|
1
|
Hammel A, Cucos LM, Caras I, Ionescu I, Tucureanu C, Tofan V, Costache A, Onu A, Hoepfner L, Hippler M, Neupert J, Popescu CI, Stavaru C, Branza-Nichita N, Bock R. The red alga Porphyridium as a host for molecular farming: Efficient production of immunologically active hepatitis C virus glycoprotein. Proc Natl Acad Sci U S A 2024; 121:e2400145121. [PMID: 38833465 PMCID: PMC11181018 DOI: 10.1073/pnas.2400145121] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2024] [Accepted: 05/03/2024] [Indexed: 06/06/2024] Open
Abstract
Microalgae are promising production platforms for the cost-effective production of recombinant proteins. We have recently established that the red alga Porphyridium purpureum provides superior transgene expression properties, due to the episomal maintenance of transformation vectors as multicopy plasmids in the nucleus. Here, we have explored the potential of Porphyridium to synthesize complex pharmaceutical proteins to high levels. Testing expression constructs for a candidate subunit vaccine against the hepatitis C virus (HCV), we show that the soluble HCV E2 glycoprotein can be produced in transgenic algal cultures to high levels. The antigen undergoes faithful posttranslational modification by N-glycosylation and is recognized by conformationally selective antibodies, suggesting that it adopts a proper antigenic conformation in the endoplasmic reticulum of red algal cells. We also report the experimental determination of the structure of the N-glycan moiety that is attached to glycosylated proteins in Porphyridium. Finally, we demonstrate the immunogenicity of the HCV antigen produced in red algae when administered by injection as pure protein or by feeding of algal biomass.
Collapse
Affiliation(s)
- Alexander Hammel
- Max-Planck-Institut für Molekulare Pflanzenphysiologie, Department of Organelle Biology, Biotechnology and Molecular Ecophysiology, D-14476Potsdam-Golm, Germany
| | - Lia-Maria Cucos
- Institute of Biochemistry of the Romanian Academy, Department of Viral Glycoproteins, 060031Bucharest, Romania
| | - Iuliana Caras
- ”Cantacuzino” Medico-Military National Research Institute, 050096Bucharest, Romania
| | - Irina Ionescu
- ”Cantacuzino” Medico-Military National Research Institute, 050096Bucharest, Romania
| | - Catalin Tucureanu
- ”Cantacuzino” Medico-Military National Research Institute, 050096Bucharest, Romania
| | - Vlad Tofan
- ”Cantacuzino” Medico-Military National Research Institute, 050096Bucharest, Romania
| | - Adriana Costache
- ”Cantacuzino” Medico-Military National Research Institute, 050096Bucharest, Romania
| | - Adrian Onu
- ”Cantacuzino” Medico-Military National Research Institute, 050096Bucharest, Romania
| | - Lara Hoepfner
- Institute of Plant Biology and Biotechnology, University of Münster, D-48143Münster, Germany
| | - Michael Hippler
- Institute of Plant Biology and Biotechnology, University of Münster, D-48143Münster, Germany
- Institute of Plant Science and Resources, Okayama University, Kurashiki710-0046, Japan
| | - Juliane Neupert
- Max-Planck-Institut für Molekulare Pflanzenphysiologie, Department of Organelle Biology, Biotechnology and Molecular Ecophysiology, D-14476Potsdam-Golm, Germany
| | - Costin-Ioan Popescu
- Institute of Biochemistry of the Romanian Academy, Department of Viral Glycoproteins, 060031Bucharest, Romania
| | - Crina Stavaru
- ”Cantacuzino” Medico-Military National Research Institute, 050096Bucharest, Romania
| | - Norica Branza-Nichita
- Institute of Biochemistry of the Romanian Academy, Department of Viral Glycoproteins, 060031Bucharest, Romania
| | - Ralph Bock
- Max-Planck-Institut für Molekulare Pflanzenphysiologie, Department of Organelle Biology, Biotechnology and Molecular Ecophysiology, D-14476Potsdam-Golm, Germany
- NIBIO, Norwegian Institute of Bioeconomy Research, NO-1431 Ås, Norway
| |
Collapse
|
|
2
|
Zhao Q, He K, Zhang X, Xu M, Zhang X, Li H. Production and immunogenicity of different prophylactic vaccines for hepatitis C virus (Review). Exp Ther Med 2022; 24:474. [PMID: 35761816 PMCID: PMC9214603 DOI: 10.3892/etm.2022.11401] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2022] [Accepted: 05/12/2022] [Indexed: 11/29/2022] Open
Abstract
Hepatitis C virus (HCV) infection is a global health challenge, and prophylactic vaccines are the most effective way to eliminate the infection. To date, numerous forms of preventive vaccines have entered the clinical trial stage, including the virus-like particle (VLP) vaccine, recombinant subunit vaccine, peptide vaccine and nucleic acid vaccine. The rational design makes it easier to obtain specific vaccine structures with a broad spectrum and strong immunogenicity. Different vaccine antigens can evoke different immune responses, including humoral and T-cell immune responses, and can be produced using different expression systems, such as bacteria, yeast, mammals, plants, insects or parasites. Intracellular and insoluble production and a narrow immune spectrum are two difficulties that limit the application of vaccines. The present study summarizes the immunogenicity of different preventive vaccines, evaluates the characteristics of different expression systems used for vaccine production, and analyzes the strategies to enhance the secretion and immune spectrum of vaccine proteins.
Collapse
Affiliation(s)
- Qianqian Zhao
- Microbiology Department, Jinan Central Hospital Affiliated to Shandong First Medical University, Jinan, Shandong 250013, P.R. China
| | - Kun He
- Microbiology Department, Jinan Central Hospital Affiliated to Shandong First Medical University, Jinan, Shandong 250013, P.R. China
| | - Xiuhua Zhang
- Key Laboratory of Biological Drugs, Shandong Academy of Pharmaceutical Science, Jinan, Shandong 250101, P.R. China
| | - Mingjie Xu
- Microbiology Department, Jinan Central Hospital Affiliated to Shandong First Medical University, Jinan, Shandong 250013, P.R. China
| | - Xiuping Zhang
- Microbiology Department, Jinan Central Hospital Affiliated to Shandong First Medical University, Jinan, Shandong 250013, P.R. China
| | - Huanjie Li
- Medical Research and Laboratory Diagnostic Center, Jinan Central Hospital, Cheeloo College of Medicine, Shandong University, Jinan, Shandong 250013, P.R. China
| |
Collapse
|
|
3
|
Dobrica M, van Eerde A, Tucureanu C, Onu A, Paruch L, Caras I, Vlase E, Steen H, Haugslien S, Alonzi D, Zitzmann N, Bock R, Dubuisson J, Popescu C, Stavaru C, Liu Clarke J, Branza‐Nichita N. Hepatitis C virus E2 envelope glycoprotein produced in Nicotiana benthamiana triggers humoral response with virus-neutralizing activity in vaccinated mice. PLANT BIOTECHNOLOGY JOURNAL 2021; 19:2027-2039. [PMID: 34002936 PMCID: PMC8486241 DOI: 10.1111/pbi.13631] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/23/2020] [Revised: 04/27/2021] [Accepted: 05/13/2021] [Indexed: 05/03/2023]
Abstract
Chronic infection with hepatitis C virus (HCV) remains a leading cause of liver-related pathologies and a global health problem, currently affecting more than 71 million people worldwide. The development of a prophylactic vaccine is much needed to complement the effective antiviral treatment available and achieve HCV eradication. Current strategies focus on increasing the immunogenicity of the HCV envelope glycoprotein E2, the major target of virus-neutralizing antibodies, by testing various expression systems or manipulating the protein conformation and the N-glycosylation pattern. Here we report the first evidence of successful production of the full-length HCV E2 glycoprotein in Nicotiana benthamiana, by using the Agrobacterium-mediated transient expression technology. Molecular and functional analysis showed that the viral protein was correctly processed in plant cells and achieved the native folding required for binding to CD81, one of the HCV receptors. N-glycan analysis of HCV-E2 produced in N. benthamiana and mammalian cells indicated host-specific trimming of mannose residues and possibly, protein trafficking. Notably, the plant-derived viral antigen triggered a significant immune response in vaccinated mice, characterized by the presence of antibodies with HCV-neutralizing activity. Together, our study demonstrates that N. benthamiana is a viable alternative to costly mammalian cell cultures for the expression of complex viral antigens and supports the use of plants as cost-effective production platforms for the development of HCV vaccines.
Collapse
Affiliation(s)
| | | | - Catalin Tucureanu
- Cantacuzino” Medico‐Military National Research InstituteBucharestRomania
| | - Adrian Onu
- Cantacuzino” Medico‐Military National Research InstituteBucharestRomania
| | - Lisa Paruch
- NIBIO ‐ Norwegian Institute of Bioeconomy ResearchÅsNorway
| | - Iuliana Caras
- Cantacuzino” Medico‐Military National Research InstituteBucharestRomania
| | - Ene Vlase
- Cantacuzino” Medico‐Military National Research InstituteBucharestRomania
| | - Hege Steen
- NIBIO ‐ Norwegian Institute of Bioeconomy ResearchÅsNorway
| | | | - Dominic Alonzi
- Oxford Glycobiology InstituteDepartment of BiochemistryUniversity of OxfordOxfordUK
| | - Nicole Zitzmann
- Oxford Glycobiology InstituteDepartment of BiochemistryUniversity of OxfordOxfordUK
| | - Ralph Bock
- Max Planck Institute of Molecular Plant PhysiologyPotsdam‐GolmGermany
| | - Jean Dubuisson
- Université LilleCNRSINSERMCHU LilleInstitut Pasteur de LilleU1019‐UMR 9017‐CIIL‐Center for Infection and Immunity of LilleLilleFrance
| | | | - Crina Stavaru
- Cantacuzino” Medico‐Military National Research InstituteBucharestRomania
| | | | | |
Collapse
|
|
4
|
Abstract
Pestiviruses are members of the family Flaviviridae, a group of enveloped viruses that bud at intracellular membranes. Pestivirus particles contain three glycosylated envelope proteins, Erns, E1, and E2. Among them, E1 is the least characterized concerning both biochemical features and function. E1 from bovine viral diarrhea virus (BVDV) strain CP7 was analyzed with regard to its intracellular localization and membrane topology. Here, it is shown that even in the absence of other viral proteins, E1 is not secreted or expressed at the cell surface but localizes predominantly in the endoplasmic reticulum (ER). Using engineered chimeric transmembrane domains with sequences from E1 and vesicular stomatitis virus G protein, the E1 ER-retention signal could be narrowed down to six fully conserved polar residues in the middle part of the transmembrane domain of E1. Retention was observed even when several of these polar residues were exchanged for alanine. Mutations with a strong impact on E1 retention prevented recovery of infectious viruses when tested in the viral context. Analysis of the membrane topology of E1 before and after the signal peptide cleavage via a selective permeabilization and an in vivo labeling approach revealed that mature E1 is a typical type I transmembrane protein with a single span transmembrane anchor at its C terminus, whereas it adopts a hairpin-like structure with the C terminus located in the ER lumen when the precleavage situation is mimicked by blocking the cleavage site between E1 and E2. IMPORTANCE The shortage of specific antibodies against E1, making detection and further analysis of E1 difficult, resulted in a lack of knowledge on E1 compared to Erns and E2 with regard to biosynthesis, structure, and function. It is known that pestiviruses bud intracellularly. Here, we show that E1 contains its own ER retention signal: six fully conserved polar residues in the middle part of the transmembrane domain are shown to be the determinants for ER retention of E1. Moreover, those six polar residues could serve as a functional group that intensely affect the generation of infectious viral particles. In addition, the membrane topology of E1 has been determined. In this context, we also identified dynamic changes in membrane topology of E1 with the carboxy terminus located on the luminal side of the ER in the precleavage state and relocation of this sequence upon signal peptidase cleavage. Our work provides the first systematic analysis of the pestiviral E1 protein with regard to its biochemical and functional characteristics.
