scholarly journals Role of the Asialoglycoprotein Receptor in Binding and Entry of Hepatitis C Virus Structural Proteins in Cultured Human Hepatocytes

2003 ◽  
Vol 77 (1) ◽  
pp. 546-559 ◽  
Author(s):  
Bertrand Saunier ◽  
Miriam Triyatni ◽  
Luca Ulianich ◽  
Padma Maruvada ◽  
Paul Yen ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Fluorescent Protein ◽  
Human Hepatocytes ◽  
Asialoglycoprotein Receptor ◽  
Structural Proteins ◽  
Hepatic Cells ◽  
Genotype 1A ◽  
Green Fluorescent ◽  
Hcv Structural Proteins

ABSTRACT We used a baculovirus-based system to prepare structural proteins of hepatitis C virus (HCV) genotype 1a. Binding of this preparation to cultured human hepatic cells was both dose dependent and saturable. This binding was decreased by calcium depletion and was partially prevented by ligands of the asialoglycoprotein receptor (ASGP-R), thyroglobulin, asialothyroglobulin, and antibody against a peptide in the carbohydrate recognition domain of ASGP-R but not preimmune antibody. Uptake by hepatocytes was observed with both radiolabeled and dye-labeled HCV structural proteins. With hepatocytes expressing the hH1 subunit of the ASGP-R fused to green fluorescent protein, we could show by confocal microscopy that dye stain cointernalized with the fusion protein in an area surrounding the nucleus. Internalization was more efficient with a preparation containing p7 than with one that did not. The two preparations bound to transfected 3T3-L1 cells expressing either both (hH1 and hH2) subunits of the ASGP-R (3T3-22Z cells) or both hH1 and a functionally defective variant of hH2 (3T3-24X cells) but not to parental cells. Additionally, uptake of dye-labeled preparation containing p7 was observed with 3T3-22Z cells but not with 3T3-L1 or 3T3-24X cells or with the preparation lacking p7, suggesting that p7 regulates the internalization properties of HCV structural proteins. Our observations suggest that HCV structural proteins bind to and cointernalize with the ASGP-R in cultured human hepatocytes.

scholarly journals Suppression of short interfering RNA-mediated gene silencing by the structural proteins of hepatitis C virus

2008 ◽  
Vol 89 (11) ◽  
pp. 2761-2766 ◽  
Author(s):  
Jingmin Ji ◽  
Andrea Glaser ◽  
Marion Wernli ◽  
Jan Martin Berke ◽  
Darius Moradpour ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Gene Silencing ◽  
Fluorescent Protein ◽  
Structural Proteins ◽  
Short Interfering Rna ◽  
Argonaute 2 ◽  
Co Precipitation ◽  
Green Fluorescent ◽  
Interfering Rna

Viruses have evolved strategies to overcome the antiviral effects of the host at different levels. Besides specific defence mechanisms, the host responds to viral infection via the interferon pathway and also by RNA interference (RNAi). However, several viruses have been identified that suppress RNAi. We addressed the question of whether hepatitis C virus (HCV) suppresses RNAi, using cell lines constitutively expressing green fluorescent protein (GFP) and inducibly expressing HCV proteins. It was found that short interfering RNA-mediated GFP gene silencing was inhibited when the entire HCV polyprotein was expressed. Further studies showed that HCV structural proteins, and in particular envelope protein 2 (E2), were responsible for this inhibition. Co-precipitation assays demonstrated that E2 bound to Argonaute-2 (Ago-2), a member of the RNA-induced silencing complex, RISC. Thus, HCV E2 that interacts with Ago-2 is able to suppress RNAi.

scholarly journals A hepatitis C virus (HCV) NS3/4A protease-dependent strategy for the identification and purification of HCV-infected cells

2006 ◽  
Vol 87 (12) ◽  
pp. 3587-3598 ◽  
Author(s):  
Adrien Breiman ◽  
Damien Vitour ◽  
Myriam Vilasco ◽  
Catherine Ottone ◽  
Sonia Molina ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Fluorescent Protein ◽  
Rapid Identification ◽  
Specific Gene ◽  
Genotype 1A ◽  
Cat Assay ◽  
Infected Cells ◽  
Green Fluorescent ◽  
Cleavage Motif

