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Heuschkel MJ, Baumert TF, Verrier ER. Cell Culture Models for the Study of Hepatitis D Virus Entry and Infection. Viruses 2021; 13:v13081532. [PMID: 34452397 PMCID: PMC8402901 DOI: 10.3390/v13081532] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2021] [Revised: 07/28/2021] [Accepted: 07/29/2021] [Indexed: 12/29/2022] Open
Abstract
Chronic hepatitis D is one of the most severe and aggressive forms of chronic viral hepatitis with a high risk of developing hepatocellular carcinoma (HCC). It results from the co-infection of the liver with the hepatitis B virus (HBV) and its satellite, the hepatitis D virus (HDV). Although current therapies can control HBV infection, no treatment that efficiently eliminates HDV is available and novel therapeutic strategies are needed. Although the HDV cycle is well described, the lack of simple experimental models has restricted the study of host–virus interactions, even if they represent relevant therapeutic targets. In the last few years, the discovery of the sodium taurocholate co-transporting polypeptide (NTCP) as a key cellular entry factor for HBV and HDV has allowed the development of new cell culture models susceptible to HBV and HDV infection. In this review, we summarize the main in vitro model systems used for the study of HDV entry and infection, discuss their benefits and limitations and highlight perspectives for future developments.
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Affiliation(s)
- Margaux J. Heuschkel
- Université de Strasbourg, Inserm, Institut de Recherche sur les Maladies Virales et Hépatiques UMR_S1110, 67000 Strasbourg, France; (M.J.H.); (T.F.B.)
| | - Thomas F. Baumert
- Université de Strasbourg, Inserm, Institut de Recherche sur les Maladies Virales et Hépatiques UMR_S1110, 67000 Strasbourg, France; (M.J.H.); (T.F.B.)
- Institut Hospitalo-Universitaire, Pôle Hépato-Digestif, Nouvel Hôpital Civil, 1 Place de L’Hôpital, 67000 Strasbourg, France
| | - Eloi R. Verrier
- Université de Strasbourg, Inserm, Institut de Recherche sur les Maladies Virales et Hépatiques UMR_S1110, 67000 Strasbourg, France; (M.J.H.); (T.F.B.)
- Correspondence: ; Tel.: +33-3-68-85-37-06
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Muriungi NG, Ueda K. TIMM29 interacts with hepatitis B virus preS1 to modulate the HBV life cycle. Microbiol Immunol 2020; 64:792-809. [PMID: 32970362 DOI: 10.1111/1348-0421.12852] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2020] [Revised: 09/15/2020] [Accepted: 09/18/2020] [Indexed: 12/28/2022]
Abstract
Hepatitis B virus (HBV), a major global health problem, can cause chronic hepatitis, liver cirrhosis, and hepatocellular carcinomas in chronically infected patients. However, before HBV infection can be adequately controlled, many mysteries about the HBV life cycle must be solved. In this study, TIMM29, an inner mitochondrial membrane protein, was identified as an interaction partner of the preS1 region of the HBV large S protein. The interaction was verified by both an immunoprecipitation with preS1 peptides and a GST-pulldown assay. Immunofluorescence studies also showed colocalization of preS1 and TIMM29. Moreover, it was determined that the preS1 bound with amino acids 92-189 of the TIMM29 protein. Infection of HBV in TIMM29-overexpressing NTCP/G2 cells resulted in a significant decrease of HBeAg and both extracellular particle-associated and core particle-associated HBV DNA without affecting cccDNA formation. Comparable results were obtained with TIMM29-overexpressing HB611 cells, which constitutively produce HBV. In contrast, knockout of TIMM29 in NTCP/G2 cells led to a higher production of HBV including HBeAg expression, as did knockout of TIMM29 in HB611. Collectively, these results suggested that TIMM29 interacts with the preS1 region of the HBV large S protein and modulates HBV amplification.
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Affiliation(s)
- Nelly Gakii Muriungi
- Division of Virology, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka, 565-0871, Japan
| | - Keiji Ueda
- Division of Virology, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka, 565-0871, Japan
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Herrscher C, Roingeard P, Blanchard E. Hepatitis B Virus Entry into Cells. Cells 2020; 9:cells9061486. [PMID: 32570893 PMCID: PMC7349259 DOI: 10.3390/cells9061486] [Citation(s) in RCA: 81] [Impact Index Per Article: 16.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2020] [Revised: 06/15/2020] [Accepted: 06/16/2020] [Indexed: 02/06/2023] Open
Abstract
Hepatitis B virus (HBV), an enveloped partially double-stranded DNA virus, is a widespread human pathogen responsible for more than 250 million chronic infections worldwide. Current therapeutic strategies cannot eradicate HBV due to the persistence of the viral genome in a special DNA structure (covalently closed circular DNA, cccDNA). The identification of sodium taurocholate co-transporting polypeptide (NTCP) as an entry receptor for both HBV and its satellite virus hepatitis delta virus (HDV) has led to great advances in our understanding of the life cycle of HBV, including the early steps of infection in particular. However, the mechanisms of HBV internalization and the host factors involved in this uptake remain unclear. Improvements in our understanding of HBV entry would facilitate the design of new therapeutic approaches targeting this stage and preventing the de novo infection of naïve hepatocytes. In this review, we provide an overview of current knowledge about the process of HBV internalization into cells.
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Affiliation(s)
- Charline Herrscher
- Inserm U1259, Morphogénèse et Antigénicité du VIH et des Virus des Hépatites (MAVIVH), Université de Tours and CHRU de Tours, 37032 Tours, France;
| | - Philippe Roingeard
- Inserm U1259, Morphogénèse et Antigénicité du VIH et des Virus des Hépatites (MAVIVH), Université de Tours and CHRU de Tours, 37032 Tours, France;
- Plate-Forme IBiSA des Microscopies, PPF ASB, Université de Tours and CHRU de Tours, 37032 Tours, France
- Correspondence: (P.R.); (E.B.); Tel.: +33-2-3437-9646 (E.B.)
| | - Emmanuelle Blanchard
- Inserm U1259, Morphogénèse et Antigénicité du VIH et des Virus des Hépatites (MAVIVH), Université de Tours and CHRU de Tours, 37032 Tours, France;
- Plate-Forme IBiSA des Microscopies, PPF ASB, Université de Tours and CHRU de Tours, 37032 Tours, France
- Correspondence: (P.R.); (E.B.); Tel.: +33-2-3437-9646 (E.B.)
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Evaluation of HBV-Like Circulation in Wild and Farm Animals from Brazil and Uruguay. INTERNATIONAL JOURNAL OF ENVIRONMENTAL RESEARCH AND PUBLIC HEALTH 2019; 16:ijerph16152679. [PMID: 31357451 PMCID: PMC6695864 DOI: 10.3390/ijerph16152679] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/26/2019] [Revised: 07/02/2019] [Accepted: 07/08/2019] [Indexed: 12/20/2022]
Abstract
The origin of the hepatitis B virus is a subject of wide deliberation among researchers. As a result, increasing academic interest has focused on the spread of the virus in different animal species. However, the sources of viral infection for many of these animals are unknown since transmission may occur from animal to animal, human to human, animal to human, and human to animal. The aim of this study was to evaluate hepadnavirus circulation in wild and farm animals (including animals raised under wild or free conditions) from different sites in Brazil and Uruguay using serological and molecular tools. A total of 487 domestic wild and farm animals were screened for hepatitis B virus (HBV) serological markers and tested via quantitative and qualitative polymerase chain reaction (PCR) to detect viral DNA. We report evidence of HBsAg (surface antigen of HBV) and total anti-HBc (HBV core antigen) markers as well as low-copy hepadnavirus DNA among domestic and wild animals. According to our results, which were confirmed by partial genome sequencing, as the proximity between humans and animals increases, the potential for pathogen dispersal also increases. A wider knowledge and understanding of reverse zoonoses should be sought for an effective One Health response.
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Cao J, Luo S, Xiong Y. The Variability of Amino Acid Sequences in Hepatitis B Virus. Virol Sin 2019; 34:42-49. [PMID: 30610573 DOI: 10.1007/s12250-018-0070-x] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2018] [Accepted: 11/13/2018] [Indexed: 12/23/2022] Open
Abstract
Hepatitis B virus (HBV) is an important human pathogen belonging to the Hepadnaviridae family, Orthohepadnavirus genus. Over 240 million people are infected with HBV worldwide. The reverse transcription during its genome replication leads to low fidelity DNA synthesis, which is the source of variability in the viral proteins. To investigate the variability quantitatively, we retrieved amino acid sequences of 5,167 records of all available HBV genotypes (A-J) from the Genbank database. The amino acid sequences encoded by the open reading frames (ORF) S/C/P/X in the HBV genome were extracted and subjected to alignment. We analyzed the variability of the lengths and the sequences of proteins as well as the frequencies of amino acids. It comprehensively characterized the variability and conservation of HBV proteins at the level of amino acids. Especially for the structural proteins, hepatitis B surface antigens (HBsAg), there are potential sites critical for virus assembly and immune recognition. Interestingly, the preS1 domains in HBsAg were variable at some positions of amino acid residues, which provides a potential mechanism of immune-escape for HBV, while the preS2 and S domains were conserved in the lengths of protein sequences. In the S domain, the cysteine residues and the secondary structures of the alpha-helix and beta-sheet were likely critical for the stable folding of all HBsAg components. Also, the preC domain and C-terminal domain of the core protein are highly conserved. However, the polymerases (HBpol) and the HBx were highly variable at the amino acid level. Our research provides a basis for understanding the conserved and important domains of HBV viral proteins, which could be potential targets for anti-virus therapy.
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Affiliation(s)
- Jianhao Cao
- Institute of Antibody Engineering, School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou, 510515, China.
| | - Shuhong Luo
- Institute of Antibody Engineering, School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou, 510515, China.
| | - Yuanyan Xiong
- School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China.