Collapse
|
|
5
|
Abstract
Liver cancer is a global problem and hepatocellular carcinoma (HCC) accounts for about 85% of this cancer. In the USA, etiologies and risk factors for HCC include chronic hepatitis C virus (HCV) infection, diabetes, non-alcoholic steatohepatitis (NASH), obesity, excessive alcohol drinking, exposure to tobacco smoke, and genetic factors. Chronic HCV infection appears to be associated with about 30% of HCC. Chronic HCV infection induces multistep changes in liver, involving metabolic disorders, steatosis, cirrhosis and HCC. Liver carcinogenesis requires initiation of neoplastic clones, and progression to clinically diagnose malignancy. Tumor progression associates with profound exhaustion of tumor-antigen-specific CD8+T cells, and accumulation of PD-1hi CD8+T cells and Tregs. In this chapter, we provide a brief description of HCV and environmental/genetic factors, immune regulation, and highlight mechanisms of HCV associated HCC. We also underscore HCV treatment and recent paradigm of HCC progression, highlighted the current treatment and potential future therapeutic opportunities.
Collapse
|
|
6
|
Lee JY, Cortese M, Haselmann U, Tabata K, Romero-Brey I, Funaya C, Schieber NL, Qiang Y, Bartenschlager M, Kallis S, Ritter C, Rohr K, Schwab Y, Ruggieri A, Bartenschlager R. Spatiotemporal Coupling of the Hepatitis C Virus Replication Cycle by Creating a Lipid Droplet- Proximal Membranous Replication Compartment. Cell Rep 2020; 27:3602-3617.e5. [PMID: 31216478 DOI: 10.1016/j.celrep.2019.05.063] [Citation(s) in RCA: 81] [Impact Index Per Article: 16.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2019] [Revised: 04/05/2019] [Accepted: 05/17/2019] [Indexed: 02/08/2023] Open
Abstract
The hepatitis C virus (HCV) is a major cause of chronic liver disease, affecting around 71 million people worldwide. Viral RNA replication occurs in a membranous compartment composed of double-membrane vesicles (DMVs), whereas virus particles are thought to form by budding into the endoplasmic reticulum (ER). It is unknown how these steps are orchestrated in space and time. Here, we established an imaging system to visualize HCV structural and replicase proteins in live cells and with high resolution. We determined the conditions for the recruitment of viral proteins to putative assembly sites and studied the dynamics of this event and the underlying ultrastructure. Most notable was the selective recruitment of ER membranes around lipid droplets where structural proteins and the viral replicase colocalize. Moreover, ER membranes wrapping lipid droplets were decorated with double membrane vesicles, providing a topological map of how HCV might coordinate the steps of viral replication and virion assembly.
Collapse
Affiliation(s)
- Ji-Young Lee
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 344, 69120 Heidelberg, Germany; German Center for Infection Research, Heidelberg Partner Site, Im Neuenheimer Feld 344, 69120 Heidelberg, Germany
| | - Mirko Cortese
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 344, 69120 Heidelberg, Germany
| | - Uta Haselmann
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 344, 69120 Heidelberg, Germany
| | - Keisuke Tabata
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 344, 69120 Heidelberg, Germany
| | - Inés Romero-Brey
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 344, 69120 Heidelberg, Germany
| | - Charlotta Funaya
- Electron Microscopy Core Facility, Heidelberg University, 69120 Heidelberg, Germany
| | - Nicole L Schieber
- Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany
| | - Yu Qiang
- Biomedical Computer Vision Group, Heidelberg University, BIOQUANT, IPMB, and DKFZ Heidelberg, Im Neuenheimer Feld 267, 69120 Heidelberg, Germany
| | - Marie Bartenschlager
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 344, 69120 Heidelberg, Germany
| | - Stephanie Kallis
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 344, 69120 Heidelberg, Germany
| | - Christian Ritter
- Biomedical Computer Vision Group, Heidelberg University, BIOQUANT, IPMB, and DKFZ Heidelberg, Im Neuenheimer Feld 267, 69120 Heidelberg, Germany
| | - Karl Rohr
- Biomedical Computer Vision Group, Heidelberg University, BIOQUANT, IPMB, and DKFZ Heidelberg, Im Neuenheimer Feld 267, 69120 Heidelberg, Germany
| | - Yannick Schwab
- Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany; Electron Microscopy Core Facility, European Molecular Biology Laboratory, 69117 Heidelberg, Germany
| | - Alessia Ruggieri
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 344, 69120 Heidelberg, Germany
| | - Ralf Bartenschlager
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 344, 69120 Heidelberg, Germany; German Center for Infection Research, Heidelberg Partner Site, Im Neuenheimer Feld 344, 69120 Heidelberg, Germany; Division of Virus-Associated Carcinogenesis, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 242, Heidelberg, Germany.
| |
Collapse
|
|
7
|
Collett S, Torresi J, Earnest-Silveira L, Christiansen D, Elbourne A, Ramsland PA. Probing and pressing surfaces of hepatitis C virus-like particles. J Colloid Interface Sci 2019; 545:259-268. [DOI: 10.1016/j.jcis.2019.03.022] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2019] [Revised: 03/07/2019] [Accepted: 03/09/2019] [Indexed: 02/09/2023]
|
|
8
|
Radtke C, Tews BA. Retention and topology of the bovine viral diarrhea virus glycoprotein E2. J Gen Virol 2017; 98:2482-2494. [PMID: 28874234 DOI: 10.1099/jgv.0.000912] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Pestiviruses are enveloped viruses that bud intracellularly. They have three envelope glycoproteins, Erns, E1, and E2. E2 is the receptor binding protein and the main target for neutralizing antibodies. Both Erns and E2 are retained intracellularly. Here, E2 of the bovine viral diarrhea virus (BVDV) strain CP7 was used to study the membrane topology and intracellular localization of the protein. E2 is localized in the ER and there was no difference between E2 expressed alone or in the context of the viral polyprotein. The mature E2 protein was found to possess a single span transmembrane anchor. For the mapping of a retention signal CD72-E2 fusion proteins, as well as E2 alone were analysed. This confirmed the importance of the transmembrane domain and arginine 355 for intracellular retention, but also revealed a modulating effect on retention through the cytoplasmic tail of the E2 protein, especially through glutamine 370. Mutants with a strong impact on retention were tested in the viral context and we were able to rescue BVDV with certain mutations that in E2 alone impaired intracellular retention and lead to export of E2 to the cells surface.
Collapse
Affiliation(s)
- Christina Radtke
- Institute of Immunology, Friedrich-Loeffler-Institut, Federal Research Institute of Animal Health, Südufer 10, 17493 Greifswald - Insel Riems, Germany.,Present address: Department of Pharmacology, University Medicine of Greifswald, Center of Drug Absorption and Transport (C_DAT), Felix-Hausdorff Straße 3, 17487 Greifswald, Germany
| | - Birke Andrea Tews
- Institute of Immunology, Friedrich-Loeffler-Institut, Federal Research Institute of Animal Health, Südufer 10, 17493 Greifswald - Insel Riems, Germany
| |
Collapse
|
|
9
|
Immunogenicity and functional characterization of Leishmania-derived hepatitis C virus envelope glycoprotein complex. Sci Rep 2016; 6:30627. [PMID: 27481352 PMCID: PMC4969751 DOI: 10.1038/srep30627] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2016] [Accepted: 07/06/2016] [Indexed: 02/07/2023] Open
Abstract
Hepatitis C virus (HCV) envelope glycoproteins E1 and E2 are the main inducers of a cross-neutralizing antibody response which plays an important role in the early phase of viral infection. Correctly folded and immunologically active E1E2 complex can be expressed in mammalian cells, though the production process might still prove restrictive, even if the immunological response of a vaccine candidate is positive. Here, we report a characterization and immunogenicity study of a full-length (fE1E2) and soluble version of the E1E2 complex (tE1E2) from genotype 1a, successfully expressed in the cells of Leishmania tarentolae. In a functional study, we confirmed the binding of both Leishmania-derived E1E2 complexes to the CD-81 receptor and the presence of the major epitopes participating in a neutralizing antibody response. Both complexes were proved to be highly immunogenic in mice and elicited neutralizing antibody response. Moreover, cross-reactivity of the mouse sera was detected for all tested HCV genotypes with the highest signal intensity observed for genotypes 1a, 1b, 5 and 6. Since the development of a prophylactic vaccine against HCV is still needed to control the global infection, our Leishmania-derived E1E2 glycoproteins could be considered a potential cost-effective vaccine candidate.
Collapse
|
|
10
|
Ren Y, Min YQ, Liu M, Chi L, Zhao P, Zhang XL. N-glycosylation-mutated HCV envelope glycoprotein complex enhances antigen-presenting activity and cellular and neutralizing antibody responses. Biochim Biophys Acta Gen Subj 2016; 1860:1764-75. [DOI: 10.1016/j.bbagen.2015.08.007] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2015] [Revised: 08/07/2015] [Accepted: 08/08/2015] [Indexed: 02/08/2023]
|
|
11
|
Beaumont E, Roch E, Chopin L, Roingeard P. Hepatitis C Virus E1 and E2 Proteins Used as Separate Immunogens Induce Neutralizing Antibodies with Additive Properties. PLoS One 2016; 11:e0151626. [PMID: 26966906 PMCID: PMC4788456 DOI: 10.1371/journal.pone.0151626] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2016] [Accepted: 03/01/2016] [Indexed: 02/07/2023] Open
Abstract
Various strategies involving the use of hepatitis C virus (HCV) E1 and E2 envelope glycoproteins as immunogens have been developed for prophylactic vaccination against HCV. However, the ideal mode of processing and presenting these immunogens for effective vaccination has yet to be determined. We used our recently described vaccine candidate based on full-length HCV E1 or E2 glycoproteins fused to the heterologous hepatitis B virus S envelope protein to compare the use of the E1 and E2 proteins as separate immunogens with their use as the E1E2 heterodimer, in terms of immunogenetic potential and the capacity to induce neutralizing antibodies. The specific anti-E1 and anti-E2 antibody responses induced in animals immunized with vaccine particles harboring the heterodimer were profoundly impaired with respect to those in animals immunized with particles harboring E1 and E2 separately. Moreover, the anti-E1 and anti-E2 antibodies had additive neutralizing properties that increase the cross-neutralization of heterologous strains of various HCV genotypes, highlighting the importance of including both E1 and E2 in the vaccine for an effective vaccination strategy. Our study has important implications for the optimization of HCV vaccination strategies based on HCV envelope proteins, regardless of the platform used to present these proteins to the immune system.
Collapse
Affiliation(s)
- Elodie Beaumont
- INSERM U966, Université François Rabelais and CHRU de Tours, Tours, France
| | - Emmanuelle Roch
- INSERM U966, Université François Rabelais and CHRU de Tours, Tours, France
| | - Lucie Chopin
- INSERM U966, Université François Rabelais and CHRU de Tours, Tours, France
| | - Philippe Roingeard
- INSERM U966, Université François Rabelais and CHRU de Tours, Tours, France
| |
Collapse
|
|
12
|
Sato Y. [Structure and Function of a Novel Class of High Mannose-binding Proteins with Anti-viral or Anti-tumor Activity]. YAKUGAKU ZASSHI 2015; 135:1281-9. [PMID: 26521877 DOI: 10.1248/yakushi.15-00217] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The recently discovered high mannose (HM)-binding lectin family in lower organisms such as bacteria, cyanobacteria, and marine algae represents a novel class of anti-viral or anti-tumor compounds. This lectin family shows unique carbohydrate binding properties with exclusive high specificity for HM glycans with core trisaccharide comprising Manα(1-3)Manα(1-6)Man at the D2 arm. At low nanomolar levels, these lectins exhibit potent antiviral activity against HIV and influenza viruses through the recognition of HM glycans on virus spike glycoproteins. In addition, some of these lectins, such as bacterial PFL, show cytotoxicity for various cancer cells at low micromolar levels. Cell surface molecules to which PFL bound were identified as integrin alpha 2 and epidermal growth factor receptor (EGFR) by peptide mass finger printing with MALDI-TOF MS. Upon PFL binding, these molecules were rapidly internalized to cytoplasm. EGFR was time dependently degraded in the presence of PFL, and this process was largely responsible for autophagy. Furthermore, PFL sensitizes cancer cells to the EGFR kinase inhibitor, gefitinib. In vivo experiments showed that intratumoral injection of PFL significantly inhibited the growth of tumors in nude mice. PFL-mediated down regulation of integrin/EGFR ultimately contributed to the inhibition of tumor growth both in vitro and in vivo. Thus, the novel anti-cancer mechanism of PFL suggests that this lectin is potentially useful as an anti-cancer drug or as an adjuvant for other drugs. This class of proteins will likely have beneficial impact as a tool for biochemical and biomedical research because of its unique carbohydrate specificity and various biological activities.
