Enterovirus (EV) infection has been shown to cause a marked shutoff of host protein synthesis, an event mainly achieved through the cleavages of eukaryotic translation initiation factors eIF4GI and eIF4GII that are mediated by viral 2A protease (2A(pro)). Using fluorescence resonance energy transfer (FRET), we developed genetically encoded and FRET-based biosensors to visualize and quantify the specific proteolytic process in intact cells. This was accomplished by stable expression of a fusion substrate construct composed of the green fluorescent protein 2 (GFP(2)) and red fluorescent protein 2 (DsRed2), with a cleavage motif on eIF4GI or eIF4GII connected in between. The FRET biosensor showed a real-time and quantifiable impairment of FRET upon EV infection. Levels of the reduced FRET closely correlated with the cleavage kinetics of the endogenous eIF4Gs isoforms. The FRET impairments were solely attributed to 2A(pro) catalytic activity, irrespective of other viral-encoded protease, the activated caspases or general inhibition of protein synthesis in the EV-infected cells. The FRET biosensors appeared to be a universal platform for several related EVs. The spatiotemporal and quantitative imaging enabled by FRET can shed light on the protease-substrate behaviors in their normal milieu, permitting investigation into the molecular mechanism underlying virus-induced host translation inhibition. Biotechnol. Bioeng. 2009; 104: 1142-1152. (C) 2009 Wiley Periodicals, Inc.