c-Cbl-mediated ubiquitination of IRF3 negatively regulates IFN-β production and cellular antiviral response

Cellular Signalling 28 (2016) 1683–1693

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c-Cbl-mediated ubiquitination of IRF3 negatively regulates IFN-β production and cellular antiviral response Xibao Zhao 1, Huihui Zhu 1, Juan Yu, Hongrui Li, Jiafeng Ge, Weilin Chen ⁎ Institute of Immunology, Zhejiang University School of Medicine, Hangzhou 310058, China

a r t i c l e

i n f o

Article history: Received 16 May 2016 Received in revised form 29 July 2016 Accepted 4 August 2016 Available online 05 August 2016 Keywords: c-Cbl IRF3 Ubiquitination Antiviral response

a b s t r a c t Induction of type I interferon is a fundamental cellular response to viral infection. Interferon regulatory factor 3 (IRF3) plays an essential role in Toll-like receptor (TLR) and retinoic acid–inducible gene I (RIG-I) mediated induction of type I interferon and host antiviral responses. However, posttranslational regulation of IRF3 remains to be fully understood. In this study, we identified E3 ubiquitin ligase Casitas B-lineage lymphoma (c-Cbl) as a negative regulator for IRF3 protein stability and IFN-β signal pathway. Knockdown of c-Cbl expression by small interfering RNA enhanced virus-induced IFN-β production as well as cellular antiviral response, whereas overexpression of c-Cbl inhibited virus-induced IFN-β signaling. Coimmunoprecipitation experiments demonstrated that c-Cbl interacted with IRF3 via TKB domain of c-Cbl and IRF association domain of IRF3, promoting K48-linked polyubiquitination and proteasomal degradation of IRF3. Therefore, our findings suggest that c-Cbl negatively regulates IFN-β signaling and cellular antiviral response by promoting IRF3 ubiquitination and degradation, providing a new mechanism for control of type I interferon induction. © 2016 Elsevier Inc. All rights reserved.

1. Introduction TLR, RLR and NLR are very important receptors recognizing pathogenic microorganisms and initialing the innate immune response. The innate immune system constitutes the first line of host defense against virus infection. Upon recognizing invading viruses infection, host cells activate downstream signaling pathways that lead to production of type I interferon (IFN), including IFN-β and IFN-α family members, these cytokines mediated induction of both the innate immune response and subsequent development of adaptive immunity to viruses [1,2]. Expression of type I interferon is transcriptionally regulated through coordinated activation of latent transcription factors including nuclear factor-kappa B (NF-κB), interferon regulatory factor 3 (IRF3) and IRF7, in these factors, IRF3 and IRF7 in particular are activated in response to viral infection and are mainly involved in type I interferon induction [2,3]. However, previous research demonstrates that overproduction of type I IFNs results in adverse pathogenic effects characteristic of many autoimmune disorders, such as systemic lupus erythematous (SLE) [4]. So, understanding the mechanisms that maintaining proper low amounts of type I IFN under physiological conditions, or restraining its excessive magnitude when the antiviral innate response which is critical to protecting against such harmful effects.

⁎ Corresponding author. E-mail address: [email protected] (W. Chen). 1 These authors contributed equally.

http://dx.doi.org/10.1016/j.cellsig.2016.08.002 0898-6568/© 2016 Elsevier Inc. All rights reserved.

Studies have revealed a working model on virus how to trigger type I IFN signaling in the past decades. Retinoic acid-inducible gene I (RIG-I) or melanoma differentiation-associated gene 5 (MDA5) plays an important role in recognition of virus-derived double-stranded RNA and 5′triphosphorylated single-stranded RNA in the cytoplasm, and leads to the production of type I IFN to clean virus [5,6]. Cytosolic DNA is detected by cyclic GMP-AMP synthase (cGAS) to activate several convergent signaling pathways to produce type I IFN [7]. After ligand binding, cGAS and RIG-I signal through respective adaptor proteins stimulator of interferon genes (STING) and mitochondrial antiviral signaling protein (MAVS) to recruit the kinases I kappa B kinase (IKK) and TANKbinding kinase 1 (TBK1), which then activate the transcription factors NF-κB and IRF3, respectively [8]. Recent studies show that activated RIG-I forms oligomers to convert MAVS into prion-like polymers and recruit ubiquitin E3 ligase tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2), TRAF3 TRAF5 and TRAF6 to synthesize polyubiquitin chains, following activating kinases TBK1 and IKK α/β, leading to activation of the critical transcription factors IRF3 and NF-κB [9–12]. In addition, Toll-like receptors 3 and 4 (TLR3 and TLR4) signal can active TBK1 and IRF3 through the adaptor protein TIR-domain-containing adapter-inducing interferon-β (TRIF) [13]. Phosphorylated IRF3 and NF-κB translocate into the nucleus and directly induce production of an array of cytokines including type I IFNs to help host defense virus infection [14]. IRF3 is an important member of the IRF family that is directly implicated in the transcriptional induction of type I IFN. It is tightly controlled by posttranscriptional modifications, such as phosphorylation,

