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 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 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 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 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.

scholarly journals Analysis of the subcellular localization of hepatitis C virus E2 glycoprotein in live cells using EGFP fusion proteins

2003 ◽  
Vol 84 (3) ◽  
pp. 561-566 ◽  
Author(s):  
François Kien ◽  
Jean-Daniel Abraham ◽  
Catherine Schuster ◽  
Marie Paule Kieny
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Fusion Protein ◽  
Subcellular Localization ◽  
Fluorescent Protein ◽  
Live Cells ◽  
Target Cells ◽  
E2 Glycoprotein ◽  
Covalently Linked ◽  
Green Fluorescent

Hepatitis C virus (HCV) E1 and E2 glycoproteins assemble intracellularly to form a non-covalently linked heterodimer, which is retained in the endoplasmic reticulum (ER). To study the subcellular localization of E2 in live cells, the enhanced green fluorescent protein (EGFP) was fused to the N terminus of E2. Using fluorescence and confocal microscopy, we have confirmed that E2 is located in the ER, where budding of HCV virions is thought to occur. Immunoprecipitation experiments using a conformation-sensitive antibody and a GST pull-down assay showed that fusion of EGFP to E2 interferes neither with its heterodimeric assembly with E1, nor with proper folding of the ectodomain, nor with the capacity of E2 to interact with human CD81, indicating that the EGFP–E2 fusion protein is functional. As a tool to study binding of E2 to target cells, we also described the expression of an EGFP–E2 fusion protein at the cell surface.

scholarly journals Infectious Hepatitis C Virus Pseudo-particles Containing Functional E1–E2 Envelope Protein Complexes

2003 ◽  
Vol 197 (5) ◽  
pp. 633-642 ◽  
Author(s):  
Birke Bartosch ◽  
Jean Dubuisson ◽  
François-Loïc Cosset
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Fluorescent Protein ◽  
Protein Complexes ◽  
Marker Gene ◽  
Infectious Hepatitis ◽  
Protein Marker ◽  
A Cell ◽  
Green Fluorescent

The study of hepatitis C virus (HCV), a major cause of chronic liver disease, has been hampered by the lack of a cell culture system supporting its replication. Here, we have successfully generated infectious pseudo-particles that were assembled by displaying unmodified and functional HCV glycoproteins onto retroviral and lentiviral core particles. The presence of a green fluorescent protein marker gene packaged within these HCV pseudo-particles allowed reliable and fast determination of infectivity mediated by the HCV glycoproteins. Primary hepatocytes as well as hepato-carcinoma cells were found to be the major targets of infection in vitro. High infectivity of the pseudo-particles required both E1 and E2 HCV glycoproteins, and was neutralized by sera from HCV-infected patients and by some anti-E2 monoclonal antibodies. In addition, these pseudo-particles allowed investigation of the role of putative HCV receptors. Although our results tend to confirm their involvement, they provide evidence that neither LDLr nor CD81 is sufficient to mediate HCV cell entry. Altogether, these studies indicate that these pseudo-particles may mimic the early infection steps of parental HCV and will be suitable for the development of much needed new antiviral therapies.

scholarly journals Superinfection Exclusion in Cells Infected with Hepatitis C Virus

2007 ◽  
Vol 81 (8) ◽  
pp. 3693-3703 ◽  
Author(s):  
Donna M. Tscherne ◽  
Matthew J. Evans ◽  
Thomas von Hahn ◽  
Christopher T. Jones ◽  
Zania Stamataki ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
General Mechanism ◽  
Hcv Genotype ◽  
Superinfection Exclusion ◽  
Genotype 1A ◽  
Antiviral Compounds ◽  
Specific Protease ◽  
Infected Cells ◽  
Hcvcc Infection

ABSTRACT Superinfection exclusion is the ability of an established virus infection to interfere with infection by a second virus. In this study, we found that Huh-7.5 cells acutely infected with hepatitis C virus (HCV) genotype 2a (chimeric strain J6/JFH) and cells harboring HCV genotype 1a, 1b, or 2a full-length or subgenomic replicons were resistant to infection with cell culture-produced HCV (HCVcc). Replicon-containing cells became permissive for HCVcc infection after treatment with an HCV-specific protease inhibitor. With the exception of cells harboring a J6/JFH-FLneo replicon, infected or replicon-containing cells were permissive for HCV pseudoparticle (HCVpp) entry, demonstrating a postentry superinfection block downstream of primary translation. The surprising resistance of J6/JFH-FLneo replicon-containing cells to HCVpp infection suggested a defect in virus entry. This block was due to reduced expression of the HCV coreceptor CD81. Further analyses indicated that J6/JFH may be toxic for cells expressing high levels of CD81, thus selecting for a CD81low population. CD81 down regulation was not observed in acutely infected cells, suggesting that this may not be a general mechanism of HCV superinfection exclusion. Thus, HCV establishes superinfection exclusion at a postentry step, and this effect is reversible by treatment of infected cells with antiviral compounds.

Sign in / Sign up

Export Citation Format

Share Document