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

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.


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

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 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-α and IFN-β 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-α and IFN-β 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. 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 cells that did not alter the TLR7 expression. Quantitative real-time PCR signified TLR7 agonist treatment stimulating higher transcriptional levels of IFN-α and IFN-β 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-α and IFN-β 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.

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-κB and AP1 promoters, as well as the production of pro-inflammatory cytokines (IL-6, IL-8 and TNF-α) 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-κB and AP1 promoters, which was associated with reducing the stimulated upregulation of IL-6, IL-8 and TNF-α mRNA ( Figures 3A,B and 4A-C). To explore the inhibitory effect of PLPro on NF-κB and AP1 signals, the levels of NF-κB 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-κB, 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-κB p65, p38 MAPK and c-Jun, resulting in suppressing of the NF-κB and AP-1 signaling pathway. . 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.

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-κB and AP1 promoters, as well as the production of pro-inflammatory cytokines (IL-6, IL-8 and TNF-α) 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-κB and AP1 promoters, which was associated with reducing the stimulated upregulation of IL-6, IL-8 and TNF-α mRNA ( Figures 3A,B and 4A-C). To explore the inhibitory effect of PLPro on NF-κB and AP1 signals, the levels of NF-κB 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-κB, 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-κB p65, p38 MAPK and c-Jun, resulting in suppressing of the NF-κB and AP-1 signaling pathway.  Both types of cells were treated with(out) IMQ for 4 h, and then, NF-κB-driven luciferase reporter activity and the TNF-α mRNA level were determined using the dual luciferase reporter assay (A) and quantitative PCR (B), respectively. For determining NF-κB activation, the lysates were also analyzed using Western blot with anti-phospho-NF-κB p65 antibodies (C). ** p-Value < 0.01 by Student's t-test.

Ubiquitin Removal of TRAF3 and TRAF6 Responsible for the TLR7 Antagonism of SARS-CoV PLPro
To examine the inhibitory mechanism of TLR7 antagonism by SARS-CoV PLPro, differential profiles of ubiquitin-conjugated proteins in the vector control and PLPro-expressing cells in the absence or presence of IMQ were analyzed using immune-precipitation ( Figure 5A,B). Because Lys63-linked auto-ubiquitination of TRAF is required for its E3 ubiquitin ligase activity and SARS-CoV PLPro proceeds the de-ubiquitination enzymatic activity, the ubiquitination status of TRAF3 and TRAF6 was subsequently analyzed for whether they were involved in the TLR7 signaling pathway induced by IMQ ( Figure 5). The immunoprecipitation assay indicated that PLPro reduced Lys48-and Lys63-linked poly-ubiquitination of TRAF3 and TRAF6 induced by IMQ ( Figure 5A,B, Lane 2 vs. 4). Subsequently, Western blotting analysis indicated that IMQ-induced activation of TBK1 was suppressed in PLPro-expressing cells ( Figure 5C, Lane 3 vs. Lane 4; Figure 5D). Therefore, the results indicated that PLPro reduced Lys63-linked poly-ubiquitination of TRAF3 and TRAF6, which correlated with the activation of TBK1, p38 MAPK, NF-κB and IRF3 in the TLR7 signaling pathway.

Ubiquitin Removal of TRAF3 and TRAF6 Responsible for the TLR7 Antagonism of SARS-CoV PLPro
To examine the inhibitory mechanism of TLR7 antagonism by SARS-CoV PLPro, differential profiles of ubiquitin-conjugated proteins in the vector control and PLPro-expressing cells in the absence or presence of IMQ were analyzed using immune-precipitation ( Figure 5A,B). Because Lys63-linked auto-ubiquitination of TRAF is required for its E3 ubiquitin ligase activity and SARS-CoV PLPro proceeds the de-ubiquitination enzymatic activity, the ubiquitination status of TRAF3 and TRAF6 was subsequently analyzed for whether they were involved in the TLR7 signaling pathway induced by IMQ ( Figure 5). The immunoprecipitation assay indicated that PLPro reduced Lys48-and Lys63-linked poly-ubiquitination of TRAF3 and TRAF6 induced by IMQ ( Figure 5A,B, Lane 2 vs. 4). Subsequently, Western blotting analysis indicated that IMQ-induced activation of TBK1 was suppressed in PLPro-expressing cells ( Figure 5C, Lane 3 vs. Lane 4; Figure 5D). Therefore, the results indicated that PLPro reduced Lys63-linked poly-ubiquitination of TRAF3 and TRAF6, which correlated with the activation of TBK1, p38 MAPK, NF-κB and IRF3 in the TLR7 signaling pathway.

Discussion
This study was the first report in which SARS-CoV PLPro showed antagonistic activity against TLR7 agonist-induced production of IFN-α, IFN-β, TNF-α, IL-6 and IL-8 via reducing TLR7 agonist-induced phosphorylation of transcription factors IRF3, NF-κB and c-Jun (Figures 1, 3 and 4). TLR7 agonist-induced IFN-β stimulated the transcriptional activity of STAT1 to upregulate 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 Figure 5. Detecting the Lys48-and Lys63-linked ubiquitination of TRAF3 and TRAF6 measured by the immunoprecipitation assay. Vector control and PLPro-expressing cells were treated with(out) IMQ for 1 day; cell lysates were immunoprecipitated with anti-TRAF3 (A) or anti-TRAF6 (B) followed by Western blotting probed with either anti-Lys48 ubiquitin or anti-Lys63 ubiquitin antibodies. Phospho-TBK1 levels were detected by Western blot (C). The relative band intensity of phospho-TBK1 was normalized by TBK1, compared to the mock vector control cells, and quantified using ImageJ based on triplicate replicates of each experiment (D).

Discussion
This study was the first report in which SARS-CoV PLPro showed antagonistic activity against TLR7 agonist-induced production of IFN-α, IFN-β, TNF-α, IL-6 and IL-8 via reducing TLR7 agonist-induced phosphorylation of transcription factors IRF3, NF-κB and c-Jun (Figures 1, 3 and 4). TLR7 agonist-induced IFN-β stimulated the transcriptional activity of STAT1 to upregulate 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 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 ( Figure 5). 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 ( Figure 5A,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 ( Figures 1D and 3C) 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.

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 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].

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 2ˆSDS-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.

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.