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ubiquitination and deubiquitination. Previous studies showed that many molecules involved in regulation IRF3 activity, including WDR5 [15], TRIM32 [16], Siglec-G [17], DAPK1 [18], and Pin1 [19]. IRF3 ubiquitination and deubiquitination by virus or host components can influence its activity and stability. TRIM26, FoxO1 and RBCK1 are E3 ligases that induce K48-linked ubiquitination and degradation of IRF3 [20–22]. PLpro is a deubiquitination enzyme that deubiquitinate IRF3 to negatively regulate production of IFN-β [23]. However, whether and how other E3 ligases molecules are involved in regulating this process is of great interest. Casitas B-lineage lymphoma (c-Cbl) is a proto-oncogene with widespread mutations identified in hematopoietic malignancies [24] and it have identified that c-Cbl can regulate nuclear β-catenin and angiogenesis through its Wnt-mediated phosphorylation [25]. Acting mainly as an ubiquitin E3 ligase, c-Cbl is a critical negative regulator of receptor tyrosine kinases (RTKs). Recently study have demonstrated that c-Cbl could entrap and promote degradation of the active tyrosine kinase Src, enabling tumor cell survival [26]. The tyrosine kinase binding (TKB) domain of c-Cbl is required for its binding with RTKs, and the RING finger domain is essential for its E3 ubiquitin ligase activity [27]. In previous reports, c-Cbl also plays an important role in antiviral innate immunity by regulated RIG-I and IRF8 etc. [17,28]. In this study, we identified E3 ubiquitin ligase c-Cbl as a negative regulator targeting IRF3 in antiviral innate immunity signaling pathways. We also showed that c-Cbl was associated with IRF3 and promoted K48-linked ubiquitination and degradation of IRF3. These findings suggest a negative regulation in antiviral response mechanism involving c-Cbl-mediated posttranslational modification of IRF3.

10 ng of the Renilla luciferase construct phRL-TK (Promega, for normalization of transfection efficiency) and various other expression plasmids or a control plasmid. In some experiments, cells were knockdown c-Cbl for 24 h, and then transfected luciferase reporter plasmid, further infected or mock infected with VSV at 24 h after the luciferase reporter plasmid transfection. Twelve hours after VSV infection, cells were harvested. Luciferase assays were performed using a dual-specific luciferase assay kit (Promega) according to the manufacturer's protocol. The relative luciferase activity was calculated by dividing the Firefly luciferase activity by the Renilla luciferase activity.

2. Materials and methods

2.5. Quantitative PCR

2.1. Mice and cell culture

Total cellular RNA was isolated from cells using Trizol regent and subjected to quantitative PCR analysis to measure expression of mRNA. Data were determined by normalization of expression of β-actin or GAPDH in each sample. Bio-Rad CFX-touch Real-Time PCR system was used for quantitative PCR. Gene-specific primer sequences were as following: mIFN-β: 5′-ATGAGTGGTGGTTGCAGGC-3, 5′-TGACCTTTCA AATGCAGTAGATTCA-3; hIFN-β: 5′-TTGTTGAGAACCTCCTGGCT-3, 5′TGACTATGGTCCAGGCACAG-3; hIRF3: 5′-CACGACAGCTCTTTCCATGA-3, 5′-AGCCAGTGCTCGATGAATCT-3; hGAPDH: 5′-GAGTCAACGGATTTGGT CGT-3, 5′-GACAAGCTTCCCGTTCTCAG-3; mβ-actin: 5′-AGTGTGACGTT GACATCCGT-3, 5′-GCAGCTCAGTAACAGTCCGC-3.

C57BL/6 mice (6–8 weeks, female) were purchased from Joint Ventures Sipper BK Experimental Animals, Shanghai. Mice were kept and bred in pathogen-free condition. All animal experiments were undertaken in according with the National Institute of Health Guide for the Care and Use of Laboratory Animals with approval of the Zhejiang University, Hangzhou. Mouse peritoneal macrophages were collected from C57BL/6 mice after 3 days following injection of 2 ml of 3% thioglycolate broth and isolated by adhering cells to tissue plates at 37 °C for 3 h. The mouse peritoneal macrophages were cultured in 1640 with 10% FCS. Mouse macrophage cell line RAW264.7 cells and HEK-293T cells were purchased from ATCC and cultured in DMEM medium containing 10% FBS. 2.2. Plasmids, antibodies and reagents Myc-tagged RIG-I (N), Flag-tagged MAVS, Myc-tagged TBK1, Flagtagged IRF3, V5-c-Cbl, IRF3-5D, HA-Ub, HA-K48-Ub and HA-K63-Ub plasmids were constructed by standard molecular biology techniques by cloning into the pcDNA3.1 vector. Antibodies against c-Myc (#sc40), HA (#sc-805) and Flag (OctA-Probe Antibody, #sc-807) were purchased from Santa Cruz Biotechnology. Antibodies specific to p-IRF3 (#4947), IRF3 (#4302) and c-Cbl (#8447) were obtained from Cell Signaling Technology. Antibodies specific to β-actin (#AA128) and GAPDH (#AG019) were purchased from Beyotime Biotechnology of China. V5tagged antibody (#M100812) was purchased from Hua An Biotechnology of China. Reagents used in this study included the following: cycloheximide (#C4218), MG132 (#M8699) and chloroquine (#C6628) were obtained from Sigma-Aldrich.

