Noncanonical Role of FBXO6 in Regulating Antiviral Immunity

Noncanonical Role of FBXO6 in Regulating Antiviral Immunity Xiaohong Du, Fang Meng, Di Peng, Zining Wang, Wei Ouyang, Yu Han, Yayun Gu, Lingbo Fan, Fei Wu, Xiaodong This information is current as Jiang, Feng Xu and F. Xiao-Feng Qin of October 2, 2021. J Immunol published online 15 July 2019 http://www.jimmunol.org/content/early/2019/07/12/jimmun ol.1801557 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published July 15, 2019, doi:10.4049/jimmunol.1801557 The Journal of Immunology

Noncanonical Role of FBXO6 in Regulating Antiviral Immunity

Xiaohong Du,*,†,1 Fang Meng,*,†,1 Di Peng,*,†,1 Zining Wang,‡,1 Wei Ouyang,x Yu Han,x Yayun Gu,*,† Lingbo Fan,*,† Fei Wu,*,† Xiaodong Jiang,{ Feng Xu,x,2 and F. Xiao-Feng Qin*,†,2

The evolutionarily conserved F-box family of proteins are well known for their role as the key component of SKP1–Cullin1–F-box (SCF) E3 ligase in controlling cell cycle, cell proliferation and cell death, carcinogenesis, and cancer metastasis. However, thus far, there is only limited investigation on their involvement in antiviral immunity. In contrast to the canonical function of FBXO6 associated with SCF E3 ligase complex, we report, in this study, that FBXO6 can also potently regulate the activation of IFN-I signaling during host response to viral infection by targeting the key transcription factor IFN-regulatory factor 3 (IRF3) for

accelerated degradation independent of SCF in human embryonic kidney cells (HEK293T) and human lung cancer epithelial cells Downloaded from (A549). Structure and function delineation has further revealed that FBXO6 interacts with IAD domain of IRF3 through its FBA region to induce ubiquitination and degradation of IRF3 without the involvement of SCF. Thus, our studies have identified a general but, to our knowledge, previously unrecognized role and a novel noncanonical mechanism of FBXO6 in modulating IFN-I–mediated antiviral immune responses, which may protect the host from immunopathology of overreactive and harmful IFN-I production. The Journal of Immunology, 2019, 203: 000–000. http://www.jimmunol.org/ ype I IFNs (IFN-I), including IFN-a and IFN-b, are The homeostasis of IFN-I and balanced IFN-I response is critical critical antiviral innate cytokines that can activate both for the health of the body. When production of IFN-I is insufficient, T innate and adaptive immune responses. Upon viral in- the virus is difficult to be eliminated, leading to chronic infectious fection, host pattern-recognition receptors (PRRs), including diseases (4–6). However, prolonged activation of cellular antiviral TLRs, RLRs, and DNA sensors, sense pathogen-associated mo- responses might produce aberrant and unwanted amounts of lecular patterns (PAMPs), therefore triggering the activation of IFN-I, leading to autoimmune inflammatory diseases (6–9). As the antiviral immunity (1). The activation signals emanated from essential component of IFN-I signaling, regulation of IRF3 is PRRs are subsequently transduced by multiple adaptor molecules, critical for maintaining the homeostasis of IFN-I production in

like TIR domain–containing, adapter-inducing IFN-b (TRIF), host cells (10). Phosphatase PP2A has been reported to recruit its by guest on October 2, 2021 mitochondrial antiviral signaling protein (MAVS), and stimulator adaptor protein RACK1 to promote the dephosphorylation of IRF3 of IFN genes (STING), and converged on the activation of TANK- (11). Furthermore, Phosphatase and tensin homolog (PTEN) with binding kinase 1 (TBK1) or IKB kinase ε (IKKε or IKKi). Further phosphatase activity can negatively regulate IRF3 phosphorylation downstream of the pathway, TBK1 coordinates with IKKi to phos- at Ser-97 to block its import to nucleus (12). In addition to phorylate IFN-regulatory factor 3 (IRF3), leading to the translocation phosphorylation, nuclear receptor-binding SET domain 3 (NSD3) of phosphorylated IRF3 from cytoplasm to nucleus to induce IFN-I has been identiﬁed as the lysine methyltransferase that methylate gene expression (1–3). IRF3 at K366, enhancing the transcription activity of IRF3 (13).