Collapse
Affiliation(s)
- Yuichiro Sato
- Department of Medical Pharmacy, Faculty of Pharmacy, Yasuda Women's University
| |
Collapse
|
|
13
|
Sato Y, Morimoto K, Kubo T, Sakaguchi T, Nishizono A, Hirayama M, Hori K. Entry Inhibition of Influenza Viruses with High Mannose Binding Lectin ESA-2 from the Red Alga Eucheuma serra through the Recognition of Viral Hemagglutinin. Mar Drugs 2015; 13:3454-65. [PMID: 26035023 PMCID: PMC4483639 DOI: 10.3390/md13063454] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2015] [Revised: 05/07/2015] [Accepted: 05/22/2015] [Indexed: 11/16/2022] Open
Abstract
Lectin sensitivity of the recent pandemic influenza A virus (H1N1-2009) was screened for 12 lectins with various carbohydrate specificity by a neutral red dye uptake assay with MDCK cells. Among them, a high mannose (HM)-binding anti-HIV lectin, ESA-2 from the red alga Eucheuma serra, showed the highest inhibition against infection with an EC50 of 12.4 nM. Moreover, ESA-2 exhibited a wide range of antiviral spectrum against various influenza strains with EC50s of pico molar to low nanomolar levels. Besides ESA-2, HM-binding plant lectin ConA, fucose-binding lectins such as fungal AOL from Aspergillus oryzae and AAL from Aleuria aurantia were active against H1N1-2009, but the potency of inhibition was of less magnitude compared with ESA-2. Direct interaction between ESA-2 and a viral envelope glycoprotein, hemagglutinin (HA), was demonstrated by ELISA assay. This interaction was effectively suppressed by glycoproteins bearing HM-glycans, indicating that ESA-2 binds to the HA of influenza virus through HM-glycans. Upon treatment with ESA-2, no viral antigens were detected in the host cells, indicating that ESA-2 inhibited the initial steps of virus entry into the cells. ESA-2 would thus be useful as a novel microbicide to prevent penetration of viruses such as HIV and influenza viruses to the host cells.
Collapse
Affiliation(s)
- Yuichiro Sato
- Faculty of Pharmacy, Yasuda Women's University, 6-13-1 Yasuhigashi, Asaminami-Ku, Hiroshima 731-0153, Japan.
| | - Kinjiro Morimoto
- Faculty of Pharmacy, Yasuda Women's University, 6-13-1 Yasuhigashi, Asaminami-Ku, Hiroshima 731-0153, Japan.
| | - Takanori Kubo
- Faculty of Pharmacy, Yasuda Women's University, 6-13-1 Yasuhigashi, Asaminami-Ku, Hiroshima 731-0153, Japan.
| | - Takemasa Sakaguchi
- Department of Virology, Institute of Biomedical & Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-Ku, Hiroshima 734-8551, Japan.
| | - Akira Nishizono
- Department of Microbiology, Faculty of Medicine, Oita University, 1-1 Idaigaoka, Hasama-machi, Yufu, Oita 879-5593, Japan.
| | - Makoto Hirayama
- Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima 739-8528, Japan.
| | - Kanji Hori
- Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima 739-8528, Japan.
| |
Collapse
|
|
14
|
Abstract
The past decade has witnessed steady and rapid progress in HCV research, which has led to the recent breakthrough in therapies against this significant human pathogen. Yet a deeper understanding of the life cycle of the virus is required to develop more affordable treatments and to advance vaccine design. HCV entry presents both a challenge for scientific research and an opportunity for alternative intervention approaches, owning to its highly complex nature and the myriad of players involved. More than half a dozen cellular proteins are implicated in HCV entry; and a more definitive picture regarding the structures of the glycoproteins is emerging. A role of apolipoproteins in HCV entry has also been established. Still, major questions remain, and the answers to these, which we summarize in this review, will hopefully close the gaps in our understanding and complete the puzzle that is HCV entry.
Collapse
Affiliation(s)
- Sarah C Ogden
- Department of Biological Science, Florida State University, Tallahassee, FL 32306-4295, USA
| | - Hengli Tang
- Department of Biological Science, Florida State University, Tallahassee, FL 32306-4295, USA ; Institute of Health Sciences, Anhui University, Hefei, 230601, PR China
| |
Collapse
|
|
15
|
Douglas DN, Kneteman NM. Generation of improved mouse models for the study of hepatitis C virus. Eur J Pharmacol 2015; 759:313-25. [PMID: 25814250 DOI: 10.1016/j.ejphar.2015.03.022] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2015] [Revised: 03/06/2015] [Accepted: 03/12/2015] [Indexed: 12/15/2022]
Abstract
Approximately 3% of the world׳s population suffers from chronic infections with hepatitis C virus (HCV). Although current treatment regimes are capable of effectively eradicating HCV infection from these patients, the cost of these combinations of direct-acting antivirals are prohibitive. Approximately 80% of untreated chronic HCV carriers will be at high risk for developing severe liver disease, including fibrosis, cirrhosis, and hepatocellular carcinoma. A vaccine is urgently needed to lessen this global burden. Besides humans, HCV infection can be experimentally transmitted to chimpanzees, and this is the best model for studies of HCV infection and related innate and adaptive immune responses. Although the chimpanzee model yielded valuable insight, limited availability, high cost and ethical considerations limit their utility. The only small animal models of robust HCV infection are highly immunodeficient mice with human chimeric livers. However, these mice cannot be used to study adaptive immune responses and therefore a more relevant animal model is needed to assist in vaccine development. Novel strains of immunodeficient mice have been developed that allow for the engraftment of human hepatopoietic stem cells, as well as functional human lymphoid cells and tissues, effectively creating human immune systems in otherwise immunodeficient mice. These humanized mice are rapidly emerging as pre-clinical bridges for numerous pathogens that, like HCV, only cause infectious disease in humans. This review highlights the potential these new models have for changing the current landscape for HCV research and vaccine development.
Collapse
Affiliation(s)
- Donna N Douglas
- Department of Surgery, University of Alberta, Edmonton, Alberta, Canada T6G 2E1; Li Ka Shing Institute of Virology, University of Alberta, Edmonton, Alberta, Canada T6G 2E1.
| | - Norman M Kneteman
- Department of Surgery, University of Alberta, Edmonton, Alberta, Canada T6G 2E1; Li Ka Shing Institute of Virology, University of Alberta, Edmonton, Alberta, Canada T6G 2E1; KMT Hepatech Inc., Edmonton, Alberta, Canada T6G 2M9
| |
Collapse
|
|
16
|
Cheung RCF, Wong JH, Pan W, Chan YS, Yin C, Dan X, Ng TB. Marine lectins and their medicinal applications. Appl Microbiol Biotechnol 2015; 99:3755-73. [PMID: 25794876 PMCID: PMC7080081 DOI: 10.1007/s00253-015-6518-0] [Citation(s) in RCA: 51] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2014] [Revised: 03/01/2015] [Accepted: 03/02/2015] [Indexed: 12/16/2022]
Abstract
Marine organisms have been extensively explored for the last several decades as potential sources of novel biologically active compounds, and extensive research has been conducted on lectins. Lectins derived from marine organisms are structurally diverse and also differ from those identified from terrestrial organisms. Marine lectins appear to be particularly useful in some biological applications. They seem to induce negligible immunogenicity because they have a relatively small size, are more stable due to extensive disulfide bridge formation, and have high specificity for complex glyco-conjugates and carbohydrates instead of simple sugars. It is clear that many of them have not yet been extensively studied when compared with their terrestrial counterparts. Marine lectins can be used to design and develop new potentially useful therapeutic agents. This review encompasses recent research on the isolation and identification of marine lectins with potential value in medicinal applications.
Collapse
Affiliation(s)
- Randy Chi Fai Cheung
- School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China
| | | | | | | | | | | | | |
Collapse
|
|
17
|
Hamed MR, Brown RJ, Zothner C, Urbanowicz RA, Mason CP, Krarup A, McClure CP, Irving WL, Ball JK, Harris M, Hickling TP, Tarr AW. Recombinant human L-ficolin directly neutralizes hepatitis C virus entry. J Innate Immun 2014; 6:676-84. [PMID: 24854201 PMCID: PMC6741592 DOI: 10.1159/000362209] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2013] [Revised: 03/16/2014] [Accepted: 03/16/2014] [Indexed: 12/25/2022] Open
Abstract
L-ficolin is a soluble pattern recognition molecule expressed by the liver that contributes to innate immune defense against microorganisms. It is well described that binding of L-ficolin to specific pathogen-associated molecular patterns activates the lectin complement pathway, resulting in opsonization and lysis of pathogens. In this study, we demonstrated that in addition to this indirect effect, L-ficolin has a direct neutralizing effect against hepatitis C virus (HCV) entry. Specific, dose-dependent binding of recombinant L-ficolin to HCV glycoproteins E1 and E2 was observed. This interaction was inhibited by soluble L-ficolin ligands. Interaction of L-ficolin with E1 and E2 potently inhibited entry of retroviral pseudoparticles bearing these glycoproteins. L-ficolin also inhibited entry of cell-cultured HCV in a calcium-dependent manner. Neutralizing concentrations of L-ficolin were found to be circulating in the serum of HCV-infected individuals. This is the first description of direct neutralization of HCV entry by a ficolin and highlights a novel role for L-ficolin as a virus entry inhibitor.
Collapse
Affiliation(s)
- Mohamed R. Hamed
- School of Life Sciences, and Nottingham Digestive Diseases Biomedical Research Unit, University of Nottingham, Nottingham, UK
- Medical Microbiology and Immunology Department, Faculty of Medicine, Mansoura University, Mansoura, Egypt
| | - Richard J.P. Brown
- School of Life Sciences, and Nottingham Digestive Diseases Biomedical Research Unit, University of Nottingham, Nottingham, UK
| | - Carsten Zothner
- School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK
| | - Richard A. Urbanowicz
- School of Life Sciences, and Nottingham Digestive Diseases Biomedical Research Unit, University of Nottingham, Nottingham, UK
| | - Christopher P. Mason
- School of Life Sciences, and Nottingham Digestive Diseases Biomedical Research Unit, University of Nottingham, Nottingham, UK
| | - Anders Krarup
- Biochemistry Department, University of Oxford, Oxford, UK
| | - C. Patrick McClure
- School of Life Sciences, and Nottingham Digestive Diseases Biomedical Research Unit, University of Nottingham, Nottingham, UK
| | - William L. Irving
- School of Life Sciences, and Nottingham Digestive Diseases Biomedical Research Unit, University of Nottingham, Nottingham, UK
| | - Jonathan K. Ball
- School of Life Sciences, and Nottingham Digestive Diseases Biomedical Research Unit, University of Nottingham, Nottingham, UK
| | - Mark Harris
- School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK
| | - Timothy P. Hickling
- School of Life Sciences, and Nottingham Digestive Diseases Biomedical Research Unit, University of Nottingham, Nottingham, UK
| | - Alexander W. Tarr
- School of Life Sciences, and Nottingham Digestive Diseases Biomedical Research Unit, University of Nottingham, Nottingham, UK
| |
Collapse
|
|
18
|
Incorporation of hepatitis C virus E1 and E2 glycoproteins: the keystones on a peculiar virion. Viruses 2014; 6:1149-87. [PMID: 24618856 PMCID: PMC3970144 DOI: 10.3390/v6031149] [Citation(s) in RCA: 50] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2013] [Revised: 02/21/2014] [Accepted: 02/27/2014] [Indexed: 12/13/2022] Open
Abstract
Hepatitis C virus (HCV) encodes two envelope glycoproteins, E1 and E2. Their structure and mode of fusion remain unknown, and so does the virion architecture. The organization of the HCV envelope shell in particular is subject to discussion as it incorporates or associates with host-derived lipoproteins, to an extent that the biophysical properties of the virion resemble more very-low-density lipoproteins than of any virus known so far. The recent development of novel cell culture systems for HCV has provided new insights on the assembly of this atypical viral particle. Hence, the extensive E1E2 characterization accomplished for the last two decades in heterologous expression systems can now be brought into the context of a productive HCV infection. This review describes the biogenesis and maturation of HCV envelope glycoproteins, as well as the interplay between viral and host factors required for their incorporation in the viral envelope, in a way that allows efficient entry into target cells and evasion of the host immune response.
