SARS Coronavirus Papain-Like Protease Inhibits the TLR7 Signaling Pathway Through Removing Lys63-Linked Polyubiquitination of TRAF3 and TRAF6

International Journal of Molecular Sciences

Article SARS Coronavirus Papain-Like Protease Inhibits the TLR7 Signaling Pathway through Removing Lys63-Linked Polyubiquitination of TRAF3 and TRAF6

Shih-Wen Li 1, Ching-Ying Wang 1, Yu-Jen Jou 1, Su-Hua Huang 2, Li-Hsin Hsiao 1, Lei Wan 3, Ying-Ju Lin 3, Szu-Hao Kung 4 and Cheng-Wen Lin 1,2,* 1 Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung 404, Taiwan; [email protected] (S.-W.L.); [email protected] (C.-Y.W.); [email protected] (Y.-J.J.); [email protected] (L.-H.H.) 2 Department of Biotechnology, College of Health Science, Asia University, Wufeng, Taichung 413, Taiwan; [email protected] 3 Department of Medical Genetics and Medical Research, China Medical University Hospital, Taichung 404, Taiwan; [email protected] (L.W.); [email protected] (Y.-J.L.) 4 Department of Biotechnology and Laboratory Science in Medicine, National Yang Ming University, Taipei 112, Taiwan; [email protected] * Correspondence: [email protected]; Tel.: +886-4-2205-3366 (ext. 7210); Fax: +886-4-2205-7414

Academic Editor: Atsushi Matsuzawa Received: 14 March 2016; Accepted: 26 April 2016; Published: 5 May 2016

Abstract: Severe acute respiratory syndrome coronavirus (SARS-CoV) papain-like protease (PLPro) reportedly inhibits the production of type I interferons (IFNs) and pro-inﬂammatory cytokines in Toll-like receptor 3 (TLR3) and retinoic acid-inducible gene 1 (RIG-I) pathways. The study investigated the inhibitory effect and its antagonistic mechanism of SARS-CoV PLPro on TLR7-mediated cytokine production. TLR7 agonist (imiquimod (IMQ)) concentration-dependently induced activation of ISRE-, NF-κB- and AP-1-luciferase reporters, as well as the production of IFN-α, IFN-β, TNF-α, IL-6 and IL-8 in human promonocyte cells. However, SARS-CoV PLPro signiﬁcantly inhibited IMQ-induced cytokine production through suppressing the activation of transcription factors IRF-3, NF-κB and AP-1. Western blot analysis with anti-Lys48 and anti-Lys63 ubiquitin antibodies indicated the SARS-CoV PLPro removed Lys63-linked ubiquitin chains of TRAF3 and TRAF6, but not Lys48-linked ubiquitin chains in un-treated and treated cells. The decrease in the activated state of TRAF3 and TRAF6 correlated with the inactivation of TBK1 in response to IMQ by PLPro. The results revealed that the antagonism of SARS-CoV PLPro on TLR7-mediated innate immunity was associated with the negative regulation of TRAF3/6-TBK1-IRF3/NF-κB/AP1 signals.

Keywords: SARS-CoV; Toll-like receptor 7; imiquimod; TRAF3; TRAF6; Lys63-linked polyubiquitin chains

1. Introduction Toll-like receptor 7 (TLR7) is one of the well-known pattern recognition receptors [1], sensing single-stranded RNA viruses via the recognition of the viral RNA genome [2]. Binding of the ligand to the TLR extracellular domain causes the homodimerization of TLRs via further interaction of the intracellular Toll/interleukin-1 receptor (TIR) domain, then activates myeloid differentiation factor 88 (MyD88)-dependent and/or TIR domain-containing adaptor-inducing IFN-β (TRIF)-dependent pathways [1,3]. In the MyD88-dependent pathway, the complex of MyD88 with activated TLR dimers, except for TLR3, recruits IL-1 receptor-associated kinase (IRAK)-4, then leads IRAK4 to activate other members of the IRAK, like IRAK1 and IRAK2. The activated IRAKs directly interact with TNF

