冠状病毒PLpro蛋白酶对泛素样分子的DUB活性

SARS冠状病毒基因组中非结构基因nsp3编码的木瓜样蛋白酶 (PLpro) 在病毒基因组复制及逃避宿主天然免疫中发挥重要作用,是研发抗病毒药物的重要靶标．SARS冠状病毒PLpro是一种病毒编码的去泛素化酶 (DUB)．为深入研究SARS冠状病毒 PLpro对泛素样分子 (ubiquitin-like protein,UBL) 的DUB特性,本研究构建缺失 PLpro N末端泛素样结构域 (Ubl) 和下游跨膜结构域 (TM) 的PLpro构建体（constructs）,并构建3种缺失蛋白酶催化活性的突变体,检测PLpro对泛素样分子干扰素刺激基因15 (ISG15)及SUMO-1的作用．实验结果表明,PLpro和PLpro-TM 在细胞内具有很强的去ISG(DeISGylation) 活性;缺失PLpro N末端泛素样结构域(Ubl) 对PLpro 的去ISG15 活性没有影响;对PLpro蛋白酶活性位点C1651 和 H1812 突变后,PLpro-TM的去ISG15活性消失,而对D1826位点突变后不影响此活性．PLpro 不具有去SUMO (DeSUMOylation)活性,而PLpro-TM具有一定的去SUMO活性;PLpro催化活性相关的3个关键氨基酸残基 Cys-His-Asp突变后对去SUMO活性有一定的影响．研究结果提示,SARS PLpro除了具有DUB的活性,还具有体内去ISG活性和去SUMO活性;PLpro蛋白酶活性与其去ISG活性之间有一定相关性;PLpro去SUMO-1 活性具有TM 依赖性．SARS冠状病毒PLpro 对泛素样分子作用特性的研究为阐明病毒逃避宿主天然免疫机制和开发新型抗病毒药物提供重要的理论依据．     

SARS 冠状病毒非结构蛋白质NSP3突变体构建及其对类泛素分子DUB活性

SARS冠状病毒(SARS-CoV) 非结构蛋白NSP3编码的木瓜蛋白酶样蛋白酶(PLpro)对泛素样分子(Ubl) 具有去泛素化酶(DUB)活性,但目前有关NSP3 DUB活性研究的报道甚少. 本研究构建包含Nsp3基因 N末端不同结构域的突变体,并检测NSP3及其一系列突变体对类泛素分子ISG15和SUMO所修饰蛋白质分子的作用特性. 实验结果表明,NSP3及其突变体NSP3AD,NSP3AE,NSP3AF具有一定的去ISG15活性,而其突变体NSP3AC则没有去ISG15 (DeISGylation) 活性. 研究结果提示,SARS NSP3具有一定的体内去ISG15活性,并且这种活性主要依赖于Nsp3基因编码的PLpro. 但SARS NSP3及其突变体NSP3AC,NSP3AD,NSP3AE和NSP3AF并不具有去SUMO (DeSUMOylation) 活性. SARS冠状病毒NSP3对类泛素样分子作用特性的研究为后续NSP3的生物学特性及其对干扰素通路的调控研究奠定了基础.     

