SARS冠状病毒分子生物学课件.ppt

1、Molecular Biology of CoronavirusZhongbin Chen,Ph.DDistribution of genome sizes of ssRNA+viruses Alexander E.Gorbalenya,et al.Virus Research Volume 117,Issue 1,April 2006,Pages 17-37 Morphology of the SARS coronavirusKonradStadler,etal.Nature Reviews Microbiology 1,209-218(2003)冠状病毒及其宿主和所致疾病冠状病毒及其宿主和

2、所致疾病Genetic Virus Host Group Diseases(infection sites)Respiratory Enteric OtherHCoV-229E human NL63-CoV human TGEV Pig?PRCoV pig PEDV pig FIPV cat systemicFCoV cat CCoV dog HCoV-OC43 human?SARS-CoV human HKU1-CoV human MHV mouse CNS,systemicRCoV rat HEV pig CNSBCoV cattle IBV chicken kidneyTCoV turk

3、ey 123Comparison of coronavirus genome structuresKonradStadler,etal.Nature Reviews Microbiology 1,209-218(2003)Life cycle of coronaviruses KonradStadler,etal.Nature Reviews Microbiology 1,209-218(2003)Enzymatic activities and characteristics of coronavirus nsp protein domainsActivityLocation and des

4、ignationProtein familyArchitectureCommentsPapain-like proteinaseOne or two PLpro domains within nsp3Deubiquitinating enzyme familyFinger-palm-thumbFingertips contain zinc-binding domainADP-ribose 1-phosphataseADRP(or X)domain within nsp3Macro-HS2A-fold familyThree-layered /Also found in other plus-s

5、trand RNA viruses3C-like cysteine proteinasensp5(3CLpro or main protease,Mpro)Two-barrel proteinase familyTwelve antiparallel-strandsExtra,-helical,carboxyl-terminal domain IIIRNA-dependent RNA polymeraseRdRp domain within nsp12Viral RdRp familyFinger-palm-thumb(predicted)RdRp motif VI signature GDD

6、 replaced with SDD5-to-3 helicase(associated,NTPase,and RNA 5-triphosphatase activities)HEL domain within nsp13Superfamily 1 helicaseModeled to Escherichia coli Rep helicaseAdjacent to amino-proximal,binuclear,zinc-binding domain3-to-5 exonucleaseExoN domain within nsp14DEDD superfamilyHexahelical b

7、undle(predicted)Unable to cleave ribose 2-O-methylated RNA substratesUridylate-specific endoribonucleaseNendoU domain within nsp15XendoU familyWing-body-wing(butterfly fold)Active in hexameric form;RNA bound to internal cavityS-adenosylmethionine-dependent 2-O-methyltransferasensp16(MT)Rrmj methyltr

8、ansferase familyMethyltransferase fold(predicted)Activity yet to be determinedStanley G.Sawicki,et al.Journal of Virology,January 2007,p.20-29,Vol.81,No.1Multiple alignments of nidovirus main proteinases(3CLpro)coronavirus 3CLpro domains(A)and arterivirus 3CLSPs domains(B)Conservation of sites cleav

9、ed by nidovirus main proteinases John Ziebuhr,Eric J.Snijder and Alexander E.Gorbalenya.Journal of General Virology(2000),81,853-879.人类新发冠状病毒：人类新发冠状病毒：NL63 2004年首次发现，人类第种冠状病毒年首次发现，人类第种冠状病毒 主要感染婴幼儿主要感染婴幼儿(0-3岁岁)，并引起严重呼吸道，并引起严重呼吸道症状：发热、气管炎、支气管炎和肺炎症状：发热、气管炎、支气管炎和肺炎.全球性感染！全球性感染！与小孩的重度咳嗽与小孩的重度咳嗽(croup)高度

10、相关。高度相关。NL63 使用与使用与SARS 冠状病毒一样的细胞受体冠状病毒一样的细胞受体(ACE2)。蛋白酶是蛋白酶是NL63冠状病毒复制周期的关键蛋白冠状病毒复制周期的关键蛋白PLP1PLP2Replicase gene3CLproHel Y()nsp4nsp6PolX5(A)n-3NEORF3SM1a1bnsp1nsp2Acnsp3nsp5.基因组复制基因组复制/转录转录ER-anchored RCnsp1-nsp16nsp1nsp2nsp3nsp4nsp5nsp6病毒粒子病毒粒子:Detection of nsp3 and nsp4 in HCoV-NL63-infected cel

11、lsMockNL63 nsp3nsp31 2 32041319038MockNL63 nsp4nsp4131903831 1 2 3WBZhongbin Chen,et al.J.Virol,2007,81(11):6007-6018IF detection of nsp3 and nsp4nsp3Cells:Infected Infected Infected Mock-infectedSerum:Immune Immune Pre-immune ImmuneMagnifiednsp4Green:nsp3 or nsp4Red:Cytox(Nuclear staining)IP:Flag W