2.4. Transfection and RNA interference assay 1 × 106 mouse peritoneal macrophages or 2 × 105 293T cells or 2 × 105 RAW 264.7 cells were seeded into each well of 12-well plates and incubated overnight, and then transfected with c-Cbl siRNA or control siRNA using INTERFERin, according to the manufacturer's instructions. 2 × 105 RAW 264.7 cells or 293T cells were seeded into each well of 12-well plates or 5 × 105 293T cells were seeded into each well of 6-well plates, and incubated overnight, JetPEI polyplus we used for the transfection of plasmids, according to the manufacturer's instructions. c-Cbl-specific siRNA oligonucleotides were obtained from GenePharma. The following sequences were targeted for c-Cbl siRNA: 1: CCAGGAACAAUAUGAAUUATT, 2: CCUCCGGGAAUUUGUUUCUTT, 3: GGAGACACUUUCCGGAUUATT. The transfected siRNA was a complex that mixed with same amount of siRNA 1, siRNA 2 and siRNA 3 respectively.

2.6. Immunoblot analysis and coimmunoprecipitation 293T cells were transfected expression plasmid for 24 h or transfected siRNA for 48 h. In some experiments, cells further infected or mock infected with VSV after transfection. Cells lysed with 1 × cell lysis buffer containing protease inhibitor mixture. For coimmunoprecipitation, whole-cell extracts were collected 24 h after transfection and were lysed in immunoprecipitation buffer and containing protease inhibitor mixture. After centrifugation for 5 min at 12,000 g, supernatants were collected and incubated with protein A/G Plus-Agarose Immunoprecipitation reagent (Santa Cruz Biotechnology) together with 1 μg anti-Flag or anti-HA or IRF3 antibody. After 6 h of incubation, beads were washed five times with immunoprecipitation buffer. Samples were boiled with 1% (wt/vol) SDS sample buffer. Equal amounts of protein were subjected to 10%–12% SDS-PAGE and transferred onto PVDF membranes, and immunoblot was performed, as described previously [29].

2.3. Luciferase assay

2.7. ELISA

HEK-293T cells grown in 96-well plates were co-transfected with 90 ng of luciferase reporter plasmid (IFN-β or ISRE luciferase reporter),

Mouse IFN-β ELISA was performed according to the manufacturer's instructions (Biolegend).

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2.8. Virus manipulation Cells were grown in 12-well plates and transfected with the indicated plasmids prior to virus infection. The culture medium was replaced by serum-free DMEM containing VSV-GFP viruses. One hour later, the cells were washed with PBS and then fed with DMEM containing 10% FBS. VSV-GFP replication was visualized by monitoring the GFP expression level in fluorescence microscopy or by Flow cytometry analysis. 2.9. Data analysis All data are presented as the mean ± SD of three independent experiments performed in duplicate. GraphPad Prism 5.0 was used for plotting data. Treatment effects were statistically analyzed using the twotailed, paired Student's t-test, where P b 0.05 was considered to be significant. 3. Results 3.1. c-Cbl negatively regulates virus-induced IFN-β production c-Cbl is a critical ubiquitin E3 ligase to negative regulate RTKs and involved in many tumor initiation and development. However, whether c-Cbl interacting other proteins in the immune response needs to be further studied. To explore the role of c-Cbl in antiviral innate immunity, we used vesicular stomatitis virus (VSV) to demonstrate the effect of cCbl on the activation of IFN-β expression. We synthesized specific c-Cbl siRNA and used it to knock down the endogenous expression of c-Cbl in primary peritoneal macrophages. Immunoblot analysis showed that endogenous c-Cbl protein was obvious decreased in mouse peritoneal macrophages (Fig. 1A). We also got V5-tagged expression plasmid encoding c-Cbl and confirmed the protein expression level of plasmid by immunoblot (Fig. 1B). RLRs can recognize RNA virus and lead to the expression of IFN-β. Q-PCR and ELISA analysis showed that VSV infection induced IFN-β mRNA expression (Fig. 1C) and production (Fig. 1D) were increased in mouse peritoneal macrophages transfected with c-Cbl siRNA, compared to that transfected with control siRNA. We also found that silencing of c-Cbl could slightly increase IFN-α4 mRNA expression after VSV infection in mouse peritoneal macrophages (Fig. S1), and our results showed that the expression of IFN-β had an obvious change than IFN-α, so we focused on IFN-β expression in our study. The poly (I:C) present in the cytosol has been shown to activate IFN-β production through RIG-I and MDA-5. We showed that IFN-β expression induced by poly (I:C) transfection was also promoted upon cCbl silence (Fig. 1E). Similarly, Q-PCR experiments indicated that silencing of c-Cbl could increase IFN-β mRNA expression after VSV infection in RAW264.7 cells (Fig. 1F). We also got the similarly conclusion that overexpression of c-Cbl apparently inhibited VSV induced IFN-β mRNA expression in RAW264.7 cells (Fig. 1G). Furthermore, Luciferase reporter assays show that silence of c-Cbl strongly promoted VSV induced activation of IFN-β (Fig. 1H) and ISRE (Fig. 1I) promoter in 293T cells. These results demonstrate that c-Cbl negatively regulates VSV-induced IFN-β production. 3.2. c-Cbl suppresses cellular antiviral response IFN-β plays critical roles in the innate immune responses against viral infection and we showed that c-Cbl negatively regulates VSV-triggered IFN-β production. Next, we used flow cytometry to confirm this result and the results revealed that 30.2% cells were infected (GFP+) in cells transfected with c-Cbl siRNA, compared to 60.1% of GFP+ cells in cells transfected with control siRNA 12 hours post infection in mouse peritoneal macrophages (Fig. 2A). Correspondingly, virus replication was more significantly inhibited in c-Cbl-knockdown mouse peritoneal macrophages infected with VSV (Fig. 2B). To further investigate, we transfected c-Cbl expression plasmid or control plasmid into

