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Li X, Cheng N, Shi D, Li Y, Li C, Zhu M, Jin Q, Wu Z, Zhu L, He Y, Yao H, Ji J. Sulfated liposome-based artificial cell membrane glycocalyx nanodecoys for coronavirus inactivation by membrane fusion. Bioact Mater 2024; 33:1-13. [PMID: 38024234 PMCID: PMC10660003 DOI: 10.1016/j.bioactmat.2023.10.021] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2023] [Revised: 10/21/2023] [Accepted: 10/22/2023] [Indexed: 12/01/2023] Open
Abstract
As a broad-spectrum antiviral nanoparticle, the cell membrane nanodecoy is a promising strategy for preventing viral infections. However, most of the cell membrane nanodecoys can only catch virus and cannot induce inactivation, which may bring about a considerably high risk of re-infection owing to the possible viral escape from the nanodecoys. To tackle this challenge, sulfated liposomes are employed to mimic the cell membrane glycocalyx for constructing an artificial cell membrane glycocalyx nanodecoy that exhibits excellent anti-coronavirus activity against HCoV-OC43, wild-type SARS-CoV-2, Alpha and Delta variant SARS-CoV-2 pseudovirus. In addition, this nanodecoy, loaded with surface sulfate groups as SARS-CoV-2 receptor arrays, can enhance the antiviral capability to virus inactivation through destroying the virus membrane structure and transfer the spike protein to postfusion conformation. Integrating bio-inspired recognition and inactivation of viruses in a single supramolecular entity, the artificial cell membrane nanodecoy opens a new avenue for the development of theranostic antiviral nanosystems, whose mass production is favored due to the facile engineering of sulfated liposomes.
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Affiliation(s)
- Xu Li
- MOE Key Laboratory of Macromolecule Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou, 310027, China
| | - Ningtao Cheng
- School of Medicine, Zhejiang University, Hangzhou, 310058, China
| | - Danrong Shi
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310003, China
| | - Yutong Li
- MOE Key Laboratory of Macromolecule Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou, 310027, China
| | - Chen Li
- College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027, China
| | - Miaojin Zhu
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310003, China
| | - Qiao Jin
- MOE Key Laboratory of Macromolecule Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou, 310027, China
| | - Zhigang Wu
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310003, China
| | - Linwei Zhu
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310003, China
| | - Yi He
- College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027, China
| | - Hangping Yao
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310003, China
| | - Jian Ji
- MOE Key Laboratory of Macromolecule Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou, 310027, China
- Shanxi-Zheda Institute of Advanced Materials and Chemical Engineering, Taiyuan, 030032, China
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2
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Pérez-Vargas J, Teppa E, Amirache F, Boson B, Pereira de Oliveira R, Combet C, Böckmann A, Fusil F, Freitas N, Carbone A, Cosset FL. A fusion peptide in preS1 and the human protein disulfide isomerase ERp57 are involved in hepatitis B virus membrane fusion process. eLife 2021; 10:64507. [PMID: 34190687 PMCID: PMC8282342 DOI: 10.7554/elife.64507] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2020] [Accepted: 06/29/2021] [Indexed: 12/13/2022] Open
Abstract
Cell entry of enveloped viruses relies on the fusion between the viral and plasma or endosomal membranes, through a mechanism that is triggered by a cellular signal. Here we used a combination of computational and experimental approaches to unravel the main determinants of hepatitis B virus (HBV) membrane fusion process. We discovered that ERp57 is a host factor critically involved in triggering HBV fusion and infection. Then, through modeling approaches, we uncovered a putative allosteric cross-strand disulfide (CSD) bond in the HBV S glycoprotein and we demonstrate that its stabilization could prevent membrane fusion. Finally, we identified and characterized a potential fusion peptide in the preS1 domain of the HBV L glycoprotein. These results underscore a membrane fusion mechanism that could be triggered by ERp57, allowing a thiol/disulfide exchange reaction to occur and regulate isomerization of a critical CSD, which ultimately leads to the exposition of the fusion peptide.