*Center of Systems Medicine, Institute of Basic Medical Sciences, Chinese Academy Drug Development of China (Grant 2015ZX09102023), and the National Grand of Medical Sciences and Peking Union Medical College, Beijing 100005, China; Foreign Experts Projects (Culture and Education) (Grant GDW20181100054). †Suzhou Institute of Systems Medicine, Suzhou, Jiangsu 215123, China; ‡Collaborative X.D., F.M., D.P., Z.W., W.O., Y.H., Y.G., L.F., and F.W. performed the experiments; Innovation Center of Cancer Medicine, Department of Experimental Medicine, State Key X.D., F.M., D.P., Z.W., X.J., F.X., and F.X.-F.Q. analyzed data and wrote the man- Laboratory of Oncology in South China, Sun Yat-sen University, Guangzhou, Guangdong x uscript; and F.X. and F.X.-F.Q. were responsible for research design, strategy, and 510275, China; Department of Infectious Diseases, Second Affiliated Hospital of Zhejiang { supervision. University School of Medicine, Hangzhou, Zhejiang 310009, China; and Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06520 Address correspondence and reprint requests to Prof. F. Xiao-Feng Qin or Prof. Feng Xu, Suzhou Institute of Systems Medicine, No. 100 Chongwen Road, Suzhou, 1X.D., F.M., D.P., and Z.W. contributed equally to this work. Jiangsu 215123, China (F.X.-F.Q.) or Department of Infectious Diseases, Second 2F.X. and F.X.-F.Q. contributed equally to this work. Affiliated Hospital of Zhejiang University School of Medicine, No. 88 Jiefang Road, Hangzhou, Zhejiang 310009, China (F.X.). E-mail addresses: [email protected] ORCIDs: 0000-0003-2856-9058 (Z.W.); 0000-0001-5764-6628 (F.X.); 0000-0002- (F.X.-F.Q.) or [email protected] (F.X.) 5395-0304 (F.X.-F.Q.). The online version of this article contains supplemental material. Received for publication November 26, 2018. Accepted for publication June 11, 2019. Abbreviations used in this article: BiLC, bimolecular luminescence complementation; CHX, cycloheximide; Co-IP, coimmunoprecipitation; HA, hemagglutinin; This work was supported by the Chinese Academy for Medical Sciences Initiative IFN-I, type I IFN; IRF3, IFN-regulatory factor 3; ISG, IFN-stimulated gene; ISRE, for Innovative Medicine (Grant 2016-I2M-1-005), the National Natural Science IFN stimulation response element; KO, knockout; poly(dA:dT), poly(deoxyadenylic- Foundation of China (Grants 81701567, 81773058, and 31800726), the Jiangsu deoxythymidylic) acid; poly(I:C), polyinosinic:polycytidylic acid; SCF, SKP1– Provincial Natural Science Foundation (Grants BK20151252 and BK20171232), Cullin1–F-box; SeV, Sendai virus; sgRNA, short-guide RNA; siRNA, small interfering the Fund of Jiangsu Provincial Science and Technology Department (Grant RNA; TBK1, TANK-binding kinase 1; VSV, vesicular stomatitis virus; VSV-eGFP, BM2016006), the Fund of the Suzhou Municipal Science and Technology Bureau VSV with enhanced GFP; WT, wild-type. (Grant SZS201716), the Non-profit Central Research Institute Fund of the Chinese Academy of Medical Sciences (Grant 2017NL31002), the Key Program for Innovative Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1801557 2 FBXO6 AND ANTIVIRAL IMMUNITY