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Smc5/6 Antagonism by HBx Is an Evolutionarily Conserved Function of Hepatitis B Virus Infection in Mammals. J Virol 2018; 92:JVI.00769-18. [PMID: 29848586 DOI: 10.1128/jvi.00769-18] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2018] [Accepted: 05/09/2018] [Indexed: 12/14/2022] Open
Abstract
Chronic infection with hepatitis B virus (HBV) is a major cause of liver disease and cancer in humans. HBVs (family Hepadnaviridae) have been associated with mammals for millions of years. Recently, the Smc5/6 complex, known for its essential housekeeping functions in genome maintenance, was identified as an antiviral restriction factor of human HBV. The virus has, however, evolved to counteract this defense mechanism by degrading the complex via its regulatory HBx protein. Whether the antiviral activity of the Smc5/6 complex against hepadnaviruses is an important and evolutionarily conserved function is unknown. In this study, we used an evolutionary and functional approach to address this question. We first performed phylogenetic and positive selection analyses of the Smc5/6 complex subunits and found that they have been conserved in primates and mammals. Yet, Smc6 showed marks of adaptive evolution, potentially reminiscent of a virus-host "arms race." We then functionally tested the HBx proteins from six divergent hepadnaviruses naturally infecting primates, rodents, and bats. We demonstrate that despite little sequence homology, these HBx proteins efficiently degraded mammalian Smc5/6 complexes, independently of the host species and of the sites under positive selection. Importantly, all HBx proteins also rescued the replication of an HBx-deficient HBV in primary human hepatocytes. These findings point to an evolutionarily conserved requirement for Smc5/6 inactivation by HBx, showing that Smc5/6 antiviral activity has been an important defense mechanism against hepadnaviruses in mammals. It will be interesting to investigate whether Smc5/6 may further be a restriction factor of other, yet-unidentified viruses that may have driven some of its adaptation.IMPORTANCE Infection with hepatitis B virus (HBV) led to 887,000 human deaths in 2015. HBV has been coevolving with mammals for millions of years. Recently, the Smc5/6 complex, which has essential housekeeping functions, was identified as a restriction factor of human HBV antagonized by the regulatory HBx protein. Here we address whether the antiviral activity of Smc5/6 is an important evolutionarily conserved function. We found that all six subunits of Smc5/6 have been conserved in primates, with only Smc6 showing signatures of an "evolutionary arms race." Using evolution-guided functional analyses that included infections of primary human hepatocytes, we demonstrated that HBx proteins from very divergent mammalian HBVs could all efficiently antagonize Smc5/6, independently of the host species and sites under positive selection. These findings show that Smc5/6 antiviral activity against HBV is an important function in mammals. They also raise the intriguing possibility that Smc5/6 may restrict other, yet-unidentified viruses.
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Abstract
Viral hepatitis in poultry is a complex disease syndrome caused by several viruses belonging to different families including avian hepatitis E virus (HEV), duck hepatitis B virus (DHBV), duck hepatitis A virus (DHAV-1, -2, -3), duck hepatitis virus Types 2 and 3, fowl adenoviruses (FAdV), and turkey hepatitis virus (THV). While these hepatitis viruses share the same target organ, the liver, they each possess unique clinical and biological features. In this article, we aim to review the common and unique features of major poultry hepatitis viruses in an effort to identify the knowledge gaps and aid the prevention and control of poultry viral hepatitis. Avian HEV is an Orthohepevirus B in the family Hepeviridae that naturally infects chickens and consists of three distinct genotypes worldwide. Avian HEV is associated with hepatitis-splenomegaly syndrome or big liver and spleen disease in chickens, although the majority of the infected birds are subclinical. Avihepadnaviruses in the family of Hepadnaviridae have been isolated from ducks, snow geese, white storks, grey herons, cranes, and parrots. DHBV evolved with the host as a noncytopathic form without clinical signs and rarely progressed to chronicity. The outcome for DHBV infection varies by the host's ability to elicit an immune response and is dose and age dependent in ducks, thus mimicking the pathogenesis of human hepatitis B virus (HBV) infections and providing an excellent animal model for human HBV. DHAV is a picornavirus that causes a highly contagious virus infection in ducks with up to 100% flock mortality in ducklings under 6 wk of age, while older birds remain unaffected. The high morbidity and mortality has an economic impact on intensive duck production farming. Duck hepatitis virus Types 2 and 3 are astroviruses in the family of Astroviridae with similarity phylogenetically to turkey astroviruses, implicating the potential for cross-species infections between strains. Duck astrovirus (DAstV) causes acute, fatal infections in ducklings with a rapid decline within 1-2 hr and clinical and pathologic signs virtually indistinguishable from DHAV. DAstV-1 has only been recognized in the United Kingdom and recently in China, while DAstV-2 has been reported in ducks in the United States. FAdV, the causative agent of inclusion body hepatitis, is a Group I avian adenovirus in the genus Aviadenovirus. The affected birds have a swollen, friable, and discolored liver, sometimes with necrotic or hemorrhagic foci. Histologic lesions include multifocal necrosis of hepatocytes and acute hepatitis with intranuclear inclusion bodies in the nuclei of the hepatocytes. THV is a picornavirus that is likely the causative agent of turkey viral hepatitis. Currently there are more questions than answers about THV, and the pathogenesis and clinical impacts remain largely unknown. Future research in viral hepatic diseases of poultry is warranted to develop specific diagnostic assays, identify suitable cell culture systems for virus propagation, and develop effective vaccines.
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Affiliation(s)
- Danielle M Yugo
- A Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University, 1981 Kraft Drive, Blacksburg, VA 24061-0913
| | - Ruediger Hauck
- B Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA 95616
| | - H L Shivaprasad
- C California Animal Health and Food Safety Laboratory System, University of California-Davis, Tulare, CA 93274
| | - Xiang-Jin Meng
- A Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University, 1981 Kraft Drive, Blacksburg, VA 24061-0913
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Woodchuck sodium taurocholate cotransporting polypeptide supports low-level hepatitis B and D virus entry. Virology 2017; 505:1-11. [PMID: 28213271 DOI: 10.1016/j.virol.2017.02.006] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2016] [Revised: 02/07/2017] [Accepted: 02/08/2017] [Indexed: 12/15/2022]
Abstract
Sodium taurocholate cotransporting polypeptide (NTCP) is the functional receptor for human hepatitis B virus (HBV) and its satellite hepatitis D virus (HDV). Species barriers to HBV/HDV infection are mainly determined at entry level by variations in the sequences of particular NTCP orthologs. In this study, we sought to determine whether the NTCP ortholog in woodchuck (Marmota monax), woodchuck NTCP (wNTCP) supports viral infection. We found that wNTCP is capable of supporting HBV/HDV infection in HepG2 cells, but to much lower extent than human NTCP (hNTCP), which is about 90% reduction of hNTCP. Comprehensive site-directed mutagenesis mapping of hNTCP and wNTCP revealed that the residue at position 263 is a novel site crucial for viral entry. The important role of site 263 in infection is conserved among NTCP orthologs and may therefore be a potential target for blocking the viral entry.
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9
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Abstract
Hepatitis B virus (HBV) infection affects 240 million people worldwide. A liver-specific bile acid transporter named the sodium taurocholate cotransporting polypeptide (NTCP) has been identified as the cellular receptor for HBV and its satellite, the hepatitis D virus (HDV). NTCP likely acts as a major determinant for the liver tropism and species specificity of HBV and HDV at the entry level. NTCP-mediated HBV entry interferes with bile acid transport in cell cultures and has been linked with alterations in bile acid and cholesterol metabolism in vivo. The human liver carcinoma cell line HepG2, complemented with NTCP, now provides a valuable platform for studying the basic biology of the viruses and developing treatments for HBV infection. This review summarizes critical findings regarding NTCP's role as a viral receptor for HBV and HDV and discusses important questions that remain unanswered.
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Affiliation(s)
- Wenhui Li
- National Institute of Biological Sciences, Zhongguancun Life Science Park, Beijing 102206, China;
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Yan H, Li W. Sodium taurocholate cotransporting polypeptide acts as a receptor for hepatitis B and D virus. Dig Dis 2015; 33:388-96. [PMID: 26045274 DOI: 10.1159/000371692] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
Abstract
Infection of hepatitis B virus (HBV) remains a major public health problem worldwide. Understanding the viral infection and developing antivirals against HBV have been hampered by the lack of convenient culture systems and animal models for the infection. Sodium taurocholate cotransporting polypeptide (NTCP), a key bile acid transporter expressed in liver, was recently identified as a critical receptor for viral entry of HBV and its satellite virus hepatitis D virus (HDV). This finding enabled a reliable cell culture system for the viruses. Detailed studies have shown that NTCP is the major determinant for the species specificity of HBV and HDV at entry level. NTCP is responsible for most sodium-dependent bile salt uptake in liver. The molecular determinant critical for HBV/HDV infection overlaps with that for bile acids transporting on NTCP. We evaluated bile acids as potential antivirals for HBV and HDV infection, and developed bile acid derivatives that effectively block taurocholate transporting as well as viral infections. The discovery that NTCP acts as a receptor for HBV has opens a new door for future studies towards the ultimate goal of curative treatment of HBV infection.
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Affiliation(s)
- Huan Yan
- National Institute of Biological Sciences, Beijing, China
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Le Vee M, Jouan E, Noel G, Stieger B, Fardel O. Polarized location of SLC and ABC drug transporters in monolayer-cultured human hepatocytes. Toxicol In Vitro 2015; 29:938-46. [PMID: 25862123 DOI: 10.1016/j.tiv.2015.03.019] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2014] [Revised: 02/16/2015] [Accepted: 03/25/2015] [Indexed: 12/31/2022]
Abstract
Human hepatocytes cultured in a monolayer configuration represent a well-established in vitro model in liver toxicology, notably used in drug transporter studies. Polarized status of drug transporters, i.e., their coordinated location at sinusoidal or canalicular membranes, remains however incompletely documented in these cultured hepatocytes. The present study was therefore designed to analyze transporter expression and location in such cells. Most of drug transporters were first shown to be present at notable mRNA levels in monolayer-cultured human hepatocytes. Cultured human hepatocytes, which morphologically exhibited bile canaliculi-like structures, were next demonstrated, through immunofluorescence staining, to express the influx transporters organic anion transporting polypeptide (OATP) 1B1, OATP2B1 and organic cation transporter (OCT) 1 and the efflux transporter multidrug resistance-associated protein (MRP) 3 at their sinusoidal pole. In addition, the efflux transporters P-glycoprotein and MRP2 were detected at the canalicular pole of monolayer-cultured human hepatocytes. Moreover, canalicular secretion of reference substrates for the efflux transporters bile salt export pump, MRP2 and P-glycoprotein as well as sinusoidal drug transporter activities were observed. This polarized and functional expression of drug transporters in monolayer-cultured human hepatocytes highlights the interest of using this human in vitro cell model in xenobiotic transport studies.