As a tool for the identification and/or purification of hepatitis C virus (HCV)-infected cells, a chimeric form of the Gal4VP16 transcription factor was engineered to be activated only in the presence of the HCV NS3/4A protease and to induce different reporter genes [choramphenical acetyltransferase (CAT), green fluorescent protein (GFP) and the cell-surface marker H-2Kk] through the (Gal4)5-E1b promoter. For this, the NS5A/5B trans-cleavage motif of HCV of genotype 1a was inserted between Gal4VP16 and the N terminus of the endoplasmic reticulum (ER)-resident protein PERK, and it was demonstrated that it could be cleaved specifically by NS3/4A. Accordingly, transient transfection in tetracycline-inducible UHCV-11 cells expressing the HCV polyprotein of genotype 1a revealed the migration of the Gal4VP16 moiety of the chimera from the ER to the nucleus upon HCV expression. Activation of the chimera provoked specific gene induction, as shown by CAT assay, first in UHCV-11 cells and then in Huh-7 cells expressing an HCV replicon of genotype 1b (Huh-7 Rep). In addition, the GFP reporter gene allowed rapid fluorescence monitoring of HCV expression in the Huh-7 Rep cells. Finally, the chimera was introduced into Huh-7.5 cells infected with cell culture-generated HCV JFH1 (genotype 2a), allowing the purification of the HCV-infected cells by immunomagnetic cell sorting using H-2Kk as gene reporter. In conclusion, the Gal4VP16 chimera activation system can be used for the rapid identification and purification of HCV-infected cells.

scholarly journals Genetic Immunization of Wild-Type and Hepatitis C Virus Transgenic Mice Reveals a Hierarchy of Cellular Immune Response and Tolerance Induction against Hepatitis C Virus Structural Proteins

2001 ◽  
Vol 75 (24) ◽  
pp. 12121-12127 ◽  
Author(s):  
Jujin Satoi ◽  
Kazumoto Murata ◽  
Martin Lechmann ◽  
Elanchezhiyan Manickan ◽  
Zhensheng Zhang ◽  
...  
Keyword(s):  
Immune Response ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Transgenic Mice ◽  
Structural Proteins ◽  
Dna Immunization ◽  
Wild Type ◽  
Genetic Immunization ◽  
Cellular Immune ◽  
Hcv Structural Proteins

ABSTRACT To study the effect of genetic immunization on transgenic expression of hepatitis C virus (HCV) proteins, we evaluated the immunological response of HCV transgenic mice to HCV expression plasmids. FVB/n transgenic mice expressing HCV structural proteins (core, E1, and E2) and wild-type (WT) FVB/n mice were immunized intramuscularly with plasmids expressing core (pHCVcore) or core/E1/E2 (pHCVSt). After immunization, HCV-specific humoral and cellular immune response was studied. Both WT and transgenic mice immunized with either HCV construct produced antibodies and exhibited T-cell proliferative responses against core or envelope. In WT mice immunized with pHCVSt, cytotoxic T-lymphocyte (CTL) activities were detected against E2 but not against core or E1, whereas strong CTL activities against core could be detected in WT mice immunized with pHCVcore. In pHCVSt-immunized, transgenic mice, CTL activities against the core or envelope were completely absent, but core-specific CTL activities could be detected in pHCVcore-immunized transgenic mice. A similar pattern of immune responses was also observed in other mouse strains, including a transgenic line expressing human HLA-A2.1 molecules (AAD mice). Despite the presence of a peripheral cellular immunity against HCV, no liver pathology or lymphocytic infiltrate was observed in these transgenic mice. Our study suggests a hierarchy of CTL response against the HCV structural proteins (E2 > core > E1) in vivo when the proteins are expressed as a polyprotein. The HCV transgenic mice can be induced by DNA immunization to generate anti-HCV antibodies and anticore CTLs. However, they are tolerant at the CTL level against the E2 protein despite DNA immunization.

scholarly journals Robust production of infectious viral particles in Huh-7 cells by introducing mutations in hepatitis C virus structural proteins

2007 ◽  
Vol 88 (9) ◽  
pp. 2495-2503 ◽  
Author(s):  
David Delgrange ◽  
André Pillez ◽  
Sandrine Castelain ◽  
Laurence Cocquerel ◽  
Yves Rouillé ◽  
...  
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Core Protein ◽  
Potential Effect ◽  
Structural Proteins ◽  
Wild Type ◽  
Genotype 1A ◽  
Viral Particles ◽  
A Cell

