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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 violet7053 S W L spirit1126 C Y W alvajou Y J J bonny6789 L H H 2 Department of Biotechnology College of Health Science Asia University Wufeng Taichung 413 Taiwan shhuang asia edu tw 3 Department of Medical Genetics and Medical Research China Medical University Hospital Taichung 404 Taiwan lei joseph L W yjlin kath Y J L 4 Department of Biotechnology and Laboratory Science in Medicine National Yang Ming University Taipei 112 Taiwan szkung ym edu tw Correspondence cwlin mail cmu edu tw 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 B and AP 1 luciferase reporters as well as the production of IFN IFN TNF 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 B 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 B 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 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 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 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 I B kinases IKKs initiating AP 1 NF B 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 B signaling pathway by preventing I B 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 B signaling via de ubiquitination and inactivation of TNFR associated factor 2 TRAF2 and TRAF6 18 19 de ubiquitinating protease A20 inhibits NF B 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 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 B 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 B 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 Figure 1 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 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 cells that did not alter the TLR7 expression Quantitative real time PCR signified TLR7 agonist treatment stimulating higher transcriptional levels of IFN and IFN in vector controls than in PLPro expressing cells Figure 1B C Western blotting analysis of both of these cells indicated that IRF3 phosphorylation in vector control cells reached a maximum level at 30 min but disappeared at 60 min after IMQ treatment Importantly PLPro inhibited IMQ induced activation of IRF3 phosphorylation since no significant change in IRF3 phosphorylation was detectable at 30 min after IMQ treatment compared to non treatment Figure 1D To assess the functional activity of IFN and IFN induced by TLR7 agonist the interferon stimulated response element ISRE reporter of the dual luciferase assay was further performed Figure 2A The TLR7 agonist dose dependently triggered the ISRE promoter activity in vector control cells but not in PLPro expressing cells Figure 2A Quantitative real time PCR signified that the TLR7 agonist dose dependently upregulated the IFN stimulated genes ISGs including PKR and IRF7 in vector controls but not PLPro expressing cells Figure 2B C In addition TLR7 agonist induced activation of STAT1 was delayed and suppressed by SARS CoV PLPro Figure 2D Results revealed that PLPro suppresses TLR7 agonist induced production of type I IFNs through inhibiting the IRF 3 activation in the TLR7 signaling pathway Int J Mol Sci 2016 17 678 3 of 10 cells but not control s 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 cells that did not alter the TLR7 expression Quantitative real time PCR signified TLR7 agonist treatment stimulating higher transcriptional levels of IFN and IFN in vector controls than in PLPro expressing cells Figure 1B C Western blotting analysis of both of these cells indicated that IRF3 phosphorylation in vector control cells reached a maximum level at 30 min but disappeared at 60 min after IMQ trea tment Importantly PLPro inhibited IMQ induced activation of IRF3 phosphorylation since no significant change in IRF3 phosphorylation was detectable at 30 min after IMQ treatment compared to non treatment Figure 1D To assess the functional activity of IFN and IFN induced by TLR7 agoni st the interferon stimulated response element ISRE reporter of the dual luciferase assay was further performed Figure 2A The TLR7 agonist dose dependently triggered the ISR E promoter activity in vector control cells but not in PLPro expressing cells Figure 2A Quantitative real time PCR signified that the TLR7 agonist dose dependently upregulated the IFN stimulated genes ISGs including PKR and IRF7 in vector controls but not PLPro expressing cells Figure 2B C In addition TLR7 agonist induced activation of STAT1 was delayed and suppressed by SARS CoV PLPro Figure 2D Results revealed that PLPro suppresses TLR7 agonist induced production of type I IFNs th rough