Collapse
|
|
19
|
Abstract
Hepatitis C Virus (HCV) particles exhibit several unusual properties that are not found in other enveloped RNA viruses, most notably their low buoyant density and interaction with serum lipoproteins. With the advent of systems to grow HCV in cell culture, the molecular basis of HCV particle assembly and release can now be addressed. The process of virus assembly involves protein-protein interactions between viral structural and nonstructural proteins and the coordinated action of host factors. This chapter reviews our current understanding of these interactions and factors.
Collapse
Affiliation(s)
- Brett D Lindenbach
- Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT 06536, USA.
| |
Collapse
|
|
20
|
Beaumont E, Patient R, Hourioux C, Dimier-Poisson I, Roingeard P. Chimeric hepatitis B virus/hepatitis C virus envelope proteins elicit broadly neutralizing antibodies and constitute a potential bivalent prophylactic vaccine. Hepatology 2013; 57:1303-13. [PMID: 23150224 DOI: 10.1002/hep.26132] [Citation(s) in RCA: 51] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/31/2012] [Revised: 10/17/2012] [Accepted: 10/17/2012] [Indexed: 12/12/2022]
Abstract
UNLABELLED The development of a prophylactic vaccine against hepatitis C virus (HCV) has become an important medical priority, because 3-4 million new HCV infections are thought to occur each year worldwide. Hepatitis B virus (HBV) is another major human pathogen, but infections with this virus can be prevented with a safe, efficient vaccine, based on the remarkable ability of the envelope protein (S) of this virus to self-assemble into highly immunogenic subviral particles. Chimeric HBV-HCV envelope proteins in which the N-terminal transmembrane domain of S was replaced with the transmembrane domain of the HCV envelope proteins (E1 or E2) were efficiently coassembled with the wild-type HBV S protein into subviral particles. These chimeric particles presented the full-length E1 and E2 proteins from a genotype 1a virus in an appropriate conformation for formation of the E1-E2 heterodimer. Produced in stably transduced Chinese hamster ovary cells and used to immunize New Zealand rabbits, these particles induced a strong specific antibody (Ab) response against the HCV and HBV envelope proteins in immunized animals. Sera containing anti-E1 or anti-E2 Abs elicited by these particles neutralized infections with HCV pseudoparticles and cell-cultured viruses derived from different heterologous 1a, 1b, 2a, and 3 strains. Moreover, the anti-hepatitis B surface response induced by these chimeric particles was equivalent to the response induced by a commercial HBV vaccine. CONCLUSIONS Our results provide support for approaches based on the development of bivalent HBV-HCV prophylactic vaccine candidates potentially able to prevent initial infection with either of these two hepatotropic viruses.
Collapse
Affiliation(s)
- Elodie Beaumont
- INSERM U966, Université François Rabelais and CHRU de Tours, Tours, France
| | | | | | | | | |
Collapse
|
|
21
|
Beaumont E, Roingeard P. Prospects for prophylactic hepatitis C vaccines based on virus-like particles. Hum Vaccin Immunother 2013; 9:1112-8. [PMID: 23406827 DOI: 10.4161/hv.23900] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
Given the global prevalence and long-term complications of chronic hepatitis C virus (HCV) infection, HCV constitutes one of the greatest challenges to human health of this decade. Considerable efforts have focused on the development of new effective treatments, but about three to four million individuals become infected each year, adding to the world reservoir of HCV infection. The development of a prophylactic vaccine against hepatitis C virus has thus become an important medical priority. Only a few vaccine candidates have progressed to the clinical phase, and published data on both the efficacy and safety of these vaccines are limited, due to many scientific, logistic and bioethic challenges. Fortunately, new innovative vaccine formulations, modes of vaccination and delivery technologies have been developed in recent years. Several preclinical trials of virus-like particle (VLP)-based vaccination strategies are currently underway and have already generated very promising results. In this commentary, we consider the current state of prophylactic HCV vaccines, the hurdles to be overcome in the future and the various VLP-based vaccination approaches currently being developed.
Collapse
Affiliation(s)
- Elodie Beaumont
- 1 INSERM U966; Université François Rabelais and CHRU de Tours; Tours, France
| | | |
Collapse
|
|
22
|
Fichter KM, Ingle NP, McLendon PM, Reineke TM. Polymeric nucleic acid vehicles exploit active interorganelle trafficking mechanisms. ACS NANO 2013; 7:347-64. [PMID: 23234474 PMCID: PMC3586558 DOI: 10.1021/nn304218q] [Citation(s) in RCA: 60] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/21/2023]
Abstract
Materials that self-assemble with nucleic acids into nanocomplexes (e.g. polyplexes) are widely used in many fundamental biological and biomedical experiments. However, understanding the intracellular transport mechanisms of these vehicles remains a major hurdle in their effective usage. Here, we investigate two polycation models, Glycofect (which slowly degrades via hydrolysis) and linear polyethyleneimine (PEI) (which does not rapidly hydrolyze), to determine the impact of polymeric structure on intracellular trafficking. Cells transfected using Glycofect underwent increasing transgene expression over the course of 40 h and remained benign over the course of 7 days. Transgene expression in cells transfected with PEI peaked at 16 h post-transfection and resulted in less than 10% survival after 7 days. While saccharide-containing Glycofect has a higher buffering capacity than PEI, polyplexes created with Glycofect demonstrate more sustained endosomal release, possibly suggesting an additional or alternative delivery mechanism to the classical "proton sponge mechanism". PEI appeared to promote release of DNA from acidic organelles more than Glycofect. Immunofluorescence images indicate that both Glycofect and linear PEI traffic oligodeoxynucleotides to the Golgi and endoplasmic reticulum, which may be a route towards nuclear delivery. However, Glycofect polyplexes demonstrated higher co-localization with the ER than PEI polyplexes, and co-localization experiments indicate the retrograde transport of polyplexes via COP I vesicles from the Golgi to the ER. We conclude that slow release and unique trafficking behaviors of Glycofect polyplexes may be due to the presence of saccharide units and the degradable nature of the polymer, allowing more efficacious and benign delivery.
Collapse
Affiliation(s)
- Katye M. Fichter
- Department Chemistry, Missouri State University, Springfield, MO
| | - Nilesh. P. Ingle
- Department of Chemistry, University of Minnesota-Twin Cities, Minneapolis, MN
| | - Patrick M. McLendon
- Division of Molecular Cardiovascular Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH
| | - Theresa M. Reineke
- Department of Chemistry, University of Minnesota-Twin Cities, Minneapolis, MN
- Corresponding Author. Correspondence should be addressed to Professor Theresa M. Reineke, Department of Chemistry, University of Minnesota-Twin Cities, Minneapolis, MN. Phone: 612-624-8042.
| |
Collapse
|
|
23
|
Vander Heyden AB, Naismith TV, Snapp EL, Hanson PI. Static retention of the lumenal monotopic membrane protein torsinA in the endoplasmic reticulum. EMBO J 2011; 30:3217-31. [PMID: 21785409 DOI: 10.1038/emboj.2011.233] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2011] [Accepted: 06/16/2011] [Indexed: 02/06/2023] Open
Abstract
TorsinA is a membrane-associated enzyme in the endoplasmic reticulum (ER) lumen that is mutated in DYT1 dystonia. How it remains in the ER has been unclear. We report that a hydrophobic N-terminal domain (NTD) directs static retention of torsinA within the ER by excluding it from ER exit sites, as has been previously reported for short transmembrane domains (TMDs). We show that despite the NTD's physicochemical similarity to TMDs, it does not traverse the membrane, defining torsinA as a lumenal monotopic membrane protein and requiring a new paradigm to explain retention. ER retention and membrane association are perturbed by a subset of nonconservative mutations to the NTD, suggesting that a helical structure with defined orientation in the membrane is required. TorsinA preferentially enriches in ER sheets, as might be expected for a lumenal monotopic membrane protein. We propose that the principle of membrane-based protein sorting extends to monotopic membrane proteins, and identify other proteins including the monotopic lumenal enzyme cyclooxygenase 1 (prostaglandin H synthase 1) that share this mechanism of retention with torsinA.
Collapse
Affiliation(s)
- Abigail B Vander Heyden
- Department of Cell Biology and Physiology, Washington University School of Medicine, St Louis, MO, USA
| | | | | | | |
Collapse
|
|
24
|
Miyachi H, Kobayashi Y, Relja B, Fujita N, Iwasa M, Gabazza EC, Takei Y. Effect of suppressor of cytokine signaling on hepcidin production in hepatitis C virus replicon cells. Hepatol Res 2011; 41:364-74. [PMID: 21348906 DOI: 10.1111/j.1872-034x.2011.00777.x] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/24/2023]
Abstract
AIM Hepcidin is a key regulator of systemic iron metabolism and its expression is modulated by hepatitis C virus (HCV) infection. Suppressor of cytokine signaling 1 (SOCS-1) and SOCS-3 act as negative regulators of the Jak/signal transducers and activators of transcription signaling pathway. In this study, we investigated how HCV infection modulates SOCS-1 and SOCS-3 production and how these SOCS proteins affect hepcidin production. METHODS The effects of SOCS-1 and SOCS-3 on hepcidin production were investigated using a complete genome, HCV replicon system. RESULTS Unexpectedly, basal expression levels of hepcidin (HAMP) mRNA and the bioactive form of hepcidin protein, hepcidin-25, were significantly higher in replicon cells. Regardless of HCV infection, STAT3 was activated in response to interleukin-6 (IL-6), but this activation was greater in replicon cells than in cured cells. Basal expression of the SOCS-3 protein was enhanced, but basal expression of SOCS-1 protein was reduced, in replicon cells. Expression of SOCS-3 increased dramatically in response to IL-6 stimulation but expression of SOCS-1 was not induced by IL-6. Interestingly, silencing of SOCS-1 and SOCS-3 gene expression enhanced STAT3 activation and HAMP gene expression. In addition, overexpression of SOCS-1 protein strongly suppressed STAT3 activation and HAMP gene expression. CONCLUSIONS This in vitro study shows that SOCS-3 expression was enhanced but SOCS-1 expression was reduced by HCV infection. The upregulation of hepcidin induced by IL-6 was found to be negatively regulated by SOCS-1 and SOCS-3. The modulation of SOCS1 and SOCS3 in HCV-infected hepatocytes may explain, at least in part, the relative shortage of hepcidin production in CH-C.
Collapse
Affiliation(s)
- Hirohide Miyachi
- Department of Gastroenterology and Hepatology, Mie University Graduate school of Medicine Center for Physical and Mental Health, Mie University Graduate School of Medicine Department of Immunology, Mie University Graduate School of Medicine, Mie, Japan Klinik für Unfall-, Hand- und Wiederherstellungschirurgie, J.W. Goethe-Universität Frankfurt, Frankfurt am Main, Germany
| | | | | | | | | | | | | |
Collapse
|
|
25
|
Sato Y, Morimoto K, Hirayama M, Hori K. High mannose-specific lectin (KAA-2) from the red alga Kappaphycus alvarezii potently inhibits influenza virus infection in a strain-independent manner. Biochem Biophys Res Commun 2011; 405:291-6. [PMID: 21219864 PMCID: PMC7092952 DOI: 10.1016/j.bbrc.2011.01.031] [Citation(s) in RCA: 56] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2010] [Accepted: 01/06/2011] [Indexed: 01/22/2023]
Abstract
The carbohydrate binding profile of the red algal lectin KAA-2 from Kappaphycus alvarezii was evaluated by a centrifugal ultrafiltration–HPLC method using pyridylaminated oligosaccharides. KAA-2 bound exclusively to high mannose type N-glycans, but not to other glycans such as complex type, hybrid type, or the pentasaccharide core of N-glycans. This lectin exhibited a preference for an exposed α1–3 Man on a D2 arm in a similar manner to Eucheuma serra agglutinin (ESA-2), which shows various biological activities, such as anti-HIV and anti-carcinogenic activity. We tested the anti-influenza virus activity of KAA-2 against various strains including the recent pandemic H1N1-2009 influenza virus. KAA-2 inhibited infection of various influenza strains with EC50s of low nanomolar levels. Immunofluorescence microscopy using an anti-influenza antibody demonstrated that the antiviral activity of KAA-2 was exerted by interference with virus entry into host cells. This mechanism was further confirmed by the evidence of direct binding of KAA-2 to a viral envelope protein, hemagglutinin (HA), using an ELISA assay. These results indicate that this lectin would be useful as a novel antiviral reagent for the prevention of infection.
