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

Huijuan Zhong,Rui Luo,Liurong Fang,Huanchun Chen,Dang Wang,Min Shang,Huan Zhang,Chao Ouyang,Haiping Ouyang,Shaobo Xiao,Wenzhe Ho

doi:10.1371/journal.pone.0080976

Abstract

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.

Highlights

Innate immune responses are activated through host pattern recognition receptors (PRRs), which recognize molecular structures called pathogen-associated molecular patterns (PAMPs) that are structurally conserved within large groups of pathogens
To identify potential ubiquitin-specific proteases (USPs) that might regulate the antiviral innate immune response, we screened a pool of 162 siRNAs targeting 54 human USPs genes for their abilities to regulate Sendai virus (SEV)-induced activation of the IFN-stimulated response element (ISRE) promoter in reporter assays in HEK-293T cells
Our further analysis focused on USP25, since a previous study demonstrated that USP11, which is the topranking candidate in this study, was constitutively associated with IκBα and attenuated IκBα degradation to negatively regulate TNFα-induced NF-κB activation [34]

Summary

Introduction

Innate immune responses are activated through host pattern recognition receptors (PRRs), which recognize molecular structures called pathogen-associated molecular patterns (PAMPs) that are structurally conserved within large groups of pathogens. Upon engagement of PAMPs, PRRs initiate signaling pathways, triggering the production of type I interferons (IFNs) [1]. IFN stimulation of promoters containing ISRE results in the transcriptional induction of a large number of IFN-stimulated genes (ISGs) to evoke antiviral activity [3]. Recognition of virus-derived double-stranded RNA (dsRNA) and 5’triphosphorylated single-stranded RNA (5’pppssRNA) by retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) in the cytoplasm lead to the production of type I IFN and inflammatory cytokines [4,5,6]. After sensing cytoplasmic viral RNAs, RIG-I and/or MDA5 interacts with the caspase activation and recruitment domain (CARD)-containing protein IFN-beta promoter stimulator 1 (IPS-1, known as MAVS/VISA/Cardif) via CARD-like domains on both the RNA sensors and IPS-1 [9,10,11,12]. Phosphorylated IRF3 and NF-κB translocate into the nucleus and directly induces type I IFNs, an array of cytokines and other mediators required for host defense [13,14]

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Results

Conclusion