A Continuous Assay of Hepatitis C Virus Protease Based on Resonance Energy Transfer Depsipeptide Substrates

1996 ◽  
Vol 240 (1) ◽  
pp. 60-67 ◽  
Author(s):  
Marina Taliani ◽  
Elisabetta Bianchi ◽  
Frank Narjes ◽  
Marco Fossatelli ◽  
Andrea Urbani ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Continuous Assay

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 Probing the Active Site of the Hepatitis C Virus Serine Protease by Fluorescence Resonance Energy Transfer

2000 ◽  
Vol 275 (20) ◽  
pp. 15106-15113 ◽  
Author(s):  
Daniela Fattori ◽  
Andrea Urbani ◽  
Mirko Brunetti ◽  
Raffaele Ingenito ◽  
Antonello Pessi ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Serine Protease ◽  
Fluorescence Resonance Energy Transfer ◽  
Active Site ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Fluorescence Resonance

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 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 Characterization of the interaction between hepatitis C virus NS5B and the human oestrogen receptor alpha

2012 ◽  
Vol 93 (4) ◽  
pp. 780-785 ◽  
Author(s):  
Julia Hillung ◽  
Elena Ruiz-López ◽  
Itxaso Bellón-Echeverría ◽  
Pilar Clemente-Casares ◽  
Antonio Mas
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Oestrogen Receptor ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Host Factors ◽  
Host Proteins ◽  
Receptor Alpha ◽  
Oestrogen Receptor Alpha

The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is part of the viral replicative complex and plays a crucial role in HCV replication. It has been described that NS5B interacts with cellular proteins, and that interactions between NS5B and host proteins are crucial for viral replication. Some of the host factors involved in the HCV replication cycle include the oestrogen receptor alpha (ESR1), protein kinases (c-Src) and chaperones (Hsp70). In this report, we determine the requirements for the interplay between NS5B and the domain C of ESR1 (ESR1C) by using Förster Resonance Energy Transfer. NS5B–ESR1C and ESR1C–ESR1C interactions are dependent on ionic strength, indicating that contacts are mainly electrostatic. Additionally, NS5B residues involved in NS5B oligomerization were also essential for NS5B–ESR1C interaction. The study of the interactions among viral and host factors will provide data to establish innovative therapeutic strategies and the development of new antiviral drugs.

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