新型冠状病毒PLpro蛋白酶结构与功能的生物信息学分析

2019年12月,由新型冠状病毒(SARS-CoV-2)引起的新型冠状病毒肺炎(COVID-19)在中国武汉暴发。SARS-CoV-2的基因组编码2种病毒蛋白酶,即木瓜样蛋白酶(Papain-like protease,PLpro)和3C样蛋白酶(3C-like protease)。其中PLpro是SARS-CoV-2复制酶复合体(RC)形成的重要调节蛋白分子,对于病毒基因组转录和复制至关重要。因此,将SARS-CoV-2 PLpro作为药物的靶点对COVID-19的治疗具有积极意义。本研究应用生物信息学工具分析新型冠状病毒的木瓜样蛋白酶的结构和功能,首先利用BLAST和BioEdit获取SARS-CoV-2 PLpro蛋白酶(SC2-PLpro)及其同源蛋白的氨基酸序列,并利用BLAST和MEGA 6.0进行同源性分析。之后,利用ProParam和Proscale分别对SC2-PLpro蛋白酶的理化性质、亲水性和疏水性进行分析。然后,通过SOMPA、ScanProsite和InterPro分别预测SC2-PLpro蛋白酶的二级结构和功能区域,进一步利用SignalP 4.0和TMHMM对SC2-PLpro蛋白酶的信号肽和跨膜区进行分析。最后,通过SWISS-MODEL对SARS-CoV-2 PLpro蛋白酶进行三级结构同源建模。结果显示,对SARS-CoV-2 PLpro蛋白酶与已报道的PLpro蛋白酶进行多序列比对后,发现SARS-CoV-2 nsp3的746~1063段氨基酸与多种冠状病毒PLpro蛋白酶氨基酸序列高度相似。同时,同源性分析发现SARS-CoV-2与蝙蝠冠状病毒的PLpro蛋白酶具有同源性,其中与QHR63299、AVP78030相似性最高。对SC2-PLpro进行理化性质预测结果显示,其由318个氨基酸所组成,为稳定亲水性蛋白。二级结构预测结果显示SC2-PLpro主要含有α-螺旋、延伸链、β-转角、无规卷曲,四种结构贯穿整条氨基酸链。进一步进行功能分析,发现其具有完整的催化三联体、锌结合域、泛素样N末端结构域,故推测该蛋白具有去泛素化的功能。然后,信号肽假说和跨膜结构域分析结果表明,SC2-PLpro既不是分泌蛋白,也不属于跨膜蛋白。本研究提示,生物信息学分析SC2-PLpro为稳定性亲水蛋白,属于非跨膜蛋白,比较保守,具有去泛素化的功能,利用此功能可以进一步规避宿主的固有免疫反应。通过制备PLpro蛋白酶小分子抑制剂,可能有助于治疗新型冠状病毒肺炎。     

人类冠状病毒调节宿主抗病毒天然免疫分子机制

SARS冠状病毒和正在全球流行的猪源H1N1型流感病毒等人类新发呼吸道病毒对人类生命健康构成严重威胁.人类重要呼吸道病毒与宿主抗病毒天然免疫的关系是近年来研究热点.SARS冠状病毒等很多RNA病毒能够编码某种蛋白质,抑制干扰素表达以及干扰素介导的抗病毒信号通路.人类冠状病毒木瓜样蛋白酶(papain-like protease,PLP)利用其自身去泛素化酶(DUB)活性,使干扰素表达通路中重要调节蛋白发生去泛素化,从而抑制干扰素信号传导.同时,PLP蛋白酶通过阻碍干扰素表达信号通路中最新发现的重要调节蛋白ERIS(也称MITA/STING)二聚化,使其失活并丧失激活干扰素通路的功能,这些发现对于阐明人类重要呼吸道病毒对宿主细胞抗病毒天然免疫反应的调节作用及其机制具有重要意义,为人类新发病毒致病机理、免疫防治以及抗病毒药物研究提供新的思路.     

猪流行性腹泻病毒(PEDV)与抗病毒天然免疫

猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)是引起猪流行性腹泻病等肠道疾病的一种动物冠状病毒.PEDV与宿主系统相互作用,特别是其对宿主抗病毒天然免疫调节作用和机制是目前动物冠状病毒研究的基础科学问题之一.基于作者近几年来对人类重要冠状病毒对宿主抗病毒天然免疫系统调节作用的研究,本文对PEDV基因组与编码蛋白主要功能以及PEDV调节宿主抗病毒天然免疫反应及其可能机制的进展和现状进行了分析.与人类冠状病毒相似,PEDV编码的木瓜样蛋白酶(papain like protease,PLP)是一个多功能蛋白酶,除了蛋白酶活性外,还具有去泛素化酶(DUB)活性和宿主干扰素拮抗活性,是PEDV编码的一种新型病毒来源DUB和宿主干扰素拮抗蛋白.这些研究为阐明PEDV对宿主抗病毒天然免疫反应调节作用和其致病机制提供了重要的理论依据,为研制新型PEDV免疫防治措施提供了重要理论基础.     

