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.

scholarly journals Protein Kinase A-Dependent Step(s) in Hepatitis C Virus Entry and Infectivity

2008 ◽  
Vol 82 (17) ◽  
pp. 8797-8811 ◽  
Author(s):  
Michelle J. Farquhar ◽  
Helen J. Harris ◽  
Mandy Diskar ◽  
Sarah Jones ◽  
Christopher J. Mee ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Energy Transfer ◽  
Life Cycle ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protein Kinase ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Viral Receptor ◽  
Kinase A

ABSTRACT Viruses exploit signaling pathways to their advantage during multiple stages of their life cycle. We demonstrate a role for protein kinase A (PKA) in the hepatitis C virus (HCV) life cycle. The inhibition of PKA with H89, cyclic AMP (cAMP) antagonists, or the protein kinase inhibitor peptide reduced HCV entry into Huh-7.5 hepatoma cells. Bioluminescence resonance energy transfer methodology allowed us to investigate the PKA isoform specificity of the cAMP antagonists in Huh-7.5 cells, suggesting a role for PKA type II in HCV internalization. Since viral entry is dependent on the host cell expression of CD81, scavenger receptor BI, and claudin-1 (CLDN1), we studied the role of PKA in regulating viral receptor localization by confocal imaging and fluorescence resonance energy transfer (FRET) analysis. Inhibiting PKA activity in Huh-7.5 cells induced a reorganization of CLDN1 from the plasma membrane to an intracellular vesicular location(s) and disrupted FRET between CLDN1 and CD81, demonstrating the importance of CLDN1 expression at the plasma membrane for viral receptor activity. Inhibiting PKA activity in Huh-7.5 cells reduced the infectivity of extracellular virus without modulating the level of cell-free HCV RNA, suggesting that particle secretion was not affected but that specific infectivity was reduced. Viral particles released from H89-treated cells displayed the same range of buoyant densities as did those from control cells, suggesting that viral protein association with lipoproteins is not regulated by PKA. HCV infection of Huh-7.5 cells increased cAMP levels and phosphorylated PKA substrates, supporting a model where infection activates PKA in a cAMP-dependent manner to promote virus release and transmission.

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 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 Intracellular Hepatitis C Virus Modeling Predicts Infection Dynamics and Viral Protein Mechanisms

2018 ◽  
Vol 92 (11) ◽  
pp. e02098-17 ◽  
Author(s):  
Thomas R. Aunins ◽  
Katherine A. Marsh ◽  
Gitanjali Subramanya ◽  
Susan L. Uprichard ◽  
Alan S. Perelson ◽  
...  
Keyword(s):  
Life Cycle ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Kinetic Data ◽  
Viral Proteins ◽  
Viral Life Cycle ◽  
Hcv Infection ◽  
Viral Rna ◽  
Model Parameters ◽  
Virus Life Cycle

ABSTRACTHepatitis C virus (HCV) infection is a global health problem, with nearly 2 million new infections occurring every year and up to 85% of these infections becoming chronic infections that pose serious long-term health risks. To effectively reduce the prevalence of HCV infection and associated diseases, it is important to understand the intracellular dynamics of the viral life cycle. Here, we present a detailed mathematical model that represents the full hepatitis C virus life cycle. It is the first full HCV model to be fit to acute intracellular infection data and the first to explore the functions of distinct viral proteins, probing multiple hypotheses ofcis- andtrans-acting mechanisms to provide insights for drug targeting. Model parameters were derived from the literature, experiments, and fitting to experimental intracellular viral RNA, extracellular viral titer, and HCV core and NS3 protein kinetic data from viral inoculation to steady state. Our model predicts higher rates for protein translation and polyprotein cleavage than previous replicon models and demonstrates that the processes of translation and synthesis of viral RNA have the most influence on the levels of the species we tracked in experiments. Overall, our experimental data and the resulting mathematical infection model reveal information about the regulation of core protein during infection, produce specific insights into the roles of the viral core, NS5A, and NS5B proteins, and demonstrate the sensitivities of viral proteins and RNA to distinct reactions within the life cycle.IMPORTANCEWe have designed a model for the full life cycle of hepatitis C virus. Past efforts have largely focused on modeling hepatitis C virus replicon systems, in which transfected subgenomic HCV RNA maintains autonomous replication in the absence of virion production or spread. We started with the general structure of these previous replicon models and expanded it to create a model that incorporates the full virus life cycle as well as additional intracellular mechanistic detail. We compared several different hypotheses that have been proposed for different parts of the life cycle and applied the corresponding model variations to infection data to determine which hypotheses are most consistent with the empirical kinetic data. Because the infection data we have collected for this study are a more physiologically relevant representation of a viral life cycle than data obtained from a replicon system, our model can make more accurate predictions about clinical hepatitis C virus infections.