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Affiliation(s)
- Jimena Pérez-Vargas
- CIRI - Centre International de Recherche en Infectiologie, Univ Lyon, Université Claude Bernard Lyon 1, Inserm, U1111, CNRS, UMR5308, ENS Lyon, Lyon, France
| | - Elin Teppa
- Sorbonne Université, CNRS, IBPS, Laboratoire de Biologie Computationnelle et Quantitative (LCQB) - UMR 7238, Paris, France.,Sorbonne Université, Institut des Sciences du Calcul et des Données (ISCD), Paris, France
| | - Fouzia Amirache
- CIRI - Centre International de Recherche en Infectiologie, Univ Lyon, Université Claude Bernard Lyon 1, Inserm, U1111, CNRS, UMR5308, ENS Lyon, Lyon, France
| | - Bertrand Boson
- CIRI - Centre International de Recherche en Infectiologie, Univ Lyon, Université Claude Bernard Lyon 1, Inserm, U1111, CNRS, UMR5308, ENS Lyon, Lyon, France
| | - Rémi Pereira de Oliveira
- CIRI - Centre International de Recherche en Infectiologie, Univ Lyon, Université Claude Bernard Lyon 1, Inserm, U1111, CNRS, UMR5308, ENS Lyon, Lyon, France
| | - Christophe Combet
- Cancer Research Center of Lyon (CRCL), UMR Inserm 1052 - CNRS 5286 - Université Lyon 1 - Centre Léon Bérard, Lyon, France
| | - Anja Böckmann
- Molecular Microbiology and Structural Biochemistry, UMR5086 CNRS-Université Lyon 1, Lyon, France
| | - Floriane Fusil
- CIRI - Centre International de Recherche en Infectiologie, Univ Lyon, Université Claude Bernard Lyon 1, Inserm, U1111, CNRS, UMR5308, ENS Lyon, Lyon, France
| | - Natalia Freitas
- CIRI - Centre International de Recherche en Infectiologie, Univ Lyon, Université Claude Bernard Lyon 1, Inserm, U1111, CNRS, UMR5308, ENS Lyon, Lyon, France
| | - Alessandra Carbone
- Sorbonne Université, CNRS, IBPS, Laboratoire de Biologie Computationnelle et Quantitative (LCQB) - UMR 7238, Paris, France
| | - François-Loïc Cosset
- CIRI - Centre International de Recherche en Infectiologie, Univ Lyon, Université Claude Bernard Lyon 1, Inserm, U1111, CNRS, UMR5308, ENS Lyon, Lyon, France
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3
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Rowell CER, Dobrovolny HM. Energy Requirements for Loss of Viral Infectivity. FOOD AND ENVIRONMENTAL VIROLOGY 2020; 12:281-294. [PMID: 32757142 PMCID: PMC7405386 DOI: 10.1007/s12560-020-09439-9] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 05/21/2020] [Accepted: 07/31/2020] [Indexed: 06/11/2023]
Abstract
Outside the host, viruses will eventually lose their ability to infect cells due to conformational changes that occur to proteins on the viral capsid. In order to undergo a conformational change, these proteins require energy to activate the chemical reaction that leads to the conformational change. In this study, data from the literature is used to calculate the energy required for viral inactivation for a variety of different viruses by means of the Arrhenius equation. We find that some viruses (rhinovirus, poliovirus, human immunodeficiency virus, Alkhumra hemorrhagic fever virus, and hepatitis A virus) have high inactivation energies, indicative of breaking of a chemical double bond. We also find that several viruses (respiratory syncytial virus, poliovirus, and norovirus) have nonlinear Arrhenius plots, suggesting that there is more than a single pathway for inactivation of these viruses.
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Affiliation(s)
- Caroline E R Rowell
- Department of Chemistry, Wingate University, Hendersonville, NC, USA
- Department of Physics and Astronomy, Texas Christian University, Fort Worth, TX, USA
| | - Hana M Dobrovolny
- Department of Physics and Astronomy, Texas Christian University, Fort Worth, TX, USA.
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4
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Seitz S, Habjanič J, Schütz AK, Bartenschlager R. The Hepatitis B Virus Envelope Proteins: Molecular Gymnastics Throughout the Viral Life Cycle. Annu Rev Virol 2020; 7:263-288. [PMID: 32600157 DOI: 10.1146/annurev-virology-092818-015508] [Citation(s) in RCA: 27] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
New hepatitis B virions released from infected hepatocytes are the result of an intricate maturation process that starts with the formation of the nucleocapsid providing a confined space where the viral DNA genome is synthesized via reverse transcription. Virion assembly is finalized by the enclosure of the icosahedral nucleocapsid within a heterogeneous envelope. The latter contains integral membrane proteins of three sizes, collectively known as hepatitis B surface antigen, and adopts multiple conformations in the course of the viral life cycle. The nucleocapsid conformation depends on the reverse transcription status of the genome, which in turn controls nucleocapsid interaction with the envelope proteins for virus exit. In addition, after secretion the virions undergo a distinct maturation step during which a topological switch of the large envelope protein confers infectivity. Here we review molecular determinants for envelopment and models that postulate molecular signals encoded in the capsid scaffold conducive or adverse to the recruitment of envelope proteins.