Recently, the characterization of a cell culture system allowing the amplification of an authentic virus, named hepatitis C virus cell culture (HCVcc), has been reported by several groups. To obtain higher HCV particle productions, we investigated the potential effect of some amino acid changes on the infectivity of the JFH-1 isolate. As a first approach, successive infections of naïve Huh-7 cells were performed until high viral titres were obtained, and mutations that appeared during this selection were identified by sequencing. Only one major modification, N534K, located in the E2 glycoprotein sequence was found. Interestingly, this mutation prevented core glycosylation of E2 site 6. In addition, JFH-1 generated with this modification facilitated the infection of Huh-7 cells. In a second approach to identify mutations favouring HCVcc infectivity, we exploited the observation that a chimeric virus containing the genotype 1a core protein in the context of JFH-1 background was more infectious than wild-type JFH-1 isolate. Sequence alignment between JFH-1 and our chimera, led us to identify two major positions, 172 and 173, which were not occupied by similar amino acids in these two viruses. Importantly, higher viral titres were obtained by introducing these residues in the context of wild-type JFH-1. Altogether, our data indicate that a more robust production of HCVcc particles can be obtained by introducing a few specific mutations in JFH-1 structural proteins.

scholarly journals Insertion of Green Fluorescent Protein into Nonstructural Protein 5A Allows Direct Visualization of Functional Hepatitis C Virus Replication Complexes

2004 ◽  
Vol 78 (14) ◽  
pp. 7400-7409 ◽  
Author(s):  
Darius Moradpour ◽  
Matthew J. Evans ◽  
Rainer Gosert ◽  
Zhenghong Yuan ◽  
Hubert E. Blum ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Green Fluorescent Protein ◽  
Hepatitis C ◽  
Fusion Protein ◽  
Fluorescent Protein ◽  
Nonstructural Protein ◽  
Rna Replication ◽  
Direct Visualization ◽  
Green Fluorescent ◽  
Nonstructural Protein 5A

ABSTRACT Hepatitis C virus (HCV) replicates its genome in a membrane-associated replication complex, composed of viral proteins, replicating RNA and altered cellular membranes. We describe here HCV replicons that allow the direct visualization of functional HCV replication complexes. Viable replicons selected from a library of Tn7-mediated random insertions in the coding sequence of nonstructural protein 5A (NS5A) allowed the identification of two sites near the NS5A C terminus that tolerated insertion of heterologous sequences. Replicons encoding green fluorescent protein (GFP) at these locations were only moderately impaired for HCV RNA replication. Expression of the NS5A-GFP fusion protein could be demonstrated by immunoblot, indicating that the GFP was retained during RNA replication and did not interfere with HCV polyprotein processing. More importantly, expression levels were robust enough to allow direct visualization of the fusion protein by fluorescence microscopy. NS5A-GFP appeared as brightly fluorescing dot-like structures in the cytoplasm. By confocal laser scanning microscopy, NS5A-GFP colocalized with other HCV nonstructural proteins and nascent viral RNA, indicating that the dot-like structures, identified as membranous webs by electron microscopy, represent functional HCV replication complexes. These findings reveal an unexpected flexibility of the C-terminal domain of NS5A and provide tools for studying the formation and turnover of HCV replication complexes in living cells.

scholarly journals Tagging of NS5A expressed from a functional hepatitis C virus replicon

2006 ◽  
Vol 87 (3) ◽  
pp. 635-640 ◽  
Author(s):  
Christopher J. McCormick ◽  
Sophie Maucourant ◽  
Stephen Griffin ◽  
David J. Rowlands ◽  
Mark Harris
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Lines ◽  
Magnetic Beads ◽  
Fluorescent Protein ◽  
Coding Region ◽  
Replicon Cell ◽  
Replication Complex ◽  
Propionibacterium Shermanii ◽  
Green Fluorescent

Knowledge of how hepatitis C virus (HCV) proteins associate with components of the host cell to form a functional replication complex is still limited. To address this issue, HCV replicon constructs were generated where either green fluorescent protein (GFP) or the Propionibacterium shermanii transcarboxylase domain (PSTCD) was introduced into the NS5A coding region. Insertion of both GFP and PSTCD was tolerated well, allowing formation of stable replicon-containing cell lines that contained viral protein and transcript levels that were comparable to those of an unmodified parental replicon. Cell lines generated from the GFP-tagged NS5A replicon allowed live-cell visualization of the location of NS5A. Cell lines generated from the PSTCD-tagged replicons allowed rapid and efficient precipitation of the PSTCD-tagged NS5A, as well as other HCV non-structural proteins, using streptavidin-coated magnetic beads. Both replicons represent useful tools that offer different but complementary ways of examining replication-complex formation in cells.