scholarly journals Fluorescence Resonance Energy Transfer-Based Assay for Characterization of Hepatitis C Virus NS3-4A Protease Activity in Live Cells

2008 ◽  
Vol 53 (2) ◽  
pp. 728-734 ◽  
Author(s):  
Rosario Sabariegos ◽  
Fernando Picazo ◽  
Beatriz Domingo ◽  
Sandra Franco ◽  
Miguel-Angel Martinez ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Fluorescence Resonance Energy Transfer ◽  
Protease Activity ◽  
Cleavage Site ◽  
Mammalian Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Fluorescence Resonance

ABSTRACT The NS3/4A protease from hepatitis C virus (HCV) plays a key role in viral replication. We report a system for monitoring the activity of this enzyme in single living mammalian cells. We constructed a fluorescence resonance energy transfer (FRET) probe that consists of an enhanced cyan fluorescent protein-citrine fusion, with a cleavage site for HCV NS3/4A protease embedded within the linker between them. Expression of the biosensor in mammalian cells resulted in a FRET signal, and cotransfection with the NS3/4A expression vector produced a significant reduction in FRET, indicating that the cleavage site was processed. Western blot and spectrofluorimetry analysis confirmed the physical cleavage of the fusion probe by the NS3/4A protease. As the level of FRET decay was a function of the protease activity, the system allowed testing of NS3/4A protease variants with different catalytic efficiencies. This FRET probe could be adapted for high-throughput screening of new HCV NS3/4 protease inhibitors.

Longer wavelength fluorescence resonance energy transfer depsipeptide substrates for hepatitis C virus NS3 protease

2007 ◽  
Vol 368 (2) ◽  
pp. 156-167 ◽  
Author(s):  
Alex K. Konstantinidis ◽  
Paul L. Richardson ◽  
Kevin A. Kurtz ◽  
Rakesh Tripathi ◽  
Chih-Ming Chen ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Fluorescence Resonance ◽  
Ns3 Protease

scholarly journals Functional Analysis of Hepatitis C Virus (HCV) Envelope Protein E1 Using a trans-Complementation System Reveals a Dual Role of a Putative Fusion Peptide of E1 in both HCV Entry and Morphogenesis

2017 ◽  
Vol 91 (7) ◽  
Author(s):  
Yimin Tong ◽  
Xiaojing Chi ◽  
Wei Yang ◽  
Jin Zhong
Keyword(s):  
Cell Culture ◽  
Life Cycle ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Ectopic Expression ◽  
Fusion Peptide ◽  
Hcv Infection ◽  
Cell Culture Model ◽  
Culture Model

ABSTRACT Hepatitis C virus (HCV) is an enveloped RNA virus belonging to the Flaviviridae family. It infects mainly human hepatocytes and causes chronic liver diseases, including cirrhosis and cancer. HCV encodes two envelope proteins, E1 and E2, that form a heterodimer and mediate virus entry. While E2 has been extensively studied, less has been done so for E1, and its role in the HCV life cycle still needs to be elucidated. Here we developed a new cell culture model for HCV infection based on the trans-complementation of E1. Virus production of the HCV genome lacking the E1-encoding sequence can be efficiently rescued by the ectopic expression of E1 in trans. The resulting virus, designated HCVΔE1, can propagate in packaging cells expressing E1 but results in only single-cycle infection in naive cells. By using the HCVΔE1 system, we explored the role of a putative fusion peptide (FP) of E1 in HCV infection. Interestingly, we found that the FP not only contributes to HCV entry, as previously reported, but also may be involved in virus morphogenesis. Finally, we identified amino acid residues in FP that are critical for biological functions of E1. In summary, our work not only provides a new cell culture model for studying HCV but also provides some insights into understanding the role of E1 in the HCV life cycle. IMPORTANCE Hepatitis C virus (HCV), an enveloped RNA virus, encodes two envelope proteins, E1 and E2, that form a heterodimeric complex to mediate virus entry. Compared to E2, the biological functions of E1 in the virus life cycle are not adequately investigated. Here we developed a new cell culture model for single-cycle HCV infection based on the trans-complementation of E1. The HCV genome lacking the E1-encoding sequence can be efficiently rescued for virus production by the ectopic expression of E1 in trans. This new model renders a unique system to dissect functional domains and motifs in E1. Using this system, we found that a putative fusion peptide in E1 is a multifunctional structural element contributing to both HCV entry and morphogenesis. Our work has provided a new cell culture model to study HCV and provides insights into understanding the biological roles of E1 in the HCV life cycle.

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