Another study also reported that viral infection could enhance Angeles, CA). HRP/anti-Flag (M2) was obtained from Sigma-Aldrich. the binding of protein arginine methyltransferases 6 (PRMT6) to HRP/antihemagglutinin was obtained from Roche. HRP/anti–b-actin was IRF3, thus inhibiting the interaction between IRF3 and TBK1 obtained from GenScript. Anti-IRF3 and anti-FBXO6 were obtained from Santa Cruz Biotechnology. Ab specific to IRF3 phosphorylated at residue (14). In conjunction to the interaction, C-terminal phosphoryla- Ser396 was obtained from Cell Signaling Technology. tion of IRF-3 appears to facilitate IRF3 proteasomal degradation (15). After stimulation by dsRNA, induced phosphorylation of the Constructs and plasmids Ser339/Pro340 motif of IRF3 led to its interaction with pepti- The constructs coding for FBXO6, IRF3, and truncated mutants were cloned dylprolyl isomerase Pin1 and, finally, polyubiquitination and then into pcDNA3.1 vector. The expression plasmids encoding RIG-I (2 card), proteasome-dependent degradation of IRF3 (16). RTA-associated IKKi, IRF3, IRF3-5D, and hemagglutinin (HA)-Ub were provided by Dr. J. Cui (University of Sun Yat-sen, Guangzhou, China). ubiquitin ligase (RAUL), a HECT domain ubiquitin E3 ligase, limited IFN-I production by directly catalyzing lysine 48-linked IRF3/BiLC dimerization reporter system polyubiquitination of IRF3 followed by proteasome-dependent To monitor the dimerization of IRF3, we exploit the IRF3/bimolecular degradation (17). RBCK1 interacts with and ubiquitinates IRF3 luminescence complementation (BiLC) reporter system as we described specifically to negatively regulate antiviral response (18); FoxO1 previously (39). IRF3/GlucC and IRF3/GlucN reporters (20 ng each vec- interacts with IRF3 in the cytosol to promote K48-linked ubiq- tor) and the indicated overexpression construct were transfected into uitination and degradation of IRF3 (19); TRIM26 was found to HEK293T or A549 cells by Lipofectamine 2000 (Invitrogen). Twenty-four hours after transfection, these cells were activated with the stimuli degraded wild-type (WT) IRF3 and the constitute active mutant [poly (I:C), poly (dA:dT), and SeV] for the indicated times. Using the IRF3 5D, but not nonactive mutant 5A in nucleus (20). Collec- Renilla luciferase assay system (Promega, Madison, WI), the cells were tively, regulation of IRF3 is rather complicated and multifaceted, lysed by Renilla lysis buffer, and the Gluc activity of the IRF3/BiLC re- and many other mechanisms remain to be uncovered. porter was measured according to the manufacturer’s instructions. Downloaded from F-box proteins were originally identified as the key component Transfection and reporter assays of SKP1–Cullin1–F-box (SCF) family of multisubunit E3 ligases HEK293T cells (3 3 104) were seeded in 96-well plates and transfected (21, 22). The highly conserved F-box domain is responsible for using Lipofectamine 2000 (Invitrogen) plus an expression plasmid encoding assembling of SCF E3 complex through direct interaction with an IFN-b (20 ng) or IFN stimulation response element (ISRE; 20 ng) lu- SKP1 subunit, whereas other less-conserved and variable domains ciferase reporter, Renilla luciferase reporter (pRL-TK; 5 ng), 25 ng activating of F-box proteins appear to determine the substrate specificity for expression plasmids encoding IKKi, IRF3-5D, and 50 ng expression plasmid http://www.jimmunol.org/ ubiquitination by different members of SCF E3 ligases (21, 22). encoding FBXO6. After 24 h, the luciferase activity was measured with the Dual-Luciferase Reporter Kit (Promega) in a Synergy II luminometer The ubiquitination substrates of SCF E3 ligases are known to play (Biotech) according to the manufacturer’s protocol. important roles in broad biological processes, including circadian amplitude, apoptosis, autophagy, cell cycle, carcinogenesis, and Coimmunoprecipitation, immunoblot analysis, and cancer metastasis (23–28). native PAGE Although it was reported that some F-box proteins, such as For coimmunoprecipitation (Co-IP), cell lysates were incubated overnight bTRCP (FBXW1), might affect IFN-I–mediated functions by me- with the appropriate Abs plus Protein A/G Magnetic Beads (Pierce). For diating the ubiquitination of the IFN-a receptor 1 (IFNAR1) and Co-IP with anti-Flag or anti-HA Abs, Ab-coated beads (Sigma-Aldrich) k a b ε were used. The beads were washed with low-salt lysis buffer, eluted in by guest on October 2, 2021 I B / / in a SCF-dependent manner (29–33), no systematic study 23 SDS Loading Buffer and resolved by SDS-PAGE. Proteins were has conducted thus far to investigate other unrecognized involve- transferred to PVDF membranes (Millipore) and incubated with the ap- ment of F-box proteins and SCF E3 ligases in IFN-I signaling. propriate Abs for detection with the Immobilon Western Chemilumines- By systematically screening F-box proteins for modulating the cent HRP Substrate (Millipore) as previously reported (40). Native PAGE IFN-I signaling in reporter assays, we have identify multiple F-box was performed as described in a previous report (41). proteins could either positively or negatively affecting the ex- RNAi and CRISPR/Cas9 assay b pression of IFN- reporters. One of such candidate F-box proteins We use CRISPR/Cas9 assay, a highly effective and powerful tool for gene is FBXO6. Interestingly, FBXO6 was originally identified as a editing, to generate the FBXO6 knockout (KO) HEK293T cells (42). Small component of SCF ubiquitin ligase primarily involved in endo- interfering RNAs (siRNAs) were transfected into HEK293T cells with plasmic reticulum-associated protein degradation, presumably Lipofectamine RNAiMax (Invitrogen) according to the manufacturer’s through ubiquitination of various glycoproteins via its FBA domain instructions. The sequences of the siRNA specific for FBXO6 are as follows: 59-GCUUCAGUGAACUUGCUAA-39. The short-guide RNAs (34–36). In addition to glycoproteins, FBA domain of FBXO6 can (sgRNAs) specific for FBXO6 was designed and cloned into sgRNA mediate the ubiquitination and degradation of Chk1 and RIOK1 (37, expression plasmid. The sequence of the sgRNA specific for FBXO6 is 38). In contrast to these known functions through SCF-dependent 59-CTTCAGCACACGGGTTGCGC-39. For FBXO6, the genomic DNA canonical pathway, in this work we have uncovered a previously fragment was amplified with forward primer 59-TGGCTAATG CAGGGTTG- GAG-39 and reverse primer 59-TACAGGGCATGAAATGGGGG-39. unrecognized role of FBXO6 in regulating IFN-I production, and the novel mechanism for regulating IRF3 degradation and stability Quantitative real-time PCR by FBXO6-mediated ubiquitination in a SCF-independent manner. Total RNAwas obtained using the Ultrapure RNA Kit (CWBiotech, Beijing, China). cDNAs were transcribed from the extracted total RNA using HiFi Materials and Methods Script cDNA Synthesis Kit (CWBiotech, Beijing, China). Real-time PCR was performed using the Fast SYBR Green PCR Master Mix in the CFX96 Cells and reagents Touch Real-Time PCR Detection System (Bio-Rad Laboratories, CA). The Human embryonic kidney cells (HEK293T) and adenocarcinomic human relative expression of each gene was normalized to the expression of 2DDCt alveolar basal epithelial cells (A549) were obtained from American Type GAPDH as determined by the 2 method, as previously reported Culture Collection and maintained in DMEM medium supplemented (43, 44). The primer sequences are listed in Table I. with 10% FBS (Thermo Fisher Scientific), 2 mM L-glutamine, 100 U/ml Statistical analysis penicillin,and 100 mg/ml streptomycin at 37˚C with 5% CO2 incuba- tion. Polyinosinic:polycytidylic acid [poly(I:C)] and poly(deoxyadenylic- All data are presented as mean values 6 SEM. The statistical significance deoxythymidylic) acid [poly(dA:dT)] were obtained from InvivoGen. of differences between two groups was analyzed with the unpaired two- Cycloheximide (CHX) was purchased from Sigma-Aldrich. Sendai virus tailed Student t test. A p value ,0.05 was considered statistically signif- (SeV) and vesicular stomatitis virus (VSV) with enhanced GFP (VSV-eGFP) icant. All calculations were performed using the Prism software program were provided by Dr. G. Cheng’s laboratory (University of California, Los for Windows (GraphPad Software). The Journal of Immunology 3

