Detection of Human Enterovirus 71 (HEV71) 3C protease activity in HEV71- infected cells using Fluorescence Resonance Energy Transfer Wu K.X. and Chu J.J.H

A cell-based system that detects human enterovirus 71 (HEV71) 3C protease (3C) activity, using fluorescence resonance energy transfer (FRET), was established by creating a stable cell line that constitutively expresses a FRET protein construct consisting of a 3C cleavage motif flanked by a FRET pair of enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP). HEV71 infection of the cells will lead to cleavage of the motif by 3C, disrupting FRET from excited ECFP to ground state EYFP. Fluoroscopy measurements indicated a time-dependent decrease in FRET efficiency in HEV71 infected cells, which was confirmed by Western blot analysis. Inhibition of HEV71 by ribavirin prevents the cleavage of the FRET substrate in a dose-dependent nature. The rapid and sensitive nature of this system demonstrates its potential for development into a high-throughput assay for anti-HEV71 agents.