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 pro

SUBMITTER: Sabariegos R 

PROVIDER: S-EPMC2630637 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature