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

作者：Zhang, Yuan; Ke, Xianliang; Zheng, Caishang; Liu, Yan; Xie, Li; Zheng, Zhenhua; Wang, Hanzhong^*

来源：Scientific Reports, 2017, 7(1): 10385.

DOI：10.1038/s41598-017-10840-x

• IN-VIVO
• FUNCTION PREDICTION
• FIREFLY LUCIFERASE
• GAUSSIA LUCIFERASE
• MOUTH-DISEASE
• INFECTION
• CHILDREN
• BIOLUMINESCENCE
• RUPINTRIVIR
• EXPRESSION

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)-1 beta-enterovirus 3C(pro) cleavage siteGaussia 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-1 beta. 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 3C(pro) cleavage site between pro-IL-1 beta 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.

• 出版日期2017-9-4
• 单位中国科学院武汉病毒研究所; 广州市妇女儿童医疗中心