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

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)-1?-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-1?. 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-1? 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.

SUBMITTER: Zhang Y

PROVIDER: S-EPMC5583365 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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

Zhang Yuan Y Ke Xianliang X Zheng Caishang C Liu Yan Y Xie Li L Zheng Zhenhua Z Wang Hanzhong H

Scientific reports 20170904 1

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<sup>pro</sup>) 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<sup>pro</ ...[more]

PMID: 28871120

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Project description:BACKGROUND:Enterovirus 71 (EV71) is one of the major causative agents of hand, foot, and mouth disease (HFMD), which is sometimes associated with severe central nervous system disease in children. There is currently no specific medication for EV71 infection. Quercetin, one of the most widely distributed flavonoids in plants, has been demonstrated to inhibit various viral infections. However, investigation of the anti-EV71 mechanism has not been reported to date. METHODS:The anti-EV71 activity of quercetin was evaluated by phenotype screening, determining the cytopathic effect (CPE) and EV71-induced cells apoptosis. The effects on EV71 replication were evaluated further by determining virus yield, viral RNA synthesis and protein expression, respectively. The mechanism of action against EV71 was determined from the effective stage and time-of-addition assays. The possible inhibitory functions of quercetin via viral 2Apro, 3Cpro or 3Dpol were tested. The interaction between EV71 3Cpro and quercetin was predicted and calculated by molecular docking. RESULTS:Quercetin inhibited EV71-mediated cytopathogenic effects, reduced EV71 progeny yields, and prevented EV71-induced apoptosis with low cytotoxicity. Investigation of the underlying mechanism of action revealed that quercetin exhibited a preventive effect against EV71 infection and inhibited viral adsorption. Moreover, quercetin mediated its powerful therapeutic effects primarily by blocking the early post-attachment stage of viral infection. Further experiments demonstrated that quercetin potently inhibited the activity of the EV71 protease, 3Cpro, blocking viral replication, but not the activity of the protease, 2Apro, or the RNA polymerase, 3Dpol. Modeling of the molecular binding of the 3Cpro-quercetin complex revealed that quercetin was predicted to insert into the substrate-binding pocket of EV71 3Cpro, blocking substrate recognition and thereby inhibiting EV71 3Cpro activity. CONCLUSIONS:Quercetin can effectively prevent EV71-induced cell injury with low toxicity to host cells. Quercetin may act in more than one way to deter viral infection, exhibiting some preventive and a powerful therapeutic effect against EV71. Further, quercetin potently inhibits EV71 3Cpro activity, thereby blocking EV71 replication.

Dataset Information

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

Publications

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

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