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Affiliation(s)
- Stefan Seitz
- Department of Infectious Diseases, University of Heidelberg, 69120 Heidelberg, Germany;
| | - Jelena Habjanič
- Bavarian NMR Center, Department of Chemistry, Technical University of Munich, 85748 Garching, Germany.,Institute of Structural Biology, Helmholtz Zentrum München, 85764 Neuherberg, Germany
| | - Anne K Schütz
- Bavarian NMR Center, Department of Chemistry, Technical University of Munich, 85748 Garching, Germany.,Institute of Structural Biology, Helmholtz Zentrum München, 85764 Neuherberg, Germany
| | - Ralf Bartenschlager
- Department of Infectious Diseases, University of Heidelberg, 69120 Heidelberg, Germany; .,Division of Virus-Associated Carcinogenesis, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany
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5
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Intracellular trafficking of bio-nanocapsule-liposome complex: Identification of fusogenic activity in the pre-S1 region of hepatitis B virus surface antigen L protein. J Control Release 2015; 212:10-8. [PMID: 26074149 DOI: 10.1016/j.jconrel.2015.06.012] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2014] [Revised: 05/07/2015] [Accepted: 06/09/2015] [Indexed: 12/18/2022]
Abstract
Bio-nanocapsules (BNCs) are a hollow nanoparticle consisting of about 100-nm liposome (LP) embedding about 110 molecules of hepatitis B virus (HBV) surface antigen (HBsAg) L protein as a transmembrane protein. Owing to the human hepatocyte-recognizing domains on the N-terminal region (pre-S1 region), BNCs have recently been shown to attach and enter into human hepatic cells using the early infection mechanism of HBV. Since BNCs could form a complex with an LP containing various drugs and genes, BNC-LP complexes have been used as a human hepatic cell-specific drug and gene-delivery system in vitro and in vivo. However, the role of BNCs in cell entry and intracellular trafficking of payloads in BNC-LP complexes has not been fully elucidated. In this study, we demonstrate that low pH-dependent fusogenic activity resides in the N-terminal part of pre-S1 region (NPLGFFPDHQLDPAFG), of which the first FF residues are essential for the activity, and which facilitates membrane fusion between LPs in vitro. Moreover, BNC-LP complexes can bind human hepatic cells specifically, enter into the cells via clathrin-mediated endocytosis, and release their payloads mostly into the cytoplasm. Taken together, the BNC portion of BNC-LP complexes can induce membrane fusion between LPs and endosomal membranes under low pH conditions, and thereby facilitate the endosomal escape of payloads. Furthermore, the fusogenic domain of the pre-S1 region of HBsAg L protein may play a pivotal role in the intracellular trafficking of not only BNC-LP complexes but also of HBV.
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6
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Urban S, Bartenschlager R, Kubitz R, Zoulim F. Strategies to inhibit entry of HBV and HDV into hepatocytes. Gastroenterology 2014; 147:48-64. [PMID: 24768844 DOI: 10.1053/j.gastro.2014.04.030] [Citation(s) in RCA: 235] [Impact Index Per Article: 21.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/22/2013] [Revised: 03/11/2014] [Accepted: 04/21/2014] [Indexed: 02/07/2023]
Abstract
Although there has been much research into the pathogenesis and treatment of hepatitis B virus (HBV) and hepatitis D virus (HDV) infections, we still do not completely understand how these pathogens enter hepatocytes. This is because in vitro infection studies have only been performed in primary human hepatocytes. Development of a polarizable, HBV-susceptible human hepatoma cell line and studies of primary hepatocytes from Tupaia belangeri have provided important insights into the viral and cellular factors involved in virus binding and infection. The large envelope (L) protein on the surface of HBV and HDV particles has many different functions and is required for virus entry. The L protein mediates attachment of virions to heparan sulfate proteoglycans on the surface of hepatocytes. The myristoylated N-terminal preS1 domain of the L protein subsequently binds to the sodium taurocholate cotransporting polypeptide (NTCP, encoded by SLC10A1), the recently identified bona fide receptor for HBV and HDV. The receptor functions of NTCP and virus entry are blocked, in vitro and in vivo, by Myrcludex B, a synthetic N-acylated preS1 lipopeptide. Currently, the only agents available to treat chronic HBV infection target the viral polymerase, and no selective therapies are available for HDV infection. It is therefore important to study the therapeutic potential of virus entry inhibitors, especially when combined with strategies to induce immune-mediated killing of infected hepatocytes.
