformation and may form induced secondary structures upon binding to target proteins or under hydrophobic environment.
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Affiliation(s)
- C Y Maeng
- Antibody Engineering Research Laboratory, Korea Research Institute of Bioscience and Biotechnology, Yusong, Taejon, 305-600
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50
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Capila I, Hernáiz MJ, Mo YD, Mealy TR, Campos B, Dedman JR, Linhardt RJ, Seaton BA. Annexin V--heparin oligosaccharide complex suggests heparan sulfate--mediated assembly on cell surfaces. Structure 2001; 9:57-64. [PMID: 11342135 DOI: 10.1016/s0969-2126(00)00549-9] [Citation(s) in RCA: 100] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022]
Abstract
BACKGROUND Annexin V, an abundant anticoagulant protein, has been proposed to exert its effects by self-assembling into highly ordered arrays on phospholipid membranes to form a protective anti-thrombotic shield at the cell surface. The protein exhibits very high-affinity calcium-dependent interactions with acidic phospholipid membranes, as well as specific binding to glycosaminoglycans (GAGs) such as heparin and heparan sulfate, a major component of cell surface proteoglycans. At present, there is no structural information to elucidate this interaction or the role it may play in annexin V function at the cell surface. RESULTS We report the 1.9 A crystal structure of annexin V in complex with heparin-derived tetrasaccharides. This structure represents the first of a heparin oligosaccharide binding to a protein where calcium ions are essential for the interaction. Two distinct GAG binding sites are situated on opposite protein surfaces. Basic residues at each site were identified from the structure and site-directed mutants were prepared. The heparin binding properties of these mutants were measured by surface plasmon resonance. The results confirm the roles of these mutated residues in heparin binding, and the kinetic and thermodynamic data define the functionally distinct character of each distal binding surface. CONCLUSION The annexin V molecule, as it self-assembles into an organized array on the membrane surface, can bind the heparan sulfate components of cell surface proteoglycans. A novel model is presented in which proteoglycan heparan sulfate could assist in the localization of annexin V to the cell surface membrane and/or stabilization of the entire molecular assembly to promote anticoagulation.
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Affiliation(s)
- I Capila
- Division of Medicinal and Natural Products Chemistry, Department of Chemistry, University of Iowa, Iowa City, IA 52242, USA
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