Development of a luciferase-based biosensor to assess enterovirus 71 3C protease activity in living cells.
Development of a luciferase-based biosensor to assess enterovirus 71 3C protease activity in living cells.
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开发基于荧光素酶的生物传感器来评估活细胞中肠道病毒 71 3C 蛋白酶活性
DOI：
10.1038/s41598-017-10840-x
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发表时间：
2017-09-04
影响因子：
4.6
通讯作者：
Wang H
中科院分区：
文献类型：
--
作者：
Zhang Y;Ke X;Zheng C;Liu Y;Xie L;Zheng Z;Wang H
肠病毒71型（EV71）是手足口病的主要病原体。到目前为止，还没有抗病毒药物被批准用于治疗EV71感染。由于EV71 C蛋白酶（3Cpro）在病毒生命周期中发挥着重要的作用，因此被认为是抗病毒药物开发的一个极具吸引力的靶点。在这项研究中，我们提出了一种转基因编码的生物传感器，可以准确、灵敏、定量地报告EV71 3Cpro的蛋白水解活性。这种生物传感器是基于一种前白细胞介素(IL)-1β-肠病毒3cproleavage位点- gaussialuciferase （GLuc）融合蛋白的催化活性，我们将其命名为i-3CS-GLuc。由于前il -1β引起的聚集，GLuc酶在融合蛋白中失活。然而，3proof EV71和其他肠道病毒，如柯萨奇病毒A9 （CVA9）、柯萨奇病毒B3 （CVB3）和脊髓灰质炎病毒可以识别和加工典型肠道病毒33cproleavage位点，在前il -1β和GLuc之间，从而释放和激活GLuc，导致荧光素酶活性增加。i-3CS-GLuc生物传感器的高灵敏度、易用性和作为转基因在细胞检测中的适用性使其成为研究活细胞中病毒蛋白酶蛋白水解事件和实现抗病毒药物高通量筛选的有力工具。
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 3Cprocleavage site-GaussiaLuciferase (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 3Cproof EV71 and other enteroviruses, such as coxsackievirus A9 (CVA9), coxsackievirus B3 (CVB3), and poliovirus can recognize and process the canonical enterovirus 3Cprocleavage 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.
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