scholarly journals Development of NS3/4A Protease-Based Reporter Assay Suitable for Efficiently Assessing Hepatitis C Virus Infection

2009 ◽  
Vol 53 (11) ◽  
pp. 4825-4834 ◽  
Author(s):  
Kao-Lu Pan ◽  
Jin-Ching Lee ◽  
Hsing-Wen Sung ◽  
Teng-Yuang Chang ◽  
John T.-A. Hsu
Keyword(s):  
Cell Culture ◽  
Life Cycle ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Infection ◽  
High Throughput ◽  
High Throughput Screening ◽  
Reporter System ◽  
Viral Protease ◽  
Peptide Cleavage

ABSTRACT A cell culture system for the production of hepatitis C virus (HCV) whole virions has greatly accelerated studies of the virus life cycle and the discovery of anti-HCV agents. However, the quantification of the HCV titers in a whole-virus infection/replication system currently relies mostly on reverse transcription-PCR or immunofluorescence assay, which would be cumbersome for high-throughput drug screening. To overcome this problem, this study has generated a novel cell line, Huh7.5-EG(Δ4B5A)SEAP, that carries a dual reporter, EG(Δ4B5A)SEAP. The EG(Δ4B5A)SEAP reporter is a viral protease-cleavable fusion protein in which the enhanced green fluorescence protein is linked to secreted alkaline phosphatase (SEAP) in frame via Δ4B5A, a short peptide cleavage substrate for NS3/4A viral protease. This study demonstrates that virus replication/infection in the Huh7.5-EG(Δ4B5A)SEAP cells can be quantitatively indicated by measuring the SEAP activity in cell culture medium. The levels of SEAP released from HCV-infected Huh7.5-EG(Δ4B5A)SEAP cells correlated closely with the amounts of HCV in the inocula. The Huh7.5-EG(Δ4B5A)SEAP cells were also shown to be a suitable host for the discovery of anti-HCV inhibitors by using known compounds that target multiple stages of the HCV life cycle. The Z′-factor of this assay ranged from 0.64 to 0.74 in 96-well plates, indicating that this reporter system is suitable for high-throughput screening of prospective anti-HCV agents.

scholarly journals Development of a Cell-Based Hepatitis C Virus Infection Fluorescent Resonance Energy Transfer Assay for High-Throughput Antiviral Compound Screening

2009 ◽  
Vol 53 (10) ◽  
pp. 4311-4319 ◽  
Author(s):  
Xuemei Yu ◽  
Bruno Sainz ◽  
Susan L. Uprichard
Keyword(s):  
Cell Culture ◽  
Energy Transfer ◽  
Life Cycle ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
High Throughput ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Viral Life Cycle ◽  
Hcv Infection

ABSTRACT A major obstacle in the treatment of chronic hepatitis C virus (HCV) infection has been the lack of effective, well-tolerated therapeutics. Notably, the recent development of the HCV cell culture infection system now allows not only for the study of the entire viral life cycle, but also for the screening of inhibitors against all aspects of HCV infection. However, in order to screen libraries of potential antiviral compounds, it is necessary to develop a highly reproducible, accurate assay for HCV infection adaptable for high-throughput screening (HTS) automation. Using an internally quenched 5-FAM/QXL 520 fluorescence resonance energy transfer (FRET) substrate containing the HCV NS3 peptide cleavage sequence, we report the development of a simple, mix-and-measure, homogenous, cell-based HCV infection assay amendable for HTS. This assay makes use of synchronized, nondividing human hepatoma-derived Huh7 cells, which support more-reproducible long-term HCV infection and can be readily scaled down to a 96-well-plate format. We demonstrate that this stable cell culture method eliminates common problems associated with standard cell-based HTS, such as cell culture variability, poor reproducibility, and low signal intensity. Importantly, this HCV FRET assay not only can identify inhibitors that act throughout the viral life cycle as effectively as more-standard HCV assays, such as real-time quantitative PCR and Western blot analysis, but also exhibits a high degree of accuracy with limited signal variation (i.e., Z′ ≥ 0.6), providing the basis for a robust HTS campaign for screening compound libraries and identifying novel HCV antivirals.

