Ubiquitin-Specific Proteases 25 Negatively Regulates Virus-Induced Type I Interferon Signaling

Ubiquitination and deubiquitination have emerged as critical regulatory processes in the virus-triggered type I interferon (IFN) induction pathway. In this study, we carried out a targeted siRNA screen of 54 ubiquitin-specific proteases (USPs) and identified USP25 as a negative regulator of the virus-triggered type I IFN signaling pathway. Overexpression of USP25 inhibited virus-induced activation of IFN-β, interferon regulation factor 3 (IRF3) and nuclear factor-kappa B (NF-κB), as well as the phosphorylation of IRF3 and NF-κB subunit p65. Furthermore, Knockdown of USP25 potentiated virus-induced induction of the IFN-β. In addition, detailed analysis demonstrated that USP25 cleaved lysine 48- and lysine 63-linked polyubiquitin chains in vitro and in vivo, and its deubiquitinating enzyme (DUB) activity, were dependent on a cysteine residue (Cys178) and a histidine residue (His607). USP25 mutants lacking DUB activity lost the ability to block virus-induced type I IFN to some degree. Mechanistically, USP25 deubiquitinated retinoic acid-inducible gene I (RIG-I), tumornecrosis factor (TNF) receptor-associated factor 2 (TRAF2), and TRAF6 to inhibit RIG-I-like receptor-mediated IFN signaling. Our findings suggest that USP25 is a novel DUB negatively regulating virus-induced type I IFN production.

泛素化与去泛素化（Ubiquitination and deubiquitination）已被证实为病毒触发的I型干扰素（type I interferon, IFN）诱导通路中的关键调控过程。本研究针对54种泛素特异性蛋白酶（ubiquitin-specific proteases, USPs）开展了靶向小干扰RNA（small interfering RNA, siRNA）筛选，鉴定出USP25为病毒触发的I型干扰素信号通路的负调控因子。过表达USP25可抑制病毒诱导的干扰素β（IFN-β）、干扰素调节因子3（interferon regulatory factor 3, IRF3）以及核因子κB（nuclear factor-kappa B, NF-κB）的活化，同时可阻断IRF3与核因子κB亚基p65的磷酸化过程。进一步实验显示，敲低USP25可增强病毒诱导的干扰素β表达。此外，详细的机制分析表明，USP25在体内外均可切割赖氨酸48位与赖氨酸63位连接的多聚泛素链，且其去泛素化酶（deubiquitinating enzyme, DUB）活性依赖于半胱氨酸残基（cysteine residue, Cys178）与组氨酸残基（histidine residue, His607）。丧失去泛素化酶活性的USP25突变体在一定程度上失去了阻断病毒诱导I型干扰素产生的能力。机制分析显示，USP25可通过对视黄酸诱导基因I（retinoic acid-inducible gene I, RIG-I）、肿瘤坏死因子（tumor necrosis factor, TNF）受体相关因子2（TRAF2）以及TRAF6进行去泛素化修饰，从而抑制视黄酸诱导基因I样受体介导的干扰素信号通路。本研究结果表明，USP25是一种新型的去泛素化酶，可负调控病毒诱导的I型干扰素产生。