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Affiliation(s)
- Marc Le Vee
- Institut de Recherches en Santé, Environnement et Travail (IRSET), UMR INSERM U1085, Faculté de Pharmacie, 2 Avenue du Pr Léon Bernard, 35043 Rennes, France
| | - Elodie Jouan
- Institut de Recherches en Santé, Environnement et Travail (IRSET), UMR INSERM U1085, Faculté de Pharmacie, 2 Avenue du Pr Léon Bernard, 35043 Rennes, France
| | - Gregory Noel
- Institut de Recherches en Santé, Environnement et Travail (IRSET), UMR INSERM U1085, Faculté de Pharmacie, 2 Avenue du Pr Léon Bernard, 35043 Rennes, France
| | - Bruno Stieger
- Department of Clinical Pharmacology and Toxicology, University Hospital, 8091 Zurich, Switzerland
| | - Olivier Fardel
- Institut de Recherches en Santé, Environnement et Travail (IRSET), UMR INSERM U1085, Faculté de Pharmacie, 2 Avenue du Pr Léon Bernard, 35043 Rennes, France; Pôle Biologie, Centre Hospitalier Universitaire, 2 rue Henri Le Guilloux, 35033 Rennes, France.
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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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Li H, Zhuang Q, Wang Y, Zhang T, Zhao J, Zhang Y, Zhang J, Lin Y, Yuan Q, Xia N, Han J. HBV life cycle is restricted in mouse hepatocytes expressing human NTCP. Cell Mol Immunol 2014; 11:175-83. [PMID: 24509445 DOI: 10.1038/cmi.2013.66] [Citation(s) in RCA: 81] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2013] [Revised: 12/09/2013] [Accepted: 12/10/2013] [Indexed: 02/08/2023] Open
Abstract
Recent studies have revealed that human sodium taurocholate cotransporting polypeptide (SLC10A1 or NTCP) is a functional cellular receptor for hepatitis B virus (HBV). However, whether human NTCP can support HBV infection in mouse hepatocyte cell lines has not been clarified. Because an HBV-permissible mouse model would be helpful for the study of HBV pathogenesis, it is necessary to investigate whether human NTCP supports the susceptibility of mouse hepatocyte cell lines to HBV. The results show that exogenous human NTCP expression can render non-susceptible HepG2 (human), Huh7 (human), Hepa1-6 (mouse), AML-12 (mouse) cell lines and primary mouse hepatocyte (PMH) cells susceptible to hepatitis D virus (HDV) which employs HBV envelope proteins. However, human NTCP could only introduce HBV susceptibility in human-derived HepG2 and Huh7 cells, but not in mouse-derived Hepa1-6, AML-12 or PMH cells. These data suggest that although human NTCP is a functional receptor that mediates HBV infection in human cells, it cannot support HBV infection in mouse hepatocytes. Our study indicated that the restriction of HBV in mouse hepatocytes likely occurs after viral entry but prior to viral transcription. We have excluded the role of mouse hepatocyte nuclear factors in the restriction of the HBV life cycle and showed that knockdown or inhibition of Sting, TBK1, IRF3 or IRF7, the components of the anti-viral signaling pathways, had no effect on HBV infection in mouse hepatocytes. Therefore, murine restriction factors that limit HBV infection need to be identified before a HBV-permissible mouse line can be created.
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Affiliation(s)
- Hanjie Li
- State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Biology, and School of Life Sciences, Xiamen University, Xiamen 361005, China
| | - Qiuyu Zhuang
- State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Biology, and School of Life Sciences, Xiamen University, Xiamen 361005, China
| | - Yuze Wang
- 1] State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Biology, and School of Life Sciences, Xiamen University, Xiamen 361005, China [2] School of Chemical Engineering, Huaqiao University, Xiamen 361005, China
| | - Tianying Zhang
- National Institute of Diagnostics and Vaccine Development in Infectious Disease, School of Public Health, Xiamen University, Xiamen 361005, China
| | - Jinghua Zhao
- National Institute of Diagnostics and Vaccine Development in Infectious Disease, School of Public Health, Xiamen University, Xiamen 361005, China
| | - Yali Zhang
- National Institute of Diagnostics and Vaccine Development in Infectious Disease, School of Public Health, Xiamen University, Xiamen 361005, China
| | - Junfang Zhang
- National Institute of Diagnostics and Vaccine Development in Infectious Disease, School of Public Health, Xiamen University, Xiamen 361005, China
| | - Yi Lin
- School of Chemical Engineering, Huaqiao University, Xiamen 361005, China
| | - Quan Yuan
- National Institute of Diagnostics and Vaccine Development in Infectious Disease, School of Public Health, Xiamen University, Xiamen 361005, China
| | - Ningshao Xia
- National Institute of Diagnostics and Vaccine Development in Infectious Disease, School of Public Health, Xiamen University, Xiamen 361005, China
| | - Jiahuai Han
- State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Biology, and School of Life Sciences, Xiamen University, Xiamen 361005, China
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Sodium taurocholate cotransporting polypeptide mediates woolly monkey hepatitis B virus infection of Tupaia hepatocytes. J Virol 2013; 87:7176-84. [PMID: 23596296 DOI: 10.1128/jvi.03533-12] [Citation(s) in RCA: 54] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Primary Tupaia hepatocytes (PTHs) are susceptible to woolly monkey hepatitis B virus (WMHBV) infection, but the identity of the cellular receptor(s) mediating WMHBV infection of PTHs remains unclear. Recently, sodium taurocholate cotransporting polypeptide (NTCP) was identified as a functional receptor for human hepatitis B virus (HBV) infection of primary human and Tupaia hepatocytes. In this study, a synthetic pre-S1 peptide from WMHBV was found to bind specifically to cells expressing Tupaia NTCP (tsNTCP) and it efficiently blocked WMHBV entry into PTHs; silencing of tsNTCP in PTHs significantly inhibited WMHBV infection. Ectopic expression of tsNTCP rendered HepG2 cells susceptible to WMHBV infection. These data demonstrate that tsNTCP is a functional receptor for WMHBV infection of PTHs. The result also indicates that NTCP's orthologs likely act as a common cellular receptor for all known primate hepadnaviruses.
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Yan H, Zhong G, Xu G, He W, Jing Z, Gao Z, Huang Y, Qi Y, Peng B, Wang H, Fu L, Song M, Chen P, Gao W, Ren B, Sun Y, Cai T, Feng X, Sui J, Li W. Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus. eLife 2012; 3:e00049. [PMID: 23150796 PMCID: PMC3485615 DOI: 10.7554/elife.00049] [Citation(s) in RCA: 1542] [Impact Index Per Article: 118.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2012] [Accepted: 09/05/2012] [Indexed: 02/06/2023] Open
Abstract
Human hepatitis B virus (HBV) infection and HBV-related diseases remain a major public health problem. Individuals coinfected with its satellite hepatitis D virus (HDV) have more severe disease. Cellular entry of both viruses is mediated by HBV envelope proteins. The pre-S1 domain of the large envelope protein is a key determinant for receptor(s) binding. However, the identity of the receptor(s) is unknown. Here, by using near zero distance photo-cross-linking and tandem affinity purification, we revealed that the receptor-binding region of pre-S1 specifically interacts with sodium taurocholate cotransporting polypeptide (NTCP), a multiple transmembrane transporter predominantly expressed in the liver. Silencing NTCP inhibited HBV and HDV infection, while exogenous NTCP expression rendered nonsusceptible hepatocarcinoma cells susceptible to these viral infections. Moreover, replacing amino acids 157–165 of nonfunctional monkey NTCP with the human counterpart conferred its ability in supporting both viral infections. Our results demonstrate that NTCP is a functional receptor for HBV and HDV. DOI:http://dx.doi.org/10.7554/eLife.00049.001 Liver diseases related to the human hepatitis B virus (HBV) kill about 1 million people every year, and more than 350 million people around the world are infected with the virus. Some 15 million of these people are also infected with the hepatitis D virus (HDV), which is a satellite virus of HBV, and this places them at an even higher risk of liver diseases, including cancer. The viruses are known to enter liver cells by binding to receptors on their surface before being engulfed. Both HBV and HDV have outer coats that consist of three kinds of envelope proteins, and a region called the pre-S1 domain in one of them is known to have a central role in the interaction between the viruses and the receptors and, therefore, in infecting the cells. However, the identity of the HBV receptor has remained a mystery. Now Yan et al. have identified this receptor to be sodium taurocholate cotransporting polypeptide. This protein, known as NTCP for short, is normally involved in the circulation of bile acids in the body. In addition to humans, only two species are known to be susceptible to infection by human HBV and HDV—chimpanzees and a small mammal known as the treeshrew. Yan et al. started by isolating primary liver cells from treeshrews, and then used a combination of advanced purification and mass spectrometry analysis to show that the NTCP on the surface of the cells interacts with the pre-S1 domain in HBV. The authors then performed a series of gene knockdown experiments on liver cells of both human and treeshrew origin: when the gene that codes for NTCP was silenced, HBV infection was greatly reduced. Moreover, they were able to transfect HepG2 cells—which are widely used in research into liver disease, but are not susceptible to HBV and HDV infection—with NTCP from humans and treeshrews to make them susceptible. Similarly, although monkeys are not susceptible to HBV, replacing just five amino acids in monkey NTCP with their human counterparts was enough to make the monkey NTCP a functional receptor for the viruses. In the past, basic research into HBV and the development of antiviral therapeutics have both been hindered by the lack of suitable in vitro infection systems and animal models. Now, the work of Yan et al. means that it will be possible to use NTCP-complemented HepG2 cells for challenges as diverse as fundamental studies of basic viral entry/replication mechanisms and large-scale drug screening. It is also possible that HBV and HDV infection might interfere with some of the important physiological functions carried out by NTCP, so the latest work could also be of interest to medical scientists working on other diseases related to these infections. DOI:http://dx.doi.org/10.7554/eLife.00049.002
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Affiliation(s)
- Huan Yan
- Graduate program in School of Life Sciences , Peking University , Beijing , China ; National Institute of Biological Sciences , Beijing , China
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16
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Entry of hepatitis B virus into immortalized human primary hepatocytes by clathrin-dependent endocytosis. J Virol 2012; 86:9443-53. [PMID: 22740403 DOI: 10.1128/jvi.00873-12] [Citation(s) in RCA: 123] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
The lack of a suitable in vitro hepatitis B virus (HBV) infectivity model has limited examination of the early stages of the virus-cell interaction. In this study, we used an immortalized cell line derived from human primary hepatocytes, HuS-E/2, to study the mechanism of HBV infection. HBV infection efficiency was markedly increased after dimethyl sulfoxide (DMSO)-induced differentiation of the cells. Transmission electron microscopy demonstrated the presence of intact HBV particles in DMSO-treated HBV-infected HuS-E/2 cells, which could be infected with HBV for up to at least 50 passages. The pre-S1 domain of the large HBsAg (LHBsAg) protein specifically interacted with clathrin heavy chain (CHC) and clathrin adaptor protein AP-2. Short hairpin RNA knockdown of CHC or AP-2 in HuS-E/2 cells significantly reduced their susceptibility to HBV, indicating that both are necessary for HBV infection. Furthermore, HBV entry was inhibited by chlorpromazine, an inhibitor of clathrin-mediated endocytosis. LHBsAg also interfered with the clathrin-mediated endocytosis of transferrin by human hepatocytes. This infection system using an immortalized human primary hepatocyte cell line will facilitate investigations into HBV entry and in devising therapeutic strategies for manipulating HBV-associated liver disorders.
