新出现的严重急性呼吸综合征(SARS)冠状病毒-2(SARS-CoV-2)可导致干扰素(IFN)反应失调,从而导致疾病严重程度。SARS-CoV-2的木瓜蛋白酶样蛋白酶(SCoV2-PLpro)先前已被报道减弱IFN应答,但是潜在的机制还没有完全理解。在这项研究中,我们发现SCoV2-PLpro能有效抑制仙台病毒诱导的IFN产生和信号传导以及RIG-I样受体(RLR)信号通路成分,包括RIG-I,MAVS,TBK1、TRAF3、TRAF6和IRF3。SCoV2-PLpro表现出与SARS-CoVPLpro不同的特异性和效率,前者对RIG-I和TRAF3介导的IFN应答具有较大的抑制作用,但对MAVS介导的IFN应答具有较弱的抑制作用。此外,我们发现SCoV2-PLpro显著降低RIG-I的K63泛素化,MAVS,TBK1、TRAF3、TRAF6、IRF3和K48-IκBα的泛素化,这是已知的先天免疫信号转导的关键。SCoV2-PLpro的去泛素化(DUB)活性需要催化残基半胱氨酸111(C111)而不是UBL结构域。值得注意的是,通过利用DUB缺陷的C111突变体,我们证明SCoV2-PLpro通过去泛素化依赖性和非依赖性机制靶向RLR信号通路调节因子,RIG-I和TBK1的抑制活性与DUB功能相关,而对MAVS的拮抗作用,TRAF3、TRAF6和IRF3不依赖于DUB活性。总的来说,我们的结果表明,SCoV2-PLpro从SCoV1-PLpro进化出不同的IFN拮抗活性,它通过各种机制靶向多个关键的RLR信号通路成分,提供对SARS-CoV-2发病机制的见解,并为开发COVID-19的抗病毒治疗提供线索。
The newly emerged severe acute respiratory syndrome (SARS) coronavirus-2 (SARS-CoV-2) can result in dysregulated interferon (IFN) responses that contribute to disease severity. The papain-like protease of SARS-CoV-2 (SCoV2-PLpro) has been previously reported to attenuate IFN responses, but the underlying mechanism is not fully understood. In this study, we found that SCoV2-PLpro potently suppressed IFN production and signaling induced by Sendai virus as well as RIG-I-like receptor (RLR) signaling pathway components, including RIG-I, MAVS, TBK1, TRAF3, TRAF6, and IRF3. SCoV2-PLpro exhibited different specificity and efficiency than SARS-CoV PLpro, with the former exerting a greater inhibitory effect on the RIG-I- and TRAF3-mediated IFN response but a weaker effect on the MAVS-mediated IFN response. Furthermore, we showed that SCoV2-PLpro significantly reduced K63-ubiquitination of RIG-I, MAVS, TBK1, TRAF3, TRAF6, and IRF3 and K48-ubiquitination of IκBα, which are known critical for the innate immune signal transduction. The deubiquitinating (DUB) activity of SCoV2-PLpro required a catalytic residue cysteine 111 (C111) but not the UBL domain. Notably, by utilizing the DUB-defective C111 mutant, we demonstrated that SCoV2-PLpro targeted RLR signaling pathway regulators via deubiquitination-dependent and -independent mechanisms, with the inhibitory activities of RIG-I and TBK1 correlating with DUB function, whereas the antagonism effects on MAVS, TRAF3, TRAF6, and IRF3 independent on DUB activity. Overall, our results reveal that SCoV2-PLpro evolves differential IFN antagonism activity from SCoV1-PLpro and it targets multiple key RLR signaling pathway components via various mechanisms, providing insights into SARS-CoV-2 pathogenesis and clues for developing antiviral therapies for COVID-19.