Int. J. Mol. Sci. 2016, 17, 678; doi:10.3390/ijms17050678 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2016, 17, 678 2 of 10 receptor-associated factor (TRAF) 6 (E3 ubiquitin ligase) plus E2 ubiquitin-conjugating enzymes Ubc13 and Uev1A, resulting in Lys63-linked ubiquitination of TRAF6, IRAKs and TGF-β-activated kinase 1 (TAK1) [4]. In the TRIF-dependent pathway, activated TLR3 directly binds TRIF, resulting in the activation of TRIF connecting with TRAF3 to turn on TANK-binding kinase 1 (TBK1) and IKKs for IRF3/7 phosphorylation [4,5]. Alternatively, TRIF associates with RIP1 and then forms the complex along with TRAF6 for activation of TAK1. Ubiquitin-activated TAK1 phosphorylates mitogen-activated protein kinases (MAPKs) and IκB-kinases (IKKs), initiating AP-1, NF-κB and IRF3/7 signaling on the production of cytokines, chemokines and type I interferons (IFNs). Severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) causes the pro-inflammatory cytokine storms, recruitment of immune responder cells into the lungs, acute respiratory distress syndrome (ARDS) and even lung fibrosis in the late phase [6,7]. Among 14 ORFs encoded by SARS-CoV [8,9], ORF1a/ORF1ab is the biggest one encoding polyprotein replicases 1a and 1ab, primarily involved in SARS-CoV replication. Specifically, ORF1a-encoded papain-like protease (PLPro) and 3C-like protease (3CLpro) process cis- and trans-cleavage activities on replicases 1a and 1ab for creating 16 nonstructural (NS) proteins (termed NS 1 through NS16). PLPro, recognizing a consensus motif LXGG as a de-ubiquitinating/de-ISGylating enzyme [10–13], exhibits the antagonistic activities of type I interferon (IFN). PLPro directly interacts with IFN regulatory factor 3 (IRF-3) to block IRF-3 phosphorylation and nuclear translocation [14,15]. Conversely, another study indicated that type I IFN antagonism of PLPro is not correlated to a direct interaction of PLPro with IRF-3 or affecting the phosphorylation of IRF3, but PLPro suppresses the NF-κB signaling pathway by preventing IκBα degradation [15]. Recent studies demonstrate that PLPro disrupts the stimulator of interferon gene (STING)-mediated signaling and then negatively regulates type I IFN induction [16,17]. PLPro physically interacts with the STING-TRAF3-TBK1 complex, reducing the ubiquitinated forms of STING, RIG-I, TRAF3, TBK1 and IRF-3. Similarly, de-ubiquitinating enzyme cylindromatosis (CYLD) inhibits NF-κB signaling via de-ubiquitination and inactivation of TNFR-associated factor 2 (TRAF2) and TRAF6 [18,19]; de-ubiquitinating protease A20 inhibits NF-κB activation induced by Toll-like receptor 4 (TLR4) via removing K63-linked polyubiquitin chains of TRAF6 [20]. PLPro interacts with above key regulators of TLR signal pathways; thus, characterizing the antagonistic mechanisms of TLR signal pathways by SARS-CoV PLPro could provide valuable insights into SARS pathogenesis. SARS-CoV-specific GU-rich ssRNA has been demonstrated to be recognized by TLR7, establishing a link with the induction of pro-inflammatory cytokines TNF-α, IL-6 and IL-12 [21]. SARS-CoV infection rapidly activates TLR7 signaling in plasmacytoid dendritic cells; however, some SARS-CoV proteins subsequently inhibit and/or modulate type I IFN responses in plasmacytoid dendritic cells [22]. Therefore, the effect of the unique SARS-CoV proteins on the activation of the TLR7 signaling pathway is noteworthy to elucidate. This study assesses possible effects of PLPro on TLR7 signaling pathways and the production of type I IFNs and pro-inflammatory cytokines. In this study, PLPro-expressing and vector control cells were treated with TLR7 agonist (imiquimod (IMQ)) and then examined on IRF3, STAT1, NF-κB, p38 MAPK and c-Jun regulation using dual luciferase reporters, Western blotting and real-time PCR assays. Analysis of ubiquitin-modified proteins reveals that the change in ubiquitination of TRAF3 and TRFA6 played the crucial role in the antagonistic mechanisms of TLR7-mediated type I IFN induction and NF-κB activation by PLPro.

2. Results

2.1. PLPro Suppressed TLR7 Agonist-Induced Production of Type I IFNs in Human Promonocytes To examine whether SARS-CoV PLPro modulates the TLR7 signaling pathway, stable transfected promonocyte cells expressing PLPro and vector control cells were established, treated with TLR7 agonist (imiquimod (IMQ)), then further analyzed for activation of type I IFN production (Figure1). Western blot analysis showed a 60-kDa band recombinant PLPro protein in transfected cells, but not Int. J. Mol. Sci. 2016, 17, 678 3 of 10 Int. J. Mol. Sci. 2016, 17, 678 3 of 10 cells, but not controls, as well as the similar expression level of TLR7 in both types of cells (Figure 1A), demonstrating that SARS-CoV PLPro was stably expressed in human promonocyte cellscontrols, that asdid well not as alter the similar the TLR7 expression expression. level of Qu TLR7antitative in both typesreal-time of cells PCR (Figure signified1A), demonstrating TLR7 agonist treatmentthat SARS-CoV stimulating PLPro higher was stablytranscriptional expressed levels in human of IFN- promonocyteα and IFN-β cells in vector that did controls not alter than the in PLPro-expressingTLR7 expression. cells Quantitative (Figure 1B,C). real-time Western PCR blotting signified analysis TLR7 agonist of both treatment of these stimulatingcells indicated higher that α β IRF3transcriptional phosphorylation levels of in IFN- vectorand control IFN- cellsin vector reached controls a maximum than in PLPro-expressing level at 30 min, but cells disappeared (Figure1B,C). at 60Western min after blotting IMQ analysis treatment. of both Importantly, of these cells indicatedPLPro inhibited that IRF3 IMQ-induced phosphorylation activation in vector of control IRF3 phosphorylationcells reached a maximum since no significant level at 30 min,change but in disappeared IRF3 phosphorylation at 60 min after was IMQ detectable treatment. at Importantly,30 min after PLPro inhibited IMQ-induced activation of IRF3 phosphorylation since no significant change in IRF3 IMQ treatment compared to non-treatment (Figure 1D). To assess the functional activity of IFN-α and phosphorylation was detectable at 30 min after IMQ treatment compared to non-treatment (Figure1D). IFN-β induced by TLR7 agonist, the interferon-stimulated response element (ISRE) reporter of the To assess the functional activity of IFN-α and IFN-β induced by TLR7 agonist, the interferon-stimulated dual-luciferase assay was further performed (Figure 2A). The TLR7 agonist dose-dependently response element (ISRE) reporter of the dual-luciferase assay was further performed (Figure2A). triggered the ISRE promoter activity in vector control cells, but not in PLPro-expressing cells The TLR7 agonist dose-dependently triggered the ISRE promoter activity in vector control cells, but (Figure 2A). Quantitative real-time PCR signified that the TLR7 agonist dose-dependently not in PLPro-expressing cells (Figure2A). Quantitative real-time PCR signified that the TLR7 agonist upregulated the IFN-stimulated genes (ISGs), including PKR and IRF7, in vector controls, but not dose-dependently upregulated the IFN-stimulated genes (ISGs), including PKR and IRF7, in vector PLPro-expressing cells (Figure 2B,C). In addition, TLR7 agonist-induced activation of STAT1 was controls, but not PLPro-expressing cells (Figure2B,C). In addition, TLR7 agonist-induced activation of delayed and suppressed by SARS-CoV PLPro (Figure 2D). Results revealed that PLPro suppresses STAT1 was delayed and suppressed by SARS-CoV PLPro (Figure2D). Results revealed that PLPro TLR7 agonist-induced production of type I IFNs through inhibiting the IRF-3 activation in the TLR7 suppresses TLR7 agonist-induced production of type I IFNs through inhibiting the IRF-3 activation in signaling pathway. the TLR7 signaling pathway.