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N-terminal myristoylation-dependent masking of neutralizing epitopes in the preS1 attachment site of hepatitis B virus. J Hepatol 2011; 55:29-37. [PMID: 21145866 DOI: 10.1016/j.jhep.2010.10.019] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/12/2010] [Revised: 08/29/2010] [Accepted: 10/30/2010] [Indexed: 12/20/2022]
Abstract
BACKGROUNDS & AIMS The N-terminally myristoylated preS1 domain of the large hepatitis B surface protein (LHBs) mediates specific attachment of hepatitis B virus (HBV) to hepatocytes. Its B-cell epitopes leading to neutralization of infectivity are not yet characterized. METHODS We inserted C- and N-terminal preS1 peptides into the most immunogenic region of HBV core particles, therewith immunized Balb/c mice and determined binding properties and neutralization potential of resulting antibodies in vitro. RESULTS The particles with preS1 inserts were highly immunogenic and the corresponding anti-preS antibodies strongly bound to HBV particles from chronic carriers infected with different HBV genotypes A-F. However, antibodies binding to the C-terminal part of preS1 did not neutralize HBV infectivity for susceptible hepatocyte cultures. In contrast, antibodies elicited by the complete N-terminal attachment site of preS1(2-48) strongly neutralized with an IC50<3μg/ml of total immunoglobulin. Interestingly, antibodies against the very N-terminal part of preS1(1-21) could not neutralize infectivity although this sequence contains the most conserved and essential part of the attachment site. These antibodies reacted well with non-myristoylated preS1 peptides but only weakly with myristoylated preS1 peptides, natural HBsAg or HBV. CONCLUSIONS N-terminal myristic acid obviously favors a topology of LHBs that makes the most essential part of the preS1 attachment site inaccessible for neutralizing antibodies, whereas antibodies to neighbouring sequences neutralized very well. Thus, addition of the preS1(2-48) peptide in a highly immunogenic form to the current hepatitis B vaccine may improve protective immunity and reduce selection of escape mutations.
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18
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Fine mapping of pre-S sequence requirements for hepatitis B virus large envelope protein-mediated receptor interaction. J Virol 2009; 84:1989-2000. [PMID: 20007265 DOI: 10.1128/jvi.01902-09] [Citation(s) in RCA: 185] [Impact Index Per Article: 11.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Previous studies showed that the N-terminal 75 amino acids of the pre-S1 domain of the hepatitis B virus (HBV) L protein are essential for HBV and hepatitis delta virus (HDV) infectivity. Consistently, synthetic lipopeptides encompassing this sequence or only parts of it efficiently block HBV and HDV infection, presumably through specific interference with a cellular receptor. Crucial for both virus infectivity and the inhibitory activity of the peptides are N-terminal myristoylation and a highly conserved motif within the N-terminal 48 amino acids. To refine the sequence requirements, we synthesized a series of HBV pre-S1 peptides containing deletions, point mutations, d-amino acid exchanges, or genotype-specific sequence permutations. Using the HepaRG cell line and a genotype D-derived virus, we determined the specific inhibitory activities of the peptides and found that (i) lipopeptides with an artificial consensus sequence inhibit HBV genotype D infection more potently than the corresponding genotype D peptides; (ii) point mutations, d-amino acid exchanges, or deletions introduced into the highly conserved part of the pre-S1 domain result in an almost complete loss of activity; and (iii) the flanking sequences comprising amino acids 2 to 8, 16 to 20, and, to a less pronounced extent, 34 to 48 gradually increase the inhibitory activity, while amino acids 21 to 33 behave indifferently. Taken together, our data suggest that HBV pre-S1-mediated receptor interference and, thus, HBV receptor recognition form a highly specific process. It requires an N-terminal acyl moiety and a highly conserved sequence that is present in primate but not rodent or avian hepadnaviruses, indicating different entry pathways for the different family members.
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Le Vee M, Lecureur V, Moreau A, Stieger B, Fardel O. Differential regulation of drug transporter expression by hepatocyte growth factor in primary human hepatocytes. Drug Metab Dispos 2009; 37:2228-35. [PMID: 19661216 DOI: 10.1124/dmd.109.028035] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023] Open
Abstract
Hepatocyte growth factor (HGF) is known to down-regulate expression of drug-detoxifying proteins such as cytochromes P450 (P450s) in human hepatocytes. The present study was designed to determine whether HGF may also impair expression of uptake and efflux drug transporters, which constitute important determinants of the liver detoxification pathway, such as P450s. Exposure of primary human hepatocytes to 20 ng/ml HGF for 48 h was found to down-regulate mRNA levels of major sinusoidal uptake transporters, including sodium taurocholate-cotransporting polypeptide (NTCP), organic anion-transporting polypeptide (OATP) 2B1, OATP1B1, organic cation transporter (OCT) 1, and organic anion transporter 2. HGF concomitantly reduced NTCP, OATP2B1, and OATP1B1 protein expression and NTCP, OATP, and OCT1 transport activities. With respect to efflux pumps, HGF decreased mRNA expression of the canalicular bile salt export pump, whereas that of the multidrug resistance (MDR) 1 gene was transiently increased. Moreover, Western blot analysis indicated that HGF up-regulated expressions of MDR1/P-glycoprotein and breast cancer resistance protein in human hepatocytes, whereas those of multidrug resistance gene-associated protein (MRP) 2 and MRP3 were unchanged. However, HGF prevented constitutive androstane receptor-related up-regulation of MRP2 occurring in phenobarbital-treated hepatocytes. Taken together, these data demonstrate that HGF differentially regulates transporter expression in human hepatocytes, i.e., it represses most of the sinusoidal uptake transporters, whereas expression of most of the efflux transporters is unchanged or increased. Such changes probably contribute to alterations of pharmacokinetics in patients with diseases associated with increased plasma levels of HGF such as fulminant hepatitis.
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Affiliation(s)
- Marc Le Vee
- Equipe d'Accueil 4427, SeRAIC/Institut National de la Santé et de la Recherche Médicale U620, Institut Fédératif de Recherches 140, University of Rennes 1, Rennes, France
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20
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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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21
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Le Vee M, Lecureur V, Stieger B, Fardel O. Regulation of drug transporter expression in human hepatocytes exposed to the proinflammatory cytokines tumor necrosis factor-alpha or interleukin-6. Drug Metab Dispos 2009; 37:685-93. [PMID: 19074973 DOI: 10.1124/dmd.108.023630] [Citation(s) in RCA: 196] [Impact Index Per Article: 12.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 are proinflammatory cytokines known to alter expression of drug transporters in rodent liver. However, their effects toward human hepatic transporters remain poorly characterized. Therefore, this study was designed to analyze the effects of these cytokines on drug transporter expression in primary human hepatocytes. Exposure to 100 ng/ml TNF-alpha or 10 ng/ml IL-6 for 48 h was found to down-regulate mRNA levels of major sinusoidal influx transporters, including sodium-taurocholate cotransporting polypeptide (NTCP), organic anion-transporting polypeptide (OATP) 1B1, OATP1B3, OATP2B1, organic cation transporter (OCT) 1, and organic anion transporter 2. TNF-alpha and IL-6 concomitantly reduced NTCP and OATP1B1 protein expression and NTCP, OATP, and OCT1 transport activities. IL-6, but not TNF-alpha, was also found to decrease mRNA expression of the canalicular transporters multidrug resistance 1 gene, multidrug resistance gene-associated protein (MRP) 2, and breast cancer resistance protein (BCRP); it concomitantly decreased MRP2 and BCRP protein expression. TNF-alpha, unlike IL-6, markedly reduced bile salt export pump mRNA levels and increased BCRP protein expression. Expression of the sinusoidal MRP3 efflux pump was found to be up-regulated at protein level by both TNF-alpha and IL-6. Taken together, these data show that TNF-alpha and IL-6 similarly altered expression of sinusoidal drug transporters and rather differentially that of canalicular efflux transporters. Such pronounced changes in hepatic transporter expression are likely to contribute to both cholestasis and alterations of pharmacokinetics caused by inflammation in humans.