Polyanionic carbosilane dendrimers prevent hepatitis C virus infection in cell culture

2017 ◽  
Vol 13 (1) ◽  
pp. 49-58 ◽  
Author(s):  
Daniel Sepúlveda-Crespo ◽  
José Luis Jiménez ◽  
Rafael Gómez ◽  
Francisco Javier De La Mata ◽  
Pedro L. Majano ◽  
...  
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Infection ◽  
Hepatitis C Virus Infection ◽  
Carbosilane Dendrimers

scholarly journals Novel Cell-Based Hepatitis C Virus Infection Assay for Quantitative High-Throughput Screening of Anti-Hepatitis C Virus Compounds

2013 ◽  
Vol 58 (2) ◽  
pp. 995-1004 ◽  
Author(s):  
Zongyi Hu ◽  
Keng-Hsin Lan ◽  
Shanshan He ◽  
Manju Swaroop ◽  
Xin Hu ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
High Throughput ◽  
High Throughput Screening ◽  
Dose Range ◽  
Hcv Infection ◽  
Replication Cycle ◽  
Screening Methods ◽  
Functional Features ◽  
Pharmacologically Active

ABSTRACTTherapy for hepatitis C virus (HCV) infection has advanced with the recent approval of direct-acting antivirals in combination with peginterferon and ribavirin. New antivirals with novel targets are still needed to further improve the treatment of hepatitis C. Previously reported screening methods for HCV inhibitors either are limited to a virus-specific function or apply a screening method at a single dose, which usually leads to high false-positive or -negative rates. We developed a quantitative high-throughput screening (qHTS) assay platform with a cell-based HCV infection system. This highly sensitive assay can be miniaturized to a 1,536-well format for screening of large chemical libraries. All candidates are screened over a 7-concentration dose range to give EC50s (compound concentrations at 50% efficacy) and dose-response curves. Using this assay format, we screened a library of pharmacologically active compounds (LOPAC). Based on the profile of dose-dependent curves of HCV inhibition and cytotoxicity, 22 compounds with adequate curves and EC50s of <10 μM were selected for validation. In two additional independent assays, 17 of them demonstrated specific inhibition of HCV infection. Ten potential candidates with efficacies of >70% and CC50s (compound concentrations at 50% cytotoxicity) of <30 μM from these validated hits were characterized for their target stages in the HCV replication cycle. In this screen, we identified both known and novel hits with diverse structural and functional features targeting various stages of the HCV replication cycle. The pilot screen demonstrates that this assay system is highly robust and effective in identifying novel HCV inhibitors and that it can be readily applied to large-scale screening of small-molecule libraries.

scholarly journals Entry and Release of Hepatitis C Virus in Polarized Human Hepatocytes

2017 ◽  
Vol 91 (18) ◽  
Author(s):  
Sandrine Belouzard ◽  
Adeline Danneels ◽  
Lucie Fénéant ◽  
Karin Séron ◽  
Yves Rouillé ◽  
...  
Keyword(s):  
Cell Culture ◽  
Life Cycle ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Polarity ◽  
Cell Polarization ◽  
Cell Culture System ◽  
Viral Particles ◽  
Polarized Cells ◽  
Lipoprotein Secretion

