Yucan Zhang,Chenxi Shi,Jin Yuan,Yue Zhang,Xiaohui Jin,Shaopo Zu,Honglei Zhang,Hui Hu
Porcine deltacoronavirus (PDCoV) is an emerging enteropathogenic coronavirus that mainly causes diarrhea and death in suckling piglets and also has the potential for cross-species transmission, threatening public health. However, there is still no effective vaccine or drug to prevent PDCoV infection. In order to accelerate the development of antiviral drugs, we established a high-throughput screening platform using a novel genome editing technology called transformation-associated recombination cloning in yeast. The recombinant PDCoV and PDCoV reporter virus expressing enhanced green fluorescent protein were both rapidly rescued with stable genealogical characteristics during passage. Further study demonstrated that the reporter virus can be used for high-throughput screening of antiviral drugs with a Z-factor of 0.821–0.826. Then, a medicine food homology compound library was applied, and we found that three compounds were potential antiviral reagents. In summary, we have established a fast and efficient reverse genetic system of PDCoV, providing a powerful platform for the research of antiviral drugs.
Simple SummaryThe helicase NSP13, encoded by porcine deltacoronavirus (PDCoV), regulates viral replication and is considered to bean ideal target for antiviral drugs. In our work, the wild-type NSP13WTand various deletion mutants were generated through expression and purification, and we tested the activities of these proteins using multiple methods. The results revealed that both the Stalk and 1B domains were necessary for NSP13WTto separate the double-stranded nucleic acids, and the deletion of the ZBD impaired this unwinding activity. Our study is the first to explore the role of each domain in the activities of PDCoV NSP13, providing mechanistic insights into helicase-regulated viral replication and laying the foundation for the development of antiviral drugs.The nonstructural protein 13 (NSP13) of PDCoV is a highly conservative helicase and plays key roles in viral replication. NSP13 contains a zinc-binding domain (ZBD), a helical Stalk domain, a beta-barrel 1B domain, and a core helicase domain. However, the specific functions of these domains of PDCoV NSP13 remain largely unknown. Here, we expressed and purified the wild-type NSP13WTand various mutants with domain deletions, and the activities of these proteins were analyzed using multiple methods. We found that NSP13ΔZBDpossessed the abilities to hydrolyze ATP and unwind double-stranded nucleic acids, but the unwinding efficiency was lower than that of NSP13WT. In contrast, NSP13ΔZBD-Stalk, NSP13Δ1B, and NSP13ΔZBD-Stalk-1Ball lost their unwinding activity, but not their ATPase activity. These results revealed that the deletion of the ZBD impaired the unwinding activity of PDCoV helicase NSP13, and the Stalk and 1B domains were critical for NSP13 to separate the duplexes. The identification of the roles of each domain in this study was helpful to gain an in-depth understanding of the overall functions of helicase NSP13, providing a theoretical basis for the development of antiviral drugs targeting helicase.
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