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 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.

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
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