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Affiliation(s)
- Stephan Urban
- Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, Heidelberg, Germany; German Center for Infection Research, Heidelberg University, Heidelberg, Germany.
| | - Ralf Bartenschlager
- Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, Heidelberg, Germany; German Center for Infection Research, Heidelberg University, Heidelberg, Germany
| | - Ralf Kubitz
- Department of Gastroenterology, Hepatology and Infectious Diseases, Medical Faculty, Heinrich Heine University, Düsseldorf, Germany
| | - Fabien Zoulim
- INSERM Unité 1052, Cancer Research Center of Lyon, Lyon University, Lyon, France
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7
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The first transmembrane domain of the hepatitis B virus large envelope protein is crucial for infectivity. J Virol 2009; 83:11819-29. [PMID: 19740987 DOI: 10.1128/jvi.01026-09] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
The early steps of the hepatitis B virus (HBV) life cycle are still poorly understood. Indeed, neither the virus receptor at the cell surface nor the mechanism by which nucleocapsids are delivered to the cytosol of infected cells has been identified. Extensive mutagenesis studies in pre-S1, pre-S2, and most of the S domain of envelope proteins revealed the presence of two regions essential for HBV infectivity: the 77 first residues of the pre-S1 domain and a conformational motif in the antigenic loop of the S domain. In addition, at the N-terminal extremity of the S domain, a putative fusion peptide, partially overlapping the first transmembrane (TM1) domain and preceded by a PEST sequence likely containing several proteolytic cleavage sites, was identified. Since no mutational analysis of these two motifs potentially implicated in the fusion process was performed, we decided to investigate the ability of viruses bearing contiguous deletions or substitutions in the putative fusion peptide and PEST sequence to infect HepaRG cells. By introducing the mutations either in the L and M proteins or in the S protein, we demonstrated the following: (i) that in the TM1 domain of the L protein, three hydrophobic clusters of four residues were necessary for infectivity; (ii) that the same clusters were critical for S protein expression; and, finally, (iii) that the PEST sequence was dispensable for both assembly and infection processes.
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8
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HBV life cycle: entry and morphogenesis. Viruses 2009; 1:185-209. [PMID: 21994545 PMCID: PMC3185491 DOI: 10.3390/v1020185] [Citation(s) in RCA: 77] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2009] [Revised: 07/31/2009] [Accepted: 08/13/2009] [Indexed: 02/07/2023] Open
Abstract
Hepatitis B virus (HBV) is a major cause of liver disease. HBV primarily infects hepatocytes by a still poorly understood mechanism. After an endocytotic process, the nucleocapsids are released into the cytoplasm and the relaxed circular rcDNA genome is transported towards the nucleus where it is converted into covalently closed circular cccDNA. Replication of the viral genome occurs via an RNA pregenome (pgRNA) that binds to HBV polymerase (P). P initiates pgRNA encapsidation and reverse transcription inside the capsid. Matured, rcDNA containing nucleocapsids can re-deliver the RC-DNA to the nucleus, or be secreted via interaction with the envelope proteins as progeny virions.
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9
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Chojnacki J, Grgacic EVL. Enveloped viral fusion: insights into the fusion of hepatitis B viruses. Future Virol 2008. [DOI: 10.2217/17460794.3.6.543] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
Viral fusion, the mechanism by which viruses gain entry into the host cell, is a key step in the replication cycle and an important new target in antiviral therapy and vaccine strategies owing to the conservation of the envelope domains involved and their resistance to immune pressure. The fusion domains of HIV-1 have been studied intensively resulting in the potent antiviral agent T20 and the identification of broadly neutralizing antibody epitopes for vaccine development. Another chronic disease-causing virus, HBV, requires the identification of new antiviral agents to deal with the disease burden of 350 million chronically-infected individuals worldwide, 20% of whom will develop liver cancer. The aim of this review is to bring together basic knowledge on the envelope signatures, mechanisms and strategies for the study of viral fusion and how that knowledge has been applied to the study of hepadnaviral fusion.