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293T cells, then infected the cells with VSV-GFP. The data demonstrated that c-Cbl-overexpression cells compared with control cells were more susceptible to viral as monitored by GFP expression (Fig. 2C). Taken together, these data suggest that c-Cbl suppresses cellular antiviral response. 3.3. c-Cbl negatively regulates IRF3-mediated IFN-β signaling Host defense against virus infection and causes a series of proteins activation involved in innate signaling pathways, which is critical for production of IFN-β and innate antiviral immune response. Many components have reported to participate in this process, such as RIG-I, MAVS, TBK1 and IRF3. We got the RIG-I activated form plasmid (RIGI(N)) which only had two CARD domain in its N terminal and we also generated IRF3 activated form plasmid (IRF3-5D) which substituted the 396, 398, 402, 404 and 405 sites Ser-Thr cluster with the phosphomimetic Asp in IRF-3. To determine the molecular mechanisms responsible for c-Cbl-mediated negatively regulation of virus-triggered type I IFN signaling, we transfected plasmids encoding RIG-I (N), MAVS, TBK1 or IRF3-5D together with IFN-β promoter in presence or absence c-Cbl, and we found that c-Cbl restrained IFN-β promoter activation by overexpression of IRF3-5D and the upstream adaptors or kinases, including RIG-I (N), MAVS and TBK1 (Fig. 3A). Similarly, RIG-I (N), MAVS, TBK1 and IRF3-5D induced activation of ISRE reporter was also inhibited by cCbl overexpression (Fig. 3B). Additionally, the IFN-β and ISRE promoter activation levels were reduced as increasing amounts of c-Cbl were expressed (Fig. 3C and D). Furthermore, we proved that knockdown of c-Cbl potentiated IFN-β and ISRE promoter activation mediated by IRF3-5D (Fig. 3E). RT-PCR also showed that overexpression of c-Cbl obviously restrained IFN-β mRNA expression after activation by IRF3-5D (Fig. 3F). These results show that c-Cbl may function at the level of downstream of IRF3 for regulation of virus-triggered IFN-β expression. To further identify whether c-Cbl targets IRF3 to negatively regulate IFN-β signaling, we used poly (I:C) and LPS to stimulate c-Cbl-knockdown mouse peritoneal macrophages. We found that the expression of IFN-β induced by poly (I:C) or LPS was markedly increased in c-Cblsilenced macrophages than that in control macrophages (Fig. 3G and H). Collectively, these results strongly demonstrate that c-Cbl negatively regulate IRF3-mediated IFN-β signaling. 3.4. c-Cbl targets IRF3 at posttranscriptional level To confirm that c-Cbl targets IRF3 to negatively regulate virus-triggered signaling, we transfected c-Cbl together with RIG-I (N), MAVS, TBK1 or IRF3 into 293T cells, immunoblot analysis showed that c-Cbl promoted RIG-I (N) degradation (Fig. 4A) and this result agreed with previous study [17], but not influence MAVS and TBK1 stability (Fig. 4B and C). In addition to these results, we found that c-Cbl also markedly promoted IRF3 degradation (Fig. 4D) and this process was involved in a c-Cbl dose dependent manner (Fig. 4E). In addition, we found that overexpression of c-Cbl promoted degradation of endogenous IRF3 in 293T cells (Fig. 4F), whereas knockdown of c-Cbl increased the stability of IRF3 after VSV infection in 293T cells, however, we didn't find that cCbl could affect the stability of IRF7 (Fig. 4G) and interact with IRF7 (Fig. S2). Besides, phosphorylation of IRF3 was increased after VSV infection in c-Cbl knockdown mouse peritoneal macrophages compared to control cells and we also found that IRF7 protein expression was not changed (Fig. 4H). To confirm whether c-Cbl affected other cellular signal pathways, we transfected with c-Cbl siRNA into mouse peritoneal macrophages, and then infected with VSV, immunoblot analysis indicated that silencing of c-Cbl could slightly increase IKK-α/β phosphorylation in NF-κB signal pathway (Fig. S3). However, c-Cbl regulated IRF3 at mRNA transcriptional level or posttranscriptional protein level was not clear. Because c-Cbl is an E3 ligase, thus we hypothesized that cCbl might regulate the stability of IRF3 protein at posttranscriptional.

