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一种基于细胞的报告基因检测法用于筛选中东呼吸综合征冠状病毒RNA依赖的RNA聚合酶活性抑制剂。

A Cell-Based Reporter Assay for Screening Inhibitors of MERS Coronavirus RNA-Dependent RNA Polymerase Activity.

作者信息

Min Jung Sun, Kim Geon-Woo, Kwon Sunoh, Jin Young-Hee

机构信息

Herbal Medicine Research Division, Korea Institute of Oriental Medicine, Daejeon 34054, Korea.

Center for Convergent Research of Emerging Virus Infection, Korea Research Institute of Chemical Technology, Daejeon 34114, Korea.

出版信息

J Clin Med. 2020 Jul 27;9(8):2399. doi: 10.3390/jcm9082399.

Abstract

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.

摘要

严重急性呼吸综合征(SARS)、中东呼吸综合征(MERS)和冠状病毒病2019(COVID-19)是由冠状病毒(CoV)感染引起的新发人畜共患病。病毒的RNA依赖性RNA聚合酶(RdRp)被认为是抗病毒治疗的一个有价值的靶点,因为CoV RdRp的序列同源性高度保守。我们建立了一种基于细胞的报告基因检测方法来检测MERS-CoV RdRp活性,以测试病毒聚合酶抑制剂。该基于细胞的报告基因系统由双顺反子报告基因构建体和MERS-CoV nsp12质粒构建体组成。在测试的9种病毒聚合酶抑制剂中,利巴韦林、索磷布韦、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中,只有瑞德西韦表现出剂量依赖性抑制作用。同时,该用于筛选MERS-CoV RdRp活性抑制剂的检测方法的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,非核苷类HCV RdRp抑制剂达沙布韦部分抑制MERS-CoV RdRp活性。综上所述,基于细胞的MERS-CoV RdRp活性报告基因检测方法证实瑞德西韦是细胞中MERS-CoV RdRp的直接抑制剂。基于细胞的MERS-CoV RdRp活性报告基因检测方法对于筛选MERS-CoV RdRp特异性抑制剂是可靠且准确的。它可能为开发针对新发CoV感染的抗病毒药物提供一个有价值的平台。

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