【作者】 杨宇东；

【导师】 陈忠斌；

【作者基本信息】 中国人民解放军军事医学科学院 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 SARS冠状病毒基因组由29.7kb的正义、单链RNA组成,基因组的前2/3部分翻译两个大的复制酶多聚蛋白ppla和pplab。在木瓜样蛋白酶(Papain-like protease, PLpro)和3C样蛋白酶(Chymotrypsin-like protease,3CLpro)的作用下,将其切割成16个非结构蛋白(nsp1-16),16个非结构蛋白在内质网膜上组装成一个多功能的复制酶复合体。此复合体介导完成病毒基因组的复制和结构基因的转录翻译。不同于其他已知冠状病毒编码两个木瓜样蛋白(PLP1和PLP2), SARS冠状病毒只编码一个木瓜样结构域PLpro。SARS PLpro特异性识别多聚蛋白ppla(pp1ab)N端nsp1-2、nsp2-3和nsp3-4之间的LXGG序列,参与1a(1ab)复制酶蛋白N端的切割加工并释放成熟产物nsp1, nsp2和nsp3。SARS冠状病毒PLpro蛋白酶结构与功能研究是近几年冠状病毒分子生物学研究的热点之一SARS PLpro是由非结构蛋白nsp3编码,nsp3编码7个小的结构域,在PLpro的C端编码一跨膜域TM使PLpro定位在膜上。在PLpro与TM之间编码两个小的结构域NAB-G2M。PLpro由四个不同结构域组成,最开始的62个氨基酸形成一个独立的泛素样N末端结构域(Ubl)。其余三个组成一个伸展的右手结构包括掌心、拇指和指形结构域。本研究利用基因重组技术首先构建了带有TM跨膜域的PLpro-TM重组质粒,并构建了缺失泛素样结构域(Ubl)和NAB-G2M结构域的重组质粒。其次,利用PCR定点突变技术将PLpro中与蛋白酶催化活性相关的三个关键氨基酸残基Cys-His-Asp突变为Ala,构建出三种酶活性缺失突变体。经生物信息学预测发现,SARS PLpro蛋白酶的高级结构与细胞内去泛素化酶HAUSP高度相似,因此,推测PLpro同样具有去泛素活性。为了验证此推测,本研究以SARS冠状病毒PLpro蛋白酶为研究对象,将HA-Ub与PLpro基因共转染HEK293T细胞,发现PLpro能显著减少细胞内蛋白的泛素化修饰(包括K63、K48修饰泛素形式)作用,此实验证实了PLpro具有体内DUB活性,利用同样方法,检测到带有跨膜域TM的PLpro同样具有DUB活性。并且缺失泛素样结构域(Ubl)和NAB-G2M的PLpro也都具有很强的DUB活性,提示Ubl和NAB-G2M结构域与PLpro的DUB活性无关；而PLpro蛋白酶催化活性相关位点突变后对其DUB活性有一定影响,提示PLpro的蛋白酶活性位点同样为去泛素活性的重要位点。病毒感染宿主细胞后,病毒核酸可被细胞膜上受体(TLR3)及胞内受体(RIG-I)所识别,募集下游衔接蛋白(IPS-1、ERIS、TRAF3等),活化的衔接蛋白再将信号传递给下游激酶IKKβ/IKKα激活NF-κB或通过激酶IKKε/TBK1激活1IRF3、IRF7等核转录因子,核转录因子入核启动干扰素基因的转录、翻译,从而上调细胞中的IFNβ表达。表达的干扰素与其受体结合,激活下游干扰素信号通路,最后翻译出上百种抗病毒蛋白来抑制病毒复制。以SARS冠状病毒为研究对象,将PLpro与IFNβ-luc报告基因质粒共转细胞,转染24h后利用Sendai病毒激活IFNβ通路,或利用共转通路中调节蛋白(如RIG-IN、IPS-1、IKKε、IRF3等)激活IFNβ通路,裂解细胞检测荧光素酶报告基因活性,发现Sendai病毒或通路中调节蛋白都可明显激活IFNβ通路,而共转PLpro以后,荧光素酶活性明显降低,证明PLpro对IFNβ通路具有明显的负调节作用,是一种IFN拮抗分子。利用同样方法,检测到缺失泛素样结构域Ubl或NAB-G2M结构域的PLpro对干扰素通路仍具有很强的抑制作用；PLpro-TM的三种突变体C1651A、H1812A、D1826A抑制干扰素通路也很明显并具有剂量依赖性。我们前期研究发现,SARS PLpro可通过与转录因子IRF3相互作用并抑制其磷酸化和阻断其向胞核转运,从而抑制宿主干扰素表达。但最新研究证实,PLpro并不与IRF3直接相互作用,因此,SARS PLpro抑制宿主干扰素通路分子机制还不清楚。针对这一问题,本研究进一步对PLpro抑制干扰素通路的抑制机理方面进行探索。首先,利用Co-IP技术检测PLpro-TM与通路中蛋白的相互作用情况,将带有Flag标签的TRAF3、ERIS、TBK1、IKKε等通路中分子与带有V5标签的PLpro-TM共转HEK293T细胞,利用鼠抗Flag进行IP,兔抗V5进行检测,结果提示PLpro-TM可与TRAF3、ERIS、TBK1、IKKε相互作用。将两种带有不同的标签的TRAF3复合物中蛋白共转到HEK293T细胞中,在PLpro-TM存在与不存在的情况下,检测两蛋白的相互作用情况,以此方法检测到,PLpro-TM可以破坏TRAF3与TBK1、TRAF3与IKKε、ERIS与IRF3之间的相互作用,此结果提示,PLpro可通过阻断TRAF3复合体的形成而抑制IRF3的活化,从而抑制其二聚化入核上调干扰素的表达。除此之外,蛋白泛素化修饰是调节天然免疫应答的关键机制。泛素化修饰同磷酸化一样,是一种可逆的共价修饰,通过泛素化修饰可调节靶蛋白的稳定性、活性及细胞内定位等。泛素化修饰存在于天然免疫应答的各个时期。通路中许多分子如RIG-I、TRAF3、ERIS、IRF3等都可进行泛素化修饰,RIG-I的K63多聚泛素化修饰是活化下游IPS-1衔接蛋白的基础；TRAF3的K63多聚泛素修饰可介导激酶TBK1、IKKε活化,从而激活IRF3并上调干扰素的表达。那么,PLpro作为病毒编码的新型DUB对这些泛素化分子是否具有作用活性?本研究将通路中泛素化修饰分子RIG-I、TRAF3、ERIS、IRF3与泛素共转HEK 293T细胞,通过Co-IP实验可检测到几种蛋白的泛素化修饰,而共转PLpro-TM后,泛素化条带消失,提示PLpro-TM对上述分子都具有DUB活性。因此,本研究证明PLpro通过阻断TRAF3复合物的形成和对通路中关键调节蛋白的DUB活性而负调节抗病毒夫然免疫干扰素通路。综上所述,本研究通过一系列实验证实,SARS冠状病毒PLpro除了具有经典的蛋白酶活性之外还具有许多新功能。第一,SARS冠状病毒PLpro蛋白酶是一种新型病毒编码的DUB。第二,SARS PLpro蛋白酶对宿主抗病毒天然免疫反应具有负调节作用,是一种新型干扰素拮抗分子。第三,SARS PLpro蛋白酶通过阻断TRAF3复合物的形成和对干扰素通路中调节蛋白的DUB活性而抑制干扰素的表达。本研究揭示了SARS PLpro抑制抗病毒天然免疫干扰素通路的新机制,为阐明病毒致病机理和研发新型抗病毒药物提供重要的理论依据。更多还原