Result monitor the dimerization of IRF3 (39). If the IFN-I signaling is FBXO6 negatively regulates IFN-I signaling and activated, the IRF3/GlucN and IRF3/GlucC will form dimer and antiviral responses exhibit the complete activity of luciferase. With this IRF3/BiLC reporter system, we found that ectopically expressed FBXO6 To assess the role of FBXO6 in regulation of IFN-I signaling, we use ISRE luciferase reporter as a tool to monitor the activity of IFN-I– inhibited the IRF3-dimer luciferase activities triggered by SeV or induced JAK/STAT signaling pathway. HEK293T cells were trans- poly(dA:dT) (Fig. 1B). Furthermore, we assessed the effect of fected with an ISRE luciferase reporter, internal control Renilla FBXO6 on IRF3 dimerization and found that the level of both luciferase reporter, and the FBXO6 expression plasmid, and the dimerized and total IRF3 was decreased with elevated FBXO6 transfected cells were subsequently infected by SeV for 12 h. We expression (Fig. 1C). To evaluate whether IFN-I play a critical role found that FBXO6 inhibited SeV-induced ISRE luciferase activities in defending virus invasion, we next transfected HEK293T cells (Fig. 1A). The similar results were obtained from these cells treated with FBXO6 expression or control plasmids, and infected cells with poly(I:C) and poly(dA:dT) (Fig. 1A). In addition, FBXO6 was with VSV-eGFP at a multiplicity of infection of 0.05. Twelve also shown to inhibit the activation of ISRE luciferase activities hours later, more GFP-positive HEK293T cells transfected with stimulated by IFN-b (Supplemental Fig. 1A), suggesting that FBXO6 FBXO6 expression vector were observed (Fig. 1D), suggesting might be able to regulate the IFN-I receptor signaling as well. that HEK293T cells with FBXO6 expression vector are much As phosphorylation and dimerization of IRF3 is the hallmark of more easily infected than those cells with control vector. Taken its activation, we developed an IRF3/BiLC reporter system to together, these results clearly show that FBXO6 acts as a negative Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 1. FBXO6 negatively regulates IFN-I signaling and antiviral responses. (A) HEK293T cells were transfected with FBXO6 expression plasmid and internal control Renilla luciferase reporter, plus ISRE luciferase reporter, and followed by treatment with SeV for 12 h, poly(I:C), or poly(dA:dT) for 18 h before the luciferase reporter assays were performed. (B) HEK293T cells were transfected with FBXO6 expression plasmid and internal control firefly luciferase reporter, plus IRF3 dimer luciferase reporter, and followed by treatment with SeV for 12 h or poly(dA:dT) for 14 h before the luciferase reporter assays were performed. (C) HEK293T cells were transfected with the indicated plasmids and followed by SeV infection for 12 h. The cell lysates were separated by native (top) or SDS (bottom) PAGE and analyzed by immunoblots with the indicated Abs. Numbers above the lanes indicate the relative accumulation of dimerized IRF3. (D) Fluorescence microscopy assessing the VSV-eGFP infection in HEK293T cells transfected with FBXO6 expression plasmid and followed with VSV-eGFP at multiplicity of infection (MOI) = 0.05 for 12 h. Original magnification 310. All results are representative of three replicate experiments. *p # 0.05, **p # 0.01, ***p # 0.001. 4 FBXO6 AND ANTIVIRAL IMMUNITY regulator in activation of IFN-I signaling triggered by dsDNA, found that knockdown of FBXO6 enhanced SeV-induced IFN-b, RNA, or viral infection through inhibiting IRF3 dimerization. ISRE luciferase reporter activities, and the total IRF3 protein level and its dimerization (Fig. 2A, 2B). Meanwhile, we examined the Knockdown of FBXO6 enhances IRF3 activation and antiviral effects of FBXO6 knockdown on IRF3 dimerization in A549 cells gene expression with IRF3/BiLC reporter system, and found that FBXO6 knock- We next investigated whether FBXO6 knockdown would potentiates down promoted IRF3-dimer luciferase activities triggered by IRF3 activation as well as IFN-I production and IFN-stimulated poly(I:C), poly(dA:dT), and SeV, respectively (Fig. 2C). genes (ISG) expression. We first used FBXO6 specific siRNA to To identify the effects of FBXO6 knockdown on the expression interfere FBXO6 expression and observed that siRNA interfered of IFN-I and ISG, we transfected HEK293T cells with FBXO6 FBXO6 protein expression by 86% (Supplemental Fig. 1B). We specific or scramble siRNA, followed by treatment with poly(dA:dT) Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 2. Knockdown of FBXO6 enhances IRF3 activation and antiviral gene expression. (A) HEK293T cells transfected with FBXO6 specific or scramble siRNA, together with IFN-b luciferase reporter and internal control Renilla luciferase reporter, followed by treatment with SeV for 12 h before the luciferase reporter assays were performed. (B) HEK293T cells were transfected with the indicated siRNA and followed by SeV infection for 12 h. The cell lysates were separated by native (top) or SDS (bottom) PAGE and analyzed by immunoblots with the indicated Abs. Numbers above the lanes indicate the relative accumulation of dimerized IRF3. (C) A549 cells were transfected with IRF3/BiLC reporter vectors and FBXO6 specific or scramble siRNA and treated by poly(I:C), poly(dA:dT), and SeV for 10 h before the luciferase reporter assays were performed. (D and E) Real-time PCR analysis of IFNB1, CCL5,and ISG54 mRNA in HEK293T cells, which were transfected with FBXO6 specific or scramble siRNA and followed by poly(dA:dT) treatment or SeV infection for 12 h. All results are representative of three replicate experiments. ***p # 0.001, ****p # 0.0001. The Journal of Immunology 5 or SeV. We found that HEK293T cells with FBXO6 specific siRNA FBXO6 could form a complex with IRF3 (Fig. 4C). To further have higher IFNB1 mRNA expression than those with scramble address how FBXO6 interacts with IRF3, we constructed a series siRNA (Fig. 2D, 