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Fig. 1. c-Cbl negatively regulates virus-induced IFN-β production. (A) Mouse peritoneal macrophages were transfected with control siRNA or c-Cbl siRNA for 48 h. c-Cbl protein expression was analyzed by immunoblot. (B) Control vector or V5-c-Cbl expression plasmid transiently transfected into 293T cells for 24 h, immunoblot analysis of c-Cbl expression with V5. (C and D) Mouse peritoneal macrophages were transfected with control siRNA or c-Cbl siRNA for 48 h and then infected with VSV (MOI = 1) for the indicated hours. Q-PCR (C) and ELISA (D) were used to analysis of IFN-β induction in mouse peritoneal macrophages. (E) Mouse peritoneal macrophages were transfected with control siRNA or c-Cbl siRNA for 48 h and then transfected with poly (I:C) (1 μg/ml) for the indicated hours. Q-PCR was used to analysis of IFN-β mRNA expression in mouse peritoneal macrophages. (F) RAW 264.7 cells were transfected with control siRNA or c-Cbl siRNA, 48 h later, cells were infected with VSV (MOI = 1) for indicated hours and Q-PCR was used to analysis of IFN-β mRNA expression. (G) RAW 264.7 cells were transfected with expression plasmid of c-Cbl or control vector. Twenty-four hours later, cells were infected with VSV (MOI = 1) for indicated hours and Q-PCR was used to analysis of IFN-β mRNA expression. (H and I) 293T cells were transfected with control siRNA or c-Cbl siRNA for 24 h, and then transfected with the IFN-β or ISRE luciferase reporter plasmid (90 ng), phRLTK plasmid (10 ng). Twenty-four hours later, cells were infected with VSV (MOI = 0.05) for 12 h and luciferase reporter assay was used to analysis of IFN-β (H) and ISRE (I) promoter activity. Similar results were obtained from three independent experiments (A, B). Data are shown as mean ± SD of one representative experiment in (C–I). *P b 0.05, **P b 0.01, ***P b 0.001.

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Fig. 2. c-Cbl suppresses cellular antiviral response. (A) Mouse peritoneal macrophages were transfected with control siRNA or c-Cbl siRNA, and then infected with VSV-GFP (MOI = 1) for 12 h. Flow cytometry assessed the VSV infection of mouse peritoneal macrophages. Numbers above bracketed lines indicate the percentage of cells, expressing GFP (infected cells). (B) Determination of VSV titers in supernatants of c-Cbl-knockdown mouse peritoneal macrophages infected for indicated hours with VSV by TCID50 assay. (C) 293T cells were overexpression of c-Cbl for 24 h, then infected with VSV-GFP (MOI = 0.01) for 12 h and imaged by microscopy. Similar results were obtained from three independent experiments in (A–C).

To test this hypothesis, we transiently transfected c-Cbl siRNA or c-Cbl expression plasmid to knockdown or overexpression c-Cbl in 293T cells, results from Q-PCR experiment confirmed that silencing or overexpression of c-Cbl was not influenced the IRF3 expression in mRNA level (Fig. 4I and J). Therefore, we conclude that c-Cbl negatively regulates IRF3 at posttranscriptional level. 3.5. c-Cbl is associated with IRF3 Our studies indicated that c-Cbl regulated IRF3 in protein level, so we hypothesis c-Cbl interact with IRF3. To address this hypothesis, we co-

transfected V5 tagged c-Cbl and HA tagged IRF3 into 293T cells, coimmunoprecipitation experiments revealed that c-Cbl interacted with IRF3 in a physiological state (Fig. 5A). Furthermore, interaction of endogenous c-Cbl with endogenous IRF3 was also detected in 293T cells (Fig. 5B). Previous study showed that Pin1 could interact with IRF3 through its phosphorylated motif [19], to confirm whether phosphorylated of IRF3 could influence c-Cbl interacting with IRF3, we cotransfected V5 tagged c-Cbl and Flag tagged IRF3 into 293T cells, and then infected with VSV, coimmunoprecipitation experiments revealed that c-Cbl interacting with IRF3 had a slightly increase (Fig. 5C). These results demonstrated that c-Cbl interacted with IRF3 in a non-

