SARS冠状病毒主蛋白酶抑制剂的筛选及抑制动力学研究

对SARS冠状病毒主蛋白酶(SARS-CoV M^pro)进行异源重组表达与提纯,并以其为靶点,利用基于荧光共振能量转移(FRET)技术的体外药物筛选模型,对蛋白酶抑制剂聚焦库96种化合物进行了体外抑制活性的评价,并从动力学的角度探讨筛选出的阳性化合物对SARS-CoV M^pro的抑制能力与机制。结果表明:通过筛选获得抑制率>80%、淬灭率<20%的化合物5种,为P-1-08、P-1-19、P-2-24、P-2-28、P-2-54,其半数有效抑制浓度(IC₅₀)分别为:0.69±0.05μmol/L、1.19±0.41μmol/L、0.14±0.01μmol/L、1.36±0.07μmol/L、0.36±0.03μmol/L。其中化合物P-1-08、P-1-19、P-2-24、P-2-54对SARS冠状病毒主蛋白酶的抑制作用为不可逆抑制,化合物P-2-28的抑制作用为可逆抑制。根据Lineweaver-Burk图和Dixon图的研究,发现化合物P-2-28对SARS冠状病毒主蛋白酶呈竞争性抑制,抑制常数Ki为0.81μmol/L。通过对底物浓度,IC₅₀值及Ki值关系的研究,进一步验证了P-2-28的抑制作用为竞争性抑制。该抑制剂的发现为SARS冠状病毒主蛋白酶抑制剂的研究打下基础,为抗SARS病毒药物开发提供了先导化合物。     

Critical Assessment of the Important Residues Involved in the Dimerization and Catalysis of MERS Coronavirus Main Protease

Background

A highly pathogenic human coronavirus (CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), has emerged in Jeddah and other places in Saudi Arabia, and has quickly spread to European and Asian countries since September 2012. Up to the 1^st October 2015 it has infected at least 1593 people with a global fatality rate of about 35%. Studies to understand the virus are necessary and urgent. In the present study, MERS-CoV main protease (M^pro) is expressed; the dimerization of the protein and its relationship to catalysis are investigated.

Methods and Results

The crystal structure of MERS-CoV M^pro indicates that it shares a similar scaffold to that of other coronaviral M^pro and consists of chymotrypsin-like domains I and II and a helical domain III of five helices. Analytical ultracentrifugation analysis demonstrated that MERS-CoV M^pro undergoes a monomer to dimer conversion in the presence of a peptide substrate. Glu169 is a key residue and plays a dual role in both dimerization and catalysis. The mutagenesis of other residues found on the dimerization interface indicate that dimerization of MERS-CoV M^pro is required for its catalytic activity. One mutation, M298R, resulted in a stable dimer with a higher level of proteolytic activity than the wild-type enzyme.

Conclusions

MERS-CoV M^pro shows substrate-induced dimerization and potent proteolytic activity. A critical assessment of the residues important to these processes provides insights into the correlation between dimerization and catalysis within the coronaviral M^pro family.     

SARS冠状病毒PLpro蛋白酶的结构与功能

SARS冠状病毒基因组编码2种病毒蛋白酶,即木瓜样蛋白酶（PLpro）和3C样蛋白酶(3CLpro).其中,PLpro蛋白酶结构与功能研究是近年来冠状病毒分子生物学研究的热点之一. PLpro蛋白酶参与SARS冠状病毒1a(1ab)复制酶多聚蛋白N端部分的切割加工,是SARS冠状病毒复制酶复合体(RC)形成的重要调节蛋白分子;最新研究表明,SARS冠状病毒PLpro蛋白酶是一种病毒编码的去泛素化酶（DUB）,对细胞蛋白具有明显去泛素化作用;而且对泛素(Ub)和泛素样分子ISG15均具有活性. PLpro蛋白酶对宿主抗病毒天然免疫反应具有负调节作用,是SARS冠状病毒的一种重要干扰素拮抗分子.PLpro蛋白酶是一种多功能病毒蛋白酶.本文结合作者课题组研究工作,对SARS冠状病毒PLpro蛋白酶结构和功能研究最新进展进行综述.     

