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

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.

Screening of Hepatitis C Virus Inhibitors Using Genotype 1a HCV Replicon Cell Lines

2011 ◽  
Vol 22 (1) ◽  
Author(s):  
Margaret Robinson ◽  
Yang Tian ◽  
Nikos Pagratis ◽  
William E. Delaney
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Lines ◽  
Replicon Cell ◽  
Genotype 1A

scholarly journals Effect of the 5′ non-translated region on self-assembly of hepatitis C virus genotype 1a structural proteins produced in insect cells

2004 ◽  
Vol 85 (12) ◽  
pp. 3659-3670 ◽  
Author(s):  
Christel Girard ◽  
Marc Ravallec ◽  
Marcel Mariller ◽  
Jean-Pierre Bossy ◽  
Annie Cahour ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Self Assembly ◽  
Insect Cells ◽  
Structural Proteins ◽  
Virus Genotype ◽  
Genotype 1A

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 Hepatitis C virus non-structural proteins responsible for suppression of the RIG-I/Cardif-induced interferon response

2007 ◽  
Vol 88 (12) ◽  
pp. 3323-3333 ◽  
Author(s):  
Megumi Tasaka ◽  
Naoya Sakamoto ◽  
Yoshie Itakura ◽  
Mina Nakagawa ◽  
Yasuhiro Itsui ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Lines ◽  
Type I Interferons ◽  
Hek293 Cells ◽  
Structural Proteins ◽  
Interferon Response ◽  
Type I ◽  
Cellular Expression ◽  
Expression Response

Viral infections activate cellular expression of type I interferons (IFNs). These responses are partly triggered by RIG-I and mediated by Cardif, TBK1, IKKϵ and IRF-3. This study analysed the mechanisms of dsRNA-induced IFN responses in various cell lines that supported subgenomic hepatitis C virus (HCV) replication. Transfection of dsRNA into Huh7, HeLa and HEK293 cells induced an IFN expression response as shown by IRF-3 dimerization, whilst these responses were abolished in corresponding cell lines that expressed HCV replicons. Similarly, RIG-I-dependent activation of the IFN-stimulated response element (ISRE) was significantly suppressed by cells expressing the HCV replicon and restored in replicon-eliminated cells. Overexpression analyses of individual HCV non-structural proteins revealed that NS4B, as well as NS34A, significantly inhibited RIG-I-triggered ISRE activation. Taken together, HCV replication and protein expression substantially blocked the dsRNA-triggered, RIG-I-mediated IFN expression response and this blockade was partly mediated by HCV NS4B, as well as NS34A. These mechanisms may contribute to the clinical persistence of HCV infection and could constitute a novel antiviral therapeutic target.

scholarly journals Novel Hepatitis C Virus Reporter Replicon Cell Lines Enable Efficient Antiviral Screening against Genotype 1a

2010 ◽  
Vol 54 (8) ◽  
pp. 3099-3106 ◽  
Author(s):  
Margaret Robinson ◽  
Huiling Yang ◽  
Siu-Chi Sun ◽  
Betty Peng ◽  
Yang Tian ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Drug Discovery ◽  
Cell Lines ◽  
Luciferase Reporter ◽  
Replicon Cell ◽  
Adaptive Mutations ◽  
Discovery Research ◽  
Genotype 1A ◽  
Drug Discovery Research

ABSTRACT The hepatitis C virus (HCV) subgenomic replicon is the primary tool for evaluating the activity of anti-HCV compounds in drug discovery research. Despite the prevalence of HCV genotype 1a (∼70% of U.S. HCV patients), few genotype 1a reporter replicon cell lines have been described; this is presumably due to the low replication capacity of such constructs in available Huh-7 cells. In this report, we describe the selection of highly permissive Huh-7 cell lines that support robust replication of genotype 1a subgenomic replicons harboring luciferase reporter genes. These novel cell lines support the replication of multiple genotype 1a replicons (including the H77 and SF9 strains), are significantly more permissive to genotype 1a HCV replication than parental Huh7-Lunet cells, and maintain stable genotype 1a replication levels suitable for antiviral screening. We found that the sensitivity of genotype 1a luciferase replicons to known antivirals was highly consistent between individual genotype 1a clonal cell lines but could vary significantly between genotypes 1a and 1b. Sequencing of the nonstructural region of 12 stable replicon cell clones suggested that the enhanced permissivity is likely due to cellular component(s) in these new cell lines rather than the evolution of novel adaptive mutations in the replicons. These new reagents will enhance drug discovery efforts targeting genotype 1a and facilitate the profiling of compound activity among different HCV genotypes and subtypes.