2E). Consistently, knockdown of FBXO6 also truncated mutants of FBXO6 and IRF3 (Fig. 4D, 4E). FBXO6 induced higher mRNA expressions of ISG, including ISG54 and comprises a highly conserved F-box domain and another defined CCL5 (Fig. 2D, 2E). Together, we demonstrate that knockdown of structure reported for protein/protein interaction called FBA do- FBXO6 enhance IRF3 dimerization and IFN-I gene expression. main, whereas IRF3 can be dissected into two major function The primer sequences are listed in Table I. domains namely DBD and IAD. We investigated the interaction between FBXO6- and IRF3-truncated mutants and found that FBA FBXO6 KO promotes the activation of IFN-I signaling domain of FBXO6 interacts with IRF3 IAD domain (Fig. 4D, 4E). To determine the effects of FBXO6 KO on the activation of IFN-I FBXO6 promotes IRF3 degradation by proteasome-mediated signaling, we next designed the sgRNA specific for FBXO6 and pathway of ubiquitin modification cloned the sgRNA into a sgRNA-expressing plasmid. Then, we generated the FBXO6 KO HEK293T clones by transfection of Previous studies have demonstrated that SCF E3 ligase complex sgRNA expression plasmid, selection with puromycin, and sub- used F-box proteins to recognize and bind targeted substrates for cloning of the resistant cells. The immunoblot analysis con- ubiquitin-mediated protein destruction. In this study, we won- firmed the complete loss of FBXO6 protein in the KO HEK293T dered whether FBXO6 degrades the key adaptor protein IRF3 in cells (Supplemental Fig. 1C). Compared with those in WT IFN-I signaling. To test this, HEK293T cells were transfected HEK293T cells, the IFN-b luciferase activities were significantly with IRF3 expression plasmids, together with FBXO6 in a dosage- elevated in FBXO6 KO HEK293T cells triggered by poly(I:C), increasing manner. We observed that FBXO6 affects the stability poly(dA:dT), and SeV, respectively (Fig. 3A). As expected, we of both endogenous and exogenous IRF3 (Fig. 5A). Consistently, Downloaded from found that the ISRE luciferase activities were also signifi- IRF3 expression was more stable in A549 cells with FBXO6 cantly enhanced in FBXO6 KO HEK293T cells by various stimuli specific siRNA than those in A549 cells with scramble siRNA, (Fig. 3B). In addition, the IRF3-dimer luciferase activities were resulting in elevated phosphorylated IRF3 accumulation after also enhanced in FBXO6 KO HEK293T cells compared with stimulated by SeV infection in A549 with FBXO6 specific siRNA those in WT HEK293T cells triggered by RIG-I (2 card) (Fig. 3C). (Fig. 5B). To confirm the effect of FBXO6 on IRF3 stability, To further confirm that the phenotype of FBXO6 KO cells is due we tested the degradation of IRF3 by FBXO6 in the presence of http://www.jimmunol.org/ to FBXO6 deletion, FBXO6 KO HEK293T cells were transfected translation inhibitor CHX. HEK293T cells were cotransfected with FBXO6 expression plasmids, and their ISRE luciferase ac- with plasmids of IRF3 and FBXO6. Then cells were treated with tivities triggered by RIG-I (2 card) were found to be repressed to 100 ng/ml CHX and harvested for immunoblot analysis. The re- WT cells level (Fig. 3D). Real-time PCR further confirmed sults showed that FBXO6 could strongly degraded IRF3 with the that FBXO6 KO HEK293T cells produced more IFNB1 mRNA treatment of CHX (Fig. 5C). Interestingly, both WT and F-box expression than those in WT HEK293T cells when the cells domain–deleted FBXO6 affect the stability of IRF3 and inhibit were treated with poly(I:C), poly(dA:dT), and SeV, respectively the IFN-b luciferase reporter stimulated by transfection of RIG-I (Fig. 3E–G). Consistently, FBXO6 KO also improved mRNA (2 card), implying that FBXO6 regulates stability of IRF3 inde- by guest on October 2, 2021 expression level of CCL5 and ISG54 genes (Fig. 3E–G). According pendent of SCF E3 ligase activity (Fig. 5D, 5E). We further found to these results, we conclude that FBXO6 KO enhance the activa- that proteasome inhibitor MG132, which inhibits the degradation of IFN-I signaling, resulting in elevated IFN-I production and tion of ubiquitin-conjugated proteins by proteasome degradation ISG expression. pathway, almost abrogated IRF3 degradation mediated by FBXO6 in HEK293T cells, suggesting that IRF3 destruction induced by FBXO6 interacts with IRF3 downstream of FBXO6 is through proteasome-dependent pathway (Fig. 5F). IFN-I–signaling pathway Moreover, the Co-IP experiment showed that knockdown of To delineate the mechanism how FBXO6 regulates IFN-I signaling, FBXO6 inhibit the ubiquitination of IRF3, suggesting that FBXO6 we transfected HEK293T TBK1 KO versus WT cells with ex- could promote IRF3 ubiquitination (Fig. 5G). Taken together, these pression plasmids containing IKKi or IRF3-5D (a constitutively results reveal that FBXO6 promotes IRF3 destruction through activated form of IRF3 mutation) together with FBXO6 expression ubiquitination-mediated proteasome degradation pathway. plasmid, ISRE luciferase reporter, and internal control Renilla luciferase reporter. We found that ISRE activation was significantly Discussion reduced in both TBK1 WT and KO HEK293T cells transfected with F-box proteins, as a key component of SCF E3 ligase, play im- IKKi (but not IRF3-5D) and FBXO6 plasmid (Fig. 4A). These portant roles in multiple critical cellular signaling, including cir- results suggested that FBXO6 could inhibit IKKi and might cadian amplitude, apoptosis, autophagy, cell cycle, carcinogenesis, function at the downstream of TBK1/IKKi in IFN-I–signaling and cancer metastasis (23–28). It is reported that some F-box pathway. To narrow down the target proteins, we then performed proteins, such as bTRCP, also regulate the IFN-I signaling by Co-IP assays and found that FBXO6 was associated with IRF3 mediating the ubiquitination of the IFNAR1 and IkBa/b/ε in a (Fig. 4B). Co-IP of the endogenous proteins further confirmed that SCF-dependent manner (29–33). Moreover, some viruses use the