Collapse
Affiliation(s)
- Yuichiro Sato
- Faculty of Pharmacy, Yasuda Women's University, 6-13-1 Yasuhigashi, Asaminami, Hiroshima 731-0153, Japan
| | | | | | | |
Collapse
|
|
26
|
Ma Y, Anantpadma M, Timpe JM, Shanmugam S, Singh SM, Lemon SM, Yi M. Hepatitis C virus NS2 protein serves as a scaffold for virus assembly by interacting with both structural and nonstructural proteins. J Virol 2011; 85:86-97. [PMID: 20962101 PMCID: PMC3014171 DOI: 10.1128/jvi.01070-10] [Citation(s) in RCA: 123] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2010] [Accepted: 10/05/2010] [Indexed: 11/20/2022] Open
Abstract
Many aspects of the assembly of hepatitis C virus (HCV) remain incompletely understood. To characterize the role of NS2 in the production of infectious virus, we determined NS2 interaction partners among other HCV proteins during productive infection. Pulldown assays showed that NS2 forms complexes with both structural and nonstructural proteins, including E1, E2, p7, NS3, and NS5A. Confocal microscopy also demonstrated that NS2 colocalizes with E1, E2, and NS5A in dot-like structures near lipid droplets. However, NS5A did not coprecipitate with E2 and interacted only weakly with NS3 in pulldown assays. Also, there was no demonstrable interaction between p7 and E2 or NS3 in such assays. Therefore, NS2 is uniquely capable of interacting with both structural and nonstructural proteins. Among mutations in p7, NS2, and NS3 that prevent production of infectious virus, only p7 mutations significantly reduced NS2-mediated protein interactions. These p7 mutations altered the intracellular distribution of NS2 and E2 and appeared to modulate the membrane topology of the C-terminal domain of NS2. These results suggest that NS2 acts to coordinate virus assembly by mediating interactions between envelope proteins and NS3 and NS5A within replication complexes adjacent to lipid droplets, where virus particle assembly is thought to occur. p7 may play an accessory role by regulating NS2 membrane topology, which is important for NS2-mediated protein interactions and therefore NS2 function.
Collapse
Affiliation(s)
- Yinghong Ma
- Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Galveston, Texas 77555-0610
| | - Manu Anantpadma
- Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Galveston, Texas 77555-0610
| | - Jennifer M. Timpe
- Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Galveston, Texas 77555-0610
| | - Saravanabalaji Shanmugam
- Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Galveston, Texas 77555-0610
| | - Sher M. Singh
- Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Galveston, Texas 77555-0610
| | - Stanley M. Lemon
- Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Galveston, Texas 77555-0610
| | - MinKyung Yi
- Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Galveston, Texas 77555-0610
| |
Collapse
|
|
27
|
Bivalkar-Mehla S, Vakharia J, Mehla R, Abreha M, Kanwar JR, Tikoo A, Chauhan A. Viral RNA silencing suppressors (RSS): novel strategy of viruses to ablate the host RNA interference (RNAi) defense system. Virus Res 2011; 155:1-9. [PMID: 20951748 PMCID: PMC3042272 DOI: 10.1016/j.virusres.2010.10.003] [Citation(s) in RCA: 81] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2010] [Revised: 09/28/2010] [Accepted: 10/05/2010] [Indexed: 12/13/2022]
Abstract
Pathogenic viruses have developed a molecular defense arsenal for their survival by counteracting the host anti-viral system known as RNA interference (RNAi). Cellular RNAi, in addition to regulating gene expression through microRNAs, also serves as a barrier against invasive foreign nucleic acids. RNAi is conserved across the biological species, including plants, animals and invertebrates. Viruses in turn, have evolved mechanisms that can counteract this anti-viral defense of the host. Recent studies of mammalian viruses exhibiting RNA silencing suppressor (RSS) activity have further advanced our understanding of RNAi in terms of host-virus interactions. Viral proteins and non-coding viral RNAs can inhibit the RNAi (miRNA/siRNA) pathway through different mechanisms. Mammalian viruses having dsRNA-binding regions and GW/WG motifs appear to have a high chance of conferring RSS activity. Although, RSSs of plant and invertebrate viruses have been well characterized, mammalian viral RSSs still need in-depth investigations to present the concrete evidences supporting their RNAi ablation characteristics. The information presented in this review together with any perspective research should help to predict and identify the RSS activity-endowed new viral proteins that could be the potential targets for designing novel anti-viral therapeutics.
Collapse
Affiliation(s)
- Shalmali Bivalkar-Mehla
- Dept of Pathology, Microbiology and Immunology, University of South Carolina School of Medicine, Columbia, SC 29209
| | - Janaki Vakharia
- Dept of Pathology, Microbiology and Immunology, University of South Carolina School of Medicine, Columbia, SC 29209
| | - Rajeev Mehla
- Dept of Pathology, Microbiology and Immunology, University of South Carolina School of Medicine, Columbia, SC 29209
| | - Measho Abreha
- Dept of Pathology, Microbiology and Immunology, University of South Carolina School of Medicine, Columbia, SC 29209
| | - Jagat Rakesh Kanwar
- Laboratory of Immunology and Molecular Biomedical Research (LIMBR), BioDeakin, Institute for Technology & Research Innovation (ITRI), Deakin University, Geelong, Victoria 3217. Australia
| | - Akshay Tikoo
- Medical University of Americas, Charlestown, Nevis, WI
| | - Ashok Chauhan
- Dept of Pathology, Microbiology and Immunology, University of South Carolina School of Medicine, Columbia, SC 29209
| |
Collapse
|
|
28
|
Production of hepatitis C virus lacking the envelope-encoding genes for single-cycle infection by providing homologous envelope proteins or vesicular stomatitis virus glycoproteins in trans. J Virol 2010; 85:2138-47. [PMID: 21159872 DOI: 10.1128/jvi.02313-10] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
Hepatitis C virus (HCV) infection is a major worldwide health problem. The envelope glycoproteins are the major components of viral particles. Here we developed a trans-complementation system that allows the production of infectious HCV particles in whose genome the regions encoding envelope proteins are deleted (HCVΔE). The lack of envelope proteins could be efficiently complemented by the expression of homologous envelope proteins in trans. HCVΔE production could be enhanced significantly by previously described adaptive mutations in NS3 and NS5A. Moreover, HCVΔE could be propagated and passaged in packaging cells stably expressing HCV envelope proteins, resulting in only single-round infection in wild-type cells. Interestingly, we found that vesicular stomatitis virus (VSV) glycoproteins could efficiently rescue the production of HCV lacking endogenous envelope proteins, which no longer required apolipoprotein E for virus production. VSV glycoprotein-mediated viral entry could allow for the bypass of the natural HCV entry process and the delivery of HCV replicon RNA into HCV receptor-deficient cells. Our development provides a new tool for the production of single-cycle infectious HCV particles, which should be useful for studying individual steps of the HCV life cycle and may also provide a new strategy for HCV vaccine development.
Collapse
|
|
29
|
The length of and nonhydrophobic residues in the transmembrane domain of dengue virus envelope protein are critical for its retention and assembly in the endoplasmic reticulum. J Virol 2010; 84:4782-97. [PMID: 20181718 DOI: 10.1128/jvi.01963-09] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023] Open
Abstract
The morphogenesis of many enveloped viruses, in which viral nucleocapsid complex interacts with envelope (E) protein, is known to take place at various sites along the secretory pathway. How viral E protein retains in a particular intracellular organelle for assembly remains incompletely understood. In this study, we investigated determinants in the E protein of dengue virus (DENV) for its retention and assembly in the endoplasmic reticulum (ER). A chimeric experiment between CD4 and DENV precursor membrane/E constructs suggested that the transmembrane domain (TMD) of E protein contains an ER retention signal. Substitutions of three nonhydrophobic residues at the N terminus of the first helix (T1) and at either the N or C terminus of the second helix of the TMD with three hydrophobic residues, as well as an increase in the length of T1, led to the release of chimeric CD4 and E protein from the ER, suggesting that short length and certain nonhydrophobic residues of the TMD are critical for ER retention. The analysis of enveloped viruses assembled at the plasma membrane and of those assembled in the Golgi complex and ER revealed a trend of decreasing length and increasing nonhydrophobic residues of the TMD of E proteins. Taken together, these findings support a TMD-dependent sorting for viral E proteins along the secretory pathway. Moreover, similar mutations introduced into the TMD of DENV E protein resulted in the increased production of virus-like particles (VLPs), suggesting that modifications of TMD facilitate VLP production and have implications for utilizing flaviviral VLPs as serodiagnostic antigens and vaccine candidates.
Collapse
|
|
30
|
Patient R, Hourioux C, Vaudin P, Pagès JC, Roingeard P. Chimeric hepatitis B and C viruses envelope proteins can form subviral particles: implications for the design of new vaccine strategies. N Biotechnol 2009; 25:226-34. [PMID: 19356608 DOI: 10.1016/j.nbt.2009.01.001] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2008] [Revised: 01/02/2009] [Accepted: 01/08/2009] [Indexed: 12/18/2022]
Abstract
The hepatitis B virus (HBV) envelope protein (S) self-assembles into subviral particles used as commercial vaccines against hepatitis B. These particles are excellent carriers for foreign epitopes, which can be inserted into the external hydrophilic loop or at the N- or C-terminal end of the HBV S protein. We show here that the N-terminal transmembrane domain (TMD) of HBV S can be replaced by the TMDs of the hepatitis C virus (HCV) envelope proteins E1 and E2, to generate fusion proteins containing the entire HCV E1 or E2 sequence that are efficiently coassembled with the HBV S into particles. This demonstrates the remarkable tolerance of the HBV S protein to sequence substitutions conserving its subviral particle assembly properties. These findings may have implications for the design of new vaccine strategies based on the use of HBV subviral particles as carriers for various transmembrane proteins and produced using the same industrial procedures that are established for the HBV vaccine.
Collapse
Affiliation(s)
- Romuald Patient
- INSERM U966, Université François Rabelais and CHRU de Tours, France
| | | | | | | | | |
Collapse
|
|
31
|
Gottwein JM, Bukh J. Cutting the gordian knot-development and biological relevance of hepatitis C virus cell culture systems. Adv Virus Res 2008; 71:51-133. [PMID: 18585527 DOI: 10.1016/s0065-3527(08)00002-x] [Citation(s) in RCA: 77] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Worldwide approximately 180 million people are chronically infected with hepatitis C virus (HCV). HCV isolates exhibit extensive genetic heterogeneity and have been grouped in six genotypes and various subtypes. Additionally, several naturally occurring intergenotypic recombinants have been described. Research on the viral life cycle, efficient therapeutics, and a vaccine has been hampered by the absence of suitable cell culture systems. The first system permitting studies of the full viral life cycle was intrahepatic transfection of RNA transcripts of HCV consensus complementary DNA (cDNA) clones into chimpanzees. However, such full-length clones were not infectious in vitro. The development of the replicon system and HCV pseudo-particles allowed in vitro studies of certain aspects of the viral life cycle, RNA replication, and viral entry, respectively. Identification of the genotype 2 isolate JFH1, which for unknown reasons showed an exceptional replication capability and resulted in formation of infectious viral particles in the human hepatoma cell line Huh7, led in 2005 to the development of the first full viral life cycle in vitro systems. JFH1-based systems now enable in vitro studies of the function of viral proteins, their interaction with each other and host proteins, new antivirals, and neutralizing antibodies in the context of the full viral life cycle. However, several challenges remain, including development of cell culture systems for all major HCV genotypes and identification of other susceptible cell lines.
