Abstract
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 (3Cpro) 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 3Cpro. This biosensor is based on the catalyzed activity of a pro–interleukin (IL)-1β-enterovirus 3Cpro 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 3Cpro of EV71 and other enteroviruses, such as coxsackievirus A9 (CVA9), coxsackievirus B3 (CVB3), and poliovirus can recognize and process the canonical enterovirus 3Cpro 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.
Highlights
Enterovirus 71 (EV71) belongs to the genus Enterovirus of the Picornaviridae family, with a positive sense, single-stranded RNA genome approximately 7400 nt in length
In 2013, scientists found that the fusion of mouse pro–interleukin (IL)-1β on the N-terminal of Gaussia Luciferase (GLuc) lacking its secretion signal peptide can inhibit its catalytic activity because pro–IL-1β possesses a strong propensity to form protein aggregates, and based on this, they developed a biosensor pro–IL-1β-GLuc that can report the proteolytic activity of caspase-1 in the course of inflammasome activation in a highly sensitive manner[21]
To generate biosensors for EV71 3 C protease (3Cpro) activity, the canonical enterovirus 3Cpro cleavage site EALFQ↓GPPK was inserted into the iGLuc construct at different positions
Summary
EV71 belongs to the genus Enterovirus of the Picornaviridae family, with a positive sense, single-stranded RNA genome approximately 7400 nt in length. A canonical enterovirus 3Cpro cleavage site EALFQ↓GPPK was inserted between mouse pro–IL-1β and GLuc lacking its secretion signal peptide to generate the biosensor pro– IL-1β-enterovirus 3Cpro cleavage site-GLuc (i-3CS-GLuc) Within this biosensor, the GLuc enzyme is normally inactive because of the protein aggregation caused by pro–IL-1β. We demonstrated that the 3Cpro of EV71 and some other enteroviruses could recognize and process the EALFQ↓GPPK site within i-3CS-GLuc, activating the GLuc enzyme and allowing the monitoring of cytosolic cleavage events and protease activity with high sensitivity and specificity. This biosensor’s ease of use and its applicability in living cells make it a powerful tool to screen antiviral drugs with high-throughput
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