The N-terminal cysteine protease domain of rice stripe tenuivirus Pc1 possesses deubiquitinating enzyme activity
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The N‑terminal cysteine protease domain of rice stripe tenuivirus Pc1 possesses deubiquitinating enzyme activity Shuling Zhao1 · Xiaomei Gu1 · Jie Li1 · Changyong Liang1 Received: 17 August 2020 / Accepted: 27 October 2020 © Springer Science+Business Media, LLC, part of Springer Nature 2020
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. Keywords Rice stripe tenuivirus · OTU domain · Pc1 · Deubiquitinating activity · Key residues Rice stripe tenuivirus (RSV) is an important pathogen of rice and mainly transmitted by the insect vector small brown planthopper in a persistent, circulative-propagative manner. Rice stripe disease caused by RSV results in severe yield losses in rice production in many East Asian countries [1]. RSV is a negative sense single-stranded RNA virus and belongs to the genus Tenuivirus in the family Phenuiviridae under the order Bunyavirales [2]. Its genome consists of four-segmented single-stranded RNAs encoding seven proteins with negative sense or ambisence coding strategy [3]. The complementary sense of RNA1 encodes a large protein Pc1 with predicted molecular weight of ~ 336.8 kDa, which is considered to be the viral RNA-dependent RNA polymerase (RdRp) [4]. RNA2, RNA3 and RNA4 employ an Edited by Karel Petrzik. * Changyong Liang [email protected] 1
College of Bioscience and Biotechnology, Yangzhou University, No. 48 Wenhui Road East, Yangzhou 225009, People’s Republic of China
ambisence coding strategy and each encodes two proteins [5]. Sequence analysis of RSV genome and predicted proteins revealed the presence of an ovarian tumor (OTU) like protease motif in the N-terminal 155 amino acid residues of Pc1 [6]. The ot
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