A. B. 2.5 0μg/ml IMQ * 5μg/ml IMQ 2 * 10μg/ml IMQ * 1.5 mRNA level

PLpro α * 1 TLR7 0.5 β-actin IFNRelative 0 Vector control cells PLpro-expressing cells C. 60 D. ** 0 μg/ml IMQ IMQ 50 5 μg/ml IMQ 0 30 60 min 10 μg/ml IMQ Vector PLPro Vector PLPro Vector PLPro 40 mRNA level

β 1 2 3 4 5 6 30 ** ** p-IRF3 20 IRF3

Relastive IFN- Relastive 10 ** β-actin 0 Vector control cells PLPro-expressing cells FigureFigure 1. 1. EffectEffect of of SARS-CoV SARS-CoV PLPro PLPro on on TLR7 TLR7 agonistagonist-inducedinduced production of type II IFNsIFNs viavia IRF3IRF3 signaling.signaling. The The expression expression level level of of PLPro PLPro and and TLR7 TLR7 in in the vector control andand PLPro-expressingPLPro-expressing cellscells waswas analyzed analyzed using WesternWestern blot blot assay assay (A ).(A Both). Both types types of transfected of transfected cells were cells treated were withtreated or withoutwith or withoutimiquimod imiquimod (IMQ) for(IMQ) 4 h, for and 4 h, then, and theirthen, mRNAtheir mRNA levels levels of IFN- of αIFN-andα IFN-and IFN-β wereβ were measured measured by byquantitative quantitative PCR. PCR. Relative Relative mRNA mRNA levels levels of of IFN- IFN-αα(B (B)) and and IFN- IFN-ββ( C(C)) were were normalized normalized by by GAPDHGAPDH mRNA,mRNA, presented presented as as a a relative relative ratio. ratio. To To determine determine IRF3 activation, the lysates werewere alsoalso analyzedanalyzed usingusing Western Western blot blot with with anti anti-phospho-IRF3-phospho-IRF3 antibodies antibodies ( D). * p-Value << 0.05;0.05; **** pp-value-value << 0.010.01 byby Student’sStudent’s t-test.t-test.

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A. B. 0 μg/ml IMQ 180 0μg/ml IMQ 3 ** 5 μg/ml IMQ 160 5μg/ml IMQ * 10 μg/ml IMQ 2.5 140 ** 10μg/ml IMQ * 120 2 100 * 1.5 * 80 ** 60 1 40 ** 0.5

20 mRNA PKR Relative level ISRE-driven luciferaseactivity 0 0 Vector control cells SARS PLpro-expressing cells Vector control cells PLpro-expressing cells C. 2.5 0μg/ml IMQ D. IMQ ** 5μg/ml IMQ 10μg/ml IMQ 0 30 60 min 2 * Vector PLPro Vector PLPro Vector PLPro 1 2 3 4 5 6 1.5 p-STAT1 (Tyr701) * 1 * STAT1

0.5 β-actin Relative IRF7 mRNA IRF7 Relative level

0 Vector control cells PLpro-expressing cells

Figure 2.2. Inhibitory effect of SARS-CoV PLPro on TLR7TLR7 agonist-induced activation of type I IFNIFN signaling.signaling. ISRE-driven ISRE-driven luciferase luciferase reporter reporter activity activity and and the the mRNA mRNA levels of PKR and IRF7 werewere determined 44 h post-IMQ treatment. ISRE-driven ﬁreﬂyfirefly luciferase activity was normalized by Renilla luciferaseluciferase activityactivity ((A). RelativeRelative mRNAmRNA levels of PKRPKR ((B)) andand IRF7IRF7 ((CC)) werewere normalizednormalized byby GAPDHGAPDH mRNA, presented as a relative ratio.ratio. In addition, the activated statusstatus of STAT1 waswas examinedexamined usingusing WesternWestern blotblot withwith anti-phospho-STAT1anti-phospho-STAT1 (Tyr701)(Tyr701) antibodiesantibodies ((DD).). **p p-Value-Value < < 0.05;0.05; **** pp-value-value << 0.010.01 byby Student’s tt-test.-test.