NL63冠状病毒木瓜样蛋白酶去泛素化酶活性和对宿主抗病毒天然免疫反应调节作用

引起人类呼吸道感染的冠状病毒已多达5种．冠状病毒与宿主相互作用决定了其致病性和免疫特性．冠状病毒感染后宿主会立即启动抗病毒天然免疫反应,而人类冠状病毒往往会编码特定蛋白逃逸或抑制宿主的天然免疫反应．NL63冠状病毒是一种新型人类冠状病毒,其非结构蛋白nsp3编码2个木瓜样蛋白酶(PLP)核心结构域PLP1和PLP2．前期研究发现,人类冠状病毒PLP2是一种病毒编码的去泛素化酶(DUB),但是对其DUB特性和功能还不清楚．研究发现,NL63冠状病毒PLP1和PLP2两个核心结构域中只有PLP2具有DUB活性,而且,PLP2的DUB活性对K48和K63连接的多聚泛素化修饰不表现明显特异性．同时,蛋白酶活性催化位点C1678和H1836突变后对其DUB活性有明显抑制作用,而蛋白酶活性催化位点D1849突变后对DUB活性无影响．其次,PLP2而非PLP1核心结构域能够明显抑制仙台病毒和重要信号蛋白(RIG-I、ERIS/STING/MITA)激活的干扰素表达,表明PLP2是一种冠状病毒编码的干扰素拮抗剂,而且PLP2的干扰素拮抗作用不完全依赖其蛋白酶活性．机制研究表明,PLP2能够与干扰素表达通路中的重要调节蛋白RIG-I和ERIS发生相互作用,通过对RIG-I和ERIS的去泛素化负调控宿主抗病毒天然免疫反应．此外,PLP2除利用DUB活性抑制干扰素表达外,很可能存在不依赖自身催化活性的其他组分共同抑制干扰素的产生．以上研究对阐明人类新发冠状病毒免疫和致病机理以及抗病毒药物研发具有重要参考价值．     

去泛素化酶USP11在细胞中的功能作用及其研究进展

Ubiquitin-specific protease 11(USP11)属于半胱氨酸蛋白酶,是去泛素化酶家族(deubiquitinating enzymes,DUBs)的重要成员之一。近年来研究表明USP11能调节细胞内众多蛋白底物的稳定性及功能,包括DNA修复蛋白、病毒RNA复制相关蛋白、TGFβ和NF-κB信号转导通路相关蛋白等,在疾病的发生发展中起着重要的作用。主要综述了USP11的结构、在细胞中的分子功能以及与肿瘤和病毒性疾病的关系,探讨了USP11作为治疗分子靶标的可能性。     

病毒编码泛素及相关蛋白的研究进展

泛素(Ubiquitin, Ub)是一种由76个氨基酸残基组成的小分子蛋白,广泛存在于各种真核生物中,不同来源的泛素蛋白具有类似的结构、功能和免疫学特征.目前所知,泛素主要通过ATP依赖性的泛素-蛋白酶复合体通路(Ubiquitin-proteasomes pathway, UPP)途径,经泛素活化酶(E1)、泛素结合酶(E2)和泛素连接酶(E3)的梯级反应,高效并高度选择性地对胞质或胞核内蛋白进行完全或部分降解,泛素依赖性蛋白质降解涉及细胞周期调控、信号转导、DNA修复和蛋白质数量控制[1].另外,泛素还可作为分子伴侣参与核糖体的生物发生、DNA转录调控及其它多种生理功能[2].近年来发现,一些真核生物病毒,如杆状病毒、疱疹病毒、痘病毒、腺病毒和反转录病毒等,均编码泛素或能与泛素相互作用的蛋白.病毒编码的泛素和泛素相关蛋白与病毒粒子形成及病毒出芽增殖有关,也与宿主细胞凋亡调控、宿主免疫系统调控及病毒侵染宿主细胞的机理相联系.本文就这一方面的研究进展进行简述.     

SARS冠状病毒中的核衣壳蛋白

严重急性呼吸综合征(SARS)的元凶是一种新冠状病毒,研究病毒结构蛋白的功能有助于了解病毒的感染、复制和包装等生理过程。其中核衣壳蛋白是SARS冠状病毒中含量最丰富和最保守的结构蛋白,自身聚合后包被病毒RNA基因组形成螺旋状核壳体是SARS冠状病毒成熟的关键步骤;核衣壳蛋白能与病毒或宿主细胞中多种蛋白质相互作用,还能影响宿主细胞的多个通路。因此核衣壳蛋白是一个重要的多功能蛋白质,参与了病毒感染、复制和病毒包装等过程。     

SARS-CoV 基因组编码蛋白研究进展

严重急性呼吸综合征(severe acute respiratory syndrome,SARS)2002年底暴发于中国广东,后蔓延至全球的传染性疾病．其病原体为一种新型的未知冠状病毒,基因组长度约30 kb,预测具有14个开放读码框．至今为止,对 SARS 冠状病毒(SARS-COV)基因组编码蛋白质的研究已取得显著进展,其研究主要集中在复制酶 1a/1b、结构蛋白及“附属”蛋白(SARS-CoV 特异性蛋白)结构与功能的研究．以 SARS-CoV 的蛋白组成及功能研究为主要内容,系统介绍了 SARS-CoV 蛋白质研究进展．     

Identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity

Gene 1 of the coronavirus associated with severe acute respiratory syndrome (SARS) encodes replicase polyproteins that are predicted to be processed into 16 nonstructural proteins (nsps 1 to 16) by two viral proteases, a papain-like protease (PLpro) and a 3C-like protease (3CLpro). Here, we identify SARS coronavirus amino-terminal replicase products nsp1, nsp2, and nsp3 and describe trans-cleavage assays that characterize the protease activity required to generate these products. We generated polyclonal antisera to glutathione S-transferase-replicase fusion proteins and used the antisera to detect replicase intermediates and products in pulse-chase experiments. We found that nsp1 (p20) is rapidly processed from the replicase polyprotein. In contrast, processing at the nsp2/3 site is less efficient, since a approximately 300-kDa intermediate (NSP2-3) is detected, but ultimately nsp2 (p71) and nsp3 (p213) are generated. We found that SARS coronavirus replicase products can be detected by 4 h postinfection in the cytoplasm of infected cells and that nsps 1 to 3 colocalize with newly synthesized viral RNA in punctate, perinuclear sites consistent with their predicted role in viral RNA synthesis. To determine if PLpro is responsible for processing these products, we cloned and expressed the PLpro domain and the predicted substrates and established PLpro trans-cleavage assays. We found that the PLpro domain is sufficient for processing the predicted nsp1/2 and nsp2/3 sites. Interestingly, expression of an extended region of PLpro that includes the downstream hydrophobic domain was required for processing at the predicted nsp3/4 site. We found that the hydrophobic domain is inserted into membranes and that the lumenal domain is glycosylated at asparagine residues 2249 and 2252. Thus, the hydrophobic domain may anchor the replication complex to intracellular membranes. These studies revealed that PLpro can cleave in trans at the three predicted cleavage sites and that it requires membrane association to process the nsp3/4 cleavage site.     

Proteolytic processing and deubiquitinating activity of papain-like proteases of human coronavirus NL63

Human coronavirus NL63 (HCoV-NL63), a common human respiratory pathogen, is associated with both upper and lower respiratory tract disease in children and adults. Currently, no antiviral drugs are available to treat CoV infections; thus, potential drug targets need to be identified and characterized. Here, we identify HCoV-NL63 replicase gene products and characterize two viral papain-like proteases (PLPs), PLP1 and PLP2, which process the viral replicase polyprotein. We generated polyclonal antisera directed against two of the predicted replicase nonstructural proteins (nsp3 and nsp4) and detected replicase proteins from HCoV-NL63-infected LLC-MK2 cells by immunofluorescence, immunoprecipitation, and Western blot assays. We found that HCoV-NL63 replicase products can be detected at 24 h postinfection and that these proteins accumulate in perinuclear sites, consistent with membrane-associated replication complexes. To determine which viral proteases are responsible for processing these products, we generated constructs representing the amino-terminal end of the HCoV-NL63 replicase gene and established protease cis-cleavage assays. We found that PLP1 processes cleavage site 1 to release nsp1, whereas PLP2 is responsible for processing both cleavage sites 2 and 3 to release nsp2 and nsp3. We expressed and purified PLP2 and used a peptide-based assay to identify the cleavage sites recognized by this enzyme. Furthermore, by using K48-linked hexa-ubiquitin substrate and ubiquitin-vinylsulfone inhibitor specific for deubiquitinating enzymes (DUBs), we confirmed that, like severe acute respiratory syndrome (SARS) CoV PLpro, HCoV-NL63 PLP2 has DUB activity. The identification of the replicase products and characterization of HCoV-NL63 PLP DUB activity will facilitate comparative studies of CoV proteases and aid in the development of novel antiviral reagents directed against human pathogens such as HCoV-NL63 and SARS-CoV.     