Table I. The primer sequences used in quantitative real-time PCR

Gene Sense (59–39) Antisense (59–39) IFNB1 59-CCTACAAAGAAGCAGCAA-39 59-TCCTCAGGGATGTCAAAG-39 ISG54 59-GGAGGGAGAAAACTCCTTGGA-39 59-GGCCAGTAGGTTGCACATTGT-39 CCL5 59-ATCCTCATTGCTACTGCCCTC-39 59-GCCACTGGTGTAGAAATACTCC-39 GAPDH 59-GAACGGGAAGCTCACTGG-39 59-GCCTGCTTCACCACCTTCT-39 Data shown are the primer sequences used in quantitative real-time PCR. 6 FBXO6 AND ANTIVIRAL IMMUNITY Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 3. FBXO6 KO promotes the activation of IFN-I signaling. (A and B) The luciferase activity in WT and FBXO6 KO HEK293T cells transfected with internal control Renilla luciferase reporter and IFN-b luciferase reporter or ISRE luciferase reporter, followed by treatment with intracellular poly(I:C) for 18 h, poly(dA:dT) for 18 h, or SeV for 12 h. (C) The WT and FBXO6 KO HEK293T cells were transfected with internal control ﬁreﬂy luciferase reporter and IRF3 dimer luciferase reporter and followed by stimulation with RIG-I (2 card) for 24 h before the luciferase reporter assays were performed. (D) The luciferase activity in WT and FBXO6 KO HEK293T cells transfected with internal control Renilla luciferase reporter and ISRE luciferase reporter, plus FBXO6 expression plasmid, followed by transfection with RIG-I (2 card). (E–G) Real-time PCR analysis of IFNB1, CCL5, and ISG54 mRNA in WT and FBXO6 KO HEK293T cells treated with poly(I:C) for 12 h, poly(dA:dT) for 12 h, or SeV for 12 h. All results are representative of three replicate experiments. *p # 0.05, **p # 0.01, ***p # 0.001, ****p # 0.0001. The Journal of Immunology 7

FIGURE 4. FBXO6 interacts with IRF3 downstream of IFN-I–signaling pathway. (A) The luciferase activity in WT and TBK1 KO HEK293T cells transfected with internal control Renilla luciferase reporter and ISRE luciferase reporter, plus FBXO6 expression plasmid, followed by transfection with IKKi or IRF3-5D for 24 h. (B) HEK293T cells were transfected with the indicated expression plasmids for 48 h. The cell lysates were immunoprecipitated with anti-Flag, and immunopre- cipitates were analyzed by immunoblot with indicated Abs. (C) The cell lysates prepared from