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Affiliation(s)
- Marc Le Vee
- Unité Propre de Recherche et de l'Enseignement Supérieur, Equipe d'Accueil, SeRAIC/Institut National de la Santé et de la Recherche Médicale U620, Institut Fédératif de Recherches 140, University of Rennes 1, Rennes, France
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Heterologous replacement of the supposed host determining region of avihepadnaviruses: high in vivo infectivity despite low infectivity for hepatocytes. PLoS Pathog 2008; 4:e1000230. [PMID: 19057662 PMCID: PMC2585059 DOI: 10.1371/journal.ppat.1000230] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2008] [Accepted: 11/05/2008] [Indexed: 12/12/2022] Open
Abstract
Hepadnaviruses, including hepatitis B virus (HBV), a highly relevant human pathogen, are small enveloped DNA viruses that replicate via reverse transcription. All hepadnaviruses display a narrow tissue and host tropism. For HBV, this restricts efficient experimental in vivo infection to chimpanzees. While the cellular factors mediating infection are largely unknown, the large viral envelope protein (L) plays a pivotal role for infectivity. Furthermore, certain segments of the PreS domain of L from duck HBV (DHBV) enhanced infectivity for cultured duck hepatocytes of pseudotyped heron HBV (HHBV), a virus unable to infect ducks in vivo. This implied a crucial role for the PreS sequence from amino acid 22 to 90 in the duck tropism of DHBV. Reasoning that reciprocal replacements would reduce infectivity for ducks, we generated spreading-competent chimeric DHBVs with L proteins in which segments 22–90 (Du-He4) or its subsegments 22–37 and 37–90 (Du-He2, Du-He3) are derived from HHBV. Infectivity for duck hepatocytes of Du-He4 and Du-He3, though not Du-He2, was indeed clearly reduced compared to wild-type DHBV. Surprisingly, however, in ducks even Du-He4 caused high-titered, persistent, horizontally and vertically transmissable infections, with kinetics of viral spread similar to those of DHBV when inoculated at doses of 108 viral genome equivalents (vge) per animal. Low-dose infections down to 300 vge per duck did not reveal a significant reduction in specific infectivity of the chimera. Hence, sequence alterations in PreS that limited infectivity in vitro did not do so in vivo. These data reveal a much more complex correlation between PreS sequence and host specificity than might have been anticipated; more generally, they question the value of cultured hepatocytes for reliably predicting in vivo infectivity of avian and, by inference, mammalian hepadnaviruses, with potential implications for the risk assessment of vaccine and drug resistant HBV variants. Hepatitis B virus (HBV) associated liver disease is a leading cause of death worldwide. Host range restrictions limit experimental HBV infections largely to chimpanzees or isolated human hepatocytes. A narrow host range is shared by the animal hepadnaviruses, e.g. from ducks (DHBV) and herons (HHBV); HHBV does not infect ducks though it can establish a low-level infection in cultured duck hepatocytes. Host tropism is thought to be mediated by the PreS domain of the large viral envelope protein, because certain duck virus PreS segments introduced into the envelope of spreading-incompetent HHBV pseudotypes enhanced infectivity for duck hepatocytes. Expecting that reciprocal exchanges in DHBV would negatively impact duck tropism, we generated chimeric DHBVs in which the PreS regions in question are derived from HHBV and which are autonomously spreading-competent; this allowed us to directly compare their infectivity for duck hepatocytes and ducks. Surprisingly, even the chimera with the largest portion of HHBV sequence was as infectious for ducks as authentic DHBV; in vitro infectivity, however, was substantially reduced. These unexpected differences question the value of cultured hepatocytes to reliably predict in vivo infectivity of avihepadnaviruses and, by inference, also that of vaccine escape and therapy resistant HBV variants.
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Le Vee M, Gripon P, Stieger B, Fardel O. Down-regulation of organic anion transporter expression in human hepatocytes exposed to the proinflammatory cytokine interleukin 1beta. Drug Metab Dispos 2008; 36:217-22. [PMID: 17991769 DOI: 10.1124/dmd.107.016907] [Citation(s) in RCA: 96] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/13/2025] Open
Abstract
Interleukin (IL) 1beta is a proinflammatory cytokine known to markedly alter expression of major organic anion transporters in rodent hepatocytes. However, its effects toward human hepatic transporters remain poorly characterized. Therefore, the present study was aimed at determining IL-1beta effects on expression of organic anion transporters in primary human hepatocytes and highly differentiated human hepatoma HepaRG cells. Exposure to 1 ng/ml IL-1beta was first shown to markedly repress mRNA expression of sodium-taurocholate cotransporting polypeptide (NTCP), a major sinusoidal transporter handling bile acids, in both human hepatocytes and HepaRG cells. It concomitantly reduced NTCP protein levels and NTCP-mediated cellular uptake of taurocholate in HepaRG cells. Other transporters such as the influx transporters organic anion transporting polypeptide (OATP)-B, OATP-C, and OATP8 and the efflux pumps multidrug resistance-associated protein (MRP) 2, MRP3, MRP4, and breast cancer resistance protein were also down-regulated at mRNA levels in human hepatocytes treated by IL-1beta for 24 h, and most of these transporters were similarly repressed in IL-1beta-exposed HepaRG cells; the cytokine also reduced bile salt export pump (BSEP) and OATP-C protein expression in human hepatocytes. IL-1beta was further shown to activate the extracellular signal-regulated protein kinase (ERK) in human hepatocytes and HepaRG cells; however, chemical inhibition of this kinase failed to counteract repressing effects of IL-1beta toward NTCP, BSEP, OATP-B, and OATP-C. Taken together, these data indicate that IL-1beta treatment reduced expression of major organic anion transporters in human hepatic cells in an ERK-independent manner. Such IL-1beta effects may likely participate in both cholestasis and alterations of hepatic detoxification pathways caused by inflammation in humans.
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Affiliation(s)
- Marc Le Vee
- Institut National de la Santé et de la Recherche Médicale U620, Faculty of Pharmacy, Rennes, France
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24
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Abstract
Hepatitis B viruses are small enveloped DNA viruses referred to as Hepadnaviridae that cause transient or persistent (chronic) infections of the liver. This family is divided into two genera, orthohepadnavirus and avihepadnavirus, which infect mammals or birds as natural hosts, respectively. They possess a narrow host range determined by the initial steps of viral attachment and entry. Hepatitis B virus is the focus of biomedical research owing to its medical significance. Approximately 2 billion people have serological evidence of hepatitis B, and of these approximately 350 million people have chronic infections (World Health Organisation, Fact Sheet WHO/204, October 2000). Depending on viral and host factors, the outcomes of infection with hepatitis B virus vary between acute hepatitis, mild or severe chronic hepatitis or cirrhosis. Chronic infections are associated with an increased risk for the development of hepatocellular carcinoma.
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Affiliation(s)
- Hans-Jürgen Netter
- Monash University, Department of Microbiology, Clayton Campus, Victoria 3800, Australia
| | - Shau-Feng Chang
- Industrial Technology Research Institute, Biomedical Engineering Laboratories, 300 Hsinchu, Taiwan
| | - Michael Bruns
- Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie an der Universität Hamburg, 20251 Hamburg, Germany
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25
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Abstract
Host range describes the range of species that a virus can infect to productively propagate itself. Productive infection requires compatibility between virus and host molecules. Thus host range may be restricted by lack of appropriate permissivity factors;alternatively, hosts may actively counteract infection using restriction factors. Incompatibility between virus and host can manifest on the level of individual cells,of tissues or organs,and of the entire organism. All hepatitis B viruses are hepatotropic,but individual viruses infect the livers of only selected mammalian (orthohepadnaviruses) and avian (avihepadnaviruses) hosts. Hence a narrow host range is thought to be a salient feature of hepadnaviruses. Here we briefly review general mechanisms of host range restriction,and summarise older as well as recent data pertaining to hepadnaviral host range. Clearly,the term species-specific is inadequate for many hepadnaviruses because they can infect different species from one genus,and even species from different genera. For a few others,only a single species,or genus,has been identified that supports efficient infection;however,this could as well relate to the restricted number of experimentally addressable test species. Together with the uncertainty about quantitative phylogenetic relationships between species,still largely based on morphological rather than molecular criteria,this leaves the term narrow open to interpretation. Finally,few if any of the host molecules enabling productive infection by a hepadnavirus have unambiguously been identified,the role of restriction factors has not yet been assessed,and even on the virus side the so-called host determining regions in the PreS domains of the large envelope proteins appear to be relevant only under specialised experimental conditions. Hence this important aspect of hepadnavirus biology is still far from being understood.
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Yang J, Xi Q, Deng R, Wang J, Hou J, Wang X. Identification of interspecies recombination among hepadnaviruses infecting cross-species hosts. J Med Virol 2007; 79:1741-50. [PMID: 17854046 DOI: 10.1002/jmv.20983] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
Members of the family Hepadnaviridae are divided into two genera, Orthohepadnavirus (from mammalian) and Avihepadnavirus (from avian). Recombination had been found to occur among human hepatitis B virus (HBV) strains of different genotypes, or between hepadnavirus strains from human and nonhuman primate. To reach a comparatively complete inspection of interspecies recombination events among hepadnavirus strains from various hosts, 837 hepadnavirus complete genome sequences from human and 112 from animals were analyzed by using fragment typing to scan for potential interspecies recombinants. Further bootscanning and phylogenetic analyses of the potential recombinants revealed six genome sequences as interspecies recombinants. Interspecies recombination events were found to occur among HBV strains from human and nonhuman primates, from gibbons of different genera, from chimpanzee and an unknown host, and between two avian hepadnavirus strains from birds of different subfamilies, which was identified for the first time. HBV interspecies recombinants were found to have recombination hot spots similar to that of human HBV intergenotype recombinants, breakpoints frequently locating near gene boundaries. Interspecies recombination found in this study may alter current views on hepadnavirus host specificity.
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Affiliation(s)
- Jie Yang
- Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou, People's Republic of China
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Lepère C, Régeard M, Le Seyec J, Gripon P. The translocation motif of hepatitis B virus envelope proteins is dispensable for infectivity. J Virol 2007; 81:7816-8. [PMID: 17494068 PMCID: PMC1933348 DOI: 10.1128/jvi.00224-07] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023] Open
Abstract
The early events of hepatitis B virus (HBV) infection remain unclear. In 2006, Stoeckl et al. proposed a new entry mechanism involving a translocation motif (TLM) present in the pre-S2 domain of envelope proteins (L. Stoeckl, A. Funk, A. Kopitzki, B. Brandenburg, S. Oess, H. Will, H. Sirma, and E. Hildt, Proc. Natl. Acad. Sci. USA 103:6730-6734, 2006). After receptor binding and internalization into the endosomal compartment, this motif would allow the translocation of HBV particles through the endosomal membrane into the cytosol. In this study we have used two different mutated viruses containing a truncated TLM and showed their ability to infect human hepatocytes in primary culture, thus demonstrating the dispensability of the TLM for HBV infectivity.