2.2. Inhibition of TLR7 Agonist-Induced Pro-InflammatoryPro-Inflammatory CytokinesCytokines byby SARS-CoVSARS-CoV PLProPLPro Besides type II IFNIFN production,production, we checkedchecked whetherwhether thethe TLR7TLR7 agonistagonist upregulatesupregulates pro-inflammatorypro-inflammatory cytokines. The The effect effect of of SARS-CoV PLPro on the activation of NF-κB and AP1 promoters,promoters, as wellwell asas thethe productionproduction ofof pro-inflammatorypro-inflammatory cytokinescytokines (IL-6,(IL-6, IL-8IL-8 andand TNF-TNF-α)) waswas furtherfurther characterized via via dual-lucif dual-luciferaseerase reporter reporter assay assay and and SYBR SYBR green green real-time real-time PCR PCR (Figures (Figures 3 and3 and4). Results4). Results indicated indicated that thatSARS-CoV SARS-CoV PLPro PLPro inhibits inhibits TLR7 TLR7 agonist-induced agonist-induced activation activation of NF- of NF-κB andκB andAP1 AP1promoters, promoters, which which was was associated associated with with reducing reducing the the stimulated stimulated upregulation upregulation of of IL-6, IL-6, IL-8 and TNF-TNF-αα mRNAmRNA (Figures3 3A,BA,Band and4 A–C).4A–C). To To explore explore the the inhibitory inhibitory effect effect of of PLPro PLPro on on NF- NF-κB and AP1 signals,signals, thethe levelslevels ofof NF-NF-κκBB p65, p65, p38 p38 MAPK MAPK and and c-Jun c-Jun phosphorylation phosphorylation were were subsequently subsequently detected detected in responsesin responses toTLR7 to TLR7 agonist agonist using using Western Western blotting blotting (Figures (Figures3C and 43CD, and respectively). 4D, respectively). Western blottingWestern demonstratedblotting demonstrated that PLPro that suppressed PLPro suppressed TLR7 agonist-induced TLR7 agonist-induced phosphorylation phosphorylation of NF-κB, p38of MAPKNF-κB, andp38 c-JunMAPK compared and c-Jun to compared those in the to vectorthose controlin the vector at 30 mincontrol post-treatment. at 30 min post-treatment. The results demonstrated The results thatdemonstrated SARS-CoV that PLPro SARS-CoV suppressed PL TLR7-inducedPro suppressed cytokines TLR7-induced through decreasingcytokines through the phospho-NF- decreasingκB p65,the phospho-NF- p38 MAPK andκB c-Jun,p65, p38 resulting MAPK in and suppressing c-Jun, resulting of the NF- inκ Bsuppressing and AP-1 signaling of the NF- pathway.κB and AP-1 signaling pathway.

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A. ** B. A. 70 ** 0μg/ml IMQ B. 4.5 70 0μg/ml IMQ 4.5 0 μg/ml IMQ 4 ** 5μg/ml IMQ ** 05 μ μg/mlg/ml IMQ IMQ 60 5μg/ml IMQ 4 60 ** 10μg/ml IMQ 3.5 510 μg/ml μg/ml IMQ IMQ 50 ** 10μg/ml IMQ 3.5 ** 10 μg/ml IMQ 50 3 ** ** 3 40 ** 2.5 40 ** 2.5 ** ** level mRNA 2 ** 30 level mRNA α ** 30 2 α 1.5 ** 20 1.5

20 TNF- 1

10 TNF- 1 10NFkB activity luciferase driven 0.5 NFkB activity luciferase driven 0.5 0 0 0 Vector control cells PLpro-expressing cells 0 Vector control cells PLpro-expressing cells Vector control cells PLpro-expressing cells Vector control cells PLpro-expressing cells IMQ C. IMQ C. 0 30 60 min 0 30 60 min Vector PLPro Vector PLPro Vector PLPro Vector PLPro Vector PLPro Vector PLPro 1 2 3 4 5 6 Phospho-NFκBp65 1 2 3 4 5 6 Phospho-NFκBp65 NFκBp65 NFκBp65 β-actin β-actin