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Affiliation(s)
- Jakub Chojnacki
- Abteilung Virologie, Universitätsklinikum Heidelberg, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany
| | - Elizabeth VL Grgacic
- Macfarlane Burnet Institute for Medical Research & Public Health, 85 Commercial Road, Melbourne, 3004, Australia
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10
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Characterization of the Putative Membrane Fusion Peptides in the Envelope Proteins of Human Hepatitis B Virus. B KOREAN CHEM SOC 2007. [DOI: 10.5012/bkcs.2007.28.10.1756] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
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Abstract
Hepadnaviridae is a family of hepatotropic DNA viruses that is divided into the genera orthohepadnavirus of mammals and avihepadnavirus of birds. All members of this family can cause acute and chronic hepatic infection, which in the case of human hepatitis B virus (HBV) constitutes a major global health problem. Although our knowledge about the molecular biology of these highly liver-specific viruses has profoundly increased in the last two decades, the mechanisms of attachment and productive entrance into the differentiated host hepatocytes are still enigmatic. The difficulties in studying hepadnaviral entry were primarily caused by the lack of easily accessible in vitro infection systems. Thus, for more than twenty years, differentiated primary hepatocytes from the respective species were the only in vitro models for both orthohepadnaviruses (e.g. HBV) and avihepadnaviruses (e.g. duck hepatitis B virus [DHBV]). Two important discoveries have been made recently regarding HBV: (1) primary hepatocytes from tree-shrews; i.e., Tupaia belangeri, can be substituted for primary human hepatocytes, and (2) a human hepatoma cell line (HepaRG) was established that gains susceptibility for HBV infection upon induction of differentiation in vitro. A number of potential HBV receptor candidates have been described in the past, but none of them have been confirmed to function as a receptor. For DHBV and probably all other avian hepadnaviruses, carboxypeptidase D (CPD) has been shown to be indispensable for infection, although the exact role of this molecule is still under debate. While still restricted to the use of primary duck hepatocytes (PDH), investigations performed with DHBV provided important general concepts on the first steps of hepadnaviral infection. However, with emerging data obtained from the new HBV infection systems, the hope that DHBV utilizes the same mechanism as HBV only partially held true. Nevertheless, both HBV and DHBV in vitro infection systems will help to: (1) functionally dissect the hepadnaviral entry pathways, (2) perform reverse genetics (e.g. test the fitness of escape mutants), (3) titrate and map neutralizing antibodies, (4) improve current vaccines to combat acute and chronic infections of hepatitis B, and (5) develop entry inhibitors for future clinical applications.
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Affiliation(s)
- Dieter Glebe
- Institute of Medical Virology, Justus-Liebig University of Giessen, Frankfurter Strasse 107, D-35392 Giessen, Germany.
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12
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Franke C, Matschl U, Bruns M. Enzymatic treatment of duck hepatitis B virus: topology of the surface proteins for virions and noninfectious subviral particles. Virology 2006; 359:126-36. [PMID: 17045625 DOI: 10.1016/j.virol.2006.09.006] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2006] [Revised: 08/14/2006] [Accepted: 09/02/2006] [Indexed: 12/29/2022]
Abstract
The large surface antigen L of duck hepatitis B virus exhibits a mixed topology with the preS domains of the protein alternatively exposed to the particles' interior or exterior. After separating virions from subviral particles (SVPs), we compared their L topologies and showed that both particle types exhibit the same amount of L with the following differences: 1--preS of intact virions was enzymatically digested with chymotrypsin, whereas in SVPs only half of preS was accessible, 2--phosphorylation of L at S118 was completely removed by phosphatase treatment only in virions, 3--iodine-125 labeling disclosed a higher ratio of exposed preS to S domains in virions compared to SVPs. These data point towards different surface architectures of virions and SVPs. Because the preS domain acts in binding to a cellular receptor of hepatocytes, our findings implicate the exclusion of SVPs as competitors for the receptor binding and entry of virions.
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Affiliation(s)
- Claudia Franke
- Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie an der Universität Hamburg, Martinistrasse 52, D-20251 Hamburg, Germany
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13
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Whitbeck JC, Zuo Y, Milne RSB, Cohen GH, Eisenberg RJ. Stable association of herpes simplex virus with target membranes is triggered by low pH in the presence of the gD receptor, HVEM. J Virol 2006; 80:3773-80. [PMID: 16571794 PMCID: PMC1440471 DOI: 10.1128/jvi.80.8.3773-3780.2006] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023] Open
Abstract
Using a liposome-binding assay, we investigated the requirements for activation of herpes simplex virus (HSV) into a state capable of membrane interaction. Virions were mixed with liposomes along with the ectodomain of one of three gD receptors (HVEMt, nectin-1t, or nectin-2t) and incubated under different pH and temperature conditions. Virions failed to associate with liposomes in the presence of nectin-1 or nectin-2 at any temperature or pH tested. In contrast, HVEMt triggered association of HSV with liposomes at pH 5.3 or 5.0 when incubated at 37 degrees C, suggesting that HVEM binding and mildly acidic pH at a physiological temperature provide coactivation signals, allowing virus association with membranes. Virions incubated with HVEMt at 37 degrees C without liposomes rapidly lost infectivity upon exposure to pH 5.0, suggesting that these conditions lead to irreversible virus inactivation in the absence of target membranes. Consistent with the idea that soluble receptor molecules provide a trigger for HSV entry, HVEMt promoted virus entry into receptor-deficient CHO K1 cells. However, in B78H1 cells, HVEMt promoted virus entry with markedly lower efficiency. Interestingly, HSV entry into receptor-bearing CHO K1 cells has been shown to proceed via a pH-dependent manner, whereas HSV entry into receptor-bearing B78H1 cells is pH independent. Based on these observations, we propose that the changes triggered by HVEM and mildly acidic pH that allow liposome association are similar or identical to changes that occur during pH-dependent HSV entry.