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．     

Substrate-Induced Dimerization of Engineered Monomeric Variants of Triosephosphate Isomerase from Trichomonas vaginalis

The dimeric nature of triosephosphate isomerases (TIMs) is maintained by an extensive surface area interface of more than 1600 Ų. TIMs from Trichomonas vaginalis (TvTIM) are held in their dimeric state by two mechanisms: a ball and socket interaction of residue 45 of one subunit that fits into the hydrophobic pocket of the complementary subunit and by swapping of loop 3 between subunits. TvTIMs differ from other TIMs in their unfolding energetics. In TvTIMs the energy necessary to unfold a monomer is greater than the energy necessary to dissociate the dimer. Herein we found that the character of residue I45 controls the dimer-monomer equilibrium in TvTIMs. Unfolding experiments employing monomeric and dimeric mutants led us to conclude that dimeric TvTIMs unfold following a four state model denaturation process whereas monomeric TvTIMs follow a three state model. In contrast to other monomeric TIMs, monomeric variants of TvTIM1 are stable and unexpectedly one of them (I45A) is only 29-fold less active than wild-type TvTIM1. The high enzymatic activity of monomeric TvTIMs contrast with the marginal catalytic activity of diverse monomeric TIMs variants. The stability of the monomeric variants of TvTIM1 and the use of cross-linking and analytical ultracentrifugation experiments permit us to understand the differences between the catalytic activities of TvTIMs and other marginally active monomeric TIMs. As TvTIMs do not unfold upon dimer dissociation, herein we found that the high enzymatic activity of monomeric TvTIM variants is explained by the formation of catalytic dimeric competent species assisted by substrate binding.     

SARS冠状病毒的变异特性分析

通过对目前所发表或公布的CARS相关资料进行分析,结合所做的一些工作,对SARS-CoV的变异特性、影响变异的可能因素以及变异与Ⅰ临床病程发展的联系进行了探讨。结果显示,SARS．CoV在变异发生频率和动态变化上具有RNA病毒的普遍特性,但由于其在人类的传播时间较短,相对于常见的引起慢性感染的RNA病毒,如丙型肝炎病毒(HCV)、人类免疫缺陷病毒(HIV)等,得到鉴定的变异序列数量相对较少。SARS-CoV5基因尤其是S1区变异程度较高,Ⅳ基因较为保守。就目前有限的研究结果分析,没有发现病毒变异与Ⅰ临床病程发展之间有明确的联系。     

SARS冠状病毒的起源研究进展

SARS冠状病毒（SARS-CoV）是一种新现病毒,可感染多种动物,果子狸是人类sARs-CoV重要的动物宿主之一,是已知较理想的实验动物模型。遗传因素在SARS-CoV的出现过程中起重要作用,它很可能是哺乳动物和鸟类冠状病毒之间重组产生的新物种,但发生基因重组不是SARS在人群中暴发的原因。     

冠状病毒的新成员--SARS-CoV的基因组特性

2003年3月,人类发现一种新的冠状病毒SARS-CoV,这种病毒是非典型性肺炎(SARS)的病原体。SARS-CoV的基因组序列已经由包括中国科学家在内的全世界的科研人员测定完成。该文对国际报道的SARS病源的基因序列进行了收录,阐述了SARS-CoV基因组的基本特性：SARS-CoV的基因组长约28-30kb,与冠状病毒科的基因组长度相符合,其中包括11个编码序列,基因组的组织方式也与其他冠状病毒类似,从表面蛋白(S蛋白)、外膜蛋白(M蛋白)和核蛋白(N蛋白)上看,SARS病毒与其他冠状病毒的对应蛋白进化关系接近。同时发现,在某些区域,SARS病毒的基因序列与其他冠状病毒存在相当大的差异,具有自身比较保守的基因组序列结构。而且氨基酸的序列也与其他冠状病毒有很大程度的不同。基因信息的冗余分析表明,SARS-CoV具有较低的冗余度,即发生变异的可能性比较大。虽然SARS-CoV外表与冠状病毒类似,亲缘关系未超出冠状病毒科界限,但由于蛋白基因与氨基酸的序列与其他冠状病毒有本质不同,因此可能不是其他冠状病毒的变异体,而是一种与冠状病毒类似、但早已独立存在、此前未被人类所认识的新病毒。     

SARS冠状病毒基因组全序列二碱基指纹分析

二碱基指纹是指全部16种二碱基组合在一个序列内的相对丰度.对病毒、线粒体、细菌、真菌、哺乳动物等上百个物种的基因组序列数据,以及可转座元件序列的分析显示[1-8]:二碱基指纹在基因组内序列之间基本一致,亲缘越近的物种其二碱基指纹越相似.根据这个标准重新进行的分类,与目前通行的基于核酸(或者蛋白质)序列相似性比较所形成的分类基本一致,但也有若干值得注意的差别[9].     

