病毒学-病毒粒子的装配和释放ppt课件

21:58:31,第六章病毒粒子的装配和释放,1,21:58:31,第一节病毒粒子的装配,定义：在病毒感染的细胞中，由病毒基因组指导合成的病毒结构蛋白和子代病毒核酸基因组组装，生成完整的子代病毒粒子的过程称为装配(assembly)或叫做成熟(maturation)。,2,21:58:31,第一节病毒粒子的装配,2.部位真核DNA病毒:细胞核RNA病毒:细胞质装配效率：复杂的过程，效率不高DNA噬菌体:50的DNA和结构蛋白不能组装。,3,21:58:31,装配错误，出现空壳体或其它畸形颗粒。病毒混合感染，装配错误可能产生：具有不同病毒结构蛋白构成的壳体、包膜病毒。一种病毒基因组被包闭在另一种病毒编码的外壳中形成假型病毒。3)将宿主的核酸包被进去。,4,21:58:31,研究方法：体外的病毒重建实验将一个病毒粒子拆开为核酸基因结构蛋白在一定的条件下进行体外重组形成具有感染性的病毒粒子。,5,21:58:31,2010年8月27日ScienceUCLA大学（加利福尼亚洛杉矶分校）分子和医学药理学LilyWu教授试图操纵腺病毒用于基因治疗，“我们操纵病毒，传递基因用于治疗癌症，但先前的显微技术无法看见修饰过的病毒，这就像在黑暗中组装汽车一样，检验组装是否正确的唯一方法就是开动汽车进行试验。”UCLA大学微生物学免疫学分子遗传学教授HongZhou求助。Zhou使用低温电子显微镜技术制作出了准确的病毒原子分辨三维生物模型,水中的样本可直接成像。（ScienceDOI:10.1126/science.1187433）,6,21:58:31,Fig.1.OverallstructureoftheAd5capsid.(A)Radiallycoloredsurfaceofareconstructionofthecapsid,centeredonathreefoldaxis.(B)Viewsoftheoutersurface(top)showingminorproteinIXand,followingrotationtheinnersurface(bottom)ofafacetshowingminorproteinsIIIa,VI,andVIII.Allhexons,pentonbases,andpentonfibersareshownsemitransparentlyexceptforonehexonmonomer(+)andonepenton-basemonomer(*).(Topleftinset)SideviewofproteinIXamonghexons.(Bottomrightinset)SideviewofproteinsIIIaandVIIIcenteredonapentonbase.(C)Atomicmodel(sticks)ofanhelixfromahexonmonomersuperimposedonitsdensitymap(mesh)withsomesidechainslabeled.,7,21:58:31,Fig.2.Interactionsamongminorandmajorproteinsontheinnersurface.(A)(Left)GlobalviewofthearrangementofproteinIIIa(red)andproteinVIII(blue).(Middle)OrganizationofhexontrimersintoaGON(grayshade),peripentonalhexontrimers(lightblueshade),andapenton-basepentamer(orangeshade)intoaGOS.(Topinsets)augmentationattheVCregionsoftheH3hexontrimerbythebody(left)andthehead(right)domainsofproteinVIII.(Rightinsets)ThetopinsetshowsaugmentationattheVIII-bindingdomainofproteinIIIabythebodydomainofproteinVIII;thebottominsetshowsinteractionsamongtheN-armofapentonbaseandtwoadjacentproteinsIIIa.(B)RibbonmodelofproteinIIIa(aminoacids7to300)withfourdomains.(C)RibbonmodelofproteinVIIIwiththreedomains.(Bottominset)Headdomaindensity(semitransparentgray)anditsatomicmodel(ribbon),showingcleavagesitesG110andR159betweentheN-terminalportion(blue)andtheC-terminalportion(red).,8,21:58:31,Fig.3.Interactionsamongminorandmajorproteinsontheoutersurface.(A)ThephysicalnetworkofproteinIXontheoutersurfacelasheshexonstogetherintoGONtilesbutavoidsGOStilesthatareeachcenteredonavertex.Insets:(Centerleft)RibbonmodelsoftheN-terminaldomainsofthreeproteinIXmonomers(blue,green,andred),overlyingthemodelsofthreeadjacenthexon(H2,H3,andH4)monomers(gray)atalocalthreefoldaxis.(Topleftinsets)N-jointofthreeproteinIXmonomersanditssideview,showingahydrophobiccorecontainingatripletoftyrosines(Y14)andatripletofleucines(L15).(Bottomleftinset)augmentationattheFG2regionofahexonH2bytheNtermimusofproteinIX.(Topright)Four-helixbundlewiththreeparallelandoneantiparallelheliceslinkedbyaladderofhydrophobicresidues(leucinesandvalines,magenta).