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1
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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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2
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Wetzel D, Rolf T, Suckow M, Kranz A, Barbian A, Chan JA, Leitsch J, Weniger M, Jenzelewski V, Kouskousis B, Palmer C, Beeson JG, Schembecker G, Merz J, Piontek M. Establishment of a yeast-based VLP platform for antigen presentation. Microb Cell Fact 2018; 17:17. [PMID: 29402276 PMCID: PMC5798182 DOI: 10.1186/s12934-018-0868-0] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/25/2017] [Accepted: 01/27/2018] [Indexed: 12/26/2022] Open
Abstract
Background Chimeric virus-like particles (VLP) allow the display of foreign antigens on their surface and have proved valuable in the development of safe subunit vaccines or drug delivery. However, finding an inexpensive production system and a VLP scaffold that allows stable incorporation of diverse, large foreign antigens are major challenges in this field. Results In this study, a versatile and cost-effective platform for chimeric VLP development was established. The membrane integral small surface protein (dS) of the duck hepatitis B virus was chosen as VLP scaffold and the industrially applied and safe yeast Hansenula polymorpha (syn. Pichia angusta, Ogataea polymorpha) as the heterologous expression host. Eight different, large molecular weight antigens of up to 412 amino acids derived from four animal-infecting viruses were genetically fused to the dS and recombinant production strains were isolated. In all cases, the fusion protein was well expressed and upon co-production with dS, chimeric VLP containing both proteins could be generated. Purification was accomplished by a downstream process adapted from the production of a recombinant hepatitis B VLP vaccine. Chimeric VLP were up to 95% pure on protein level and contained up to 33% fusion protein. Immunological data supported surface exposure of the foreign antigens on the native VLP. Approximately 40 mg of chimeric VLP per 100 g dry cell weight could be isolated. This is highly comparable to values reported for the optimized production of human hepatitis B VLP. Purified chimeric VLP were shown to be essentially stable for 6 months at 4 °C. Conclusions The dS-based VLP scaffold tolerates the incorporation of a variety of large molecular weight foreign protein sequences. It is applicable for the display of highly immunogenic antigens originating from a variety of pathogens. The yeast-based production system allows cost-effective production that is not limited to small-scale fundamental research. Thus, the dS-based VLP platform is highly efficient for antigen presentation and should be considered in the development of future vaccines.
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Affiliation(s)
- David Wetzel
- ARTES Biotechnology GmbH, Elisabeth-Selbert-Straße 9, 40764, Langenfeld, Germany. .,Laboratory of Plant and Process Design, Technical University of Dortmund, Emil-Figge-Straße 70, 44227, Dortmund, Germany.
| | - Theresa Rolf
- ARTES Biotechnology GmbH, Elisabeth-Selbert-Straße 9, 40764, Langenfeld, Germany
| | - Manfred Suckow
- ARTES Biotechnology GmbH, Elisabeth-Selbert-Straße 9, 40764, Langenfeld, Germany
| | - Andreas Kranz
- ARTES Biotechnology GmbH, Elisabeth-Selbert-Straße 9, 40764, Langenfeld, Germany
| | - Andreas Barbian
- Institute for Anatomy I, Düsseldorf University Hospital, Moorenstraße 5, 40225, Düsseldorf, Germany
| | - Jo-Anne Chan
- Burnet Institute for Medical Research and Public Health, 85 Commercial Road, Melbourne, VIC, 3004, Australia
| | - Joachim Leitsch
- ARTES Biotechnology GmbH, Elisabeth-Selbert-Straße 9, 40764, Langenfeld, Germany
| | - Michael Weniger
- ARTES Biotechnology GmbH, Elisabeth-Selbert-Straße 9, 40764, Langenfeld, Germany
| | - Volker Jenzelewski
- ARTES Biotechnology GmbH, Elisabeth-Selbert-Straße 9, 40764, Langenfeld, Germany
| | - Betty Kouskousis
- Burnet Institute for Medical Research and Public Health, 85 Commercial Road, Melbourne, VIC, 3004, Australia
| | - Catherine Palmer
- Burnet Institute for Medical Research and Public Health, 85 Commercial Road, Melbourne, VIC, 3004, Australia
| | - James G Beeson
- Burnet Institute for Medical Research and Public Health, 85 Commercial Road, Melbourne, VIC, 3004, Australia
| | - Gerhard Schembecker
- Laboratory of Plant and Process Design, Technical University of Dortmund, Emil-Figge-Straße 70, 44227, Dortmund, Germany
| | - Juliane Merz
- Laboratory of Plant and Process Design, Technical University of Dortmund, Emil-Figge-Straße 70, 44227, Dortmund, Germany
| | - Michael Piontek
- ARTES Biotechnology GmbH, Elisabeth-Selbert-Straße 9, 40764, Langenfeld, Germany
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3
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Abstract
