School of Biomedical Sciences, The University of Hong Kong, PokFuLam, Hong Kong.
Department of Molecular and Cell Biology, Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas (CNB-CSIC), Madrid, Spain.
FASEB J. 2019 Aug;33(8):8865-8877. doi: 10.1096/fj.201802418R. Epub 2019 Apr 29.
Severe acute respiratory syndrome coronavirus (SARS-CoV) is capable of inducing a storm of proinflammatory cytokines. In this study, we show that the SARS-CoV open reading frame 3a (ORF3a) accessory protein activates the NLRP3 inflammasome by promoting TNF receptor-associated factor 3 (TRAF3)-mediated ubiquitination of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). SARS-CoV and its ORF3a protein were found to be potent activators of pro-IL-1β gene transcription and protein maturation, the 2 signals required for activation of the NLRP3 inflammasome. ORF3a induced pro-IL-1β transcription through activation of NF-κB, which was mediated by TRAF3-dependent ubiquitination and processing of p105. ORF3a-induced elevation of IL-1β secretion was independent of its ion channel activity or absent in melanoma 2 but required NLRP3, ASC, and TRAF3. ORF3a interacted with TRAF3 and ASC, colocalized with them in discrete punctate structures in the cytoplasm, and facilitated ASC speck formation. TRAF3-dependent K63-linked ubiquitination of ASC was more pronounced in SARS-CoV-infected cells or when ORF3a was expressed. Taken together, our findings reveal a new mechanism by which SARS-CoV ORF3a protein activates NF-κB and the NLRP3 inflammasome by promoting TRAF3-dependent ubiquitination of p105 and ASC.-Siu, K.-L., Yuen, K.-S., Castaño-Rodriguez, C., Ye, Z.-W., Yeung, M.-L., Fung, S.-Y., Yuan, S., Chan, C.-P., Yuen, K.-Y., Enjuanes, L., Jin, D.-Y. Severe acute respiratory syndrome coronavirus ORF3a protein activates the NLRP3 inflammasome by promoting TRAF3-dependent ubiquitination of ASC.
严重急性呼吸系统综合征冠状病毒（SARS-CoV）能够引发炎症细胞因子风暴。在本研究中，我们发现 SARS-CoV 开放阅读框 3a（ORF3a）辅助蛋白通过促进肿瘤坏死因子受体相关因子 3（TRAF3）介导的凋亡相关斑点样蛋白（ASC）含半胱氨酸的天冬氨酸蛋白水解酶募集域（caspase recruitment domain，CARD）的泛素化，激活 NLRP3 炎性小体。SARS-CoV 和其 ORF3a 蛋白被发现是促白细胞介素-1β（IL-1β）基因转录和蛋白成熟的有效激活剂，这是激活 NLRP3 炎性小体所必需的 2 个信号。ORF3a 通过 TRAF3 依赖性泛素化和 p105 的加工，激活 NF-κB，从而诱导前白细胞介素-1β（pro-IL-1β）转录。ORF3a 诱导的 IL-1β 分泌的增加不依赖于其离子通道活性或在黑色素瘤 2 中缺失，但需要 NLRP3、ASC 和 TRAF3。ORF3a 与 TRAF3 和 ASC 相互作用，在细胞质中离散的点状结构中与它们共定位，并促进 ASC 斑点的形成。在 SARS-CoV 感染的细胞中或表达 ORF3a 时，TRAF3 依赖性 K63 连接的 ASC 泛素化更为明显。综上所述，我们的研究结果揭示了 SARS-CoV ORF3a 蛋白通过促进 TRAF3 依赖性 p105 和 ASC 的泛素化，激活 NF-κB 和 NLRP3 炎性小体的新机制。