【Abstract】 The genome of severe acute respiratory syndrome coronavirus (SARS-CoV) is a positive-sense, single-stranded,29.7kb RNA. The first 2/3 of the genome of SARS-CoV is translated into two large replicase polyproteins called as ppla and pplab. Papain-like protease (PLpro) and 3C-like protease domains present within these polyproteins direct their processing into 16 nonstructural proteins (nsp1-16) that assemble to generate a multifunctional, membrane-associated replicase complex. This complex is responsible for the copy of virus genome and transcription and translation of structural genes. Unlike other coronaviruses that encode two different PLPs(PLP1 and PLP2), SARS-CoV encodes only one PLP domain called PLpro. SARS PLpro recognizes the consensus cleavage sequence LXGG between nsp1-2, nsp2-3, nsp3-4 of the amino terminus of pp1a(1ab) to release nspl, nsp2 and nsp3. Recently, the functions of PLpro of SARS coronavirus is becoming a research focus in coronavirus molecular Biology.SARS PLpro is encoded by nsp3. nsp3 has been annotated as a multidomain protein consisting of a minimum of seven domains. It encodes a transmembrane domain (TM) after PLpro, which locate PLpro on the ER membrane. And there are two small domains of NAB-G2M between PLpro and TM. The PLpro monomer consists of four distinct domains, three of which form an extended right-hand architecture with distinct palm, thumb, and finger domains; the first 62 aminos form an independent N-terminal domain, termed the Ubl domain. In this study, we first constructed the recombination plasmids of PLpro with tansmembrane and PLpro delete N-terminal Ubl domain or NAB-G2M domain. Secondly, the Site-directed mutagenesis was performed to change the catalytic sites (Cys-His-Asp) of PLpro to Ala.We found that the crystal structure of PLpro is significant matched with a cellular deubiquitinase (DUB) HAUSP with the analyzed by Bioinformation method. So, we predict that the PLpro has a potential DUB activity. In order to experimentally test our previous prediction, HEK293T cells were co-transfected with HA-tagged ubiquitin (or K48-linked, K63-linked ubiquitin) and PLpro, constructs or mutants. We found that PLpro and constructs both possesses Deubiquiting activity in vivo, suggesting that the Ubl and NAB-G2M domains are dispensable for the Deubiquiting activity. But the Deubiquitinaion of SARS-CoV PLpro was dependent on Cys-His-Asp catalytic residues.Innate antiviral interferon response can be triggered when virus infection. Viral nucleic acids comprising viral genomes or generated during viral replication present major PAMPs that can be recognized by two different classes of PRRs, the membranebound Toll-like receptors (TLRs) or intracellular receptor retinoic acidinducible gene I (RIG-Ⅰ). Upon engagement of their respective ligands, these PRRs recruit different adaptor molecules, relaying signals to downstream kinases that activate IFN regulatory factor3 (IRF3), nuclear factorκB (NF-κB), transcription factors that coordinately up regulate IFNP transcription and expression. And then, IFN-βbinds to the IFNα/βreceptor (IFNAR), IFNARs trigger the activation of downstream signaling and initiates the transcription of several interferon stimulated genes (ISGs). The ISGs inhibit different stages of virus replication and elicit an anti-viral state in the host. In this study, PLpro, constructs or mutants were co-transfected with IFNβ-Luc and pRL-TK reporters into HEK293T cells. At 24 hours post transfection, the cells were either mock treated or treated with 100HAU/ml Sendai virus for 16 h to stimulate IFNβpathway. Or the RIG-IN, IPS-1, IKKεor IRF3 was used to stimulate IFNβinduction. Cell lysates were harvested and assayed for luciferase activity via the Dual Luciferase Reporter Assay. We demonstrated that the PLpro, contructs or mutants were all interferon antagonist. PLpro and the catalytic mutants inhibit IFNβinduction in a dose-dependent manner.This paper further explores the molecular mechanisms that PLpro inhibits the IFNβpathway. First, to determine if PLpro associate with TRAF3/TBK1/IKKs/ERIS, co-immunoprecipitation experiments were performed. HEK 293T cells were co-transfected with plasmid DNA expressing a Flag-tagged version of TRAF3/TBK1/IKKε/ERIS in the presence of PLpro-TM and cell lysates were subjected to immunoprecipitation with anti-Flag antibody. The results show that PLpro was detected in association with TRAF3/TBK1/IKKε/ERIS. Then, we demonstrated that PLpro-TM disrupts TRAF3 interaction with IKKs, TRAF3 with TBK1 and ERIS with IRF3. It suggested that PLpro inhibit the IFNβexpression by blocking the TRAF3 complex. In addition, Protein ubiquitylation has emerged as a key mechanism that regulates immune responses. Much like phosphorylation, ubiquitylation is a reversible covalent modification that regulates the stability, activity and localization of target proteins. Many pathway molecules (RIG-Ⅰ, ERIS, TRAF3 and IRF3) can be modified by ubiquitin. RIG-Ⅰand TRAF3 are both modified by K63 poly-ubiquitin. HEK 293T cells were transfected with HA-Ub and epitope-tagged versions of either RIG-Ⅰ, ERIS, TRAF3 or IRF3 and cell lysates were subjected to immunoblotting to determine the status of ubiquitination of each target. The results indicate that the PLpro-TM is capable of removing ubiquitin from molecules that play key signaling roles in the induction of interferon.Summarize all the experiments. We get the following conclusions:First, SARS PLpro is a novel DUB encoded by virus. Second, SARS PLpro inhibits IFN response of host antiviral innate immunity. Thirdly, SARS PLpro suppressing of IFNβpathway via blocking the form of TRAF3 complex and removing ubiquitin from molecules that play key signaling roles in the interferon pathway. Our study expounds the new mechanisms of inhibiting innate immunity pathway by SARS PLpro, which provide the theoretical base for the development of new antiviral drugs.更多还原