HEK293T WT and FBXO6 KO cells were Downloaded from immunoprecipitated with anti-IRF3. The immu- noprecipitates were analyzed by immunoblot with indicated Abs. (D) HA-IRF3 was cotransfected with Flag/FBXO6-truncated mutants into HEK293T cells for 48 h. The Co-IP was performed and analyzed by immunoblot with anti-HA Ab. Numbers above the lanes indicate http://www.jimmunol.org/ therelativeratioofHA/Flag.(E) HA/FBXO6 was cotransfected with Flag/IRF3-truncated mutants into HEK293T cells for 48 h. The Co- IP was performed and analyzed by immunoblot with anti-HA Ab. All results are representative of three replicate experiments. ****p # 0.0001. by guest on October 2, 2021

viral protein containing the F-box domain or hijack the F-box of IRF3 activity. IRF3 activation is finely controlled by interac- protein of host cells to promote their infection process, suggest- tion with inhibitory proteins and multiple posttranscriptional ing that F-box proteins may play an important role in antiviral modifications including phosphorylation, dephosphorylation, immune response (45–48). Therefore, it is needed to carry out the polyubiquitination, and methylation (11, 13, 14, 50). Among these systematic research on the role of F-box proteins in regulating regulations, dephosphorylation and polyubiquitination play a IFN-I signaling, a critical process in antiviral immune response, major negative regulatory role in controlling the magnitude and during virus infection. By systematically screening the F-box duration of IRF3 activation (11, 12, 16–20). In our previous study, proteins that can regulate the IFN-I–signaling pathway, we iden- we identified FBXO17 as a negative regulator of IFN-I signaling tify several F-box proteins, such as FBXO17, regulating the IFN-I by recruiting phosphatase PP2A for IRF3 dephosphorylation and signaling (49). In this study, our results demonstrate FBXO6- deactivation, suggesting F-box protein family is involved in the inhibited antiviral response through interfering IFN-I production. regulation of IFN-I signaling (49). In this study, we identified Although the regulation of IFN-I production by FBXO6 needs another F-box, FBXO6, as a negative regulator of IFN-I signaling. to be verified in vivo, both in vitro overexpression and KO ex- Considering that IFN could promote the infection of certain periments show that FBXO6 negatively regulates transcriptional pathogenic bacteria, negative regulation of IFN-I production could expression of IFN-I and ISG, resulting in an enhanced antiviral im- balance the risk between viral and bacterial infections (51). munity. Further study of molecular mechanism reveals that FBXO6 Our results demonstrated that FBXO6 interacts IRF3 with FBA interacts with IAD domain of IRF3 in FBA region, therefore inducing domain (Fig. 4D). FBA domain of FBXO6 also mediate the in- IRF3 degradation by ubiquitination modification. These findings re- teraction with its substrate, including glycosylated substrates veal a previously unknown role and mode of action of FBXO6 in Chk1 and RIOK1 (37, 38). In F-box proteins family, FBXO2, antiviral immunity. FBXO6, FBXO17, FBXO27, and FBXO44 have similar structures IRF3 is the key downstream molecule of various IFN-I–signaling comprised of an F-box motif and an FBA domain, which is related pathways involved in IFN-I production. To protect the host from closely to its interaction with glycosylated substrates (22). Given the damaging effects of an overproduction of IFN-I, there exists a that the IAD domain is blocked by the autoinhibitory domains in complex negatively regulatory network to maintain the homeostasis the inactive state of IRF3, the interaction of FBA domain and IAD 8 FBXO6 AND ANTIVIRAL IMMUNITY

FIGURE 5. FBXO6 promotes IRF3 degradation by proteasome-mediated pathway of ubiquitin modiﬁcation. (A) HEK293T cells were transfected with the indicated plasmids for 48 h before the immunoblot assay was performed with the indicated Abs. (B) A549 cells were transfected with FBXO6 speciﬁc or scramble siRNA and followed by SeV infection for the indicated times before immunoblot analysis were performed with the indicated Abs. Numbers above the lanes indicate the relative accumulation of phosphorylated IRF3. (C) Immunoblot of lysates from HEK293T cells transiently expressing Flag/FBXO6, HA/IRF3, andHA/mCherryfor24handthenculturedin the presence of 100 ng/ml CHX (time, above lanes). Expression of HA/IRF3 was normalized to that of HA/mCherry (graphed below). (D) HEK293T cells were transfected with the

indicated plasmids for 48 h before the im- Downloaded from munoblot assay was performed with the indicated Abs. (E) The luciferase activity in HEK293T cells transfected with internal control Renilla luciferase reporter and IFN-b luciferase reporter, plus FBXO6 or FBXO6 mutation expression plasmid, followed by