SARS冠状病毒M基因的克隆及其表达

从SARS冠状病毒(GD322株)组织培养上清中提取RNA,进行RTPCR扩增其M基因并克隆到巴氏毕赤酵母表达载体pPICZaB,电穿孔法将重组体整合人酵母菌P．pastoris,经抗生素Zeocin筛选产生的转化子进行表型鉴定,取Mut^-菌用甲醇诱导表达,SDS-PAGE检测其表达产物。Mut^-酵母转化菌经甲醇诱导可分泌表达约65kDa和42kDa的蛋白质,与SARS恢复期病人血清的免疫印迹证实它们为特异的重组M蛋白质,且获得的重组M蛋白质具有免疫反应性。     

Reversal of the Progression of Fatal Coronavirus Infection in Cats by a Broad-Spectrum Coronavirus Protease Inhibitor

Coronaviruses infect animals and humans causing a wide range of diseases. The diversity of coronaviruses in many mammalian species is contributed by relatively high mutation and recombination rates during replication. This dynamic nature of coronaviruses may facilitate cross-species transmission and shifts in tissue or cell tropism in a host, resulting in substantial change in virulence. Feline enteric coronavirus (FECV) causes inapparent or mild enteritis in cats, but a highly fatal disease, called feline infectious peritonitis (FIP), can arise through mutation of FECV to FIP virus (FIPV). The pathogenesis of FIP is intimately associated with immune responses and involves depletion of T cells, features shared by some other coronaviruses like Severe Acute Respiratory Syndrome Coronavirus. The increasing risks of highly virulent coronavirus infections in humans or animals call for effective antiviral drugs, but no such measures are yet available. Previously, we have reported the inhibitors that target 3C-like protease (3CLpro) with broad-spectrum activity against important human and animal coronaviruses. Here, we evaluated the therapeutic efficacy of our 3CLpro inhibitor in laboratory cats with FIP. Experimental FIP is 100% fatal once certain clinical and laboratory signs become apparent. We found that antiviral treatment led to full recovery of cats when treatment was started at a stage of disease that would be otherwise fatal if left untreated. Antiviral treatment was associated with a rapid improvement in fever, ascites, lymphopenia and gross signs of illness and cats returned to normal health within 20 days or less of treatment. Significant reduction in viral titers was also observed in cats. These results indicate that continuous virus replication is required for progression of immune-mediated inflammatory disease of FIP. These findings may provide important insights into devising therapeutic strategies and selection of antiviral compounds for further development for important coronaviruses in animals and humans.     

SARS冠状病毒M基因的克隆及其表达

从SARS冠状病毒(GD322株)组织培养上清中提取RNA,进行RT-PCR扩增其M基因并克隆到巴氏毕赤酵母表达载体pPICZαB,电穿孔法将重组体整合入酵母菌P.pastoris,经抗生素Zeocin筛选产生的转化子进行表型鉴定,取Mut+菌用甲醇诱导表达,SDS-PAGE检测其表达产物.Mut+酵母转化菌经甲醇诱导可分泌表达约65kDa和42 kDa的蛋白质,与SARS恢复期病人血清的免疫印迹证实它们为特异的重组M蛋白质,且获得的重组M蛋白质具有免疫反应性.     