ABSTRACT Hepatitis C virus (HCV) primarily infects hepatocytes, which are highly polarized cells. The relevance of cell polarity in the HCV life cycle has been addressed only in distantly related models and remains poorly understood. Although polarized epithelial cells have a rather simple morphology with a basolateral and an apical domain, hepatocytes exhibit complex polarization structures. However, it has been reported that some selected polarized HepG2 cell clones can exhibit a honeycomb pattern of distribution of the tight-junction proteins typical of columnar polarized epithelia, which can be used as a simple model to study the role of cell polarization in viral infection of hepatocytes. To obtain similar clones, HepG2 cells expressing CD81 (HepG2-CD81) were used, and clones were isolated by limiting dilutions. Two clones exhibiting a simple columnar polarization capacity when grown on a semipermeable support were isolated and characterized. To test the polarity of HCV entry and release, our polarized HepG2-CD81 clones were infected with cell culture-derived HCV. Our data indicate that HCV binds equally to both sides of the cells, but productive infection occurs mainly when the virus is added at the basolateral domain. Furthermore, we also observed that HCV virions are released from the basolateral domain of the cells. Finally, when polarized cells were treated with oleic acid and U0126, a MEK inhibitor, to promote lipoprotein secretion, a higher proportion of infectious viral particles of lower density were secreted. This cell culture system provides an excellent model to investigate the influence of cell polarization on the HCV life cycle. IMPORTANCE Hepatitis C is a major health burden, with approximately 170 million persons infected worldwide. Hepatitis C virus (HCV) primarily infects hepatocytes, which are highly polarized cells with a complex organization. The relevance of cell polarity in the HCV life cycle has been addressed in distantly related models and remains unclear. Hepatocyte organization is complex, with multiple apical and basolateral surfaces. A simple culture model of HepG2 cells expressing CD81 that are able to polarize with unique apical and basolateral domains was developed to study HCV infection. With this model, we demonstrated that HCV enters and exits hepatocytes by the basolateral domain. Furthermore, lower-density viral particles were produced under conditions that promote lipoprotein secretion. This cell culture system provides a useful model to study the influence of cell polarization on HCV infection.

scholarly journals Development of hepatitis C virus production reporter-assay systems using two different hepatoma cell lines

2012 ◽  
Vol 93 (7) ◽  
pp. 1422-1431 ◽  
Author(s):  
Midori Takeda ◽  
Masanori Ikeda ◽  
Yasuo Ariumi ◽  
Takaji Wakita ◽  
Nobuyuki Kato
Keyword(s):  
Cell Culture ◽  
Life Cycle ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Lines ◽  
Hepatoma Cell ◽  
Rna Replication ◽  
Hcv Rna ◽  
Reporter Assay ◽  
Hepatoma Cell Lines

A hepatitis C virus (HCV) infection system was developed previously using the HCV JFH-1 strain (genotype 2a) and HuH-7 cells, and this cell culture is so far the only robust production system for HCV. In patients with chronic hepatitis C, the virological effects of pegylated interferon and ribavirin therapy differ depending on the HCV strain and the genetic background of the host. Recently, we reported the hepatoma-derived Li23 cell line, in which the JFH-1 life cycle is reproduced at a level almost equal to that in HuH-7-derived RSc cells. To monitor the HCV life cycle more easily, we here developed JFH-1 reporter-assay systems using both HuH-7- and Li23-derived cell lines. To identify any genetic mutations by long-term cell culture, HCV RNAs in HuH-7 cells were amplified 130 days after infection and subjected to sequence analysis to find adaptive mutation(s) for robust virus replication. We identified two mutations, H2505Q and V2995L, in the NS5B region. V2995L but not H2505Q enhanced JFH-1 RNA replication. However, we found that H2505Q but not V2995L enhanced HCV RNA replication of strain O (genotype 1b). We also selected highly permissive D7 cells by serial subcloning of Li23 cells. The expression levels of claudin-1 and Niemann–Pick C1-like 1 in D7 cells are higher than those in parental Li23 cells. In this study, we developed HCV JFH-1 reporter-assay systems using two distinct hepatoma cell lines, HuH-7 and Li23. The mutations in NS5B resulted in different effects on strains O and JFH-1 HCV RNA replication.

scholarly journals Role of Conserved E2 Residue W420 in Receptor Binding and Hepatitis C Virus Infection

2016 ◽  
Vol 90 (16) ◽  
pp. 7456-7468 ◽  
Author(s):  
Vanessa M. Cowton ◽  
Allan G. N. Angus ◽  
Sarah J. Cole ◽  
Christina K. Markopoulou ◽  
Ania Owsianka ◽  
...  
Keyword(s):  
Cell Culture ◽  
Life Cycle ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Entry ◽  
Viral Life Cycle ◽  
Tryptophan Residue ◽  
Viral Glycoprotein ◽  
Cd81 Binding