Figure 3. Inhibition of IMQ-induced TNF-αα production via NF-κκB signaling by SARS-CoV PLPro. FigureFigure 3. 3.Inhibition Inhibition of of IMQ-induced IMQ-induced TNF-α productionproduction via via NF- NF-κB Bsignaling signaling by by SARS-CoV SARS-CoV PLPro. PLPro. Both types of cells were treated with(out) IMQ for 4 h, and then, NF-κB-driven luciferase reporter BothBoth types types of of cells cells were were treated treated with(out)with(out) IMQ for 4 h, and and then, then, NF- NF-κκB-drivenB-driven luciferase luciferase reporter reporter activity and the TNF-α mRNA level were determined using the dual luciferase reporter assay (A) and activityactivity and and the the TNF- TNF-ααmRNA mRNAlevel level werewere determined using using the the dual dual luciferase luciferase reporter reporter assay assay (A ()A and) and quantitative PCR (B), respectively. For determining NF-κB activation, the lysates were also analyzed quantitativequantitative PCR PCR (B (B),), respectively. respectively. ForFor determiningdetermining NF- NF-κκBB activation, activation, the the lysates lysates were were also also analyzed analyzed using Western blot with anti-phospho-NF-κB p65 antibodies (C). ** p-Value < 0.01 by Student’s t-test. usingusing Western Western blot blot with with anti-phospho-NF- anti-phospho-NF-κκBB p65 antibodies ( (CC).). ** ** pp-Value-Value < <0.01 0.01 by by Student’s Student’s t-test.t-test.

A. 60 B. 9 A. 60 ** 0ug/ml IMQ B. 9 0μg/ml IMQ ** 0ug/ml IMQ A 8 ** 0μg/ml IMQ 50 5ug/ml IMQ A 8 ** 5μg/ml IMQ 50 5ug/ml IMQ 5μg/ml IMQ ** 10 ug/ml IMQ 7 10μg/ml IMQ ** 10 ug/ml IMQ 7 ** 10μg/ml IMQ 40 6 ** 40 6 5 30 5 ** 30 4 ** ** 4 20 ** 3 20 ** 3 10 ** 2 ** 10 2 ** 1 1 AP-1 driven luciferase activity luciferase driven AP-1 0 level mRNA IL-6 Relative 0 AP-1 driven luciferase activity luciferase driven AP-1 0 level mRNA IL-6 Relative Vector control cells PLpro-expressing cells 0 Vector control cells PLpro-expressing cells Vector control cells PLpro-expressing cells Vector control cells PLpro-expressing cells C. 5 D. C. 5 D. IMQ 0μg/ml IMQ IMQ 4.5 0μg/ml IMQ 4.5 * 5μg/ml IMQ 0 30 60 min 4 * 5μg/ml IMQ 0 30 60 min 4 10μg/ml IMQ Vect PLP Vect PLP Vect PLP 3.5 * 10μg/ml IMQ Vect PLP Vect PLP Vect PLP 3.5 * 1 2 3 4 5 6 3 1 2 3 4 5 6 3 p-p38 MAPK 2.5 p-p38 MAPK 2.5 2 P38 MAPK 2 ** P38 MAPK 1.5 ** p-c-Jun 1.5 ** 1 ** p-c-Jun Relative IL-8mRNA levels 1 c-Jun Relative IL-8mRNA levels 0.5 c-Jun 0.5 0 β-actin 0 Vector control cells PLpro-expressing cells β-actin Vector control cells PLpro-expressing cells

Figure 4. Detection of IMQ-induced AP-1-mediated production of IL-6 and IL-8 in the vector control FigureFigure 4. 4. Detection of of IMQ-induced IMQ-induced AP-1-mediated AP-1-mediated producti productionon of IL-6 of and IL-6 IL-8 and in the IL-8 vector in the control vector and PLPro-expressing cells. AP-1-driven firefly luciferase activity was normalized by Renilla controland PLPro-expressing and PLPro-expressing cells. cells.AP-1-driven AP-1-driven firefly ﬁreﬂy luciferase luciferase activity activity was wasnormalized normalized by byRenilla Renilla luciferase activity (A). Relative mRNA levels of IL-6 (B) and IL-8 (C) were normalized by GAPDH luciferaseluciferase activity activity ( A(A).). Relative Relative mRNAmRNA levelslevels of of IL-6 IL-6 ( (BB)) and and IL-8 IL-8 (C (C) )were were normalized normalized by by GAPDH GAPDH mRNA, presented as a relative ratio. In addition, the activated status of p38 MAPK and AP-1 was mRNA, presented as a relative ratio. In addition, the activated status of p38 MAPK and AP-1 was mRNA,examined presented using Western as a relative blot with ratio. anti-phospho In addition,-p38 the MAPK activated and statusanti-phospho-c-Jun of p38 MAPK antibodies and AP-1 (D was). examined using Western blot with anti-phospho-p38 MAPK and anti-phospho-c-Jun antibodies (D). examined* p-Value using < 0.05; Western ** p-value blot < 0.01 with by anti-phospho-p38 Student’s t-test. MAPK and anti-phospho-c-Jun antibodies (D). * p*-Value p-Value < 0.05;< 0.05; ** **p -valuep-value < < 0.01 0.01 byby Student’sStudent’s t-test.