scholarly journals Hepatitis C Virus Structural Proteins Assemble into Viruslike Particles in Insect Cells

1998 ◽  
Vol 72 (5) ◽  
pp. 3827-3836 ◽  
Author(s):  
Thomas F. Baumert ◽  
Susumu Ito ◽  
David T. Wong ◽  
T. Jake Liang
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Insect Cells ◽  
Vaccine Development ◽  
Cultured Cells ◽  
Recombinant Baculovirus ◽  
Structural Proteins ◽  
Particle Assembly ◽  
Viruslike Particles ◽  
Hcv Structural Proteins

ABSTRACT Hepatitis C virus (HCV) is a leading cause of chronic hepatitis in the world. The study of HCV has been hampered by the low level of viral particles in infected individuals, the inability to propagate efficiently the virus in cultured cells, and the lack of a convenient animal model. Due to these obstacles, neither the structure of the virus nor the prerequisites for its assembly have been clearly defined. In this report, we describe a model for the production and purification of HCV-like particles in insect cells using a recombinant baculovirus containing the cDNA of the HCV structural proteins. In insect cells, expressed HCV structural proteins assembled into enveloped viruslike particles (40 to 60 nm in diameter) in large cytoplasmic cisternae, presumably derived from the endoplasmic reticulum. Biophysical characterization of viruslike particles by CsCl and sucrose gradient centrifugation revealed biophysical properties similar to those of putative virions isolated from infected humans. The results suggested that HCV core and envelope proteins without p7 were sufficient for viral particle formation. Analysis of particle-associated nucleic acids demonstrated that HCV RNAs were selectively incorporated into the particles over non-HCV transcripts. The synthesis of HCV-like particles in insect cells may provide an important tool to determine the structural requirements for HCV particle assembly as well as to study viral genome encapsidation and virus-host interactions. The described system may also represent a potential approach toward vaccine development.

scholarly journals Hepatitis C virus p7 protein is localized in the endoplasmic reticulum when it is encoded by a replication-competent genome

2007 ◽  
Vol 88 (1) ◽  
pp. 134-142 ◽  
Author(s):  
G. Haqshenas ◽  
J. M. Mackenzie ◽  
X. Dong ◽  
E. J. Gowans
Keyword(s):  
Endoplasmic Reticulum ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Lipid Bilayers ◽  
Fluorescent Protein ◽  
Wild Type ◽  
Rna Transcripts ◽  
Green Fluorescent ◽  
Viral Polyprotein

p7 protein is a small protein encoded by Hepatitis C virus (HCV) that functions as an ion channel in planar lipid bilayers. The function of p7 is vital for the virus life cycle. In this study, the p7 protein of genotype 2a (strain JFH1; the only strain that replicates and produces virus progeny in vitro) was tagged with either an enhanced green fluorescent protein (eGFP) or a haemagglutinin (HA) epitope to facilitate tracking of the protein in the intracellular environment. The tagged viral polyprotein was expressed transiently in the cells after transfection with the recombinant RNA transcripts. Confocal microscopy revealed that the tagged p7 protein was localized in the endoplasmic reticulum (ER) but not associated with mitochondria. Immunoelectron microscopy confirmed the p7 localization data and, moreover, showed that intracellular virus-like particles formed in the cells transfected with the wild-type, but not the recombinant, transcripts. Following a few passages of the transfected cells, the recombinant genome with the HA tag reverted to wild-type and the entire tag was deleted. Therefore, in this study, it has been demonstrated that the p7 protein in the context of the full-length polyprotein encoded by a replication competent genome is only localized to the ER and has a possible role in HCV particle formation.

Stable Huh-7 cell lines expressing non-structural proteins of genotype 1a of hepatitis C virus

2013 ◽  
Vol 189 (1) ◽  
pp. 65-69 ◽  
Author(s):  
Imran Shahid ◽  
Sana Gull ◽  
Bushra Ijaz ◽  
Waqar Ahmad ◽  
Muhammad Ansar ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Lines ◽  
Structural Proteins ◽  
Genotype 1A
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