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Affiliation(s)
- Charlotte Lepère
- INSERM U522, Hôpital de Pontchaillou, Avenue Henri le Guilloux, Rennes Cedex 35033, France
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Blanchet M, Sureau C. Infectivity determinants of the hepatitis B virus pre-S domain are confined to the N-terminal 75 amino acid residues. J Virol 2007; 81:5841-9. [PMID: 17376925 PMCID: PMC1900317 DOI: 10.1128/jvi.00096-07] [Citation(s) in RCA: 107] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
The N-terminal pre-S domain of the large hepatitis B virus (HBV) envelope protein plays a pivotal role at the initial step of the viral entry pathway. In the present study, the entire pre-S domain was mapped for infectivity determinants, following a reverse-genetics approach and using in vitro infection assays with hepatitis delta virus (HDV) or HBV particles. The results demonstrate that lesions created within the N-terminal 75 amino acids of the pre-S region abrogate infectivity, whereas mutations between amino acids 76 and 113, overlapping the matrix domain, had no effect. In contrast to the results of a recent study (L. Stoeckl, A. Funk, A. Kopitzki, B. Brandenburg, S. Oess, H. Will, H. Sirma, and E. Hildt, Proc. Natl. Acad. Sci. 103:6730-6734, 2006), the deletion of a cell membrane translocation motif (TLM) located between amino acids 148 and 161 at the C terminus of pre-S2 did not interfere with the infectivity of the resulting HDV or HBV mutants. Furthermore, a series of large deletions overlapping the pre-S2 domain were compatible with infectivity, although the efficiency of infection was reduced when the deletions extended to the pre-S1 domain. Overall, the results demonstrate that the activity of the pre-S domain at viral entry solely depends on the integrity of its first 75 amino acids and thus excludes any function of the matrix domain or TLM.
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Affiliation(s)
- Matthieu Blanchet
- Laboratoire de Virologie Moléculaire, Institut National de la Transfusion Sanguine, 6 Rue Alexandre-Cabanel, 75739 Paris, France
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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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Sun D, Nassal M. Stable HepG2- and Huh7-based human hepatoma cell lines for efficient regulated expression of infectious hepatitis B virus. J Hepatol 2006; 45:636-45. [PMID: 16935386 DOI: 10.1016/j.jhep.2006.05.019] [Citation(s) in RCA: 100] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/24/2006] [Revised: 05/29/2006] [Accepted: 05/31/2006] [Indexed: 12/19/2022]
Abstract
BACKGROUND/AIMS Hepatitis B virus (HBV) cannot be propagated in cultured cells but two human hepatoma cell lines, HepG2 and Huh7, support virus replication when transfected with HBV DNA. If standardization is required stably transfected cell lines provide distinct advantages. One such line, HepG2.2.15, is widely used in antiviral research but HBV production is limited and difficult to control. Our aim was to establish stable, inducibly HBV producing HepG2 and Huh7 cell lines that overcome these limitations. METHODS Based on the tetracycline (Tet)-regulated TetOFF system, a Tet-responsive promoter-controlled HBV genome was introduced into separately established, well-regulatable HepG2 and Huh7 lines expressing Tet-responsive trans-activators (tTAs). Stable clones were analyzed for regulatability and levels of HBV expression, quality of the virus produced, and responsiveness towards antivirals. RESULTS HepG2- and Huh7-based cell lines were established which, Tet-controllably, produce more HBV than HepG2.2.15 cells. The secreted virions were infectious for primary tupaia hepatocytes, and the cell lines responded as well as HepG2.215 cells to different antivirals. CONCLUSIONS The new HBV cell lines should be valuable tools for academic and pharmaceutical HBV research. The parental tTA-cells will facilitate the generation of additional lines, producing HBV variants, or other genes, in an identical host cell background.
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Affiliation(s)
- Dianxing Sun
- University Hospital Freiburg, Internal Medicine II/Molecular Biology, Hugstetter Strasse 55, D-79106 Freiburg, Germany
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31
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Blanchet M, Sureau C. Analysis of the cytosolic domains of the hepatitis B virus envelope proteins for their function in viral particle assembly and infectivity. J Virol 2006; 80:11935-45. [PMID: 17020942 PMCID: PMC1676254 DOI: 10.1128/jvi.00621-06] [Citation(s) in RCA: 53] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
The hepatitis B virus (HBV) envelope proteins have the ability to assemble three types of viral particles, (i) the empty subviral particles (SVPs), (ii) the mature HBV virions, and (iii) the hepatitis delta virus (HDV) particles, in cells that are coinfected with HBV and HDV. To gain insight into the function of the HBV envelope proteins in morphogenesis of HBV or HDV virions, we have investigated subdomains of the envelope proteins that have been shown or predicted to lie at the cytosolic face of the endoplasmic reticulum membrane during synthesis, a position prone to interaction with the inner core structure. These domains, referred to here as cytosolic loops I and II (CYL-I and -II, respectively), were subjected to mutagenesis. The mutations were introduced in the three HBV envelope proteins, designated small, middle, and large (S-HBsAg, M-HBsAg, and L-HBsAg, respectively). The mutants were expressed in HuH-7 cells to evaluate their capacity for self-assembly and formation of HBV or HDV virions when HBV nucleocapsid or HDV ribonucleoprotein, respectively, was provided. We found that SVP-competent CYL-I mutations between positions 23 and 78 of the S domain were permissive to HBV or HDV virion assembly. One mutation (P29A) was permissive for synthesis of the S- and M-HBsAg but adversely affected the synthesis or stability of L-HBsAg, thereby preventing the assembly of HBV virions. Furthermore, using an in vitro infection assay based on the HepaRG cells and the HDV model, we have shown that particles coated with envelope proteins bearing CYL-I mutations were fully infectious, hence indicating the absence of an infectivity determinant in this region. Finally, we demonstrated that the tryptophan residues at positions 196, 199, and 201 in CYL-II, which were shown to exert a matrix function for assembly of HDV particles (I. Komla-Soukha and C. Sureau, J. Virol. 80:4648-4655, 2006), were dispensable for both assembly and infectivity of HBV virions.
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Affiliation(s)
- Matthieu Blanchet
- Laboratoire de Virologie, Institut National de la Transfusion Sanguine, 6 rue Alexandre Cabanel, 75739 Paris, France
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Yu D, Fukuda T, Kuroda S, Tanizawa K, Kondo A, Ueda M, Yamada T, Tada H, Seno M. Engineered bio-nanocapsules, the selective vector for drug delivery system. IUBMB Life 2006; 58:1-6. [PMID: 16596748 DOI: 10.1080/15216540500484368] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
The bio-nanocapsule (BNC) is our concept of artificial hollow nanoparticles that have been designed and produced through biotechnological procedures. We proposed an empty virus-like particle, which consists of a recombinant L envelope protein of hepatitis B virus (HBV) and a lipid derived from the host cell, as an engineered BNC. Although this BNC was first developed as an immunogen of hepatitis B vaccine, the pre-S1 region in N-terminus of L envelope protein confers hepatocyte specific infectivity of HBV on the BNC. This recombinant BNC is now being developed as a novel platform of drug delivery system (DDS) vector for selective delivery.
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Affiliation(s)
- Dongwei Yu
- Graduate School of Natural Science and Technology, Okayama University, 3.1.1 Tsushima-Naka, Okayama 700-8530, Japan
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Jaoudé GA, Sureau C. Role of the antigenic loop of the hepatitis B virus envelope proteins in infectivity of hepatitis delta virus. J Virol 2005; 79:10460-6. [PMID: 16051838 PMCID: PMC1182656 DOI: 10.1128/jvi.79.16.10460-10466.2005] [Citation(s) in RCA: 62] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
The infectious particles of hepatitis B virus (HBV) and hepatitis delta virus (HDV) are coated with the large, middle, and small envelope proteins encoded by HBV. While it is clear that the N-terminal pre-S1 domain of the large protein, which is exposed at the virion surface, is implicated in binding to a cellular receptor at viral entry, the role in infectivity of the envelope protein antigenic loop, also exposed to the virion surface and accessible to neutralizing antibodies, remains to be established. In the present study, mutations were created in the antigenic loop of the three envelope proteins, and the resulting mutants were evaluated for their capacity to assist in the maturation and infectivity of HDV. We observed that short internal combined deletions and insertions, affecting residues 109 to 133 in the antigenic loop, were tolerated for secretion of both subviral HBV particles and HDV virions. However, when assayed for infectivity on primary cultures of human hepatocytes or on the recently described HepaRG cell line, virions carrying deletions between residues 118 and 129 were defective. Single amino acid substitutions in this region revealed that Gly-119, Pro-120, Cys-121, Arg-122, and Cys-124 were instrumental in viral entry. These results demonstrate that in addition to a receptor-binding site previously identified in the pre-S1 domain of the L protein, a determinant of infectivity resides in the antigenic loop of HBV envelope proteins.
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Affiliation(s)
- Georges Abou Jaoudé
- Laboratoire de Virologie Moléculaire, Institut National de la Transfusion Sanguine, 6 Rue Alexandre-Cabanel, 75739 Paris, France
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34
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Barrera A, Guerra B, Notvall L, Lanford RE. Mapping of the hepatitis B virus pre-S1 domain involved in receptor recognition. J Virol 2005; 79:9786-98. [PMID: 16014940 PMCID: PMC1181564 DOI: 10.1128/jvi.79.15.9786-9798.2005] [Citation(s) in RCA: 103] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
Hepatitis B virus (HBV) and woolly monkey hepatitis B virus (WMHBV) are primate hepadnaviruses that display restricted tissue and host tropisms. Hepatitis D virus (HDV) particles pseudotyped with HBV and WMHBV envelopes (HBV-HDV and WM-HDV) preferentially infect human and spider monkey hepatocytes, respectively, thereby confirming host range bias in vitro. The analysis of chimeric HBV and WMHBV large (L) envelope proteins suggests that the pre-S1 domain may comprise two regions that affect infectivity: one within the amino-terminal 40 amino acids of pre-S1 and one downstream of this region. In the present study, we further characterized the role of the amino terminus of pre-S1 in infectivity by examining the ability of synthetic peptides to competitively block HDV infection of primary human and spider monkey hepatocytes. A synthetic peptide representing the first 45 residues of the pre-S1 domain of the HBV L protein blocked infectivity of HBV-HDV and WM-HDV, with a requirement for myristylation of the amino terminal residue. Competition studies with truncated peptides suggested that pre-S1 residues 5 to 20 represent the minimal domain for inhibition of HDV infection and, thus, presumably represent the residues involved in virus-host receptor interaction. Recombinant pre-S1 proteins expressed in insect cells blocked infection with HBV-HDV and WM-HDV at a concentration of 1 nanomolar. The ability of short pre-S1 peptides to efficiently inhibit HDV infection suggests that they represent suitable ligands for identification of the HBV receptor and that a pre-S1 mimetic may represent a rational therapy for the treatment of HBV infection.