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2.3.2.3. Ubiquitin Ubiquitin Removal Removal of of TRAF3 TRAF3 and and TRAF6 TRAF6 Responsible Responsible for for the the TLR7 TLR7 Antagonism Antagonism of of SARS-CoV SARS-CoV PLPro PLPro ToTo examine the the inhibitory mechanism mechanism of of TLR7 TLR7 antagonism antagonism by by SARS-CoV SARS-CoV PLPro, PLPro, differential differential profilesproﬁles of of ubiquitin-conjugated ubiquitin-conjugated proteins proteins in in the the vector vector control control and and PLPro-expressing PLPro-expressing cells cells in in the the absenceabsence or or presence presence of of IMQ IMQ were were analyzed using immune-precipitation (Figure (Figure 55A,B).A,B). BecauseBecause Lys63-linkedLys63-linked auto-ubiquitinationauto-ubiquitination of of TRAF TRAF is requiredis required for its for E3 ubiquitinits E3 ubiquitin ligase activity ligase and activity SARS-CoV and SARS-CoVPLPro proceeds PLPro the proceeds de-ubiquitination the de-ubiquitination enzymatic activity,enzymatic the activity, ubiquitination the ubiquitination status of TRAF3 status and of TRAF3TRAF6 and was TRAF6 subsequently was subsequently analyzed for analyzed whether for they whet wereher they involved were ininvolved the TLR7 in the signaling TLR7 signaling pathway pathwayinduced byinduced IMQ (Figure by IMQ5). (Figure The immunoprecipitation 5). The immunoprecipitation assay indicated assay that indicated PLPro that reduced PLPro Lys48- reduced and Lys48-Lys63-linked and Lys63-linked poly-ubiquitination poly-ubiquitination of TRAF3 and of TRAF3 TRAF6 and induced TRAF6 by IMQinduced (Figure by IMQ5A,B, (Figure Lane 2 5A,B,vs. 4). LaneSubsequently, 2 vs. 4). Subsequently, Western blotting Western analysis blotti indicatedng analysis that IMQ-inducedindicated that activation IMQ-induced of TBK1 activation was suppressed of TBK1 wasin PLPro-expressing suppressed in PLPro-expressing cells (Figure5C, Lanecells (Figure 3 vs. Lane 5C, 4;Lane Figure 3 vs.5D). Lane Therefore, 4; Figure the 5D). results Therefore, indicated the resultsthat PLPro indicated reduced that Lys63-linked PLPro reduced poly-ubiquitination Lys63-linked poly-ubiquitination of TRAF3 and TRAF6, of TRAF3 which and correlated TRAF6, with which the correlatedactivation with of TBK1, the activation p38 MAPK, of NF-TBK1,κB p38 and MAPK, IRF3 in NF- the TLR7κB and signaling IRF3 in the pathway. TLR7 signaling pathway.

A. Vector PLPro B. Vector PLPro IMQ (10μg/ml) - + - + IMQ (10μg/ml) - + - + IP:TRAF3 IP:TRAF6 IB:Ubi-Lys48 IB:Ubi-Lys48

IP:TRAF3 IP:TRAF6 IB:Ubi-Lys63 IB:Ubi-Lys63

IP:TRAF3 IP:TRAF6 IB:TRAF3 IB:TRAF6 C. 1.6 IMQ D. 1.4 1.2 0 30 60 min 1 Vector PLPro Vector PLPro Vector PLPro 0.8 0.6 p-TBK1

p-TBK1/TBK1 0.4 0.2 TBK1 0 Vector PLPro Vector PLPro Vector PLPro β-actin 0 30 60 min IMQ

FigureFigure 5. 5. DetectingDetecting the the Lys48- Lys48- and and Lys63-linked Lys63-linked ubiqui ubiquitinationtination of of TRAF3 TRAF3 and and TRAF6 TRAF6 measured measured by by thethe immunoprecipitation immunoprecipitation assay.assay. VectorVector control control and an PLPro-expressingd PLPro-expressing cells cells were were treated treated with(out) with(out) IMQ IMQfor 1 for day; 1 day; cell cell lysates lysates were were immunoprecipitated immunoprecipitated with with anti-TRAF3 anti-TRAF3 ( A(A)) or or anti-TRAF6anti-TRAF6 ( B) followed followed byby Western Western blotting blotting probed probed with with either either anti-L anti-Lys48ys48 ubiquitin ubiquitin or or anti-Lys anti-Lys6363 ubiquitin ubiquitin antibodies. antibodies. Phospho-TBK1Phospho-TBK1 levels levels were were detected detected by by Western Western blot blot ( (CC).). The The relative relative band band intensity intensity of of phospho-TBK1 phospho-TBK1 waswas normalized normalized by by TBK1, TBK1, compared compared to to the the mock mock ve vectorctor control control cells, cells, and and quantified quantiﬁed using using ImageJ ImageJ basedbased on on triplicate triplicate replicates replicates of of each each experiment experiment ( (DD).).

3.3. Discussion Discussion ThisThis study study was was the the first ﬁrst report report in in which which SARS-C SARS-CoVoV PLPro PLPro showed showed antagonistic antagonistic activity activity against against TLR7TLR7 agonist-induced agonist-induced production production of of IFN- IFN-αα,, IFN- IFN-ββ, ,TNF- TNF-αα, ,IL-6 IL-6 and and IL-8 IL-8 vi viaa reducing reducing TLR7 TLR7 agonist-inducedagonist-induced phosphorylation phosphorylation of of transcription transcription factors factors IRF3, IRF3, NF- NF-κκBB and and c-Jun c-Jun (Figures (Figures 1,1, 33 andand4 4).). TLR7TLR7 agonist-induced agonist-induced IFN- IFN-ββ stimulatedstimulated the the transcriptional transcriptional activity activity of of STAT1 STAT1 to to upregulate upregulate the the expression of ISGs, such as PKR and IRF7, in vector control cells (Figure 2). However, there was no significant response of STAT1-mediated signaling due to the inhibitory effect of PLPro on the production of TLR7 agonist-induced IFN-β (Figure 2). The results were constant with previous