inhibiting the IRF 3 activation in the TLR7 signaling pathway actin 0 30 60 min Vector PLPro Vector PLPro Vector PLPro IMQ 1 2 3 4 5 6 p IRF3 IRF3 D 0 0 5 1 1 5 2 2 5 Vector control cells PLpro expressing cells 0 g ml IMQ 5 g ml IMQ 10 g ml IMQ Relat i ve I F N mRNA level B 0 10 20 30 40 50 60 Vector control cells PLPro expressing cells Re l a s t i v e I F N mR N A l e v e l 0 g ml IMQ 5 g ml IMQ 10 g ml IMQ C A TLR7 actin PLpro Figure 1 Effect of SARS CoV PLPro on T LR7 agonist induced producti on of type I IFNs via IRF3 signaling The expression level of PLPro and TLR7 in the vector control and PLPro expressing cells was analyzed using Western blot assay A Both types of transfected cells were treat ed with or without imiquimod IMQ for 4 h and then their mRNA levels of IFN and IFN were measured by quantitative PCR Relative mRNA levels o f IFN B and IFN C were normalized by GAPDH mRNA presented as a relative ratio To determine IRF3 activation the lysates were a lso analyzed using Western blot with anti phospho IRF3 antibodies D p Value 0 05 p value 0 01 by Student s t test Figure 1 Effect of SARS CoV PLPro on TLR7 agonist induced production of type I IFNs via IRF3 signaling The expression level of PLPro and TLR7 in the vector control and PLPro expressing cells was analyzed using Western blot assay A Both types of transfected cells were treated with or without imiquimod IMQ for 4 h and then their mRNA levels of IFN and IFN were measured by quantitative PCR Relative mRNA levels of IFN B and IFN C were normalized by GAPDH mRNA presented as a relative ratio To determine IRF3 activation the lysates were also analyzed using Western blot with anti phospho IRF3 antibodies Value 0 05 p value 0 01 by Student s t test Int J Mol Sci 2016 17 678 4 of 10 Int J Mol Sci 2016 17 678 4 of 10 actin 0 30 60 min Vector PLPro Vector PLPro Vector PLPro IMQ 1 2 3 4 5 6 p STAT1 Tyr701 STAT1 D 0 20 40 60 80 100 120 140 160 180 Vector control cells SARS PLpro expressing cells I S R E dr i v e n l u ci f e r a se a c ti v i t y 0 g ml IMQ 5 g ml IMQ 10 g ml IMQ A 0 0 5 1 1 5 2 2 5 3 Vector control cells PLpro expressing cells 0 g ml IMQ 5 g ml IMQ 10 g ml IMQ Rela t i v e PKR m R NA lev e l B 0 0 5 1 1 5 2 2 5 Vector control cells PLpro expressing cells 0 g ml IMQ 5 g ml IMQ 10 g ml IMQ Rel a t i ve I R F7 m RNA l e vel C Figure 2 Inhibitory effect of SARS CoV PLPro on TLR7 agonist induced activation of type I IFN signaling ISRE driven luciferase reporter activity and the mRNA levels of PKR and IRF7 were determined 4 h post IMQ treatment ISRE driven firefly luciferase activity was normalized by Renilla luciferase activity A Relative mRNA levels of PKR B and IRF7 C were normalized by GAPDH mRNA presented as a relative ratio In addition the activated status of STAT1 was examined using Western blot with anti phospho STAT1 Tyr701 antibodies D p Value 0 05 p value 0 01 by Student s t test 2 2 Inhibition of TLR7 Agonist Induced Pro Inflammatory Cytokines by SARS CoV PLPro Besides type I IFN production we checked whether the TLR7 agonist upregulates pro inflammatory cytokines The effect of SARS CoV PLPro on the activation of NF B and AP1 promoters as well as the production of pro inflammatory cytokines IL 6 IL 8 and TNF was further characterized via dual luciferase reporter assay and SYBR green real time PCR Figures 3 and 4 Results indicated that SARS CoV PLPro inhibits TLR7 agonist induced activation of NF B and AP1 promoters which was associated with reducing the stimulated upregulation of IL 6 IL 8 and TNF mRNA Figures 3A B and 4A C To explore the inhibitory effect of PLPro on NF B and AP1 signals the levels of NF B p65 p38 MAPK and c Jun phosphorylation were subsequently detected in responses to TLR7 agonist using Western blotting Figures 3C and 4D respectively Western blotting demonstrated that PLPro suppressed TLR7 agonist induced phosphorylation of NF B p38 MAPK and c Jun compared to those in the vector control at 30 min post treatment The results demonstrated that SARS CoV PLPro suppressed TLR7 induced cytokines through decreasing the phospho NF B p65 p38 MAPK and c Jun resulting in suppressing of the NF B and AP 1 signaling pathway Figure 2 Inhibitory effect of SARS CoV PLPro on TLR7 agonist induced activation of type I IFN signaling ISRE driven luciferase reporter activity and the mRNA levels of PKR and IRF7 were determined 4 h post IMQ treatment ISRE driven firefly luciferase activity was normalized by Renilla luciferase activity A Relative mRNA levels of PKR B and IRF7 C were normalized by GAPDH mRNA presented as a relative ratio In addition the activated