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Antimicrobial Agents and Chemotherapy, February 2009, p. 728-734, Vol. 53, No. 2
0066-4804/09/$08.00+0     doi:10.1128/AAC.01029-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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

Rosario Sabariegos,1 Fernando Picazo,1 Beatriz Domingo,1 Sandra Franco,2 Miguel-Angel Martinez,2 and Juan Llopis1*

Centro Regional de Investigaciones Biomédicas (CRIB) and Facultad de Medicina de Albacete, Universidad de Castilla-La Mancha, Albacete, Spain,1 Fundació irsiCaixa, Universitat Autònoma de Barcelona (UAB), Barcelona, Spain2

Received 1 August 2008/ Returned for modification 2 October 2008/ Accepted 29 November 2008

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.

* Corresponding author. Mailing address: Centro Regional de Investigaciones Biomédicas (CRIB), Universidad de Castilla-La Mancha, C/ Almansa 14, 02006 Albacete, Spain. Phone: 34-967-599-315. Fax: 34-967-599-327. E-mail: juan.llopis{at}uclm.es

{triangledown} Published ahead of print on 8 December 2008.

Antimicrobial Agents and Chemotherapy, February 2009, p. 728-734, Vol. 53, No. 2
0066-4804/09/$08.00+0     doi:10.1128/AAC.01029-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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