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Fig. 3. c-Cbl negatively regulates IRF3-mediated IFN-β signaling. (A and B) 293T cells were co-transfected with the IFN-β or ISRE luciferase reporter plasmid (90 ng), phRL-TK plasmid (10 ng), c-Cbl plasmid (200 ng) and RIG-I, MAVS, IRF3-5D and TBK1 plasmids (50 ng each). At 24 h after transfection, Luciferase reporter assay was used to analysis of IFN-β (A) and ISRE (B) promoter activity. (C and D) 293T cells were co-transfected with the IFN-β or ISRE luciferase reporter plasmid (90 ng), phRL-TK plasmid (10 ng), together with plasmid of IRF3-5D (50 ng), along with increasing amounts of expression plasmid for c-Cbl (0, 100, 200 ng), 24 h after transfection, Luciferase reporter assay was used to analysis of IFN-β (C) and ISRE (D) promoter activity. (E) 293T cells were transfected with control siRNA or c-Cbl siRNA for 24 h, and then co-transfected with the IFN-β or ISRE luciferase reporter plasmid (90 ng), phRL-TK plasmid (10 ng), together with plasmid of IRF3-5D (50 ng). At 24 h after transfection, Luciferase reporter assay was used to analysis of IFN-β and ISRE promoter activity. (F) 293T cells were transfected with IRF3-5D and c-Cbl plasmid. At 24 h after transfection, Q-PCR was used to analysis of IFN-β mRNA expression. (G and H) Mouse peritoneal macrophages were transfected with control siRNA or c-Cbl siRNA for 48 h and then stimulated with poly (I:C) (10 μg/ml) (G) or LPS (100 ng/ml) (H) for the indicated hours, Q-PCR was used to analysis of IFN-β mRNA in mouse peritoneal macrophages. Data are shown as mean ± SD of one representative experiment (A–H). *P b 0.05, **P b 0.01, ***P b 0.001.

phosphorylation-dependent manner and VSV infection could promote c-Cbl interacting with IRF3. Besides, to confirm whether c-Cbl has cross-talk with Pin1 in regulation of IRF-3 signaling, we constructed the Pin1 plasmid with Flag tag and found that both of c-Cbl and Pin1 could obviously promote IRF3 degradation after VSV infection (Fig.

S4A). The result indicated that c-Cbl might have a cross-talk with Pin1 in regulation of IRF-3 signaling after VSV infection, but we didn't find that c-Cbl could interact with Pin1 with or without VSV infection by coimmunoprecipitation experiment (Fig. S4B). To determine which domain of IRF3 was required for the interaction of IRF3 with c-Cbl, we

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Fig. 4. c-Cbl targets IRF3 at posttranscriptional level. (A–D) 293T cells were co-transfected with V5-c-Cbl plasmids and Myc-RIG-I (A), Flag-MAVS (B), Myc-TBK1 (C) and Flag-IRF3 (D) expression plasmids (1 μg each), 24 h after transfection, proteins were detected by immunoblot with indicated Myc or Flag antibodies. (E) 293T cells were transfected with Flag-IRF3 and HA-Ub expression plasmids, along with increasing amounts of expression plasmid for V5-c-Cbl, proteins were detected by immunoblot assay with indicated tag antibodies. (F) 293T cells were transfected with HA-Ub and V5-c-Cbl plasmids, stability of endogenous IRF3 were detected by immunoblot assay with indicated antibodies. (G) 293T cells were transfected with control siRNA or c-Cbl siRNA, cells were infected with VSV for indicated hours, and immunoblot assay was used to analysis of endogenous IRF3 and IRF7 stability. (H) Mouse peritoneal macrophages were transfected with control siRNA or c-Cbl siRNA, cells were infected with VSV for indicated hours, and immunoblot assay was used to analysis of pIRF3 level and IRF7 stability. (I) 293T cells were transfected with c-Cbl siRNA or control siRNA for 48 h to knockdown c-Cbl expression, then VSV infected for 12 h, Q-PCR was used to analysis of the IRF3 mRNA expression. (J) 293T cells were overexpression of c-Cbl or vector for 24 h, then VSV infected for 12 h, Q-PCR was used to analysis of the IRF3 mRNA expression. Similar results were obtained from three independent experiments in (A–H). Data are shown as mean ± SD of one representative experiment in (I and J).

constructed mutants of IRF3 with the deletion of various domains and found that IRF association domain of IRF3 (IRF3 (△N)) interacted with c-Cbl (Fig. 5D). In addition, we also constructed mutants of c-Cbl with the deletion of various domains and found that TKB domain of c-Cbl (c-Cbl(N)) interacted with IRF3 (Fig. 5E). Collectively, these results indicate that c-Cbl can interact with IRF3. In addition, IRF association domain of IRF3 and TKB domain of c-Cbl are crucial for their interaction.

3.6. c-Cbl promotes degradation of IRF3 in a proteasome-dependent manner Protein degradation mainly had two pathways, one is proteasomedependent manner, and another is lysosome-dependent manner [30]. To investigate the underlying mechanisms involved in c-Cbl promoting degradation of IRF3, we co-transfected V5 tagged c-Cbl and Flag tagged

