Li Shih-Wen, Wang Ching-Ying, Jou Yu-Jen, Huang Su-Hua, Hsiao Li-Hsin, Wan Lei, Lin Ying-Ju, Kung Szu-Hao, Lin Cheng-Wen
Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung 404, Taiwan.
Department of Biotechnology, College of Health Science, Asia University, Wufeng, Taichung 413, Taiwan.
Int J Mol Sci. 2016 May 5;17(5):678. doi: 10.3390/ijms17050678.
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.
据报道，严重急性呼吸综合征冠状病毒（SARS-CoV）木瓜样蛋白酶（PLPro）可抑制Toll样受体3（TLR3）和视黄酸诱导基因1（RIG-I）途径中I型干扰素（IFN）和促炎细胞因子的产生。本研究调查了SARS-CoV PLPro对TLR7介导的细胞因子产生的抑制作用及其拮抗机制。TLR7激动剂（咪喹莫特（IMQ））以浓度依赖性方式诱导人原单核细胞中ISRE、NF-κB和AP-1荧光素酶报告基因的激活，以及IFN-α、IFN-β、TNF-α、IL-6和IL-8的产生。然而，SARS-CoV PLPro通过抑制转录因子IRF-3、NF-κB和AP-1的激活，显著抑制IMQ诱导的细胞因子产生。用抗Lys48和抗Lys63泛素抗体进行的蛋白质印迹分析表明，SARS-CoV PLPro去除了未处理和处理细胞中TRAF3和TRAF6的Lys63连接的泛素链，但未去除Lys48连接的泛素链。TRAF3和TRAF6激活状态的降低与PLPro对IMQ反应中TBK1的失活相关。结果表明，SARS-CoV PLPro对TLR7介导的先天免疫的拮抗作用与TRAF3/6-TBK1-IRF3/NF-κB/AP1信号的负调控有关。