SARS冠状病毒复制酶蛋白nsp8的原核表达

以SARS冠状病毒(BJ01株)基因组RNA为模板,经RT-PCR扩增得到SARS-CoVnsp8基因,并克隆到原核表达载体pGEX-6p-1中,构建重组质粒pNSP8E。pNSP8E转化大肠杆菌BL21(DE3),经IPTG诱导表达出可溶性的GST-nsp8融合蛋白,经亲和层析和自剪切获得了高纯度nsp8蛋白。以nsp8为抗原免疫家兔,制备了nsp8的多克隆抗体,为下一步研究其在病毒感染的细胞中的功能奠定了基础。     

严重急性呼吸综合征(SARS)冠状病毒N蛋白基因的表达及其初步应用

SARS coronavirus N gene w as expressed by Ecoli expression systemThe expressed protein was p urified and an ELISA method was se t up for detection of SARS virus i nfected patient's serum antibodyT he recombinant SARS coronavirus N protein offers an efficient way fo r serological diagnosis and is use ful for epidemiological study and vaccine development     

严重急性呼吸综合征(SARS)患者不同组织中冠状病毒的分离和鉴定

严重急性呼吸综合征(SARS)自2002年11月在中国广东爆发后,已迅速蔓延成为全球性传染疾患。为了了解SARS冠状病毒的特征,对先前SARS冠状病毒PCR检测呈阳性的来自广东的3份尸检肺组织标本、2份尸检脾组织标本：来自北京的2份咽拭子标本和1份血清标本,利用10种不同的细胞系分离病毒。结果显示,上述标本在感染细胞后,分别可在293、Vero—E6、Vero、RD和HeLa细胞系中产生细胞病变(CPE)。不同标本在上述细胞系中致CPE的能力不同,但CPE出现的时间和病变形态学特征无显著性差异。以恢复期SARS病人血清为抗体,用间接免疫荧光法对感染后细胞培养的检测,冠状病毒RT-_PCR对感染后细胞RNA的检测,初步证明分离的病毒为冠状病毒。结果再次证明冠状病毒为SARS的病原,它具有较广泛的器官分布和细胞感染能力。血清中SARS冠状病毒的分离,高度提示在SARS发病过程中存在有病毒血症。     

SARS冠状病毒非结构蛋白Nsp的原核表达

严重急性呼吸综合征病毒,即SARS冠状病毒((Severe acute respiratory syndrome coronavirus,SARS-CoV),为具有囊膜的单股正链RNA病毒,基因组约长29～31kb.基因组从5′到3′端依次编码复制酶蛋白(Rep)、刺突蛋白(S)、囊膜蛋白(E)、膜蛋白(M)和核蛋白(N)以及其他一些辅助性蛋白[1].     

SARS冠状病毒非结构蛋白Nsp的原核表达

严重急性呼吸综合征病毒,即SARS冠状病毒((Severe acute respiratory syndrome coronavirus,SARS-CoV),为具有囊膜的单股正链RNA病毒,基因组约长29~31kb。基因组从5'到3'端依次编码复制酶蛋白(Rep)、刺突蛋白(S)、囊膜蛋白(E)、膜蛋白(M)和核蛋白(N)以及其他一些辅助性蛋白[1]。编码复制酶蛋白的基因,从基因组5'端起约占全长的2/3区域(≈21.2kb),在该区域的nt13392-13398存在保守的UUUAAAC位点,此位点含有-1位的核糖体翻译移框(frameshift),可引发自单一起始位点的蛋白翻译扩展,即由ORF1a编码的Pp1a(约486kDa)扩展为由ORF1b编…     

严重急性呼吸综合征(SARS)冠状病毒抗体的检测及其临床意义

To investigate the k inetics of antibody to SARS coronavirus in SARS patients and its clinical implication,ELISA was used to dete ct antibody to SARS coronavirus(SA RS CoV),RT-PCR was used to detect the SARS CoV RNA and,besides,the C D+4 and CD+8 T cells in peripheral blood of SARS patients and healthy controls were assayed by flowcytom etryThe results showed that SARS CoV IgM were first detected from da y 7 to day 47 after SARS onset,wit h average at day 193±101SARS Co V IgG were first detected from day 4 to day 47 after SARS onset,with average at day 207±101,and the p roduction of SARS CoV IgG was corr elated with CD+4 T cell number(P<005),but had no relationship with SARS CoV RNAMost SARS patients pr oduced SARS CoV antibody,IgM produ ced almost at the same time wit h IgGSARS CoV IgG is a protective antibody against SARS CoV and the titer of IgG may be used as an ind ex indicating the specific immunit y production in SARS patients