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 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 Differential Biophysical Properties of Infectious Intracellular and Secreted Hepatitis C Virus Particles

2006 ◽  
Vol 80 (22) ◽  
pp. 11074-11081 ◽  
Author(s):  
Pablo Gastaminza ◽  
Sharookh B. Kapadia ◽  
Francis V. Chisari
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Buoyant Density ◽  
Infection Model ◽  
Virus Particles ◽  
Viral Egress ◽  
Viral Particles ◽  
A Cell ◽  
Infected Cells

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 (∼65 to 70 nm) but different in buoyant density (∼1.15 to 1.20 g/ml) from extracellular particles (∼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.

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 RNA Synthesis in a Cell-Free System Isolated from Replicon-Containing Hepatoma Cells

2003 ◽  
Vol 77 (3) ◽  
pp. 2029-2037 ◽  
Author(s):  
Richard W. Hardy ◽  
Joseph Marcotrigiano ◽  
Keril J. Blight ◽  
John E. Majors ◽  
Charles M. Rice
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Synthesis ◽  
De Novo ◽  
Hcv Rna ◽  
Free System ◽  
Cell Free System ◽  
Huh7 Cells ◽  
A Cell

ABSTRACT A number of hepatitis C virus (HCV) proteins, including NS5B, the RNA-dependent RNA polymerase, were detected in membrane fractions from Huh7 cells containing autonomously replicating HCV RNA replicons. These membrane fractions were used in a cell-free system for the analysis of HCV RNA replication. Initial characterization revealed a reaction in which the production of replicon RNA increased over time at temperatures ranging from 25 to 40°C. Heparin sensitivity and nucleotide starvation experiments suggested that de novo initiation was occurring in this system. Both Mn2+ and Mg2+ cations could be used in the reaction; however, concentrations of Mn2+ greater than 1 mM were inhibitory. Compounds shown to inhibit recombinant NS3 and NS5B activity in vitro were found to inhibit RNA synthesis in the cell-free system. This system should be useful for biochemical analysis of HCV RNA synthesis by a multisubunit membrane-associated replicase and for evaluating potential antiviral agents identified in biochemical or cell-based screens.

scholarly journals Retrovirally Transduced CD34++ Human Cord Blood Cells Generate T Cells Expressing High Levels of the Retroviral Encoded Green Fluorescent Protein Marker In Vitro

Blood ◽  
1998 ◽  
Vol 91 (2) ◽  
pp. 431-440 ◽  
Author(s):  
Bruno Verhasselt ◽  
Magda De Smedt ◽  
Rita Verhelst ◽  
Evelien Naessens ◽  
Jean Plum
Keyword(s):  
Gene Therapy ◽  
Dendritic Cells ◽  
Green Fluorescent Protein ◽  
T Cell ◽  
Gene Transfer ◽  
Cord Blood ◽  
Fluorescent Protein ◽  
Marker Gene ◽  
Green Fluorescent

Human umbilical cord blood (UCB) hematopoietic stem cells (HSC) receive increased attention as a possible target for gene-transfer in gene therapy trials. Diseases affecting the lymphoid lineage, as adenosine deaminase (ADA) deficiency and acquired immunodeficiency syndrome (AIDS) could be cured by gene therapy. However, the T-cell progenitor potential of these HSC after gene-transfer is largely unknown and was up to now not testable in vitro. We show here that highly purified CD34++ Lineage marker-negative (CD34++Lin−) UCB cells generate T, natural killer (NK), and dendritic cells in a severe combined immunodeficient mouse fetal thymus organ culture (FTOC). CD34++Lin− and CD34++CD38−Lin− UCB cells express the retroviral encoded marker gene Green Fluorescent Protein (GFP) after in vitro transduction with MFG-GFP retroviral supernatant. Transduced cells were still capable of generating T, NK, and dendritic cells in the FTOC, all expressing high levels of GFP under control of the Moloney murine leukemia virus (MoMuLV) long terminal repeat promotor. We thus present an in vitro assay for thymic T-cell development out of transduced UCB HSC, using GFP as a marker gene.

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