ABSTRACTHepatitis C virus (HCV) enters cells via interactions with several host factors, a key one being that between the viral E2 envelope glycoprotein and the CD81 receptor. We previously identified E2 tryptophan residue 420 (W420) as an essential CD81-binding residue. However, the importance of W420 in the context of the native virion is unknown, as those previous studies predate the infectious HCV cell culture (cell culture-derived HCV [HCVcc]) system. Here, we introduced four separate mutations (F, Y, A, or R) at position 420 within the infectious HCVcc JFH-1 genome and characterized their effects on the viral life cycle. While all mutations reduced E2-CD81 binding, only two (W420A and W420R) reduced HCVcc infectivity. Further analyses of mutants with hydrophobic residues (F or Y) found that interactions with the receptors SR-BI and CD81 were modulated, which in turn determined the viral uptake route. Both mutant viruses were significantly less dependent on SR-BI, and its lipid transfer activity, for virus entry. Furthermore, these viruses were resistant to the drug erlotinib, which targets epidermal growth factor receptor (EGFR) (a host cofactor for HCV entry) and also blocks SR-BI-dependent high-density lipoprotein (HDL)-mediated enhancement of virus entry. Together, our data indicate a model where an alteration at position 420 causes a subtle change in the E2 conformation that prevents interaction with SR-BI and increases accessibility to the CD81-binding site, in turn favoring a particular internalization route. These results further show that a hydrophobic residue with a strong preference for tryptophan at position 420 is important, both functionally and structurally, to provide an additional hydrophobic anchor to stabilize the E2-CD81 interaction.IMPORTANCEHepatitis C virus (HCV) is a leading cause of liver disease, causing up to 500,000 deaths annually. The first step in the viral life cycle is the entry process. This study investigates the role of a highly conserved residue, tryptophan residue 420, of the viral glycoprotein E2 in this process. We analyzed the effect of changing this residue in the virus and confirmed that this region is important for binding to the CD81 receptor. Furthermore, alteration of this residue modulated interactions with the SR-BI receptor, and changes to these key interactions were found to affect the virus internalization route involving the host cofactor EGFR. Our results also show that the nature of the amino acid at this position is important functionally and structurally to provide an anchor point to stabilize the E2-CD81 interaction.

scholarly journals A Fluorescence-Based High-Throughput Screen to Identify Small Compound Inhibitors of the Genotype 3a Hepatitis C Virus RNA Polymerase

2013 ◽  
Vol 18 (9) ◽  
pp. 1027-1034 ◽  
Author(s):  
Auda A. Eltahla ◽  
Kurt Lackovic ◽  
Christopher Marquis ◽  
John-Sebastian Eden ◽  
Peter A. White
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
High Throughput ◽  
High Throughput Screening ◽  
De Novo ◽  
Specific Activity ◽  
Hcv Rna ◽  
High Throughput Screen ◽  
Rdrp Activity

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) plays an essential role in the replication of HCV and is a key target for novel antiviral therapies. Several RdRp inhibitors are in clinical trials and have increased response rates when combined with current interferon-based therapies for genotype 1 (G1) HCV patients. These inhibitors, however, show poor efficacy against non-G1 genotypes, including G3a, which represents ~20% of HCV cases globally. Here, we used a commercially available fluorescent dye to characterize G3a HCV RdRp in vitro. RdRp activity was assessed via synthesis of double-stranded RNA from the single-stranded RNA poly(C) template. The assay was miniaturized to a 384-well microplate format and a pilot high-throughput screen was conducted using 10,208 “lead-like” compounds, randomly selected to identify inhibitors of HCV G3a RdRp. Of 150 compounds demonstrating greatest inhibition, 10 were confirmed using both fluorescent and radioactive assays. The top two inhibitors (HAC001 and HAC002) demonstrated specific activity, with an IC50 of 12.7 µM and 1.0 µM, respectively. In conclusion, we describe simple, fluorescent-based high-throughput screening (HTS) for the identification of inhibitors of de novo RdRp activity, using HCV G3a RdRp as the target. The HTS system could be used against any positive-sense RNA virus that cannot be cultured.

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