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Fig. 5. c-Cbl is associated with IRF3. (A) Immunoblot analysis of 293T cells co-transfected with HA-IRF3 and V5-c-Cbl plasmids, followed by immunoprecipitated with antibody to HA tag and immunoblot analysis with antibody to V5 tag. IgG was as control. (B) Immunoblot analysis of endogenous IRF3 and c-Cbl interaction by immunoprecipitated with antibody to IRF3 and immunoblot analysis with antibody to c-Cbl. IgG was as control. (C) Immunoblot analysis of 293T cells co-transfected with Flag-IRF3 and V5-c-Cbl plasmids, and then infected by VSV for indicated hours, followed by immunoprecipitated with antibody to Flag tag and immunoblot analysis with antibody to V5 tag. IgG was as control. (D) Schematic structure of IRF3 and the derivatives used were shown (top). HEK293T cells were co-transfected V5-c-Cbl with control, HA-IRF3 (N), HA-IRF3 (△N), HA-IRF3 (FL). Cell lysates were used for immunoprecipitation and immunoblotting, as indicated. (E) Schematic structure of c-Cbl and the derivatives used were shown (top). HEK293T cells were co-transfected HA-IRF3 with control, Flag-c-Cbl (N), Flag-c-Cbl (△N), Flag-c-Cbl (FL). Cell lysates were used for immunoprecipitated and immunoblotting, as indicated. Similar results were obtained from three independent experiments (A–E).

IRF3 into 293T cells, then treated with proteasome inhibitor MG132 and lysosome inhibitor chloroquine. Immunoblot analysis demonstrated that only MG132 could markedly restrain degradation of IRF3 (Fig. 6A), this result suggested c-Cbl promoted degradation of IRF3 by proteasome-dependent manner. The cycloheximide chase assay of 293T cells showed that c-Cbl decreased the half-life of IRF3 (Fig. 6B). These results suggest that c-Cbl promotes proteasome-dependent degradation of IRF3. 3.7. c-Cbl promotes K48-linked ubiquitination and degradation of IRF3 As an E3 ligase, c-Cbl may have the ability to induce IRF3 ubiquitination and degradation. Since c-Cbl targets IRF3 for degradation in a proteasome-dependent manner, we next test the role of c-Cbl in regulating ubiquitination of IRF3. To directly examine c-Cbl-mediated IRF3 ubiquitination, Flag tagged IRF3, HA tagged ubiquitin and V5 tagged cCbl were co-transfected into 293T cells, coimmunoprecipitation experiments showed that IRF3 ubiquitination level was markedly increased in

the presence of c-Cbl and treated with MG132 (Fig. 7A) and we also suggested that c-Cbl promoted IRF3 ubiquitination in a dose-dependent manner (Fig. 7B). Furthermore, to investigate the form of ubiquitination chains linked to IRF3, we got WT HA tagged ubiquitin and its mutants K48 or K63 sites ubiquitin, which has only one lysine in ubiquitin at position 48 or 63, respectively. Coimmunoprecipitation experiments analysis demonstrated that overexpression of c-Cbl significantly increased K48linked but not K63-linked ubiquitination of IRF3 (Fig. 7C). We further proved that c-Cbl promoted IRF3 K48-linked ubiquitination and degradation in a dose-dependent manner (Fig. 7D). Taken together, these findings reveal that c-Cbl promotes K48-linked polyubiquitination and proteasome-dependent degradation of IRF3. 4. Discussion The host antiviral innate immune response leads to production of type I IFNs and that is responsible for inhibition of virus replication, clearance of virus-infected cells, and facilitation of adaptive immune

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Fig. 6. c-Cbl promotes degradation of IRF3 in a proteasome-dependent manner. (A) 293T cells were co-transfected with Flag-IRF3, HA-Ub and V5-c-Cbl expression plasmids, and then treated with MG132 (20 μM) or Chloroquine (50 μM), immunoblot was used to analysis of protein level with indicated tag antibodies. (B) 293T cells were transfected with Flag-IRF3, HA-Ub and V5-c-Cbl, then treated with CHX (100 μg/ml) for indicated hours after transfected 24 h, immunoblot was used to analysis of protein level with indicated antibodies. Quantification of relative Flag-IRF3 levels is shown in the bottom panel. Similar results were obtained from three independent experiments (A and B).

response. This process is regulated at distinct levels to ensure proper production of type I IFNs following virus infection, because uncontrolled and excessive immune response causes pathological immunity or autoimmune diseases to the host [31,32]. In our studies, we identified the E3 ligase c-Cbl is a major ubiquitinase of IRF3 to promote IRF3 degradation and negatively regulate antiviral response. PRRs are very important receptors in recognizing pathogenic microorganisms and initialing the innate immunity and adaptive immunity, relative studies have become the hotspots in recent years [2,33,34]. In this study, we discovered that c-Cbl could down-regulate VSV induced IFN-β production, we used specific siRNA to knockdown c-Cbl mRNA, the results demonstrated that c-Cbl decrease could promote expression of IFN-β after VSV or transfected with poly (I:C) stimulation. So we conclusion that c-Cbl negatively regulates activation of RLR signaling pathway. Thus, our studies identified a novel molecular mechanism to restrict virus-induced signaling after infection. Besides, we confirmed that knockdown of c-Cbl not only increased virus-triggered activation of IFN-β/ISRE promoter and expression of IFN-β mRNA, but also