Collapse
Affiliation(s)
- Judith M Gottwein
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre, Denmark
| | | |
Collapse
|
|
32
|
Machida K, Kondo Y, Huang JY, Chen YC, Cheng KTH, Keck Z, Foung S, Dubuisson J, Sung VMH, Lai MMC. Hepatitis C virus (HCV)-induced immunoglobulin hypermutation reduces the affinity and neutralizing activities of antibodies against HCV envelope protein. J Virol 2008; 82:6711-20. [PMID: 18417597 PMCID: PMC2447061 DOI: 10.1128/jvi.02582-07] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2007] [Accepted: 04/10/2008] [Indexed: 02/06/2023] Open
Abstract
Hepatitis C virus (HCV) often causes persistent infection despite the presence of neutralizing antibodies against the virus in the sera of hepatitis C patients. HCV infects both hepatocytes and B cells through the binding of its envelope glycoprotein E2 to CD81, the putative viral receptor. Previously, we have shown that E2-CD81 interaction induces hypermutation of heavy-chain immunoglobulin (V(H)) in B cells. We hypothesize that if HCV infects antibody-producing B cells, the resultant hypermutation of V(H) may lower the affinity and specificity of the HCV-specific antibodies, enabling HCV to escape from immune surveillance. To test this hypothesis, we infected human hybridoma clones producing either neutralizing or non-neutralizing anti-E2 or anti-E1 antibodies with a lymphotropic HCV (SB strain). All of the hybridoma clones, except for a neutralizing antibody-producing hybridoma, could be infected with HCV and support virus replication for at least 8 weeks after infection. The V(H) sequences in the infected hybridomas had a significantly higher mutation frequency than those in the uninfected hybridomas, with mutations concentrating in complementarity-determining region 3. These mutations lowered the antibody affinity against the targeting protein and also lowered the virus-neutralizing activity of anti-E2 antibodies. Furthermore, antibody-mediated complement-dependent cytotoxicity with the antibodies secreted from the HCV-infected hybridomas was impaired. These results suggest that HCV infection could cause some anti-HCV-antibody-producing hybridoma B cells to make less-protective antibodies.
Collapse
Affiliation(s)
- Keigo Machida
- Department of Molecular Microbiology and Immunology, University of Southern California, Keck School of Medicine, 2011 Zonal Avenue, Los Angeles, CA 90033, USA
| | | | | | | | | | | | | | | | | | | |
Collapse
|
|
33
|
Sheikh MY, Choi J, Qadri I, Friedman JE, Sanyal AJ. Hepatitis C virus infection: molecular pathways to metabolic syndrome. Hepatology 2008; 47:2127-2133. [PMID: 18446789 DOI: 10.1002/hep.22269] [Citation(s) in RCA: 177] [Impact Index Per Article: 10.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Chronic infection with hepatitis C virus (HCV) can induce insulin resistance (IR) in a genotype-dependent fashion, thus contributing to steatosis, progression of fibrosis and resistance to interferon therapy. The molecular mechanisms in genotype 1 patients that lead to metabolic syndrome are still ambiguous. Based on our current understanding, HCV proteins associate with mitochondria and endoplasmic reticulum and promote oxidative stress. The latter mediates signals involving the p38 mitogen-activated protein kinase and activates nuclear factor kappa B. This transcription factor plays a key role in the expression of cytokines, tumor necrosis factor alpha (TNF-alpha), interleukin 6, interleukin 8, tumor growth factor beta, and Fas ligand. TNF-alpha inhibits the function of insulin receptor substrates and decreases the expression of the glucose transporter and lipoprotein lipase in peripheral tissues, which is responsible for the promotion of insulin resistance. Furthermore, reduced adiponectin levels, loss of adiponectin receptors, and decreased anti-inflammatory peroxisome proliferator-activated receptor alpha in the liver of HCV patients may contribute to reduced fatty acid oxidation, inflammation, and eventually lipotoxicity. This chain of events may be initiated by HCV-associated IR and provides a direction for future research in the areas of therapeutic intervention.
Collapse
Affiliation(s)
- Muhammad Y Sheikh
- Division of Gastroenterology and Hepatology, University of California San Francisco Fresno Education Program, Community Regional Medical Center, Fresno, CA 93721, USA.
| | | | | | | | | |
Collapse
|
|
34
|
Poumbourios P, Drummer HE. Recent advances in our understanding of receptor binding, viral fusion and cell entry of hepatitis C virus: new targets for the design of antiviral agents. Antivir Chem Chemother 2008; 18:169-89. [PMID: 17907376 DOI: 10.1177/095632020701800402] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
Improvements to antiviral therapies for the treatment of hepatitis C virus (HCV) infections will require the use of multiple drugs that target viral proteins essential for replication. The discovery of anti-HCV compounds has been severely hampered by the lack of cell culture replication systems. Since the late 1990s, the advent of sub-genomic replicons that model the intracellular events leading to HCV genome replication have enabled the discovery of HCV protease and polymerase inhibitors, but did not allow the study of HCV entry or entry inhibitors. More recently, retroviral pseudotyping of the viral glycoproteins and the development of a cell culture-based system that recapitulates the entire HCV replication cycle were achieved. These new experimental systems have enabled a rapid advance in our knowledge of how HCV glycoproteins, E1 and E2, mediate receptor binding and viral entry. These systems have facilitated the discovery of a range of viral receptors. Evidence is emerging that CD81, scavenger receptor class B type I, claudin-1 and the low-density lipoprotein receptor are involved in viral entry. In addition, DC-SIGN and L-SIGN may function to internalize virus into dendritic or endothelial cells, facilitating the transport of virions to sites of infection such as the liver. This review focuses on the interaction between the HCV glycoproteins and cellular receptors, and our current understanding of the viral entry pathway. In addition, key questions on the role that these receptors play in viral entry are raised and potential avenues for the discovery of new antiviral agents are highlighted.
Collapse
Affiliation(s)
- Pantelis Poumbourios
- Viral Fusion Laboratory, Macfarlane Burnet Institute for Medical Research and Public Health Limited, Melbourne, Australia
| | | |
Collapse
|
|
35
|
Iacob RE, Perdivara I, Przybylski M, Tomer KB. Mass spectrometric characterization of glycosylation of hepatitis C virus E2 envelope glycoprotein reveals extended microheterogeneity of N-glycans. JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY 2008; 19:428-44. [PMID: 18187336 PMCID: PMC2287207 DOI: 10.1016/j.jasms.2007.11.022] [Citation(s) in RCA: 54] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Received: 07/20/2007] [Revised: 11/27/2007] [Accepted: 11/28/2007] [Indexed: 05/25/2023]
Abstract
Hepatitis C virus (HCV) causes acute and chronic liver disease in humans, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The polyprotein encoded in the HCV genome is co- and post-translationally processed by host and viral peptidases, generating the structural proteins Core, E1, E2, and p7, and five nonstructural proteins. The two envelope proteins E1 and E2 are heavily glycosylated. Studying the glycan moieties attached to the envelope E2 glycoprotein is important because the N-linked glycans on E2 envelope protein are involved in the interaction with some human neutralizing antibodies, and may also have a direct or indirect effect on protein folding. In the present study, we report the mass spectrometric characterization of the glycan moieties attached to the E2 glycoprotein. The mass spectrometric analysis clearly identified the nature, composition, and microheterogeneity of the sugars attached to the E2 glycopeptides. All 11 sites of glycosylation on E2 protein were characterized, and the majority of these sites proved to be occupied by high mannose glycans. However, complex type oligosaccharides, which have not been previously identified, were exclusively observed at two N-linked sites, and their identity and heterogeneity were determined.
Collapse
Affiliation(s)
- Roxana E. Iacob
- Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC 27709
| | - Irina Perdivara
- Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC 27709
- Department of Chemistry, Laboratory of Analytical Chemistry, University of Konstanz, 78457 Konstanz, Germany
| | - Michael Przybylski
- Department of Chemistry, Laboratory of Analytical Chemistry, University of Konstanz, 78457 Konstanz, Germany
| | - Kenneth B. Tomer
- Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC 27709
| |
Collapse
|
|
36
|
Iacob RE, Keck Z, Olson O, Foung SK, Tomer KB. Structural elucidation of critical residues involved in binding of human monoclonal antibodies to hepatitis C virus E2 envelope glycoprotein. BIOCHIMICA ET BIOPHYSICA ACTA 2008; 1784:530-42. [PMID: 18230369 PMCID: PMC2277214 DOI: 10.1016/j.bbapap.2007.12.015] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Received: 09/24/2007] [Revised: 12/03/2007] [Accepted: 12/24/2007] [Indexed: 02/03/2023]
Abstract
Human monoclonal antibodies derived from B cells of HCV-infected individuals provide information on the immune response to native HCV envelope proteins as they are recognized during infection. Monoclonal antibodies have been useful in the determination of the function and structure of specific immunogenic domains of proteins and should also be useful for the structure/function characterization of HCV E1 and E2 envelope glycoproteins. The HCV E2 envelope glycoprotein has at least three immunodistinctive conformation domains, designated A, B, and C. Conformational epitopes within domain B and C are neutralizing antibody targets on HCV pseudoparticles as well as from infectious cell culture virus. In this study, a combination of differential surface modification and mass spectrometric limited proteolysis followed by alanine mutagenesis was used to provide insight into potential conformational changes within the E2 protein upon antibody binding. The arginine guanidine groups in the E2 protein were modified with CHD in both the affinity bound and free states followed by mass spectrometric analysis, and the regions showing protection upon antibody binding were identified. This protection can arise by direct contact between the residues and the monoclonal antibody, or by antibody-induced conformational changes. Based on the mass spectrometric data, site-directed mutagenesis experiments were performed which clearly identified additional amino acid residues on E2 distant from the site of antibody interaction, whose change to alanine inhibited antibody recognition by inducing conformational changes within the E2 protein.
Collapse
Affiliation(s)
- Roxana E. Iacob
- Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC 27709, USA
| | - Zhenyong Keck
- Department of Pathology, Stanford University School of Medicine, Stanford, CA, 94305, USA
| | - Oakley Olson
- Department of Pathology, Stanford University School of Medicine, Stanford, CA, 94305, USA
| | - Steven K.H. Foung
- Department of Pathology, Stanford University School of Medicine, Stanford, CA, 94305, USA
| | - Kenneth B. Tomer
- Address correspondence to: Dr. Roxana E. Iacob, The Barnett Institute, 341 Mugar Life Sciences Building, Northeastern University, Boston, MA 02115-5000, USA. Fax: 617.373.2855; E-mail:
| |
Collapse
|
|
37
|
Hsieh SC, Liu IJ, King CC, Chang GJ, Wang WK. A strong endoplasmic reticulum retention signal in the stem-anchor region of envelope glycoprotein of dengue virus type 2 affects the production of virus-like particles. Virology 2008; 374:338-50. [PMID: 18252258 DOI: 10.1016/j.virol.2007.12.041] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2007] [Revised: 10/25/2007] [Accepted: 12/18/2007] [Indexed: 11/25/2022]
Abstract
Recombinant virus-like particles (VLPs) of flaviviruses have been shown to be produced efficiently by co-expressing the precursor membrane (PrM) and envelope (E) proteins with few exceptions, such as dengue virus type 2 (DENV2). It was reported previously that chimeric DENV2 PrM/E construct containing the stem-anchor region of E protein of Japanese encephalitis virus (JEV) produced VLPs efficiently (Chang, G. J., Hunt, A. R., Holmes, D. A., Springfield, T., Chiueh, T. S., Roehrig, J. T., and Gubler, D. J. 2003. Enhancing biosynthesis and secretion of premembrane and envelope proteins by the chimeric plasmid of dengue virus type 2 and Japanese encephalitis virus. Virology 306, 170-180.). We investigated the mechanisms involved and reported that compared with authentic DENV2 PrM/E-expressing cells, E protein in chimeric DENV2 PrM/E-expressing cells was also present in an endoglycosidase H (endo H)-resistant compartment and has shifted more to the pellets of the soluble fraction. Replacement of the transmembrane and cytoplasmic domains of CD4 with the stem-anchor of DENV2 (CD4D2) or JEV (CD4JEV) rendered the chimeric CD4 retained predominantly in the endoplasmic reticulum (ER). Flow cytometry revealed higher proportion of CD4JEV than CD4D2 expressed on the cell surface. Together, these findings suggested that the stem-anchor of DENV2 contained an ER retention signal stronger than that of JEV, which might contribute to the inefficient production of DENV2 VLPs. Moreover, co-expression of C protein can enhance the production of DENV2 VLPs, suggesting a mechanism of facilitating viral particle formation during DENV2 replication.