status of STAT1 was examined using Western blot with anti phospho STAT1 Tyr701 antibodies D p Value 0 05 p value 0 01 by Student s t test 2 2 Inhibition of TLR7 Agonist Induced Pro Inflammatory by SARS CoV PLPro Besides type I IFN production we checked whether the TLR7 agonist upregulates pro inflammatory cytokines The effect SARS CoV PLPro on the activation of NF B and AP1 promoters as well as the production of pro inflammatory cytokines IL 6 IL 8 and TNF was further characterized via dual luciferase reporter assay and SYBR green real time PCR Figures 3 and 4 Results indicated that SARS CoV PLPro inhibits TLR7 agonist induced activation of NF B and AP1 promoters which was associated with reducing the stimulated upregulation of IL 6 IL 8 and mRNA Figures 3A B and A C T explor the inhibitory effect of PLPr on NF B and AP1 signals the levels of NF B p65 p38 MAPK and c Jun phosphorylation were subsequently detected in responses to TLR7 agonist using Western blotting Figures 3C and 4D respectively Western blotting demonstrated that PLPro suppressed TLR7 agonist induced phosphorylation of NF B p38 MAPK and c Jun compared to those in the vector control at 30 min post treatment The esults demonstrated that SARS CoV PLPro suppressed TLR7 induced cytokines through decreasing the phospho NF B p65 p38 MAPK and c Jun resulting in suppressing of the NF B and AP 1 signaling pathway Int J Mol Sci 2016 17 678 5 of 10 Int J Mol Sci 2016 17 678 5 of 10 Figure 3 Inhibition of IMQ induced TNF production via NF B signaling by SARS CoV PLPro Both types of cells were treated with out IMQ for 4 h and then NF B driven luciferase reporter activity and the TNF mRNA level were determined using the dual luciferase reporter assay A and quantitative PCR B respectively For determining NF B activation the lysates were also analyzed using Western blot with anti phospho NF B p65 antibodies C p Value 0 01 by Student s t test 0 1 2 3 4 5 6 7 8 9 Vector control cells PLpro expressing cells R e la t i v e I L 6 m R N A le v e l A 0 g ml IMQ 5 g ml IMQ 10 g ml IMQ actin p c Jun p p38 MAPK P38 MAPK 0 30 60 min Vect PLP Vect PLP Vect PLP IMQ 1 2 3 4 5 6 B D 0 10 20 30 40 50 60 Vector control cells PLpro expressing cells A P 1 d r i v en l u ci f erase acti v i ty 0ug ml IMQ 5ug ml IMQ 10 ug ml IMQ A 0 0 5 1 1 5 2 2 5 3 3 5 4 4 5 5 Vector control cells PLpro expressing cells 0 g ml IMQ 5 g ml IMQ 10 g ml IMQ Re l a t i ve IL 8 m RNA l e ve l s C c Jun Figure 4 Detection of IMQ induced AP 1 mediated production of IL 6 and IL 8 in the vector control and PLPro expressing cells AP 1 driven firefly luciferase activity was normalized by Renilla luciferase activity A Relative mRNA levels of IL 6 B and IL 8 C were normalized by GAPDH mRNA presented as a relative ratio In addition the activated status of p38 MAPK and AP 1 was examined using Western blot with anti phospho p38 MAPK and anti phospho c Jun antibodies D p Value 0 05 p value 0 01 by Student s t test actin Phospho NF Bp65 0 30 60 min Vector PLPro Vector PLPro Vector PLPro IMQ 1 2 3 4 5 6 C 0 10 20 30 40 50 60 70 Vector control cells PLpro expressing cells 0 g ml IMQ 5 g ml IMQ 10 g ml IMQ NF k B dri v en lu ci f era se act i v i t y A 0 0 5 1 1 5 2 2 5 3 3 5 4 4 5 Vector control cells PLpro expressing cells TNF m RNA l e v e l 0 g ml IMQ 5 g ml IMQ 10 g ml IMQ B NF Bp65 Figure 3 Inhibition of IMQ induced TNF production via NF B signaling by SARS CoV PLPro Both types of cells were treated with out IMQ for 4 h and then NF B driven luciferase reporter activity and the TNF mRNA level were determined using the dual luciferase reporter assay A and quantitative PCR B respectively For determining NF B activation the lysates were also analyzed using Western blot with anti phospho NF B p65 antibodies C p Value 0 01 by Student s t test Int J Mol Sci 2016 17 678 5 of 10 Figure 3 Inhibition of IMQ induced TNF production via NF B signaling by SARS CoV PLPro Both types of cells were treated with out IMQ for 4 h and then NF B driven luciferase reporter activity and the TNF mRNA level were determined using the dual luciferase reporter assay A and quantitative PCR B respectively For determining NF B activation the lysates were also analyzed using Western blot with anti phospho NF B p65 antibodies p Value 0 01 by Student s t test 0 1 2 3 4 5 6 7 8 9 Vector control cells PLpro expressing cells R e la t i v e I L 6 m R N A le v e l A 0 g ml IMQ 5 g ml IMQ 10 g ml IMQ actin p c Jun p p38 MAPK P38 MAPK 0 30 60 min Vect PLP Vect PLP Vect PLP IMQ 1 2 3 4 5 6 B D 0 10 20 30 40 50 60 Vector control cells PLpro expressing cells A P 1 d r i v