Crystal structure of the Middle East respiratory syndrome coronavirus (MERS-CoV) papain-like protease bound to ubiquitin facilitates targeted disruption of deubiquitinating activity to demonstrate its role in innate immune suppression.
Crystal structure of the Middle East respiratory syndrome coronavirus (MERS-CoV) papain-like protease bound to ubiquitin facilitates targeted disruption of deubiquitinating activity to demonstrate its role in innate immune suppression.
复制标题
中东呼吸综合征冠状病毒（MERS-COV）类似蛋白酶的蛋白酶与泛素结合的晶体结构有助于靶向破坏去泛素化活性的破坏，以证明其在先天免疫抑制中的作用。
DOI：
10.1074/jbc.m114.609644
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发表时间：
2014-12-12
期刊：
影响因子：
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通讯作者：
Mark BL
中科院分区：
文献类型：
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作者：
Bailey-Elkin BA;Knaap RC;Johnson GG;Dalebout TJ;Ninaber DK;van Kasteren PB;Bredenbeek PJ;Snijder EJ;Kikkert M;Mark BL
背景：MERS冠状病毒类木瓜蛋白酶(PLPro)处理病毒多蛋白，具有去泛素化活性。结果：与泛素结合的MERS冠状病毒PLPro的晶体结构引导突变，在不影响多蛋白切割的情况下破坏PLPro的去泛素化活性。结论：MERS冠状病毒PLPro的去泛素化活性抑制了干扰素β的诱导表达。意义：我们选择性地禁用PLPro去泛素化活性的策略使我们能够研究其在感染中的特定功能。中东呼吸综合征冠状病毒(MERS-CoV)是2012年首次分离到的一种新出现的人类病原体。MERS冠状病毒的复制部分依赖于病毒编码的木瓜蛋白酶(PLPro)，该酶在三个位置裂解病毒复制酶多蛋白，释放非结构蛋白1(NSP1)、NSP2和NSP3。除了这种复制功能外，MERS-CoV PLPro最近被证明是一种去泛素酶(DUB)，并具有去ISGyl化活性，与先前报道的其他冠状病毒PLPro结构域一样，包括严重急性呼吸综合征冠状病毒的PLPro结构域。这些活性被认为是在感染期间抑制宿主的抗病毒反应。为了了解MERS冠状病毒PLPro识别和去结合泛素(Ub)的分子基础，我们测定了其与Ub的络合物的晶体结构。在这种结构的指导下，突变被引入PLPro，以特异性地破坏Ub结合，而不影响病毒多蛋白的切割，这是通过In Transnsp3↓4切割试验确定的。在制定了一种有选择地禁用PLPro DuB活性的策略后，我们能够专门检查这一活动对先天性免疫反应的影响。虽然野生型PLPRO结构域被发现抑制干扰素-β启动子的激活，但缺乏DUB活性的PLPRO变体不再能够做到这一点。这些发现直接表明PLPRO具有抑制干扰素-β启动子活性的DUB功能，而不是其本身的蛋白分解活性。将PLPro的复制活性与其在病毒多蛋白加工中的作用分离的能力现在为进一步剖析PLPro在MERS冠状病毒感染过程中作为病毒复制的作用提供了一种方法(S)。
Background: MERS-CoV papain-like protease (PLpro) processes viral polyproteins and has deubiquitinating activity. Results: A crystal structure of MERS-CoV PLpro bound to ubiquitin guided mutagenesis to disrupt PLpro deubiquitinating activity without affecting polyprotein cleavage. Conclusion: The deubiquitinating activity of MERS-CoV PLpro suppresses the induction of interferon-β expression. Significance: Our strategy to selectively disable PLpro deubiquitinating activity enables the study of its specific functions in infection. Middle East respiratory syndrome coronavirus (MERS-CoV) is a newly emerging human pathogen that was first isolated in 2012. MERS-CoV replication depends in part on a virus-encoded papain-like protease (PLpro) that cleaves the viral replicase polyproteins at three sites releasing non-structural protein 1 (nsp1), nsp2, and nsp3. In addition to this replicative function, MERS-CoV PLpro was recently shown to be a deubiquitinating enzyme (DUB) and to possess deISGylating activity, as previously reported for other coronaviral PLpro domains, including that of severe acute respiratory syndrome coronavirus. These activities have been suggested to suppress host antiviral responses during infection. To understand the molecular basis for ubiquitin (Ub) recognition and deconjugation by MERS-CoV PLpro, we determined its crystal structure in complex with Ub. Guided by this structure, mutations were introduced into PLpro to specifically disrupt Ub binding without affecting viral polyprotein cleavage, as determined using an in trans nsp3↓4 cleavage assay. Having developed a strategy to selectively disable PLpro DUB activity, we were able to specifically examine the effects of this activity on the innate immune response. Whereas the wild-type PLpro domain was found to suppress IFN-β promoter activation, PLpro variants specifically lacking DUB activity were no longer able to do so. These findings directly implicate the DUB function of PLpro, and not its proteolytic activity per se, in the inhibition of IFN-β promoter activity. The ability to decouple the DUB activity of PLpro from its role in viral polyprotein processing now provides an approach to further dissect the role(s) of PLpro as a viral DUB during MERS-CoV infection.