A Cell-Based Reporter Assay for Screening Inhibitors of MERS Coronavirus RNA-Dependent RNA Polymerase Activity
A Cell-Based Reporter Assay for Screening Inhibitors of MERS Coronavirus RNA-Dependent RNA Polymerase Activity
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DOI：
10.3390/jcm9082399
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发表时间：
2020-08-01
影响因子：
3.9
通讯作者：
Jin, Young-Hee
中科院分区：
文献类型：
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作者：
Min, Jung Sun;Kim, Geon-Woo;Jin, Young-Hee
严重急性呼吸综合征（SARS）、中东呼吸综合征（MERS）和2019冠状病毒病（COVID-19）是由冠状病毒（CoV）感染引起的新型人畜共患疾病。由于冠状病毒依赖RNA的RNA聚合酶（RdRp）的序列高度保守，因此RdRp被认为是抗病毒治疗的重要靶点。我们建立了一种基于细胞的MERS-CoV RdRp活性报告基因检测方法，以检测病毒聚合酶抑制剂。基于细胞的报告系统由双顺反子报告基因构建体和MERS-CoV nsp 12质粒构建体组成。在测试的九种病毒聚合酶抑制剂中，利巴韦林、索非布韦、法匹拉韦、拉米夫定、齐多夫定、伐昔洛韦、阿糖腺苷、达沙布韦和瑞德西韦，只有瑞德西韦表现出剂量依赖性抑制。同时，该方法用于筛选MERS-CoV RdRp活性抑制剂的Z因子和Z '因子分别为0.778和0.782。利巴韦林和法匹拉韦不抑制MERS-CoV RdRp活性，非核苷HCV RdRp抑制剂达沙布韦部分抑制MERS-CoV RdRp活性。总之，MERS-CoV RdRp活性的基于细胞的报告基因测定证实了remdesivir是细胞中MERS-CoV RdRp的直接抑制剂。基于细胞的MERS-CoV RdRp活性报告基因检测法用于筛选MERS-CoV RdRp特异性抑制剂是可靠和准确的。它可能为开发针对新出现的CoV感染的抗病毒药物提供有价值的平台。
Severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and coronavirus disease 2019 (COVID-19) are emerging zoonotic diseases caused by coronavirus (CoV) infections. The viral RNA-dependent RNA polymerase (RdRp) has been suggested as a valuable target for antiviral therapeutics because the sequence homology of CoV RdRp is highly conserved. We established a cell-based reporter assay for MERS-CoV RdRp activity to test viral polymerase inhibitors. The cell-based reporter system was composed of the bicistronic reporter construct and the MERS-CoV nsp12 plasmid construct. Among the tested nine viral polymerase inhibitors, ribavirin, sofosbuvir, favipiravir, lamivudine, zidovudine, valacyclovir, vidarabine, dasabuvir, and remdesivir, only remdesivir exhibited a dose-dependent inhibition. Meanwhile, the Z-factor and Z '-factor of this assay for screening inhibitors of MERS-CoV RdRp activity were 0.778 and 0.782, respectively. Ribavirin and favipiravir did not inhibit the MERS-CoV RdRp activity, and non-nucleoside HCV RdRp inhibitor, dasabuvir, partially inhibited MERS-CoV RdRp activity. Taken together, the cell-based reporter assay for MERS-CoV RdRp activity confirmed remdesivir as a direct inhibitor of MERS-CoV RdRp in cells. A cell-based MERS-CoV RdRp activity reporter assay is reliable and accurate for screening MERS-CoV RdRp-specific inhibitors. It may provide a valuable platform for developing antiviral drugs for emerging CoV infections.