The hepatitis B virus (HBV) particle consists of an envelope containing three related surface proteins and probably lipid and an icosahedral nucleocapsid of approximately 30 nm diameter enclosing the viral DNA genome and DNA polymerase. The capsid is formed in the cytosol of the infected cell during packaging of an RNA pregenome replication complex by multiple copies of a 21-kDa C protein. The capsid gains the ability to bud during synthesis of the viral DNA genome by reverse transcription of the pregenome in the lumen of the particle. The three envelope proteins S, M, and L shape a complex transmembrane fold at the endoplasmic reticulum, and form disulfide-linked homo- and heterodimers. The transmembrane topology of a fraction of the large envelope protein L changes post-translationally, therefore, the N terminal domain of L (preS) finally appears on both sides of the membrane. During budding at an intracellular membrane, a short linear domain in the cytosolic preS region interacts with binding sites on the capsid surface. The virions are subsequently secreted into the blood. In addition, the surface proteins can bud in the absence of capsids and form subviral lipoprotein particles of 20 nm diameter which are also secreted.
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Affiliation(s)
- Volker Bruss
- Department of Virology, University of Göttingen, Kreuzbergring 57, Göttingen 37075, Germany.
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4
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York J, Nunberg JH. Distinct requirements for signal peptidase processing and function in the stable signal peptide subunit of the Junín virus envelope glycoprotein. Virology 2006; 359:72-81. [PMID: 17045626 DOI: 10.1016/j.virol.2006.08.048] [Citation(s) in RCA: 54] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2006] [Revised: 08/18/2006] [Accepted: 08/29/2006] [Indexed: 11/25/2022]
Abstract
The arenavirus envelope glycoprotein (GP-C) retains a cleaved and stable signal peptide (SSP) as an essential subunit of the mature complex. This 58-amino-acid residue peptide serves as a signal sequence and is additionally required to enable transit of the assembled GP-C complex to the Golgi, and for pH-dependent membrane fusion activity. We have investigated the C-terminal region of the Junín virus SSP to study the role of the cellular signal peptidase (SPase) in generating SSP. Site-directed mutagenesis at the cleavage site (positions -1 and -3) reveals a pattern of side-chain preferences consistent with those of SPase. Although position -2 is degenerate for SPase cleavage, this residue in the arenavirus SSP is invariably a cysteine. In the Junín virus, this cysteine is not involved in disulfide bonding. We show that replacement with alanine or serine is tolerated for SPase cleavage but prevents the mutant SSP from associating with GP-C and enabling transport to the cell surface. Conversely, an arginine mutation at position -1 that prevents SPase cleavage is fully compatible with GP-C-mediated membrane fusion activity when the mutant SSP is provided in trans. These results point to distinct roles of SSP sequences in SPase cleavage and GP-C biogenesis. Further studies of the unique structural organization of the GP-C complex will be important in identifying novel opportunities for antiviral intervention against arenaviral hemorrhagic disease.
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Affiliation(s)
- Joanne York
- Montana Biotechnology Center, The University of Montana, Science Complex, Room 221, Missoula, MT 59812, USA
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5
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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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6
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Grgacic EVL, Anderson DA. St, a truncated envelope protein derived from the S protein of duck hepatitis B virus, acts as a chaperone for the folding of the large envelope protein. J Virol 2005; 79:5346-52. [PMID: 15827149 PMCID: PMC1082741 DOI: 10.1128/jvi.79.9.5346-5352.2005] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2004] [Accepted: 12/14/2004] [Indexed: 02/06/2023] Open
Abstract
Envelope proteins of hepadnaviruses undergo a unique folding mechanism which results in the posttranslational translocation of 50% of the large envelope protein (L) chains across the endoplasmic reticulum. This mechanism is essential for the eventual positioning of the receptor-binding domain on the surface of the virus particle and in duck hepatitis B virus (DHBV) is dependent on the small (S) envelope protein as part of the assembly process. In this study, we report the identification of a third envelope protein, St, derived from the S protein and carrying functions previously attributed to S. Antibody mapping and mutagenesis studies indicated St to be C terminally truncated, spanning the N-terminal transmembrane domain (TM1) plus the adjacent cysteine loop. We have previously shown that the mutation of two conserved polar residues in TM1 of S (SAA) eliminates L translocation and assembly. A plasmid expressing a functional equivalent of St was able to rescue assembly, demonstrating that this assembly defect is due to mutations of the corresponding residues in St and not in S per se. Immunofluorescence analysis showed that St directly affects L protein cellular localization. These results indicate that St acts as a viral chaperone for L folding, remaining associated with the DHBV envelope upon secretion. The presence of St at a molar ratio of half that of L suggests that it is St which regulates L translocation to 50%.
