State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, China; The Key Laboratory of Preventive Veterinary Medicine in Hubei Province, Cooperative Innovation Center for Sustainable Pig Production, Wuhan, 430070, China.
Jiangxi Provincial Key Laboratory for Animal Science and Technology, College of Animal Science and Technology, Jiangxi Agricultural University, Nanchang, 330045, China.
Vet Microbiol. 2021 Jun;257:109074. doi: 10.1016/j.vetmic.2021.109074. Epub 2021 Apr 26.
Porcine epidemic diarrhea virus (PEDV) is a reemerging Alphacoronavirus that causes lethal diarrhea in piglets. Coronavirus nonstructural protein 13 (nsp13) encodes helicase, which plays pivotal roles during viral replication by unwinding viral RNA. However, the biochemical characterization of PEDV nsp13 remains largely unknown. In this study, PEDV nsp13 was expressed in Escherichia coli and purified. The recombinant nsp13 possessed ATPase and helicase activities for binding and unwinding dsDNA/RNA substrates with 5'-overhangs, and Mg and Mn were critical for its ATPase and helicase activities. PEDV nsp13 also unwound dsDNA into ssDNA in the pH from 6.0-9.0, and used energy from all nucleoside triphosphates and deoxynucleoside triphosphates. Site-directed mutagenesis demonstrated that Lys289 (K289) of PEDV nsp13 was essential for its ATPase and helicase activities. These results provide new insights into the biochemical properties of PEDV nsp13, which is a potential target for developing antiviral drugs.
猪流行性腹泻病毒（PEDV）是一种新兴的α冠状病毒，可导致仔猪致命性腹泻。冠状病毒非结构蛋白 13（nsp13）编码解旋酶，在病毒复制过程中通过解开病毒 RNA 发挥关键作用。然而，PEDV nsp13 的生化特征在很大程度上仍不清楚。本研究在大肠杆菌中表达和纯化了 PEDV nsp13。重组 nsp13 具有 ATP 酶和解旋酶活性，可结合并解开具有 5'突出端的 dsDNA/RNA 底物，Mg 和 Mn 对其 ATP 酶和解旋酶活性至关重要。PEDV nsp13 还可在 pH 值为 6.0-9.0 的条件下将 dsDNA 解旋成 ssDNA，并利用所有核苷三磷酸和脱氧核苷三磷酸的能量。定点突变表明，PEDV nsp13 的赖氨酸 289（K289）对于其 ATP 酶和解旋酶活性是必需的。这些结果为研究 PEDV nsp13 的生化特性提供了新的见解，该蛋白可能成为开发抗病毒药物的潜在靶点。
