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

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

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-α and IFN-β were measured by quantitative PCR. Relative mRNA levels of IFN-α (B) and IFN-β (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 (D). * p-Value < 0.05; ** p-value < 0.01 by Student’s t-test.

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.

Figure 3

Inhibition of IMQ-induced TNF-α production via NF-κB signaling by SARS-CoV PLPro. 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.

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.

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).