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Affiliation(s)
- Elizabeth V L Grgacic
- Macfarlane Burnet Institute for Medical Research and Public Health, Melbourne 3004, Australia.
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7
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Abstract
The hepatitis B virus (HBV) is an enveloped DNA virus with an icosahedral capsid replicating via reverse transcription. The crystal structure of the capsid is known. It has a diameter of 36 nm and is formed by one protein species (C protein). The viral envelope contains three different coterminal proteins (S, M, and L proteins) spanning the membrane several times. These proteins are not only released from infected cells as components of the viral envelope but in 10,000-fold excess as subviral lipoprotein particles with a diameter of 22 nm containing no capsid. Assembly of the capsid occurs in the cytosol and results in packaging of a 3.5 kb RNA molecule together with viral and cellular factors. This newly formed capsid cannot be enveloped. Rather, synthesis of the viral DNA genome in the lumen of the capsid by reverse transcription is required to induce a budding competent state. Envelopment then takes place at an intracellular membrane of the pre-Golgi compartment. The S and the L protein, but not the M protein, is required for this process. The L protein forms two different transmembrane topologies. The isoform exposing the N-terminal part at the cytosolic side of the membrane is essential for budding. In this domain, a 22 amino acid (aa) long linear stretch has been mapped genetically to play a vital role in the morphogenetic process. This domain probably mediates the contact to the capsid. A second matrix domain was mapped to the cytosolic loop of the S protein. A similar genetic approach identified two small areas on the capsid surface, which might interact with the envelope proteins during envelopment.
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Affiliation(s)
- Volker Bruss
- Department of Virology, University of Göttingen, Kreuzbergring 57, 37075 Göttingen, Germany.
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8
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Schultz U, Grgacic E, Nassal M. Duck hepatitis B virus: an invaluable model system for HBV infection. Adv Virus Res 2005; 63:1-70. [PMID: 15530560 DOI: 10.1016/s0065-3527(04)63001-6] [Citation(s) in RCA: 69] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Affiliation(s)
- Ursula Schultz
- Department of Internal Medicine II/Molecular Biology, University Hospital Freiburg, D-79106 Freiburg, Germany
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9
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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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10
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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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11
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Clayton RF, Owsianka A, Patel AH. Evidence for structural differences in the S domain of L in comparison with S protein of hepatitis B virus. J Gen Virol 2001; 82:1533-1541. [PMID: 11413363 DOI: 10.1099/0022-1317-82-7-1533] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
The structures of the large (L), middle (M) and small (S) versions of the envelope proteins of hepatitis B virus remain poorly characterized due to the complex nature of their conformations. Several groups have proposed transmembrane topological models depicting the lumenally and cytosolically disposed regions of these proteins. Recently, post-translational topological changes in L have been described. However, no overall differences in the topology of the S domains of the L or M, to the S protein are predicted. In this report, we investigated a previously uncharacterized anti-S monoclonal antibody (MAb), 6B1, which recognizes a conformation-sensitive epitope in S. Unlike other anti-S MAbs tested, this MAb did not recognize its epitope in the S domain of L protein. Interestingly, however, the M protein was efficiently recognized. This unique characteristic of MAb 6B1 has allowed us to study the intracellular distribution of L and S proteins. In cells expressing both L and S, L re-localized from the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) to the membrane-associated distribution of S protein indicating that L and S interact with each other. This was confirmed by immunoprecipitation assays, which also showed that the interaction between L and S results in the secretion of L protein from cells. Overall, the ability of MAb 6B1 to selectively recognize S and M, but not L, strongly points to the existence of significant topological differences in the S domain of L. The availability of this important reagent should help further our understanding of the structure of HBV surface antigens.