transfection with RIG-I (2 card) for 24 h. (F) http://www.jimmunol.org/ HEK293T were transfected with the indicated plasmids for 24 h and followed by adding DMSO or MG132 for 24 h, and immunoblot analysis assay was performed. (G) The im- munoprecipitation and immunoblot analysis of HEK293T cells transfected with the indicated siRNA and plasmids for 24 h. All results are representative of three replicate experiments. **p # 0.01, ***p # 0.001. by guest on October 2, 2021 domain is possibly dependent of the phosphorylation state of IRF3 References (52). Moreover, compared with WT IRF3, the S386A mutation 1. Takeuchi, O., and S. Akira. 2010. Pattern recognition receptors and inflamma- interacts weakly with FBXO6, implying that FBXO6 may mainly tion. Cell 140: 805–820. 2. Akira, S., S. Uematsu, and O. Takeuchi. 2006. Pathogen recognition and innate interact with the phosphorylated IRF3 at S386 site (data not immunity. Cell 124: 783–801. shown). It was previously believed that functions of F-box pro- 3. Wu, J., and Z. J. Chen. 2014. Innate immune sensing and signaling of cytosolic teins are dependent of SCF complex E3 ligase activity (canonical nucleic acids. Annu. Rev. Immunol. 32: 461–488. mechanism). However, our study showed that the function of 4. Arzumanyan, A., H. M. Reis, and M. A. Feitelson. 2013. Pathogenic mechanisms in HBV- and HCV-associated hepatocellular carcinoma. Nat. Rev. Cancer FBXO6 in negative regulation of IFN-I production is independent 13: 123–135. of the conventional function of SCF E3 ligase activity, but due to a 5. Tan, G., Q. Xiao, H. Song, F. Ma, F. Xu, D. Peng, N. Li, X. Wang, J. Niu, P. Gao, noncanonical mechanism by promoting ubiquitination and deg- et al. 2018. Type I IFN augments IL-27-dependent TRIM25 expression to inhibit HBV replication. Cell. Mol. Immunol. 15: 272–281. radation of IRF3. Similarly, FBXO17 also negatively regulates 6. Theofilopoulos, A. N., R. Baccala, B. Beutler, and D. H. Kono. 2005. Type I IFN-I production in a SCF E3 ligase activity–independent manner interferons (alpha/beta) in immunity and autoimmunity. Annu. Rev. Immunol. 23: (49). Interestingly, our results showed that both FBXO6 and 307–336. 7. Banchereau, J., and V. Pascual. 2006. Type I interferon in systemic lupus FBXO17 could interact with IAD domain of IRF3 via FBA region erythematosus and other autoimmune diseases. Immunity 25: 383–392. (49), implying there may be a similar interaction mechanism for 8. Meyer, O. 2009. Interferons and autoimmune disorders. Joint Bone Spine 76: both FBXO6/IRF3 and FBXO17/IRF3 interactions. Our studies 464–473. 9. Gall, A., P. Treuting, K. B. Elkon, Y. M. Loo, M. Gale, Jr., G. N. Barber, and suggest that in addition to canonical functions in SCF E3 ligase, D. B. Stetson. 2012. Autoimmunity initiates in nonhematopoietic cells and FBXO6, and FBXO17 (maybe more F-box proteins) also play progresses via lymphocytes in an interferon-dependent autoimmune disease. Immunity 36: 120–131. an important role in regulating the IFN-I signaling in an SCF- 10. Honda, K., and T. Taniguchi. 2006. IRFs: master regulators of ignaling by Toll- independent manner. like receptors and cytosolic pattern-recognition receptors. Nat. Rev. Immunol. 6: In summary, we report in this study that F-box protein 644–658. 11. Long, L., Y. Deng, F. Yao, D. Guan, Y. Feng, H. Jiang, X. Li, P. Hu, X. Lu, FBXO6isinvolvedinnegativelyregulatingthehomeostasisof H. Wang, et al. 2014. Recruitment of phosphatase PP2A by RACK1 adaptor IFN-I production, and we revealed the noncanonical molecular protein deactivates transcription factor IRF3 and limits type I interferon sig- mechanism of FBXO6 by mediating IRF3 ubiquitination for naling. Immunity 40: 515–529. 12. Li, S., M. Zhu, R. Pan, T. Fang, Y. Y. Cao, S. Chen, X. Zhao, C. Q. Lei, L. Guo, degradation. Y. Chen, et al. 2016. The tumor suppressor PTEN has a critical role in antiviral innate immunity. Nat. Immunol. 17: 241–249. 13. Wang, C., Q. Wang, X. Xu, B. Xie, Y. Zhao, N. Li, and X. Cao. 2017. The Disclosures methyltransferase NSD3 promotes antiviral innate immunity via direct lysine The authors have no financial conflicts of interest. methylation of IRF3. J. Exp. Med. 214: 3597–3610. The Journal of Immunology 9

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B A 30 **** siRNA Scr FBXO6 e

g 25 EV n Flag-FBXO6 + + a 20 FBXO6 h 15

Flag/Actin 1 0.14 10

d l 1.5 o

F **** IB:Flag 35 kD

c 1.0 u

L IB:Actin 43 kD E 0.5

S I 0.0 l ) tr L C m g/ n 0 (1 ß N IF

Figure legends Figure S1

(A) HEK293T cells were transfected with FBXO6 expression plasmid and internal control renilla luciferase reporter, plus ISRE luciferase reporter, and followed by treatment with 10 ng/mL IFNβ for 24h before the luciferase reporter assays were performed. (B) HEK293T cells were transfected with indicated siRNAs and plasmids.

The lysates were analyzed by immunoblots with indicated antibodies. Numbers above the lanes indicate the relative expression of Flag-FBXO6. (C) The lysates of HEK293T

WT and FBXO6 KO cells were analyzed by immunoblots with indicated antibodies.