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Affiliation(s)
- J Charles Whitbeck
- School of Dental Medicine, University of Pennsylvania, 4010 Locust Street, Levy Building, Room 212, Philadelphia, Pennsylvania 19104, USA.
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14
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Chojnacki J, Anderson DA, Grgacic EVL. A hydrophobic domain in the large envelope protein is essential for fusion of duck hepatitis B virus at the late endosome. J Virol 2006; 79:14945-55. [PMID: 16282493 PMCID: PMC1287569 DOI: 10.1128/jvi.79.23.14945-14955.2005] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
The duck hepatitis B virus (DHBV) envelope is comprised of two transmembrane (TM) proteins, the large (L) and the small (S), that assemble into virions and subviral particles. Secondary-structure predictions indicate that L and S have three alpha-helical, membrane-spanning domains, with TM1 predicted to act as the fusion peptide following endocytosis of DHBV into the hepatocyte. We used bafilomycin A1 during infection of primary duck hepatocytes to show that DHBV must be trafficked from the early to the late endosome for fusion to occur. Alanine substitution mutations in TM1 of L and S, which lowered TM1 hydrophobicity, were used to examine the role of TM1 in infectivity. The high hydrophobicity of the TM1 domain of L, but not of S, was shown to be essential for virus infection at a step downstream of receptor binding and virus internalization. Using wild-type and mutant synthetic peptides, we demonstrate that the hydrophobicity of this domain is required for the aggregation and the lipid mixing of phospholipid vesicles, supporting the role of TM1 as the fusion peptide. While lipid mixing occurred at pH 7, the kinetics of insertion of the fusion peptide was increased at pH 5, consistent with the location of DHBV in the late-endosome compartment and previous studies of the nonessential role of low pH for infectivity. Exchange of the TM1 of DHBV with that of hepatitis B virus yielded functional, infectious DHBV particles, suggesting that TM1 of all of the hepadnaviruses act similarly in the fusion mechanism.
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Affiliation(s)
- J Chojnacki
- Macfarlane Burnet Institute for Medical Research and Public Health, Melbourne, Australia
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15
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Chen M, Cheng T, Xu CY, Wu T, Ou SH, Zhang T, Zhang J, Xia NS. Hydrophobicity of reactive site loop of SCCA1 affects its binding to hepatitis B virus. World J Gastroenterol 2005; 11:2864-8. [PMID: 15902720 PMCID: PMC4305651 DOI: 10.3748/wjg.v11.i19.2864] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [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 investigate the role of SCCA2 and other SCCA1 molecules in the process of hepatitis B virus (HBV) binding to mammalian cells.
METHODS: SCCA1 and SCCA2 were isolated from HepG2. Binding protein (BP) genes were obtained through PCR. Recombinant baculoviruses expressing SCCA1, SCCA2, BP, and different mutants were constructed and utilized to infect mammalian cells to investigate the binding ability of infected cells to HBV.
RESULTS: A SCCA1 gene (A1) was isolated from HepG2, but it appeared to lack the binding ability of infected cells to HBV. Two mutants, A1-BP and BP-A1, were constructed by interchanging the carboxyl terminal of A1 and BP. Cells expressing A1-BP showed an increased virus binding capacity, but not BP-A1. Comparison of A1 sequence with the sequence of BP indicated the presence of only three amino acid changes in the carboxyl terminal, two of them were found in the reactive site loop (RSL) of SCCA1. Primary structure assay revealed that the hydrophobicity of BP and AJ515706 in this domain was strong, but A1 was relatively weak. Changing the aa349 of A1 from low hydrophobic glutamic acid to high hydrophobic valine enhanced HBV binding. In contrast, HBV binding was reduced by changing the aa349 of BP from valine to glutamic acid.
CONCLUSION: The results suggest that the hydrophobicity of RSL of SCCA1 may play an important role in HBV binding to cells.
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Affiliation(s)
- Min Chen
- Key Laboratory of Cell Biology and Tumor Cell Engineering of Ministry of Education, Xiamen University, Xiamen 361005, Fujian Province, China
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16
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Cooper A, Paran N, Shaul Y. The earliest steps in hepatitis B virus infection. BIOCHIMICA ET BIOPHYSICA ACTA 2003; 1614:89-96. [PMID: 12873769 DOI: 10.1016/s0005-2736(03)00166-4] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
The early steps in hepatitis B virus (HBV) infection, a human hepadnavirus, initiates from cell attachment followed by entry and delivery of the genetic information to the nucleus. Despite the fact that these steps determine the virus-related pathogenesis, their molecular basis is poorly understood. Cumulative data suggest that this process can be divided to cell attachment, endocytosis, membrane fusion and post-fusion consecutive steps. These steps are likely to be regulated by the viral envelope proteins and by the cellular membrane, receptors and extracellular matrix. In the absence of animal model for HBV, the duck hepadnavirus DHBV turned out to be a fruitful animal model. Therefore data concerning the early, post-attachment steps in hepadnaviral entry are largely based on studies performed with DHBV in primary duck liver hepatocytes. These studies are now starting to illuminate the mechanisms of hepadnavirus route of cell entry and to provide some new insights on the molecular basis of the strict species specificity of hepadnavirus infection.