Collapse
Affiliation(s)
- Szu-Chia Hsieh
- Institute of Microbiology, College of Medicine, National Taiwan University, and Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan
| | | | | | | | | |
Collapse
|
|
38
|
Cellular determinants of hepatitis C virus assembly, maturation, degradation, and secretion. J Virol 2007; 82:2120-9. [PMID: 18077707 DOI: 10.1128/jvi.02053-07] [Citation(s) in RCA: 353] [Impact Index Per Article: 19.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Intracellular infectious hepatitis C virus (HCV) particles display a distinctly higher buoyant density than do secreted virus particles, suggesting that the characteristic low density of extracellular HCV particles is acquired during viral egress. We took advantage of this difference to examine the determinants of assembly, maturation, degradation, and egress of infectious HCV particles. The results demonstrate that HCV assembly and maturation occur in the endoplasmic reticulum (ER) and post-ER compartments, respectively, and that both depend on microsomal transfer protein and apolipoprotein B, in a manner that parallels the formation of very-low-density lipoproteins (VLDL). In addition, they illustrate that only low-density particles are efficiently secreted and that immature particles are actively degraded, in a proteasome-independent manner, in a post-ER compartment of the cell. These results suggest that by coopting the VLDL assembly, maturation, degradation, and secretory machinery of the cell, HCV acquires its hepatocyte tropism and, by mimicry, its tendency to persist.
Collapse
|
|
39
|
Martínez-Donato G, Capdesuñer Y, Acosta-Rivero N, Rodríguez A, Morales-Grillo J, Martínez E, González M, Alvarez-Obregon JC, Dueñas-Carrera S. Multimeric HCV E2 protein obtained from Pichia pastoris cells induces a strong immune response in mice. Mol Biotechnol 2007; 35:225-35. [PMID: 17652786 DOI: 10.1007/bf02686008] [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] [Received: 11/30/1999] [Revised: 11/30/1999] [Accepted: 11/30/1999] [Indexed: 01/08/2023]
Abstract
Production of immunogenic hepatitis C virus (HCV) envelope proteins will assist in the future development of preventive or therapeutics applications. Only properly folded monomeric E2 protein is able to bind a putative cellular co-receptor CD81, but this interaction may modulate cell immune function. Recombinant E2 proteins, similar to the native form, but lacking undesirable immunoregulatory features, might be promising components of vaccine candidates against HCV. To obtain E2 suitable for structural as well as functional studies, a recombinant E2 variant (E2680) was produced in Pichia pastoris cells. E2680, comprising amino acids 384 to 680 of the HCV polyprotein, was secreted into the culture supernatant in the N-glycosilated form and was mainly composed of disulfide-linked multimers. Both monomeric and oligomeric forms of E2680 were recognized by conformational-sensitive MAb H53. In addition, antibodies in sera from 70% of HCVpositive patients were reactive against E2680. By immunizing E2680 in BALB/c mice, both a specific cellular immune response and anti-E2680 IgG antibody titers of 1:200,000 were induced. Our data suggest that recombinant E2680 could be useful to successfully induce strong anti-HCV immunity.
Collapse
Affiliation(s)
- Gillian Martínez-Donato
- Hepatitis C Department, Biomedical Research, Center for Genetic Engineering and Biotechnology, Ave. 31 e/ 158 y 190, Cubanacán, Playa, Apdo. 6162, Habana 10600, Cuba
| | | | | | | | | | | | | | | | | |
Collapse
|
|
40
|
Régeard M, Lepère C, Trotard M, Gripon P, Le Seyec J. Recent contributions of in vitro models to our understanding of hepatitis C virus life cycle. FEBS J 2007; 274:4705-18. [PMID: 17824957 DOI: 10.1111/j.1742-4658.2007.06017.x] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Hepatitis C virus is a human pathogen responsible for liver diseases including acute and chronic hepatitis, cirrhosis and hepatocellular carcinoma. Its high prevalence, the absence of a prophylactic vaccine and the poor efficiency of current therapies are huge medical problems. Since the discovery of the hepatitis C virus, our knowledge of its biology has been largely punctuated by the development of original models of research. At the end of the 1980s, the chimpanzee model led to cloning of the viral genome and the definition of infectious molecular clones. In 1999, a breakthrough was achieved with the development of a robust in vitro replication model named 'replicon'. This system allowed intensive research into replication mechanisms and drug discovery. Later, in 2003, pseudotyped retroviruses harbouring surface proteins of hepatitis C virus were produced to specifically investigate the viral entry process. It was only in 2005 that infectious viruses were produced in vitro, enabling intensive investigations into the entire life cycle of the hepatitis C virus. This review describes the different in vitro models developed to study hepatitis C virus, their contribution to current knowledge of the virus biology and their future research applications.
Collapse
Affiliation(s)
- Morgane Régeard
- INSERM, U522, IFR 140, Hôpital de Pontchaillou, Rennes, France
| | | | | | | | | |
Collapse
|
|
41
|
Bertaux C, Daelemans D, Meertens L, Cormier EG, Reinus JF, Peumans WJ, Van Damme EJM, Igarashi Y, Oki T, Schols D, Dragic T, Balzarini J. Entry of hepatitis C virus and human immunodeficiency virus is selectively inhibited by carbohydrate-binding agents but not by polyanions. Virology 2007; 366:40-50. [PMID: 17498767 DOI: 10.1016/j.virol.2007.04.008] [Citation(s) in RCA: 56] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2006] [Revised: 01/24/2007] [Accepted: 04/05/2007] [Indexed: 01/23/2023]
Abstract
We studied the antiviral activity of carbohydrate-binding agents (CBAs), including several plant lectins and the non-peptidic small-molecular-weight antibiotic pradimicin A (PRM-A). These agents efficiently prevented hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) infection of target cells by inhibiting the viral entry. CBAs were also shown to prevent HIV and HCV capture by DC-SIGN-expressing cells. Surprisingly, infection by other enveloped viruses such as herpes simplex viruses, respiratory syncytial virus and parainfluenza-3 virus was not inhibited by these agents pointing to a high degree of specificity. Mannan reversed the antiviral activity of CBAs, confirming their association with viral envelope-associated glycans. In contrast, polyanions such as dextran sulfate-5000 and sulfated polyvinylalcohol inhibited HIV entry but were devoid of any activity against HCV infection, indicating that they act through a different mechanism. CBAs could be considered as prime drug leads for the treatment of chronic viral infections such as HCV by preventing viral entry into target cells. They may represent an attractive new option for therapy of HCV/HIV coinfections. CBAs may also have the potential to prevent HCV/HIV transmission.
Collapse
Affiliation(s)
- Claire Bertaux
- Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, USA
| | | | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
42
|
Abstract
Hepatitis C virus (HCV) encodes a single polyprotein, which is processed by cellular and viral proteases to generate 10 polypeptides. The HCV genome also contains an overlapping +1 reading frame that may lead to the synthesis of an additional protein. Until recently, studies of HCV have been hampered by the lack of a productive cell culture system. Since the identification of HCV genome approximately 17 years ago, structural, biochemical and biological information on HCV proteins has mainly been obtained with proteins produced by heterologous expression systems. In addition, some functional studies have also been confirmed with replicon systems or with retroviral particles pseudotyped with HCV envelope glycoproteins. The data that have accumulated on HCV proteins begin to provide a framework for understanding the molecular mechanisms involved in the major steps of HCV life cycle. Moreover, the knowledge accumulated on HCV proteins is also leading to the development of antiviral drugs among which some are showing promising results in early-phase clinical trials. This review summarizes the current knowledge on the functions and biochemical features of HCV proteins.
Collapse
Affiliation(s)
- Jean Dubuisson
- Hepatitis C Laboratory, CNRS-UMR8161, Institut de Biologie de Lille I & II, Université de Lille, 1 rue Calmette, BP447, 59021 Lille Cedex, France.
| |
Collapse
|
|
43
|
Ciczora Y, Callens N, Penin F, Pécheur EI, Dubuisson J. Transmembrane domains of hepatitis C virus envelope glycoproteins: residues involved in E1E2 heterodimerization and involvement of these domains in virus entry. J Virol 2006; 81:2372-81. [PMID: 17166909 PMCID: PMC1865936 DOI: 10.1128/jvi.02198-06] [Citation(s) in RCA: 73] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
The transmembrane (TM) domains of hepatitis C virus (HCV) envelope glycoproteins E1 and E2 have been shown to play multiple roles during the biogenesis of the E1E2 heterodimer. By using alanine scanning insertion mutagenesis within the TM domains of HCV envelope glycoproteins, we have previously shown that the central regions of these domains as well as the N-terminal part of the TM domain of E1 are involved in heterodimerization. Here, we used a tryptophan replacement scan of these regions to identify individual residues that participate in those interactions. Our mutagenesis study identified at least four residues involved in heterodimerization: Gly 354, Gly 358, Lys 370, and Asp 728. Interestingly, Gly 354 and Gly 358 belong to a GXXXG oligomerization motif. Our tryptophan mutants were also used to generate retrovirus-based, HCV-pseudotyped particles (HCVpp) in order to analyze the effects of these mutations on virus entry. Surprisingly, two mutants consistently displayed higher infectivity compared to that of the wild type. In contrast, HCVpp infectivity was strongly affected for many mutants, despite normal E1E2 heterodimerization and normal levels of incorporation of HCV glycoproteins into HCVpp. The characterization of some of these HCVpp mutants in the recently developed in vitro fusion assay using fluorescent-labeled liposomes indicated that mutations reducing HCVpp infectivity without altering E1E2 heterodimerization affected the fusion properties of HCV envelope glycoproteins. In conclusion, this mutational analysis identified residues involved in E1E2 heterodimerization and revealed that the TM domains of HCV envelope glycoproteins play a major role in the fusion properties of these proteins.
Collapse
Affiliation(s)
- Yann Ciczora
- Hepatitis C Laboratory, CNRS-UMR8161, Institut de Biologie de Lille, 1 rue Calmette, BP447, 59021 Lille cedex, France
| | | | | | | | | |
Collapse
|
|
44
|
Gastaminza P, Kapadia SB, Chisari FV. Differential biophysical properties of infectious intracellular and secreted hepatitis C virus particles. J Virol 2006; 80:11074-81. [PMID: 16956946 PMCID: PMC1642172 DOI: 10.1128/jvi.01150-06] [Citation(s) in RCA: 195] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
The recent development of a cell culture infection model for hepatitis C virus (HCV) permits the production of infectious particles in vitro. In this report, we demonstrate that infectious particles are present both within the infected cells and in the supernatant. Kinetic analysis indicates that intracellular particles constitute precursors of the secreted infectious virus. Ultracentrifugation analyses indicate that intracellular infectious viral particles are similar in size (approximately 65 to 70 nm) but different in buoyant density (approximately 1.15 to 1.20 g/ml) from extracellular particles (approximately 1.03 to 1.16 g/ml). These results indicate that infectious HCV particles are assembled intracellularly and that their biochemical composition is altered during viral egress.