Assessing Activity and Inhibition of Middle East Respiratory Syndrome Coronavirus Papain-Like and 3C-Like Proteases Using Luciferase-Based Biosensors

Middle East respiratory syndrome coronavirus (MERS-CoV) is associated with an outbreak of more than 90 cases of severe pneumonia with high mortality (greater than 50%). To date, there are no antiviral drugs or specific therapies to treat MERS-CoV. To rapidly identify potential inhibitors of MERS-CoV replication, we expressed the papain-like protease (PLpro) and the 3-chymotrypsin-like protease (3CLpro) from MERS-CoV and developed luciferase-based biosensors to monitor protease activity in cells. We show that the expressed MERS-CoV PLpro recognizes and processes the canonical CoV-PLpro cleavage site RLKGG in the biosensor. However, existing CoV PLpro inhibitors were unable to block MERS-CoV PLpro activity, likely due to the divergence of the amino acid sequence in the drug binding site. To investigate MERS-CoV 3CLpro activity, we expressed the protease in context with flanking nonstructural protein 4 (nsp4) and the amino-terminal portion of nsp6 and detected processing of the luciferase-based biosensors containing the canonical 3CLpro cleavage site VRLQS. Importantly, we found that a small-molecule inhibitor that blocks replication of severe acute respiratory syndrome (SARS) CoV and murine CoV also inhibits the activity of MERS-CoV 3CLpro. Overall, the protease expression and biosensor assays developed here allow for rapid evaluation of viral protease activity and the identification of protease inhibitors. These biosensor assays can now be used to screen for MERS-CoV-specific or broad-spectrum coronavirus PLpro and 3CLpro inhibitors.     

The papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity

Replication of the genomic RNA of severe acute respiratory syndrome coronavirus (SARS-CoV) is mediated by replicase polyproteins that are processed by two viral proteases, papain-like protease (PLpro) and 3C-like protease (3CLpro). Previously, we showed that SARS-CoV PLpro processes the replicase polyprotein at three conserved cleavage sites. Here, we report the identification and characterization of a 316-amino-acid catalytic core domain of PLpro that can efficiently cleave replicase substrates in trans-cleavage assays and peptide substrates in fluorescent resonance energy transfer-based protease assays. We performed bioinformatics analysis on 16 papain-like protease domains from nine different coronaviruses and identified a putative catalytic triad (Cys1651-His1812-Asp1826) and zinc-binding site. Mutagenesis studies revealed that Asp1826 and the four cysteine residues involved in zinc binding are essential for SARS-CoV PLpro activity. Molecular modeling of SARS-CoV PLpro suggested that this catalytic core may also have deubiquitinating activity. We tested this hypothesis by measuring the deubiquitinating activity of PLpro by two independent assays. SARS CoV-PLpro hydrolyzed both diubiquitin and ubiquitin-7-amino-4-methylcoumarin (AMC) substrates, and hydrolysis of ubiquitin-AMC is approximately 180-fold more efficient than hydrolysis of a peptide substrate that mimics the PLpro replicase recognition sequence. To investigate the critical determinants recognized by PLpro, we performed site-directed mutagenesis on the P6 to P2' residues at each of the three PLpro cleavage sites. We found that PLpro recognizes the consensus cleavage sequence LXGG, which is also the consensus sequence recognized by cellular deubiquitinating enzymes. This similarity in the substrate recognition sites should be considered during the development of SARS-CoV PLpro inhibitors.     

Human coronavirus 229E papain-like proteases have overlapping specificities but distinct functions in viral replication

Expression of the exceptionally large RNA genomes of CoVs involves multiple regulatory mechanisms, including extensive proteolytic processing of the large replicase polyproteins, pp1a and pp1ab, by two types of cysteine proteases: the chymotrypsin-like main protease and papain-like accessory proteases (PLpros). Here, we characterized the proteolytic processing of the human coronavirus 229E (HCoV-229E) amino-proximal pp1a/pp1ab region by two paralogous PLpro activities. Reverse-genetics data revealed that replacement of the PL2pro active-site cysteine was lethal. By contrast, the PL1pro activity proved to be dispensable for HCoV-229E virus replication, although reversion of the PL1pro active-site substitution to the wild-type sequence after several passages in cell culture indicated that there was selection pressure to restore the PL1pro activity. Further experiments showed that both PL1pro and PL2pro were able to cleave the nsp1-nsp2 cleavage site, with PL2pro cleaving the site less efficiently. The PL1pro-negative mutant genotype could be stably maintained in cell culture when the nsp1-nsp2 site was replaced by a short autoproteolytic sequence, suggesting that the major driving force for the observed reversion of the PL1pro mutation was the requirement for efficient nsp1-nsp2 cleavage. The data suggest that the two HCoV-229E PLpro paralogs have overlapping substrate specificities but different functions in viral replication. Within the tightly controlled interplay of the two protease activities, PL2pro plays a universal and essential proteolytic role that appears to be assisted by the PL1pro paralog at specific sites. Functional and evolutionary implications of the differential amino-terminal polyprotein-processing pathways among the main CoV lineages are discussed.     