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Affiliation(s)
- Azeneth Barrera
- Department of Virology and Immunology, Southwest National Primate Research Center, Southwest Foundation for Biomedical Research, TX 78227, USA
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35
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Franck N, Le Seyec J, Guguen-Guillouzo C, Erdtmann L. Hepatitis C virus NS2 protein is phosphorylated by the protein kinase CK2 and targeted for degradation to the proteasome. J Virol 2005; 79:2700-8. [PMID: 15708989 PMCID: PMC548468 DOI: 10.1128/jvi.79.5.2700-2708.2005] [Citation(s) in RCA: 67] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023] Open
Abstract
Hepatitis C virus (HCV) nonstructural 2 (NS2) protein is a hydrophobic transmembrane protein, described to be involved in different functions, such as apoptosis inhibition and gene transcription modulation. We investigated here NS2 protein turnover and found that NS2 was rapidly degraded by the proteasome in different cell lines, as in primary human hepatocytes. Since posttranslational modifications can influence protein turnover, we looked for potential phosphoacceptor sites in NS2. Computational sequence analysis in combination with screening of NS2 point mutants revealed that serine residue 168 was critical for degradation. In the quest of a protein kinase for NS2, we identified by sequence analysis that the serine residue 168 was part of a consensus casein kinase 2 (CK2) recognition site (S/TXXE). This motif was highly conserved since it could be found in the NS2 primary consensus sequences from all HCV genotypes. To verify whether CK2 is involved in NS2 phosphorylation, we showed by an in vitro kinase assay that CK2 phosphorylated NS2, as far as this CK2 motif was conserved. Interestingly, NS2 became resistant to protein degradation when the CK2 motif was modified by a single point mutation. Furthermore, inhibition of CK2 activity by curcumin decreased NS2 phosphorylation in vitro and stabilized NS2 expression in HepG2 cells. Finally, we showed in Huh-7.5 replicon cells that NS2, expressed in the context of the HCV polyprotein, was also sensitive to both proteasome-mediated degradation and CK2 inhibitor treatment. We suggest that NS2 is a short-lived protein whose degradation by the proteasome is regulated in a phosphorylation-dependent manner through the protein kinase CK2.
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Affiliation(s)
- Nathalie Franck
- INSERM, U522, Hôpital de Pontchaillou, 2, Rue Henri le Guilloux, Rennes Cedex 35033, France.
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36
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Guo H, Mason WS, Aldrich CE, Saputelli JR, Miller DS, Jilbert AR, Newbold JE. Identification and characterization of avihepadnaviruses isolated from exotic anseriformes maintained in captivity. J Virol 2005; 79:2729-42. [PMID: 15708992 PMCID: PMC548436 DOI: 10.1128/jvi.79.5.2729-2742.2005] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Five new hepadnaviruses were cloned from exotic ducks and geese, including the Chiloe wigeon, mandarin duck, puna teal, Orinoco sheldgoose, and ashy-headed sheldgoose. Sequence comparisons revealed that all but the mandarin duck viruses were closely related to existing isolates of duck hepatitis B virus (DHBV), while mandarin duck virus clones were closely related to Ross goose hepatitis B virus. Nonetheless, the S protein, core protein, and functional domains of the Pol protein were highly conserved in all of the new isolates. The Chiloe wigeon and puna teal hepatitis B viruses, the two new isolates most closely related to DHBV, also lacked an AUG start codon at the beginning of their X open reading frame (ORF). But as previously reported for the heron, Ross goose, and stork hepatitis B viruses, an AUG codon was found near the beginning of the X ORF of the mandarin duck, Orinoco, and ashy-headed sheldgoose viruses. In all of the new isolates, the X ORF ended with a stop codon at the same position. All of the cloned viruses replicated when transfected into the LMH line of chicken hepatoma cells. Significant differences between the new isolates and between these and previously reported isolates were detected in the pre-S domain of the viral envelope protein, which is believed to determine viral host range. Despite this, all of the new isolates were infectious for primary cultures of Pekin duck hepatocytes, and infectivity in young Pekin ducks was demonstrated for all but the ashy-headed sheldgoose isolate.
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Affiliation(s)
- Haitao Guo
- Fox Chase Cancer Center, 333 Cottman Ave., Philadelphia, PA 19111, USA
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37
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Gripon P, Cannie I, Urban S. Efficient inhibition of hepatitis B virus infection by acylated peptides derived from the large viral surface protein. J Virol 2005; 79:1613-22. [PMID: 15650187 PMCID: PMC544121 DOI: 10.1128/jvi.79.3.1613-1622.2005] [Citation(s) in RCA: 276] [Impact Index Per Article: 13.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
The lack of an appropriate in vitro infection system for the major human pathogen hepatitis B virus (HBV) has prevented a molecular understanding of the early infection events of HBV. We used the novel HBV-infectible cell line HepaRG and primary human hepatocytes to investigate the interference of infection by HBV envelope protein-derived peptides. We found that a peptide consisting of the authentically myristoylated N-terminal 47 amino acids of the pre-S1 domain of the large viral envelope protein (L protein) specifically prevented HBV infection, with a 50% inhibitory concentration (IC50) of 8 nM. The replacement of myristic acid with other hydrophobic moieties resulted in changes in the inhibitory activity, most notably by a decrease in the IC50 to picomolar concentrations for longer unbranched fatty acids. The obstruction of HepaRG cell susceptibility to HBV infection after short preincubation times with the peptides suggested that the peptides efficiently target and inactivate a receptor at the hepatocyte surface. Our data both shed light on the molecular mechanism of HBV entry into hepatocytes and provide a basis for the development of potent hepadnaviral entry inhibitors as a novel therapeutic concept for the treatment of hepatitis Beta.
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Affiliation(s)
- Philippe Gripon
- Institut National de la Santé et de la Recherche Médicale (INSERM) U522, Hôpital de Pontchaillou, Rennes, France
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38
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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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39
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Dandri M, Burda MR, Zuckerman DM, Wursthorn K, Matschl U, Pollok JM, Rogiers X, Gocht A, Köck J, Blum HE, von Weizsäcker F, Petersen J. Chronic infection with hepatitis B viruses and antiviral drug evaluation in uPA mice after liver repopulation with tupaia hepatocytes. J Hepatol 2005; 42:54-60. [PMID: 15629507 DOI: 10.1016/j.jhep.2004.09.021] [Citation(s) in RCA: 61] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/27/2004] [Revised: 09/09/2004] [Accepted: 09/17/2004] [Indexed: 01/18/2023]
Abstract
BACKGROUND/AIMS Transplantation of primary human hepatocytes and establishment of hepatitis B virus (HBV) infection in immunodeficient urokinase plasminogen activator (uPA) transgenic mice was shown. However, the availability of usable primary human hepatocytes is very limited. Therefore, alternative and more accessible sources of hepatocytes permissive for HBV infection are highly desirable. Here we investigated the potential of primary hepatocytes from the tree shrew Tupaia belangeri that were shown to be susceptible to HBV infection. METHODS Freshly isolated or cryopreserved primary tupaia hepatocytes were transplantated via intrasplenic injection into immunodeficient uPA/RAG-2 mice. Engrafted mice were then infected with HBV and woolly monkey (WM)-HBV positive sera. RESULTS Extensive proliferation of xenografted cells was demonstrated by the stable production of tupaia alpha1-antitrypsin in serum and liver of transplanted mice. Quantitative PCR assays demonstrated the presence of circulating viral particles as well as intracellular viral DNA, including covalently closed circular (ccc) DNA, in transplanted mice. Viral infection could be serially passaged in mice. Furthermore, viral replication was strongly inhibited by treating mice with adefovir dipivoxil. CONCLUSIONS uPA mice repopulated with tupaia hepatocytes represent a useful and more accessible model for HBV infection studies, including the evaluation of antiviral therapy and cccDNA.
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Affiliation(s)
- Maura Dandri
- Department of Medicine, University of Hamburg, Eppendorf, Martinistr. 52, 20246 Hamburg, Germany
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40
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Barrera A, Guerra B, Lee H, Lanford RE. Analysis of host range phenotypes of primate hepadnaviruses by in vitro infections of hepatitis D virus pseudotypes. J Virol 2004; 78:5233-43. [PMID: 15113905 PMCID: PMC400381 DOI: 10.1128/jvi.78.10.5233-5243.2004] [Citation(s) in RCA: 38] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023] Open
Abstract
Hepatitis B virus (HBV) and woolly monkey hepatitis B virus (WMHBV) have natural host ranges that are limited to closely related species. The barrier for infection of primates seems to be at the adsorption and/or entry steps of the viral replication cycle, since a human hepatoma cell line is permissive for HBV and WMHBV replication following transfection of cloned DNA. We hypothesized that the HBV and WMHBV envelope proteins contain the principal viral determinants of host range. As previously shown by using the hepatitis D virus (HDV) system, recombinant HBV-HDV particles were infectious in chimpanzee as well as human hepatocytes. We extended the HDV system to include HDV particles pseudotyped with the WMHBV envelope. In agreement with the natural host ranges of HBV and WMHBV, in vitro infections demonstrated that HBV-HDV and WM-HDV particles preferentially infected human and spider monkey cells, respectively. Previous studies have implicated the pre-S1 region of the large (L) envelope protein in receptor binding and host range; therefore, recombinant HDV particles were pseudotyped with the hepadnaviral envelopes containing chimeric L proteins with the first 40 amino acids from the pre-S1 domain exchanged between HBV and WMHBV. Surprisingly, addition of the human amino terminus to the WMHBV L protein increased infectivity on spider monkey hepatocytes but did not increase infectivity for human hepatocytes. Based upon these data, we discuss the possibility that the L protein may be comprised of two domains that affect infectivity and that sequences downstream of residue 40 may influence host range and receptor binding or entry.
