title: Fluorescence resonance energy transfer-based assay for characterization of hepatitis C virus NS3-4A protease activity in live cells.
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.
authors.affiliation: Centro Regional de Investigaciones Biomédicas, Universidad de Castilla-La Mancha, Albacete, Spain.
authors.forename: Rosario
authors.initials: R
authors.lastname: Sabariegos
authors.forename: Fernando
authors.initials: F
authors.lastname: Picazo
authors.forename: Beatriz
authors.initials: B
authors.lastname: Domingo
authors.forename: Sandra
authors.initials: S
authors.lastname: Franco
authors.forename: Miguel-Angel
authors.initials: MA
authors.lastname: Martinez
authors.forename: Juan
authors.initials: J
authors.lastname: Llopis
mesh_terms: D015153:Blotting, Western; D002384:Catalysis; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D016174:Hepacivirus / Q000201:enzymology*; D006801:Humans; D010957:Plasmids / Q000235:genetics; D013050:Spectrometry, Fluorescence; D017361:Viral Nonstructural Proteins / Q000235:genetics*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D017931:DNA Primers; D049452:Green Fluorescent Proteins; D017361:Viral Nonstructural Proteins; C485182:Cyan Fluorescent Protein; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases