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.