Development of a luciferase-based biosensor to assess enterovirus 71 3C protease activity in living cells

Enterovirus 71 (EV71) is a major pathogen of hand, foot, and mouth disease (HFMD). To date, no antiviral drug has been approved to treat EV71 infection. Due to the essential role that EV71 3 C protease (3C pro ) plays in the viral life cycle, it is generally considered as a highly appealing target for antiviral drug development. In this study, we present a transgene-encoded biosensor that can accurately, sensitively and quantitatively report the proteolytic activity of EV71 3C pro . This biosensor is based on the catalyzed activity of a pro–interleukin (IL)-1β-enterovirus 3C pro cleavage site- Gaussia Luciferase (GLuc) fusion protein that we named i-3CS-GLuc. GLuc enzyme is inactive in the fusion protein because of aggregation caused by pro–IL-1β. However, the 3C pro of EV71 and other enteroviruses, such as coxsackievirus A9 (CVA9), coxsackievirus B3 (CVB3), and poliovirus can recognize and process the canonical enterovirus 3C pro cleavage site between pro–IL-1β and GLuc, thereby releasing and activating GLuc and resulting in increased luciferase activity. The high sensitivity, ease of use, and applicability as a transgene in cell-based assays of i-3CS-GLuc biosensor make it a powerful tool for studying viral protease proteolytic events in living cells and for achieving high-throughput screening of antiviral agents.