Hepatitis C Virus Structural Proteins and Activation of Toll-Like Receptors

2005 ◽  
Vol 43 (01) ◽  
Author(s):  
M Hoffmann ◽  
P Henneke ◽  
S Weichert ◽  
H Barth ◽  
B Gissler ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Structural Proteins ◽  
Toll Like Receptors

scholarly journals Resistance Analysis of the Hepatitis C Virus NS3 Protease Inhibitor Asunaprevir

2012 ◽  
Vol 56 (7) ◽  
pp. 3670-3681 ◽  
Author(s):  
Fiona McPhee ◽  
Jacques Friborg ◽  
Steven Levine ◽  
Chaoqun Chen ◽  
Paul Falk ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protease Inhibitor ◽  
Clinical Studies ◽  
Replication Capacity ◽  
Replication Complex ◽  
Hcv Genotype ◽  
Genotype 1A ◽  
Genotype 1B ◽  
Ns3 Protease

ABSTRACTAsunaprevir (BMS-650032) is a potent hepatitis C virus (HCV) NS3 protease inhibitor demonstrating efficacy in alfa interferon-sparing, direct-acting antiviral dual-combination regimens (together with the NS5A replication complex inhibitor daclatasvir) in patients chronically infected with HCV genotype 1b. Here, we describe a comprehensivein vitrogenotypic and phenotypic analysis of asunaprevir-associated resistance against genotypes 1a and 1b using HCV replicons and patient samples obtained from clinical studies of short-term asunaprevir monotherapy. During genotype 1a resistance selection using HCV replicons, the primary NS3 protease substitutions identified were R155K, D168G, and I170T, which conferred low- to moderate-level asunaprevir resistance (5- to 21-fold) in transient-transfection susceptibility assays. For genotype 1b, a higher level of asunaprevir-associated resistance was observed at the same selection pressures, ranging from 170- to 400-fold relative to the wild-type control. The primary NS3 protease substitutions identified occurred predominantly at amino acid residue D168 (D168A/G/H/V/Y) and were associated with high-level asunaprevir resistance (16- to 280-fold) and impaired replication capacity. In asunaprevir single-ascending-dose and 3-day multiple-ascending-dose studies in HCV genotype 1a- or 1b-infected patients, the predominant pre-existing NS3 baseline polymorphism was NS3-Q80K. This substitution impacted initial virologic response rates in a single-ascending-dose study, but its effects after multiple doses were more ambiguous. Interestingly, for patient NS3 protease sequences containing Q80 and those containing K80, susceptibilities to asunaprevir were comparable when tested in an enzyme assay. No resistance-associated variants emerged in these clinical studies that significantly impacted susceptibility to asunaprevir. Importantly, asunaprevir-resistant replicons remained susceptible to an NS5A replication complex inhibitor, consistent with a role for asunaprevir in combination therapies.

scholarly journals Binding of Hepatitis C Virus-Like Particles Derived from Infectious Clone H77C to Defined Human Cell Lines

2002 ◽  
Vol 76 (3) ◽  
pp. 1181-1193 ◽  
Author(s):  
Sabine Wellnitz ◽  
Bettina Klumpp ◽  
Heidi Barth ◽  
Susumu Ito ◽  
Erik Depla ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Lines ◽  
Human Cell ◽  
Envelope Glycoprotein ◽  
Infectious Clone ◽  
Cell Interactions ◽  
Target Cells ◽  
Human Cell Lines ◽  
Glycoprotein E2

ABSTRACT Hepatitis C virus (HCV) is a leading cause of chronic hepatitis in the world. The study of viral entry and infection has been hampered by the inability to efficiently propagate the virus in cultured cells and the lack of a small-animal model. Recent studies have shown that in insect cells, the HCV structural proteins assemble into HCV-like particles (HCV-LPs) with morphological, biophysical, and antigenic properties similar to those of putative virions isolated from HCV-infected humans. In this study, we used HCV-LPs derived from infectious clone H77C as a tool to examine virus-cell interactions. The binding of partially purified particles to human cell lines was analyzed by fluorescence-activated cell sorting with defined monoclonal antibodies to envelope glycoprotein E2. HCV-LPs demonstrated dose-dependent and saturable binding to defined human lymphoma and hepatoma cell lines but not to mouse cell lines. Binding could be inhibited by monoclonal anti-E2 antibodies, indicating that the HCV-LP-cell interaction was mediated by envelope glycoprotein E2. Binding appeared to be CD81 independent and did not correlate with low-density lipoprotein receptor expression. Heat denaturation of HCV-LPs drastically reduced binding, indicating that the interaction of HCV-LPs with target cells was dependent on the proper conformation of the particles. In conclusion, our data demonstrate that insect cell-derived HCV-LPs bind specifically to defined human cell lines. Since the envelope proteins of HCV-LPs are presumably presented in a virion-like conformation, the binding of HCV-LPs to target cells may allow the study of virus-host cell interactions, including the isolation of HCV receptor candidates and antibody-mediated neutralization of binding.

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