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Affiliation(s)
- Arik Cooper
- Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel
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17
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Brorson K, Krejci S, Lee K, Hamilton E, Stein K, Xu Y. Bracketed generic inactivation of rodent retroviruses by low pH treatment for monoclonal antibodies and recombinant proteins. Biotechnol Bioeng 2003; 82:321-9. [PMID: 12599259 DOI: 10.1002/bit.10574] [Citation(s) in RCA: 90] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022]
Abstract
Viral safety is a predominant concern for monoclonal antibodies (mAbs) and other recombinant proteins (RPs) with pharmaceutical applications. Certain commercial purification modules, such as nanofiltration and low-pH inactivation, have been observed to reliably clear greater than 4 log(10) of large enveloped viruses, including endogenous retrovirus. The concept of "bracketed generic clearance" has been proposed for these steps if it could be prospectively demonstrated that viral log(10) reduction value (LRV) is not impacted by operating parameters that can vary, within a reasonable range, between commercial processes. In the case of low-pH inactivation, a common step in mAb purification processes employed after protein A affinity chromatography, these parameters would include pH, time and temperature of incubation, the content of salts, protein concentration, aggregates, impurities, model protein pI, and buffer composition. In this report, we define bracketed generic clearance conditions, using a prospectively defined bracket/matrix approach, where low-pH inactivation consistently achieves >or=4.6 log(10) clearance of xenotropic murine leukemia virus (X-MLV), a model for rodent endogenous retrovirus. The mechanism of retrovirus inactivation by low-pH treatment was also investigated.
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Affiliation(s)
- Kurt Brorson
- Division of Monoclonal Antibodies, Center for Biologics Evaluation and Research, Food and Drug Administration, 8800 Rockville Pike, Bethesda, Maryland 20892, USA
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18
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Butko P. Cytolytic toxin Cyt1A and its mechanism of membrane damage: data and hypotheses. Appl Environ Microbiol 2003; 69:2415-22. [PMID: 12732506 PMCID: PMC154483 DOI: 10.1128/aem.69.5.2415-2422.2003] [Citation(s) in RCA: 80] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Affiliation(s)
- Peter Butko
- Department of Chemistry and Biochemistry, University of Southern Mississippi, Hattiesburg, MS 39406-5043, USA.
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19
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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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20
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Grgacic EVL. Identification of structural determinants of the first transmembrane domain of the small envelope protein of duck hepatitis B virus essential for particle morphogenesis. J Gen Virol 2002; 83:1635-1644. [PMID: 12075081 DOI: 10.1099/0022-1317-83-7-1635] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
The envelope of duck hepatitis B virus (DHBV) consists of the small (S) and large (L) envelope proteins, which share a common C-terminal multispanning transmembrane region but differ by the long N-terminal pre-S domain of L, which is essential for interactions with both the receptor and nucleocapsid. To achieve these dual functions, L acquires mixed topologies through S-dependent post-translational translocation of its pre-S domain. This study has examined the role of S in this unusual mechanism of translocation by analysis of the alpha-helical transmembrane domains and their potential to engage in lateral interactions for envelope assembly. Through mutagenesis in constructs expressing the S and L envelope proteins independently, transmembrane domain 1 was identified as an essential structural determinant in S. Two polar residues in this helix were identified as contributing to L protein translocation through the assembly of S into particles, implying that the topological switch of L is part of the assembly and maturation process. The same domain in L was shown to be dispensable for L translocation and assembly, suggesting that transmembrane domain 1 of L and S have different functional roles and structural arrangements on the assembled particle. The conservation in all hepadnavirus envelope proteins of two polar residues at positions 24 and 27 of transmembrane domain 1, the former positively charged, points to this being a common determinant in particle morphogenesis for all hepadnaviruses.