(Bottomright)Head-onviewofthehelixbundleandthehydrophobiccore.(B)RibbonmodelofproteinIXwiththreedomainsandtheN-jointregion.(Seealsofig.S5.)(Inset)Densitymap(mesh)andatomicmodel(sticks)ofarepresentativeloopfromtheN-terminaldomain.,9,21:58:31,Fig.4.Newlyresolvedregionsinpenton-baseandhexonproteins.(A)Cryo-EMmodel(ribbons)ofthepenton-baseproteinsuperimposedonitsdensitymap(semitransparentgray).Outsidethebox,thecryo-EMatomicmodel(redribbons)isidenticaltothex-raymodel(11).InsidetheboxisournewlyresolvedN-arm(blueribbon,aminoacids37to51).(Inset)Enlargementoftheboxedregion,showingside-chaindensities(mesh)anditsatomicmodel(ribbon).(B)Cryo-EMmodelofthehexonprotein.Redribbonsshowagreementwiththex-raymodel(10).Blueribbonsshowournewlyresolvedpieces,includingtheN-terminalandC-terminalextensions.Regionnamesinthehexonmonomer(e.g.,VCandFG)follow(10).(CtoD)Conformationaladaptation.(C)TwelvehexonmonomersexhibitfivetypesofN-terminalextensioninanasymmetricunit:fouroftype1,twoeachoftypes2and3,oneoftype4,andthreeoftype5.(D)TwelvehexonmonomersexhibitsixtypesofC-terminalextension:twoeachoftypesa,b,candd,threeoftypee,andoneoftypef.Ribbonmodelssuperimposedondensity(mesh)ofthesesixtypesareshowninfig.S8A.,10,21:58:31,Fig.5.Schematicillustrationsofinteractionsamongminorandmajorproteins.Interactionsaremarkedhere,numberedinfig.S10,andlistedintableS3.(A)Contactsontheinnersurfaceofthecapsid.LettersatofdenotethepositionsofsixtypesofhexonC-extensions.Ateachvertex,fivecopiesofproteinIIIalinkfiveperipentonalhexontrimersandapenton-basepentamertomakeaGOStile(lightblueshading).ProteinVIIImediatesbindingamonghexons,linksGONtiles(grayshading)toGONtiles,andlinksGONtilestoGOStiles.(B)Contactsontheoutersurfaceamongthefourtypesofhexontrimer(H1,H2,H3,andH4)andthefourtypesofproteinIXmonomer(red,green,yellow,andblue)thatareinlaidintothecanyonsatthebordersbetweenhexons.ProteinIXlasheshexonstogethertoformGONtilesandalsolinksGONtiles.,11,21:58:31,第一节病毒粒子的装配,装配方式：完全自发或部分自发-自我装配(self-assembly)核酸和结构蛋白是经过自我装配而生成完整的病毒粒子。如TMV等轴植物病毒RNA噬菌体一些较复杂的病毒粒子也可.,12,21:58:31,第一节病毒粒子的装配,TMV的蛋白质和核酸组分pH7左右的稀盐溶液中混合,数分钟完整TMV病毒粒子:具有侵染性与天然病毒粒子无区别,13,21:58:31,指导装配,需要形态发生因子:病毒基因组编码非结构蛋白,形态发生因子有2种功能：,脚手架蛋白具有瞬时功能，但不结合入病毒结构中。一旦成熟，脚手架蛋白便从病毒结构中除去.被水解或参与另一轮的病毒装配,14,21:58:31,具有蛋白水解的切割作用。蛋白质切割是指导病毒壳体装配的另一种方式。大的前体蛋白经过切割加工，装配，一般认为，这种广泛发生于病毒装配过程中的蛋白质的翻译后切割作用，能够稳定装配步骤。,15,21:58:31,在DNA病毒中病毒DNA的合成早于壳体蛋白的合成，有的病毒要早数小时,两个步骤明显分开。裸露的二十面体RNA病毒的壳体形成和壳体与核酸的结合几乎是同时进行的。可能进化得来，防止降解。,指导装配,16,21:58:31,第一节病毒粒子的装配,病毒粒子的装配过程因病毒不同而异。TMV核酸基因组和蛋白质亚基之间自始至终存在着密切的相互作用，这是TMV自我装配的典型特征。,17,21:58:31,第一节病毒粒子的装配,球形病毒，以蛋白质亚基组装成中空的蛋白质外壳，然后再由病毒核酸基因组填入其中，形成完整的病毒粒子。如脊髓灰质炎病毒许多结构复杂的病毒粒子，装配更为复杂。,18,21:58:31,无包膜病毒装配分2个过程：壳体装配壳体与病毒核酸结合有包膜病毒，在核衣壳外包被病毒包膜。,19,21:58:31,TMV的装配：,20,21:58:31,21,21:58:31,前体池,达到一临界值,22,21:58:31,第二节病毒粒子的释放,一、定义：病毒粒子在细胞内