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 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 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 Potyvirus-induced gene silencing: the dynamic process of systemic silencing and silencing suppression

2007 ◽  
Vol 88 (8) ◽  
pp. 2337-2346 ◽  
Author(s):  
Elin Gammelgård ◽  
Maradumane Mohan ◽  
Jari P. T. Valkonen
Keyword(s):  
Gene Silencing ◽  
Fluorescent Protein ◽  
Uv Light ◽  
Deletion Mutants ◽  
Virus Induced Gene Silencing ◽  
Potato Virus A ◽  
Silencing Suppression ◽  
Dark Green ◽  
Green Fluorescent ◽  
Interfering Rna

Potato virus A (PVA; genus Potyvirus) was used for virus-induced gene silencing in a model system that included transgenic Nicotiana benthamiana (line 16c) expressing the gfp transgene for green fluorescent protein (GFP) and chimeric PVA (PVA–GFP) carrying gfp in the P1-encoding region. Infection of the 16c plants with PVA–GFP in five experiments resulted in a reproducible pattern of systemic gfp transgene silencing, despite the presence of the strong silencing-suppressor protein, HC-Pro, produced by the virus. PVA–GFP was also targeted by silencing, and virus-specific short interfering RNA accumulated from the length of the viral genome. Viral deletion mutants lacking the gfp insert appeared in systemically infected leaves and reversed silencing of the gfp transgene in limited areas. However, systemic gfp silencing continued in newly emerging leaves in the absence of the gfp-carrying virus, which implicated a systemic silencing signal that moved from lower leaves without interference by HC-Pro. Use of GFP as a visual marker revealed a novel, mosaic-like recovery phenotype in the top leaves. The leaf areas appearing red or purple under UV light (no GFP expression) contained little PVA and gfp mRNA, and corresponded to the dark-green islands observed under visible light. The surrounding green fluorescent tissues contained actively replicating viral deletion mutants that suppressed GFP silencing. Taken together, systemic progression of gene silencing and antiviral defence (RNA silencing) and circumvention of the silencing by the virus could be visualized and analysed in a novel manner.

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 Hepatitis C Virus Replicons Escape RNA Interference Induced by a Short Interfering RNA Directed against the NS5b Coding Region

2005 ◽  
Vol 79 (11) ◽  
pp. 7050-7058 ◽  
Author(s):  
Joyce A. Wilson ◽  
Christopher D. Richardson
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Interference ◽  
Point Mutations ◽  
Subsequent Treatment ◽  
Escape Mutant ◽  
Target Sequence ◽  
Short Interfering Rna ◽  
Coding Region ◽  
Interfering Rna

ABSTRACT RNA interference represents an exciting new technology that could have therapeutic applications for the treatment of viral infections. Hepatitis C virus (HCV) is a major cause of chronic liver disease and affects over 270 million individuals worldwide. The HCV genome is a single-stranded RNA that functions as both an mRNA and a replication template, making it an attractive target for therapeutic approaches using short interfering RNA (siRNA). We have shown previously that double-stranded siRNA molecules designed to target the HCV genome block gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells. However, we now show that this block is not complete. After several treatments with a highly effective siRNA, we have shown growth of replicon RNAs that are resistant to subsequent treatment with the same siRNA. However, these replicon RNAs were not resistant to siRNA targeting another part of the genome. Sequence analysis of the siRNA-resistant replicons showed the generation of point mutations within the siRNA target sequence. In addition, the use of a combination of two siRNAs together severely limited escape mutant evolution. This suggests that RNA interference activity could be used as a treatment to reduce the devastating effects of HCV replication on the liver and the use of multiple siRNAs could prevent the emergence of resistant viruses.

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.

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