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Affiliation(s)
- Elizabeth V L Grgacic
- Australian Centre for Hepatitis Virology, Macfarlane Burnet Institute for Medical Research and Public Health, Yarra Bend Road, Fairfield 3078, Victoria, Australia1
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21
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Dawe S, Duncan R. The S4 genome segment of baboon reovirus is bicistronic and encodes a novel fusion-associated small transmembrane protein. J Virol 2002; 76:2131-40. [PMID: 11836390 PMCID: PMC135948 DOI: 10.1128/jvi.76.5.2131-2140.2002] [Citation(s) in RCA: 66] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
We demonstrate that the S4 genome segment of baboon reovirus (BRV) contains two sequential partially overlapping open reading frames (ORFs), both of which are functional in vitro and in virus-infected cells. The 15-kDa gene product (p15) of the 5"-proximal ORF induces efficient cell-cell fusion when expressed by itself in transfected cells, suggesting that p15 is the only viral protein required for induction of syncytium formation by BRV. The p15 protein is a small, hydrophobic, basic, integral membrane protein, properties shared with the p10 fusion-associated small transmembrane (FAST) proteins encoded by avian reovirus and Nelson Bay reovirus. As with p10, the BRV p15 protein is also a nonstructural protein and, therefore, is not involved in virus entry. Sequence analysis indicates that p15 shares no significant sequence similarity with the p10 FAST proteins and contains a unique repertoire and arrangement of sequence-predicted structural and functional motifs. These motifs include a functional N-terminal myristylation consensus sequence, an N-proximal proline-rich motif, two potential transmembrane domains, and an intervening polybasic region. The unique structural properties of p15 suggest that this protein is a novel member of the new family of FAST proteins.
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Affiliation(s)
- Sandra Dawe
- Department of Microbiology and Immunology, Tupper Medical Building, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada
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22
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Lambert C, Prange R. Dual topology of the hepatitis B virus large envelope protein: determinants influencing post-translational pre-S translocation. J Biol Chem 2001; 276:22265-72. [PMID: 11301328 DOI: 10.1074/jbc.m100956200] [Citation(s) in RCA: 73] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
The large (L) envelope protein of the hepatitis B virus (HBV) has the peculiar capacity to form two transmembrane topologies via an as yet uncharacterized process of partial post-translational translocation of its pre-S domain across membranes. In view of a current model that predicts an HBV-specific channel generated during virion envelope assembly to enable pre-S translocation, we have examined parameters influencing L topogenesis by using protease protection analysis of wild-type and mutant L proteins synthesized in transfected cells. We demonstrate that contrary to expectation, all determinants, thought to be responsible for channel formation, are dispensable for pre-S reorientation. In particular, we observed that this process does not require (i) the helper function of the HBV S (small) and M (middle) envelope proteins, (ii) covalent dimer formation of envelope chains, or (iii) either of the three amphipathic transmembrane segments of L. Rather, the most hydrophobic transmembrane segment 2 of L was identified as a vital topogenic determinant, essential and sufficient for post-translational pre-S translocation. Cell fractionation studies revealed that pre-S refolding and thus the dual topology of L is established at the endoplasmic reticulum (ER) membrane rather than at a post-ER compartment as originally supposed. Together our data provide evidence to suggest that the topological reorientation of L is facilitated by a host cell transmembrane transport machinery such as the ER translocon.
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Affiliation(s)
- C Lambert
- Department of Medical Microbiology and Hygiene, Johannes Gutenberg-Universität Mainz, D-55101 Mainz, Germany
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23
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Lu X, Hazboun T, Block T. Limited proteolysis induces woodchuck hepatitis virus infectivity for human HepG2 cells. Virus Res 2001; 73:27-40. [PMID: 11163642 DOI: 10.1016/s0168-1702(00)00218-5] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Previous work from our laboratory has shown that digestion of hepatitis B virus (HBV) with V8 protease rendered the virus infectious for human hepatoblastoma cell line (HepG2). It was hypothesized that the cleavage exposes a 16 amino acid region that includes a consensus 'fusion' motif necessary to mediate infectivity. Since woodchuck hepatitis virus (WHV) and HBV possess significant homology in this region of their envelope proteins, including the V8 protease cleavage site, the possibility that WHV infectivity for HepG2 cells could be induced by V8 digestion was explored. WHV isolated from the serum of chronically infected woodchucks, digested with V8 protease, was shown to loose its preS domain. V8 digested WHV eluted from gel filtration columns with a size similar to that of undigested virus, suggesting that digestion with V8 protease did not cause significant changes in virion size. The amount of progeny virus secreted into the culture medium following infection of HepG2 cells with V8 digested WHV reached 2.5 pg/ml, after 8 days. Moreover, WHV DNA replicative intermediates could be detected in the cells following infection with protease digested, but not undigested, viruses. These data suggest that protease modification of WHV, a non-human virus, induced infectivity for human tissue culture cells. These results are consistent with the hypothesis that exposure of an amino acid region of the envelope polypeptide that contains a consensus fusion motif is important in Hepadnavirus entry.
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Affiliation(s)
- X Lu
- Jefferson Center for Medical Research at DVC, Thomas Jefferson University, Doylestown, PA 18901, USA.
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