Int. J. Mol. Sci. 2016, 17, 678 7 of 10 expression of ISGs, such as PKR and IRF7, in vector control cells (Figure2). However, there was no significant response of STAT1-mediated signaling due to the inhibitory effect of PLPro on the production of TLR7 agonist-induced IFN-β (Figure2). The results were constant with previous reports in that SARS-CoV PLPro inhibited TLR3- and RIG-I-mediated production of type I interferons and proinflammatory cytokines via inactivating the transcription factors IRF3 and NF-κB[15,23]. This study proposed TRAF3 and TRAF6 as the crucial regulators of PLPro antagonism via modification of the profiles of ubiquitin-conjugate proteins in the vector control and PLPro-expressing cells; thus, the ubiquitin linkage status of TRAF3 and TRAF6 was analyzed post-treatment with(out) IMQ using immunoprecipitation (Figure5). Immunoprecipitation assays indicated that PLPro decreased the Lys63-linked polyubiquitin chains of TRAF3 and TRAF6, causing the decrease in the E3 ubiquitin ligase activity of TRAF3 and TRAF6, which correlated with lower levels of TBK-1 phosphorylation in PLPro-expressing cells than vector control cells in response to IMQ. PLPro has been reported to upregulate the ubiquitin-conjugating enzyme E2-25k and proteasome subunit alpha type 5, which resulted in the increase of ERK1 poly-ubiquitination in PLPro-expressing cells as the type I IFN antagonistic mechanism of SARS PLPro via the degradation of ERK1 [24,25]. The high ubiquitination level of TRAF3 and TRAF6 in PLPro-expressing cells (Figure5A,B) might be responsible for the activation of the ubiquitin proteasome pathway, as demonstrated in our prior reports [24,25]. The finding was also similar to previous reports in that the inhibitory effect of SARS-CoV PLPro on the inactivation of the transcription factors IRF3 and NF-κB has been demonstrated to correlate with PLPro-reduced ubiquitination of RIG-I, STING TRAF3, TBK1 and IRF3 in the TLR3 and RIG-I pathways [16,17]. These previous reports proposed an antagonistic mechanism of SARS-CoV PLPro, including the inhibition of STING-MAVS-TBK1/IKKε and STING-TRAF3-TBK1 signaling in the TLR3 and RIG-I pathways. Since TBK1 has been suggested to activate protein phosphatases [26], no detectable level of IRF3 phosphorylation, as well as the decrease of NF-κB p65, p38 MAPK and c-Jun phosphorylation in the vector control and PLPro-expressing cells at 60 min post-IMQ treatment (Figures1D and3C) could be due to a the negative feedback of the TBK1-IRF3/NF- κB pathways. Therefore, we propose that the inhibition of TRAF3/6-TBK1-IRF3/NF-κB/AP1 signals by PLPro is responsible for the antagonistic mechanism of SARS-CoV PLPro against TLR7-medaited production of type I IFNs and pro-inflammatory cytokines.

4. Materials and Methods

4.1. Cell Line and Western Blot Assay SARS-CoV PLPro-expressing and empty vector control promonocyte cell lines were established, as described in our previous reports [24,25]. Both stably-transfected cell lines grew in RPMI-1640 medium containing 10% FBS and 800 µg/mL of Geneticin® Selective Antibiotic (G418 Sulfate). The expression level of PLPro and TLR7 in both stable cell lines was analyzed using Western Blotting with anti-PLPro mouse sera and anti-TLR7 mAb. To monitor the activation of the IMQ-induced TLR7 signaling pathway, PLPro-expressing and vector control cells were treated with 10 µM IMQ (InvivoGen, San Diego, CA, USA) and harvested 0, 30 and 60 min post-treatment. Lysate from IMQ-treated cells was used for the Western blotting assay; the resulting blots were probed with speciﬁc primary antibodies against IRF3, phosphor-IRF3, STAT1, phospho-STAT1(Tyr701), NF-κB p65, phospho-NF-κB p65, p38 MAPK, phospho-p38 MAPK, c-Jun, phospho-c-Jun, TBK1, phospho-TBK1, TRAF3, TRAF6 and anti-β-actin mAb (Abcam, Cambridge, UK). Immune complexes were detected with horseradish peroxidase-conjugated secondary antibodies, followed by enhanced chemiluminescence detection (Amersham Pharmacia Biotech, Piscataway, NJ, USA).

4.2. Signaling Pathway Assays with the Dual Luciferase Reporter System Cis-reporter plasmids pISRE-Luc, pAP-1-Luc and pNF-κB-Luc were used to examine the firefly luciferase activity driven from each cis-acting transcriptional element. SARS-CoV PLPro-expressing Int. J. Mol. Sci. 2016, 17, 678 8 of 10 and vector control cells were transfected with the indicated cis-reporter plasmid, plus internal control reporter pRluc-C1 (BioSignal Packard, Montréal, QC, Canada) using GenePorter reagent. After 4 h of incubation with or without IMQ, the activity of experimental firefly luciferase and control Renilla luciferase was measured by the dual Luciferase Reporter Assay System (Promega, Madison, WI, USA) and the Clarity™ Luminescence Microplate Reader (BioTek Instruments, Bad Friedrichshall, Germany) [27].