Targeting papain-like protease for broad-spectrum coronavirus inhibition

The emergence of SARS-CoV-2 variants of concern and repeated outbreaks of coronavirus epidemics in the past two decades emphasize the need for next-generation pan-coronaviral therapeutics. Drugging the multi-functional papain-like protease (PLpro) domain of the viral nsp3 holds promise. However, none of the known coronavirus PLpro inhibitors has been shown to be in vivo active. Herein, we screened a structurally diverse library of 50,080 compounds for potential coronavirus PLpro inhibitors and identified a noncovalent lead inhibitor F0213 that has broad-spectrum anti-coronaviral activity, including against the Sarbecoviruses (SARS-CoV-1 and SARS-CoV-2), Merbecovirus (MERS-CoV), as well as the Alphacoronavirus (hCoV-229E and hCoV-OC43). Importantly, F0213 confers protection in both SARS-CoV-2-infected hamsters and MERS-CoV-infected human DPP4-knockin mice. F0213 possesses a dual therapeutic functionality that suppresses coronavirus replication via blocking viral polyprotein cleavage, as well as promoting antiviral immunity by antagonizing the PLpro deubiquitinase activity. Despite the significant difference of substrate recognition, mode of inhibition studies suggest that F0213 is a competitive inhibitor against SARS2-PLpro via binding with the 157K amino acid residue, whereas an allosteric inhibitor of MERS-PLpro interacting with its 271E position. Our proof-of-concept findings demonstrated that PLpro is a valid target for the development of broad-spectrum anti-coronavirus agents. The orally administered F0213 may serve as a promising lead compound for combating the ongoing COVID-19 pandemic and future coronavirus outbreaks.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13238-022-00909-3.     

Correlation between TGF‐β1 expression and proteomic profiling induced by severe acute respiratory syndrome coronavirus papain‐like protease

Severe acute respiratory syndrome (SARS) coronavirus (SARS‐CoV) papain‐like protease (PLpro), a deubiquitinating enzyme, demonstrates inactivation of interferon (IFN) regulatory factor 3 and NF‐κB, reduction of IFN induction, and suppression of type I IFN signaling pathway. This study investigates cytokine expression and proteomic change induced by SARS‐CoV PLpro in human promonocyte cells. PLpro significantly increased TGF‐β1 mRNA expression (greater than fourfold) and protein production (greater than threefold). Proteomic analysis, Western blot, and quantitative real‐time PCR assays indicated PLpro upregulating TGF‐β1‐associated genes: HSP27, protein disulfide isomerase A3 precursor, glial fibrillary acidic protein, vimentin, retinal dehydrogenase 2, and glutathione transferase omega‐1. PLpro‐activated ubiquitin proteasome pathway via upregulation of ubiquitin‐conjugating enzyme E2–25k and proteasome subunit alpha type 5. Proteasome inhibitor MG‐132 significantly reduced expression of TGF‐β1 and vimentin. PLpro upregulated HSP27, linking with activation of p38 MAPK and ERK1/2 signaling. Treatment with SB203580 and U0126 reduced PLpro‐induced expression of TGF‐β1, vimentin, and type I collagen. Results point to SARS‐CoV PLpro triggering TGF‐β1 production via ubiquitin proteasome, p38 MAPK, and ERK1/2‐mediated signaling.     

Binding site-based classification of coronaviral papain-like proteases

The coronavirus replicase gene encodes one or two papain-like proteases (termed PL1pro and PL2pro) implicated in the N-terminal processing of the replicase polyprotein and thus contributing to the formation of the viral replicase complex that mediates genome replication. Using consensus fold recognition with the 3D-JURY meta-predictor followed by model building and refinement, we developed a structural model for the single PLpro present in the severe acute respiratory syndrome coronavirus (SCoV) genome, based on significant structural relationships to the catalytic core domain of HAUSP, a ubiquitin-specific protease (USP). By combining the SCoV PLpro model with comparative sequence analyses we show that all currently known coronaviral PLpros can be classified into two groups according to their binding site architectures. One group includes all PL2pros and some of the PL1pros, which are characterized by a restricted USP-like binding site. This group is designated the R-group. The remaining PL1pros from some of the coronaviruses form the other group, featuring a more open papain-like binding site, and is referred to as the O-group. This two-group, binding site-based classification is consistent with experimental data accumulated to date for the specificity of PLpro-mediated polyprotein processing and PLpro inhibition. It also provides an independent evaluation of the similarity-based annotation of PLpro-mediated cleavage sites, as well as a basis for comparison with previous groupings based on phylogenetic analyses.