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The N-terminal cysteine protease domain of rice stripe tenuivirus Pc1 possesses deubiquitinating enzyme activity

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Abstract

Virus encoded deubiquitinating enzyme (DUB) plays important roles in viral replication and the regulation of host innate immunity. Bioinformatics-based analysis revealed the presence of an ovarian tumor (OTU) protease domain in the N terminus of rice stripe tenuivirus (RSV) Pc1. Many viral OTU domains have been reported to possess DUB activity, which suggests that RSV OTU probably also have DUB activity. To confirm this prediction, we first expressed and purified RSV OTU domain (the N-terminal 200 amino acids of Pc1) and its three mutants (D42A, C45A and H148A) from Escherichia coli and analyzed its DUB activity in vitro. The purified RSV OTU hydrolyzed both K48-linked and K63-linked polyubiquitin chains, indicating RSV OTU domain has DUB enzyme activity in vitro. The mutations of the predicted catalytic sites (Asp42, Cys45 and His148) resulted in the loss of DUB activity, demonstrating these three residues were required for enzyme activity. Then, RSV OTU and its mutants were expressed in insect cells and assayed their DUB activities in vivo by co-transfection with HA-tagged ubiquitin. RSV OTU dramatically reduced ubiquitin-conjugated cellular proteins compared to control and the mutants, showing that RSV OTU also displays DUB activity in vivo. Characterization of RSV OTU DUB enzyme activity and its key catalytic residues will facilitate the development of novel antiviral reagents against RSV.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant No. 31600122) and Science and Technology Innovation of Yangzhou University (Grant No. 2019CXJ160).

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Authors and Affiliations

  1. College of Bioscience and Biotechnology, Yangzhou University, No. 48 Wenhui Road East, Yangzhou, 225009, People’s Republic of China

    Shuling Zhao, Xiaomei Gu, Jie Li & Changyong Liang

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  1. Shuling Zhao
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  2. Xiaomei Gu
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  3. Jie Li
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  4. Changyong Liang
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Contributions

CL and SZ conceived and designed the experiments. SZ, XG and JL carried out the experiments. JL performed the data analysis. SZ and CL wrote the manuscript. All authors read and approved the final manuscript.

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Correspondence to Changyong Liang.

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Zhao, S., Gu, X., Li, J. et al. The N-terminal cysteine protease domain of rice stripe tenuivirus Pc1 possesses deubiquitinating enzyme activity. Virus Genes 57, 117–120 (2021). https://doi.org/10.1007/s11262-020-01807-8

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  • DOI: https://doi.org/10.1007/s11262-020-01807-8

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