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Affiliation(s)
- Azeneth Barrera
- Department of Virology and Immunology, Southwest National Primate Research Center, Southwest Foundation for Biomedical Research, 7620 NW Loop 410, San Antonio, TX 78227, USA
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41
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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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42
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Schildgen O, Roggendorf M, Lu M. Identification of a glycosylation site in the woodchuck hepatitis virus preS2 protein and its role in protein trafficking. J Gen Virol 2004; 85:787-793. [PMID: 15039521 DOI: 10.1099/vir.0.19672-0] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
The middle surface antigen (M-sAg) of hepadnaviruses is one of three envelope proteins that share a common C-terminal S domain. M-sAg contains the preS2 domain in addition to the S region. The preS2 region of woodchuck hepatitis virus (WHV) contains a potential glycosylation site Asn-Gln-Thr at amino acid (aa) positions 3-5. In this study, we mutated this site by site-directed mutagenesis and confirmed that glycosylation occurs here. In in vitro translation assays, the mutation Thr to Asn at aa 5 significantly impaired glycosylation of M-sAg. The mutated M-sAg formed abnormal clustered structures in transfected cells as determined by immunofluorescent staining. Confocal microscopic analysis showed that an enrichment of this glycosylation-deficient protein in the Golgi apparatus occurred, which is not typical for the wild-type protein. These results are consistent with earlier findings that incorrect glycosylation of viral proteins may interfere with virus assembly.
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Affiliation(s)
- O Schildgen
- Institut für Virologie, Universitätsklinikum Essen, Hufelandstraße 55, 45122 Essen, Germany
| | - M Roggendorf
- Institut für Virologie, Universitätsklinikum Essen, Hufelandstraße 55, 45122 Essen, Germany
| | - M Lu
- Institut für Virologie, Universitätsklinikum Essen, Hufelandstraße 55, 45122 Essen, Germany
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Kalinina T, Riu A, Fischer L, Santantonio T, Will H, Sterneck M. Selection of a secretion-incompetent mutant in the serum of a patient with severe hepatitis B. Gastroenterology 2003; 125:1077-84. [PMID: 14517791 DOI: 10.1016/s0016-5085(03)01202-2] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/16/2023]
Abstract
BACKGROUND AND AIMS A secretion-incompetent, highly replicating hepatitis B variant was previously found as the dominant viral population in the serum of a liver transplant recipient with severe hepatitis B reinfection. The secretion block resulted from mutations in the S protein, including the Gly145Arg substitution known to emerge under antibody to hepatitis B surface antigen immunoglobulin treatment. Here we investigated the mechanisms that allow selection of a secretion-incompetent virus as the predominant strain in the serum. METHODS To reproduce the interaction of viral quasispecies occurring in vivo, cotransfection experiments were performed with full-length genomes containing wild-type or mutant sequences. In addition, the relevance of mutations in the common S part of the surface proteins for the competence of L and S protein to support viral secretion was studied. RESULTS A small amount of wild-type virus or of a wild-type S protein-expressing variant rescued secretion of the defective mutant. In the secreted virions, the high-replicating mutant genome was predominant. Selection of the defective mutant was further supported by a transdominant negative effect of mutant S protein on wild-type virion secretion. In contrast, mutant L protein with the same c-terminal mutations as mutant S protein efficiently supported virion formation and secretion. CONCLUSIONS Interaction of the variant with a small amount of wild-type virus can reverse its secretion-defective phenotype. Mutations in the common region of S and L protein have different consequences for the ability of the envelope proteins to support virion assembly and secretion.
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Affiliation(s)
- Tatyana Kalinina
- Heinrich-Pette-Institute for Experimental Virology and Immunology, University of Hamburg, Hamburg, Germany
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Glebe D, Aliakbari M, Krass P, Knoop EV, Valerius KP, Gerlich WH. Pre-s1 antigen-dependent infection of Tupaia hepatocyte cultures with human hepatitis B virus. J Virol 2003; 77:9511-21. [PMID: 12915565 PMCID: PMC187384 DOI: 10.1128/jvi.77.17.9511-9521.2003] [Citation(s) in RCA: 146] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2003] [Accepted: 06/03/2003] [Indexed: 12/17/2022] Open
Abstract
The susceptibility of the tree shrew Tupaia belangeri to human hepatitis B virus (HBV) has been demonstrated both in vivo and in vitro. In this study, we show that purified HBV infects primary T. belangeri hepatocyte cultures in a very specific manner, as detected by HBV covalently closed circular DNA, mRNA, HBV e antigen, and HBsAg production. A monoclonal antibody (MAb), MA18/7, directed against the pre-S1 domain of the large HBs protein, which has been shown to neutralize infectivity of HBV for primary human hepatocytes, also blocked infection of primary Tupaia hepatocytes. MAbs against the pre-S2 domain of HBs inhibited infection only partially, whereas an S MAb and polyvalent anti-HBs antibodies neutralized infection completely. Thus, both pre-S1 and S antigens are necessary for infection in the tupaia. Using subviral particles, >70% of primary Tupaia hepatocytes are capable of specific binding of pre-S1-rich HBsAg, showing localization in distinct membrane areas. The data show that the early steps of HBV infection in Tupaia hepatocyte cultures are comparable to those in the human system.
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Affiliation(s)
- Dieter Glebe
- Institute of Medical Virology. Institute of Anatomy and Cell Biology, Justus Liebig University Giessen, 35392 Giessen, Germany.
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Hourioux C, Kneser J, Bruss V. In vitro expression of human hepatitis B virus genomes carrying woodchuck hepatitis virus pre-S1 sequences. Intervirology 2003; 45:233-6. [PMID: 12566705 DOI: 10.1159/000067913] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
Many hepatitis B virus (HBV) in vivo experiments are unfeasible because this virus infects only humans. Former studies demonstrated that the pre-S1 domain of the viral envelope protein L determines host range. Therefore, we tried to generate HBV recombinants which might be able to infect woodchucks by exchanging different portions of the pre-S1 encoding gene with homologous parts from the related woodchuck hepatitis virus (WHV) in a cloned HBV genome. In 6 mutants, 11-92 N-terminal HBV pre-S1 codons were replaced by 20-120 codons from WHV. Four mutants carried C-terminal substitutions. The pre-S1 region overlaps with the viral polymerase gene, which is therefore also affected. After transfection of Huh7 cells, the DNA polymerase activity in cytoplasmic nucleocapsids was found to be only slightly affected. All mutants except for the largest C-terminal substitution allowed virion formation. Only the smallest N- and C-terminal substitutions had a wild-type phenotype. The remaining 7 variants allowed virion yields between 5 and 50% of that of the wild type. This demonstrated that substitution of up to 92 pre-S1 codons in an HBV genome with up to 120 codons from WHV pre- S1 was compatible with DNA replication and virion formation. Some recombinant viruses might be able to grow in woodchucks.
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Prassolov A, Hohenberg H, Kalinina T, Schneider C, Cova L, Krone O, Frölich K, Will H, Sirma H. New hepatitis B virus of cranes that has an unexpected broad host range. J Virol 2003; 77:1964-76. [PMID: 12525630 PMCID: PMC140978 DOI: 10.1128/jvi.77.3.1964-1976.2003] [Citation(s) in RCA: 51] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
All hepadnaviruses known so far have a very limited host range, restricted to their natural hosts and a few closely related species. This is thought to be due mainly to sequence divergence in the large envelope protein and species-specific differences in host components essential for virus propagation. Here we report an infection of cranes with a novel hepadnavirus, designated CHBV, that has an unexpectedly broad host range and is only distantly evolutionarily related to avihepadnaviruses of related hosts. Direct DNA sequencing of amplified CHBV DNA as well a sequencing of cloned viral genomes revealed that CHBV is most closely related to, although distinct from, Ross' goose hepatitis B virus (RGHBV) and slightly less closely related to duck hepatitis B virus (DHBV). Phylogenetically, cranes are very distant from geese and ducks and are most closely related to herons and storks. Naturally occurring hepadnaviruses in the last two species are highly divergent in sequence from RGHBV and DHBV and do not infect ducks or do so only marginally. In contrast, CHBV from crane sera and recombinant CHBV produced from LMH cells infected primary duck hepatocytes almost as efficiently as DHBV did. This is the first report of a rather broad host range of an avihepadnavirus. Our data imply either usage of similar or identical entry pathways and receptors by DHBV and CHBV, unusual host and virus adaptation mechanisms, or divergent evolution of the host genomes and cellular components required for virus propagation.
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Affiliation(s)
- Alexej Prassolov
- Heinrich Pette Institute of Experimental Virology and Immunology, Hamburg. Institute of Zoo and Wildlife Research, Berlin, Germany
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Gripon P, Rumin S, Urban S, Le Seyec J, Glaise D, Cannie I, Guyomard C, Lucas J, Trepo C, Guguen-Guillouzo C. Infection of a human hepatoma cell line by hepatitis B virus. Proc Natl Acad Sci U S A 2002; 99:15655-60. [PMID: 12432097 PMCID: PMC137772 DOI: 10.1073/pnas.232137699] [Citation(s) in RCA: 984] [Impact Index Per Article: 42.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Among numerous established human hepatoma cell lines, none has been shown susceptible to hepatitis B virus (HBV) infection. We describe here a cell line, called HepaRG, which exhibits hepatocyte-like morphology, expresses specific hepatocyte functions, and supports HBV infection as well as primary cultures of normal human hepatocytes. Differentiation and infectability are maintained only when these cells are cultured in the presence of corticoids and dimethyl sulfoxide. The specificity of this HBV infection model was ascertained by both the neutralization capacity of HBV-envelope protein-specific antibodies and the competition with an envelope-derived peptide. HepaRG cells therefore represent a tool for deciphering the mechanism of HBV entry. Moreover, their close resemblance to normal human hepatocytes makes them suitable for many applications including drug metabolism studies.
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MESH Headings
- Biomarkers
- Carcinoma, Hepatocellular/genetics
- Carcinoma, Hepatocellular/metabolism
- Carcinoma, Hepatocellular/pathology
- Carcinoma, Hepatocellular/virology
- DNA, Viral/isolation & purification
- Dimethyl Sulfoxide/pharmacology
- Female
- Hepatitis B virus/growth & development
- Hepatitis C/pathology
- Hepatocytes/virology
- Humans
- Karyotyping
- Liver/enzymology
- Liver Neoplasms/genetics
- Liver Neoplasms/metabolism
- Liver Neoplasms/pathology
- Liver Neoplasms/virology
- Organ Specificity
- RNA, Messenger/genetics
- RNA, Messenger/isolation & purification
- RNA, Viral/isolation & purification
- Tumor Cells, Cultured/metabolism
- Tumor Cells, Cultured/virology
- Virus Cultivation
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Affiliation(s)
- Philippe Gripon
- Institut National de la Santé et de la Recherche Médicale (INSERM) U522, Hôpital de Pontchaillou, 35033 Rennes, France.
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