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promoted the ability of cellular antiviral response, whereas overexpression of c-Cbl inhibited virus-triggered activation of the IFN-β/ISRE promoter and expression of IFN-β mRNA. These findings provide strong evidence that c-Cbl play an important role in virus-trigged type I IFN signaling and negatively regulate cellular antiviral response. Studies have identified that many adaptors or kinases, including RIG-I, MAVS, TBK1 and IRF3, played a significant contribution to cellular antiviral response [9,35–37]. To explore the mechanisms by which c-Cbl regulates virus-triggered type I IFN induction, we used c-Cbl to screen the key proteins in antiviral signaling pathway, contain of RIG-I, MAVS, TBK1 and IRF3 by Luciferase reporter gene system, finally we confirmed that c-Cbl could influence IRF3 stabilization in protein level, but not in mRNA level. It is well known that IRF3 is key transcription factor in the immune response to virus infection and there are many distinct mechanisms related to regulate IRF3 stability. Previously, various studies have demonstrated that several ways can be used to regulate the activation of IRF3, including phosphorylation-dependent activity, ubiquitination-dependent proteasome manner degradation and autophage-dependent lysosome manner degradation [38–40]. Ubiquitination is a crucial physiological process for the antiviral immune response in previous studies [41]. In our study, since c-Cbl is an E3 ligase, so we hypothesis that c-Cbl facilitated ubiquitination degradation of IRF3. Firstly, we confirmed that IRF3 could interact with c-Cbl by coimmunoprecipitation, we also proved that IRF association domain of IRF3 and TKB domain of c-Cbl were crucial for their interaction. Next, we found that c-Cbl targeted IRF3 for degradation in a proteasome-dependent manner. These results further confirmed our hypothesis. Further studies showed that c-Cbl mediated K48-linked ubiquitination and degradation of IRF3 by coimmunoprecipitation, and these results fully elucidated the mechanisms that c-Cbl restricted virus-triggered type I IFN induction and cellular antiviral response. The host antiviral innate immune response is regulated in a complex and distinct manner. Previous study showed that RNF26 not only mediated K11-linked polyubiquitination of MITA to up-regulate type I IFN production in the early phase, but also promoted IRF3 autophage-dependent lysosome manner degradation to suppress type I IFN production in the late phase [40]. Pin1 could direct engagement with phosphorylation of IRF3 and promote polyubiquitination and degradation of activation-induced IRF3 [19]. In our study, we found that c-Cbl could promote K48-linked polyubiquitination and degradation of IRF3. In these regulation network, c-Cbl might have crosstalk with other E3 ligases or molecular to regulate antiviral response, such as RNF26, and Pin1. In conclusion, we identified c-Cbl promoted K48-linked polyubiquitination and degradation of IRF3, which represents an important pathway in maintaining proper low amounts of type I IFN under physiological conditions, and for restraining its magnitude when the antiviral innate response intensifies. Thus, our study not only provides new insight into the molecular mechanisms for termination of excessive immune responses, but also provides a potential target for drug development against virus infection-related diseases and autoimmune diseases. Conflict of interest statement The authors declare that they have no conflicts of interest with the contents of this article. Author contributions XZ and WC conceived and designed the project. XZ and HZ conducted most of the experiments. XZ, JY, HL and JG interpreted the results. XZ and WC wrote the paper.

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Fig. 7. c-Cbl promotes K48-linked ubiquitination and degradation of IRF3. (A) 293T cells were transfected with expression plasmids of Flag-IRF3, HA-Ub, V5-c-Cbl, or control plasmid, cells were treated with MG132 (20 μM) for 6 h before harvested the cells lysates, level of transfected proteins were detected by immunoprecipitation with indicated tag antibodies. (B) 293T cells were transfected with Flag-IRF3, HA-Ub, or control plasmid, along with increasing amounts of expression plasmid for c-Cbl, cells were treated with MG132 (20 μM) for 6 h before harvested the cells lysates, level of transfected proteins were detected by immunoprecipitation with indicated tag antibodies. (C) 293T cells were transfected with expression plasmids of Flag-IRF3, HA-K48-Ub, HA-K63-Ub, V5-c-Cbl, or control plasmid, cells were treated with MG132 (20 μM) for 6 h before harvested the cells lysates, level of transfected proteins were detected by immunoprecipitation with indicated tag antibodies. (D) 293T cells were transfected with plasmids encoding Flag-IRF3, HA-K48-Ub, and increasing doses of plasmid encoding V5-c-Cbl. Half of each cell aliquot was treated with MG132 (20 mM). Cells were harvested 24 h after transfection, and protein expression was detected by immunoprecipitation (upper panel) and immunoblots (lower panel) with indicated tag antibodies. Similar results were obtained from three independent experiments (A–D).

Acknowledgments This work was supported in whole or part by grants from the National Natural Science Foundation of China (81322042, 31200682, 81273222), the Fundamental Research Funds for the Central Universities (2014XZZX003-33) and Zhejiang Provincial Natural Science Foundation under Grant no. Y2110255. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cellsig.2016.08.002. References [1] A.N. Theofilopoulos, R. Baccala, B. Beutler, D.H. Kono, Type I interferons (alpha/beta) in immunity and autoimmunity, Annu. Rev. Immunol. 23 (2005) 307–336. [2] T. Kawai, S. Akira, Innate immune recognition of viral infection, Nat. Immunol. 7 (2006) 131–137.

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