Collapse
Affiliation(s)
- Pablo Gastaminza
- The Scripps Research Institute, Maildrop SBR-1010550, North Torrey Pines Road, La Jolla, CA 92037, USA
| | | | | |
Collapse
|
|
45
|
Xiang ZH, Cai WJ, Zhao P, Kong LB, Ye LB, Wu ZH. Purification and application of bacterially expressed chimeric protein E1E2 of hepatitis C virus. Protein Expr Purif 2006; 49:95-101. [PMID: 16600629 DOI: 10.1016/j.pep.2006.02.013] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2006] [Revised: 02/15/2006] [Accepted: 02/17/2006] [Indexed: 11/28/2022]
Abstract
E1 and E2 glycoproteins are structural components of hepatitis C virus (HCV) virion. They are involved in cellular receptors interaction, neutralising antibodies elicitation, and viral morphogenesis. They are considered as major candidates for anti-HCV vaccine. In this report, we first expressed tandem E1E2 as well as C-terminally truncated E1 fragment and C-terminally truncated E2 fragment, respectively, in Escherichia coli cells and the proteins were purified to homogenesis. All the purified proteins can react specifically with patient sera. Both purified chimeric protein E1E2 and protein E2 can interact with a putative cellular receptor CD81, while purified protein E1 cannot interact with CD81. The sera of rabbit immunized with the E1E2 inhibited the binding of E2 protein to the major extracellular loop of human CD81 and reacted with both proteins E1 and E2, respectively. Anti-E1 and E2 antibodies can be generated simultaneously in the rabbit immunized with the E1E2, and the titers of antibodies were 63 or 56% higher than the titers induced by E1 or E2 alone, respectively. The results suggest that E1 and E2 can enhance their immunogenicity each other in chimeric protein E1E2 and the E. coli-derived chimeric protein E1E2 and corresponding antisera can be used as an useful tools in anti-HCV vaccine research.
Collapse
Affiliation(s)
- Zhong-Hua Xiang
- State Key Laboratory of Virology, College of Life Science, Wuhan University, Wuhan, Hubei Province 430072, PR China
| | | | | | | | | | | |
Collapse
|
|
46
|
Helle F, Wychowski C, Vu-Dac N, Gustafson KR, Voisset C, Dubuisson J. Cyanovirin-N inhibits hepatitis C virus entry by binding to envelope protein glycans. J Biol Chem 2006; 281:25177-83. [PMID: 16809348 DOI: 10.1074/jbc.m602431200] [Citation(s) in RCA: 132] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023] Open
Abstract
Inhibition of viruses at the stage of viral entry provides a route for therapeutic intervention. Because of difficulties in propagating hepatitis C virus (HCV) in cell culture, entry inhibitors have not yet been reported for this virus. However, with the development of retroviral particles pseudotyped with HCV envelope glycoproteins (HCVpp) and the recent progress in amplification of HCV in cell culture (HCVcc), studying HCV entry is now possible. In addition, these systems are essential for the identification and the characterization of molecules that block HCV entry. The lectin cyanovirin-N (CV-N) has initially been discovered based on its potent activity against human immunodeficiency virus. Because HCV envelope glycoproteins are highly glycosylated, we sought to determine whether CV-N has an antiviral activity against this virus. CV-N inhibited the infectivity of HCVcc and HCVpp at low nanomolar concentrations. This inhibition is attributed to the interaction of CV-N with HCV envelope glycoproteins. In addition, we showed that the carbohydrate binding property of CV-N is involved in the anti-HCV activity. Finally, CV-N bound to HCV envelope glycoproteins and blocked the interaction between the envelope protein E2 and CD81, a cell surface molecule involved in HCV entry. These data demonstrate that targeting the glycans of HCV envelope proteins is a promising approach in the development of antiviral therapies to combat a virus that is a major cause of chronic liver diseases. Furthermore, CV-N is a new invaluable tool to further dissect the early steps of HCV entry into host cells.
Collapse
Affiliation(s)
- François Helle
- Centre National de la Recherche Scientifique, Institut de Biologie de Lille (Unité Mixte de Recherche 8161), Institut Pasteur de Lille, 59021 Lille cedex, France
| | | | | | | | | | | |
Collapse
|
|
47
|
Callens N, Ciczora Y, Bartosch B, Vu-Dac N, Cosset FL, Pawlotsky JM, Penin F, Dubuisson J. Basic residues in hypervariable region 1 of hepatitis C virus envelope glycoprotein e2 contribute to virus entry. J Virol 2006; 79:15331-41. [PMID: 16306604 PMCID: PMC1316016 DOI: 10.1128/jvi.79.24.15331-15341.2005] [Citation(s) in RCA: 62] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023] Open
Abstract
The N terminus of hepatitis C virus (HCV) envelope glycoprotein E2 contains a hypervariable region (HVR1) which has been proposed to play a role in viral entry. Despite strong amino acid variability, HVR1 is globally basic, with basic residues located at specific sequence positions. Here we show by analyzing a large number of HVR1 sequences that the frequency of basic residues at each position is genotype dependent. We also used retroviral pseudotyped particles (HCVpp) harboring genotype 1a envelope glycoproteins to study the role of HVR1 basic residues in entry. Interestingly, HCVpp infectivity globally increased with the number of basic residues in HVR1. However, a shift in position of some charged residues also modulated HCVpp infectivity. In the absence of basic residues, infectivity was reduced to the same level as that of a mutant deleted of HVR1. We also analyzed the effect of these mutations on interactions with some potential HCV receptors. Recognition of CD81 was not affected by changes in the number of charged residues, and we did not find a role for heparan sulfates in HCVpp entry. The involvement of the scavenger receptor class B type I (SR-BI) was indirectly analyzed by measuring the enhancement of infectivity of the mutants in the presence of the natural ligand of SR-BI, high-density lipoproteins (HDL). However, no correlation between the number of basic residues within HVR1 and HDL enhancement effect was observed. Despite the lack of evidence of the involvement of known potential receptors, our results demonstrate that the presence of basic residues in HVR1 facilitates virus entry.
Collapse
Affiliation(s)
- Nathalie Callens
- Unité Hépatite C, CNRS-UPR2511, Institut de Biologie de Lille, 1 rue Calmette, BP447, 59021 Lille cedex, France
| | | | | | | | | | | | | | | |
Collapse
|
|
48
|
Rouillé Y, Helle F, Delgrange D, Roingeard P, Voisset C, Blanchard E, Belouzard S, McKeating J, Patel AH, Maertens G, Wakita T, Wychowski C, Dubuisson J. Subcellular localization of hepatitis C virus structural proteins in a cell culture system that efficiently replicates the virus. J Virol 2006; 80:2832-41. [PMID: 16501092 PMCID: PMC1395453 DOI: 10.1128/jvi.80.6.2832-2841.2006] [Citation(s) in RCA: 156] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2005] [Accepted: 12/23/2005] [Indexed: 12/25/2022] Open
Abstract
Due to the recent development of a cell culture model, hepatitis C virus (HCV) can be efficiently propagated in cell culture. This allowed us to reinvestigate the subcellular localization of HCV structural proteins in the context of an infectious cycle. In agreement with previous reports, confocal immunofluorescence analysis of the subcellular localization of HCV structural proteins indicated that, in infected cells, the glycoprotein heterodimer is retained in the endoplasmic reticulum. However, in contrast to other studies, the glycoprotein heterodimer did not accumulate in other intracellular compartments or at the plasma membrane. As previously reported, an association between the capsid protein and lipid droplets was also observed. In addition, a fraction of labeling was consistent with the capsid protein being localized in a membranous compartment that is associated with the lipid droplets. However, in contrast to previous reports, the capsid protein was not found in the nucleus or in association with mitochondria or other well-defined intracellular compartments. Surprisingly, no colocalization was observed between the glycoprotein heterodimer and the capsid protein in infected cells. Electron microscopy analyses allowed us to identify a membrane alteration similar to the previously reported "membranous web." However, no virus-like particles were found in this type of structure. In addition, dense elements compatible with the size and shape of a viral particle were seldom observed in infected cells. In conclusion, the cell culture system for HCV allowed us for the first time to characterize the subcellular localization of HCV structural proteins in the context an infectious cycle.
Collapse
Affiliation(s)
- Yves Rouillé
- CNRS-UPR2511, Institut de Biologie de Lille, 1 Rue Calmette, BP447, 59021 Lille Cedex, France
| | | | | | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
49
|
Ciczora Y, Callens N, Montpellier C, Bartosch B, Cosset FL, De Beeck AO, Dubuisson J. Contribution of the charged residues of hepatitis C virus glycoprotein E2 transmembrane domain to the functions of the E1E2 heterodimer. J Gen Virol 2005; 86:2793-2798. [PMID: 16186234 DOI: 10.1099/vir.0.81140-0] [Citation(s) in RCA: 42] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
The envelope glycoproteins of Hepatitis C virus (HCV), E1 and E2, form a heterodimer that is retained in the endoplasmic reticulum (ER). The transmembrane (TM) domains play a major role in E1E2 heterodimerization and in ER retention. Two fully conserved charged residues in the middle of the TM domain of E2 (Asp and Arg) are crucial for these functions. Replacement of the Asp residue by a Leu impaired E1E2 heterodimerization, whereas the Arg-to-Leu mutation had a milder effect. Both Asp and Arg residues were shown to contribute to the ER retention function of E2. In addition, the entry function of HCV envelope glycoproteins was affected by these mutations. Together, these data indicate that the charged residues present in the TM domain of E2 play a major role in the biogenesis and the entry function of the E1E2 heterodimer. However, the Asp and Arg residues do not contribute equally to these functions.
Collapse
Affiliation(s)
- Yann Ciczora
- CNRS-UPR2511, Unité Hépatite C, Institut de Biologie de Lille - Institut Pasteur de Lille, 1 rue Calmette, BP 447, 59021 Lille cedex, France
| | - Nathalie Callens
- CNRS-UPR2511, Unité Hépatite C, Institut de Biologie de Lille - Institut Pasteur de Lille, 1 rue Calmette, BP 447, 59021 Lille cedex, France
| | - Claire Montpellier
- CNRS-UPR2511, Unité Hépatite C, Institut de Biologie de Lille - Institut Pasteur de Lille, 1 rue Calmette, BP 447, 59021 Lille cedex, France
| | - Birke Bartosch
- Laboratoire de Vectorologie Rétrovirale et Thérapie Génique, INSERM U412, IFR74, Ecole Normale Supérieure de Lyon, Lyon, France
| | - François-Loïc Cosset
- Laboratoire de Vectorologie Rétrovirale et Thérapie Génique, INSERM U412, IFR74, Ecole Normale Supérieure de Lyon, Lyon, France
| | - Anne Op De Beeck
- CNRS-UPR2511, Unité Hépatite C, Institut de Biologie de Lille - Institut Pasteur de Lille, 1 rue Calmette, BP 447, 59021 Lille cedex, France
| | - Jean Dubuisson
- CNRS-UPR2511, Unité Hépatite C, Institut de Biologie de Lille - Institut Pasteur de Lille, 1 rue Calmette, BP 447, 59021 Lille cedex, France
| |
Collapse
|
|
50
|
Yagi S, Mori K, Tanaka E, Matsumoto A, Sunaga F, Kiyosawa K, Yamaguchi K. Identification of novel HCV subgenome replicating persistently in chronic active hepatitis C patients. J Med Virol 2005; 77:399-413. [PMID: 16173026 DOI: 10.1002/jmv.20469] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
In an effort to clarify the life cycle of HCV, the HCV genome in liver biopsies taken from chronic active hepatitis C patients undergoing interferon treatment was investigated. Molecular cloning by long distance reverse-transcription polymerase chain reaction (RT-PCR) revealed that the HCV genome in two patients with high viral loads in the liver had in-frame deletions of approximately 2 kb between E1 and NS2, which encode the E1-NS2 fusion protein and six other HCV proteins: core, NS3, NS4A, NS4B, NS5A, and NS5B. Among the remaining 21 chronic active hepatitis C patients, these types of deletion were found in another two patients and in two hepatocellular carcinoma patients. Out-of-frame deletions in the structural region were isolated from the other five patients, but the dominant RT-PCR products were non-truncated genomes. Retrospective analysis of a series of serum samples taken from a patient carrying the subgenome with the in-frame deletion revealed that both the subgenome and the full genome persisted through the 2-year period of investigation, with the subgenome being predominant during this period. Sequence analysis of the isolated cDNA suggested that both the subgenome and the full genome evolved independently. Western blotting analysis of HCV proteins from the HCV subgenome indicated that they were processed in the same way as those from the full genome. HCV subgenomes thus appear to be involved in the HCV life cycle.
Collapse
Affiliation(s)
- Shintaro Yagi
- R&D Group, Advanced Life Science Institute, Inc., Saitama, Japan
| | | | | | | | | | | | | |
Collapse
|