12、hich protease cleaves at the CS1 and CS2?ReplicasePLP1PLP2nsp1nsp2nsp3nsp4AcX TM Y?CS1CS2nsp1nsp2 Ac PLP 1 Xnsp1nsp2Ac PLP 1 Xnsp1nsp2 Ac PLP 2 PLP 2V5T7 Promoter3XFlagC1062AC1062AC1678AC1678A123CS1CS222094664614P1 MutWTP2 MutP1 MutWTP2 MutWT123precursorCleaved Products3XFlag-nsp1IP:V5Zhongbin Chen,

13、et al.J.Virol,2007,81(11):6007-6018PLP2 cleaves at the CS3?Zhongbin Chen,et al.J.Virol,2007,81(11):6007-6018Identification of cleavage sites of PLP21.Biochemical method：PLP2Zhongbin Chen,et al.J.Virol,2007,81(11):6007-6018Proteolytic processing of 1a(1ab)by PLP1 and PLP2Zhongbin Chen,et al.J.Virol,2

14、007,81(11):6007-6018Protein ubiquitionation and deubiquitination E1,E2,E3DUBUbUbUbUbUbUbUbUbUbUbProteinProtein26S proteasomeDegradationCell cycle regulation;Transcription regulation;DNA repair;Signaling transduction;Endosomal sorting;Nuclear export;Virus budding;Sulea et al,2005Modeled structure of

15、SCoV-PLproStructure of HAUSP,a known DUB SCoV-PLpro 具有与细胞具有与细胞HAUSP相似的高级结构相似的高级结构PLP2 has polyubiquitin-cleavage activity B.A.2.PLP23.PLP2 C1678AZhongbin Chen,et al.J.Virol,2007,81(11):6007-6018NL 63 PLP2 reacts with HA-Ub-VSA.B.Zhongbin Chen,et al.J.Virol,2007,81(11):6007-6018NL63 PLP2,but not PLP1

16、,reacts with DUB-specific HA-Ub-VS probes90KD38KD38KDWB:HAWB:V5PLP2-HA-UbPLP1/PLP2 Vector WT C1062A H1212A WT C1678A H1836ANL PLP1(N)NL PLP2(N)38WB:V5PLP1/PLP2WB:Flag204383190131Ub-conjugated proteins Vector vector WT C1062A H1212A WT C1678A H1836ANL PLP1(N)NL PLP2(N)3xFlag-Ub -+NL63 PLP2,but not PL

17、P1,deubiquitinates Ub-conjugated proteins in vivocalnexinNL63冠状病毒冠状病毒PLP2的去泛素化作用具有剂量依赖性的去泛素化作用具有剂量依赖性3190calnexinPLP2204131903831Ub-conjugated proteinsFlag-Ub(0.4 g)NL PLP2(N)(g)-+-+-0.1 0.004 0.02 0.1 0.5 WB:FlagWB:V5 NL63 PLP1 和和 PLP2活性位点具有相似的高级结构活性位点具有相似的高级结构PLP1PLP2PLP1PLP2问题问题：什么因素决定了PLP1无DUB活性

18、，而 PLP2有DUB活性？尚未解决的问题尚未解决的问题 为什么为什么PLP2 具有具有DUB活性活性,而而PLP1 无无DUB活性活性?PLP2 DUB的靶蛋白是什么？的靶蛋白是什么？宿主病毒蛋白 PLP2 DUB的底物特异性？的底物特异性？Ub,UBl(SUMO,ISG15,Nedd8,NEMO,FAT10 等)PLP2 DUB 生物学功能？生物学功能？细胞生命活动病毒复制与致病性Summary of multiple functions of papain-like proteases of human coronavirus NL63 DUBUbUbUbUbUbUbUbUbUbUbPr

19、oteinProtein IFN expressionIFN signaling pathwayPapain-Like Proteasesnsp1nsp2nsp3nsp4PLP1PLP2Replicase polyproteinIFN antagonist?Protease activity复习题 简述冠状病毒基因组复制、转录和蛋白质翻译和加工的特点。简述冠状病毒复制周期。总复习题总复习题名词名词:1.复制酶复合体(Replicase complex,RC)2.(病毒)多聚蛋白（polyprotein）3.(病毒)RNA依赖RNA聚合酶（RNA-dependent RNA polymerase

20、,RdRp）4.抗病毒药物靶标(Antiviral target)5.病毒蛋白酶(Viral protease)简答题简答题:1.简述乙型肝炎病毒(HBV)的复制周期以及哪些过程(阶段)可以作为 抗病毒药物研究靶标.2.简述丙型肝炎病毒(HCV)的复制周期以及哪些过程(阶段)可以作为 抗病毒药物研究靶标.3.简述冠状病毒(coronavirus)的复制周期以及病毒基因组转录的特点.Steps of HBV Replication(DNA Virus)N Engl J Med 359:1486,October 2,2008 Life cycle of hepatitis C virus(HCV)Darius Moradpour,Franois Penin&Charles M.Rice.Nature Reviews Microbiology 5,453-463(June 2007)Life cycle of coronaviruses KonradStadler,etal.Nature Reviews Microbiology 1,209-218(2003)the life cycles of human immunodeficiency virus(HIV)Nature Reviews Drug Discovery 6,1001-1018(December 2007)