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Affiliation(s)
- Reginald F Clayton
- MRC Virology Unit, Institute of Virology, Church Street, Glasgow G11 5JR, UK1
| | - Ania Owsianka
- MRC Virology Unit, Institute of Virology, Church Street, Glasgow G11 5JR, UK1
| | - Arvind H Patel
- MRC Virology Unit, Institute of Virology, Church Street, Glasgow G11 5JR, UK1
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12
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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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13
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Grgacic EV, Schaller H. A metastable form of the large envelope protein of duck hepatitis B virus: low-pH release results in a transition to a hydrophobic, potentially fusogenic conformation. J Virol 2000; 74:5116-22. [PMID: 10799586 PMCID: PMC110864 DOI: 10.1128/jvi.74.11.5116-5122.2000] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
We have examined the structure and fusion potential of the duck hepatitis B virus (DHBV) envelope proteins by treating subviral particles with deforming agents known to release envelope proteins of viruses from a metastable to a fusion-active state. Exposure of DHBV particles to low pH triggered a major structural change in the large envelope protein (L), resulting in exposure of trypsin sites within its S domain but without affecting the same region in the small surface protein (S) subunits. This conformational change was associated with increased hydrophobicity of the particle surface, most likely arising from surface exposure of the hydrophobic first transmembrane domain (TM1). In the hydrophobic conformation, DHBV particles were able to bind to liposomes and intact cells, while in their absence these particles aggregated, resulting in viral inactivation. These results suggests that some L molecules are in a spring-loaded metastable state which, when released, exposes a previously hidden hydrophobic domain, a transition potentially representing the fusion-active state of the envelope.
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Affiliation(s)
- E V Grgacic
- Macfarlane Burnet Centre for Medical Research and Australian Centre for Hepatitis Virology, Fairfield 3078, Victoria, Australia.
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14
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Urban S, Schwarz C, Marx UC, Zentgraf H, Schaller H, Multhaup G. Receptor recognition by a hepatitis B virus reveals a novel mode of high affinity virus-receptor interaction. EMBO J 2000; 19:1217-27. [PMID: 10716922 PMCID: PMC305663 DOI: 10.1093/emboj/19.6.1217] [Citation(s) in RCA: 48] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
The duck hepatitis B virus model system was used to elucidate the characteristics of receptor (carboxypeptidase D, gp180) interaction with polypeptides representing the receptor binding site in the preS part of the large viral surface protein. We demonstrate the pivotal role of carboxypeptidase D for virus entry and show its C-domain represents the virus attachment site, which binds preS with extraordinary affinity. Combining results from surface plasmon resonance spectroscopy and two-dimensional NMR analysis we resolved the contribution of preS sequence elements to complex stability and show that receptor binding potentially occurs in two steps. Initially, a short alpha-helix in the C-terminus of the receptor binding domain facilitates formation of a primary complex. This complex is stabilized sequentially, involving approximately 60 most randomly structured amino acids preceding the helix. Thus, hepadnaviruses exhibit a novel mechanism of high affinity receptor interaction by conserving the potential to adapt structure during binding rather than to preserve it per se. We propose that this process represents an alternative strategy to escape immune surveillance and the evolutionary pressure inherent in the compact hepadnaviral genome organization.
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MESH Headings
- Amino Acid Sequence
- Animals
- Binding Sites
- Carboxypeptidases/chemistry
- Carboxypeptidases/immunology
- Carboxypeptidases/isolation & purification
- Carboxypeptidases/metabolism
- Cells, Cultured
- Ducks/metabolism
- Ducks/virology
- Hepatitis B virus/chemistry
- Hepatitis B virus/drug effects
- Hepatitis B virus/metabolism
- Hepatitis B virus/physiology
- Immune Sera/immunology
- Immune Sera/pharmacology
- Kinetics
- Liver/cytology
- Liver/drug effects
- Liver/enzymology
- Liver/virology
- Molecular Sequence Data
- Mutation/genetics
- Nuclear Magnetic Resonance, Biomolecular
- Peptide Fragments/chemistry
- Peptide Fragments/genetics
- Peptide Fragments/isolation & purification
- Peptide Fragments/metabolism
- Protein Conformation
- Protein Structure, Tertiary
- Receptors, Antigen/chemistry
- Receptors, Antigen/genetics
- Receptors, Antigen/metabolism
- Receptors, Virus/chemistry
- Receptors, Virus/immunology
- Receptors, Virus/isolation & purification
- Receptors, Virus/metabolism
- Solubility
- Surface Plasmon Resonance
- Thermodynamics
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Affiliation(s)
- S Urban
- Zentrum für Molekulare Biologie (ZMBH), Universität Heidelberg, Im Neuenheimer Feld 282, 69120 Heidelberg.
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