Min Jung Sun, Kim Geon-Woo, Kwon Sunoh, Jin Young-Hee
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.
J Clin Med. 2020 Jul 27;9(8):2399. doi: 10.3390/jcm9082399.
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.
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。