4.3. Quantitative Expression Analysis of Type I IFNs and Pro-Inflammatory Cytokines Using Real-Time PCR PLPro-expressing or empty vector cells were incubated for 4 h in the presence or absence of IMQ and harvested for isolation of total RNAs by a PureLink Micro-to-Midi Total RNA Purification System Kit (Invitrogen, Carlsbad, CA, USA), following quantification of gene expression by a two-step real-time PCR using SYBR Green I. First, cDNA was synthesized from 1 µg total RNA, using oligonucleotide d(T) primer and a SuperScript III reverse transcriptase kit (Invitrogen). Each real-time PCR reaction contained 5 µL of a cDNA mixture, 1 µL of primer pair (200 nM) and 12.5 µL Smart Quant Green Master Mix with Dutp and ROX (Protech, Vancouver, WA, USA). Primer pairs included (1) 51-CAACCAGCGGTTGACTTTTT-31 and 51-ATCCAGGAAGGCAAACTGAA-31 for PKR; (2) 51-GTGAGGAAATACTTCCAAAGAATCAC-31 and 51-TCTCATGATTTCTGCTCTGACAA-31 for IFN-α; (3) 51-AACTGCAACCTTTCGAAGCC-31 and 51-TGTCGCCTACTACCTGTTGTGC-31 for IFN-β; (4) 51-CGCGGCACTAACGACAGGCGAG-31 and 51-GCTGCCGTGCCCGGAATTCCAC-31 for IRF7; (5) 51-CTTCTCCTTCCTGATCGTGG-31 and 51-GCTGGTTATCTCTCAGCTCCA-31 for TNF-α; (6) 51-GATGGATGCTTCCAATCTGGAT-31 and 51-AGTTCTCCATAGAGAACAACATA-31 for IL-6; (7) 51-CGATGTCAGTGCATAAAGACA-31 and 51-TGAATTCTCAGCCCTCTTCAAAAA-31 for IL-8; and (8) 51-AGCCACATCGCTCAGACAC-31 and 51-GCCCAATACGACCAAATCC-31 for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Real-time PCR was performed as described in our previous reports [23,24]. The amplification and detection of specific products were conducted in an ABI Prism 7900 sequence detection system (PE Applied Biosystems, Carlsbad, CA, USA). Relative changes in the mRNA level of indicated genes were normalized relative to GAPDH mRNA.

4.4. Immunoprecipitation Assays To detect ubiquitination of TRAF3 and TRAF6, lysates from PLPro-expressing or vector cells with or without IMQ were collected, incubated with speciﬁc monoclonal antibodies anti-TRAF3 or anti-TRAF6 for 4 h at 4 ˝C and then followed by the addition of protein A-Sepharose beads and an additional 2 h of incubation. After centrifugation, pellets were washed four times with NET buffer (150 mM NaCl, 0.1 mM EDTA, 30 mM Tris-HCl, pH 7.4); immunoprecipitated proteins were dissolved in 2ˆ SDS-PAGE sample buffers without 2-mercaptoethanol, boiled for 10 min, separated by SDS-PAGE and then transferred to nitrocellulose. Resulting blots were blocked with 5% skim milk, reacted with speciﬁc monoclonal antibodies anti-TRAF3, anti-TRAF6, anti-Lys48- or anti-Lys63-linked ubiquitin and then followed by enhanced chemiluminescence detection.

4.5. Statistical Analysis Data were from three independent experiments; error bars represent the standard error of the mean. Chi-square and Student’s t-test were used to analyze all data, with statistical signiﬁcance between types of cells noted at p < 0.05.

5. Conclusions In summary, SARS-CoV PLPro inhibits TLR7-mediated signaling, leading to reducing of the cytokine production during antiviral responses. PLPro diminishes Lys63-linked ubiquitination of TRAF3 and TRAF6 and then inactivates their downstream molecules, such as kinases (TBK1 and p38 MAPK) and transcription factors (IRF3, NF-κB and AP-1). The results let us conclude that SARS-CoV Int. J. Mol. Sci. 2016, 17, 678 9 of 10

PLPro negatively regulates the TRAF3/6-TBK1-IRF3/NF-κB/AP1 signals in TLR7-mediated antiviral and inﬂammatory responses.

Acknowledgments: This work was supported by the Ministry of Science and Technology, Taiwan (MOST 101-2320-B-039-036-MY3, MOST 102-2628-B-039-044-MY3) and China Medical University (CMU101-ASIA-05, CMU102-ASIA-15 and CMU103-ASIA-07). Author Contributions: Cheng-Wen Lin conceived the project, contributed to experimental designs, performed experiments, interpreted the results, generated figures and wrote the manuscript; Shih-Wen Li, Ching-Ying Wang, Yu-Jen Jou, and Li-Hsin Hsiao performed the experiments, interpreted the results, and generated figures. Su-Hua Huang, Lei Wan, Ying-Ju Lin, and Szu-Hao Kung interpreted the results and wrote the manuscript. All authors approved the manuscript. Conflicts of Interest: The authors declare no conflict of interest.

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