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.

病毒编码的去泛素化酶 (DUB) 在病毒复制和宿主先天免疫调节中起重要作用。基于生物信息学的分析揭示了水稻细条纹病毒 (RSV) Pc1 的 N 末端存在卵巢肿瘤 (OTU) 蛋白酶结构域。据报道，许多病毒 OTU 域具有 DUB 活性，这表明 RSV OTU 可能也具有 DUB 活性。为了证实这一预测，我们首先从大肠杆菌中表达并纯化了 RSV OTU 结构域（Pc1 的 N 端 200 个氨基酸）及其三个突变体（D42A、C45A 和 H148A）并在体外分析了其 DUB 活性。纯化的 RSV OTU 水解 K48 连接和 K63 连接的多聚泛素链，表明 RSV OTU 域在体外具有 DUB 酶活性。预测的催化位点（Asp42、Cys45 和 His148）的突变导致 DUB 活性的丧失，表明这三个残基是酶活性所必需的。然后，RSV OTU 及其突变体在昆虫细胞中表达，并通过与 HA 标记的泛素共转染测定其体内 DUB 活性。与对照和突变体相比，RSV OTU 显着减少了泛素结合的细胞蛋白，表明 RSV OTU 在体内也显示出 DUB 活性。RSV OTU DUB 酶活性及其关键催化残基的表征将促进针对 RSV 的新型抗病毒试剂的开发。