EffectsofrecombinantbaculovirusAcMNPV

EffectsofrecombinantbaculovirusAcMNPV-BmKITontheformationofearlycablesandnuclearpolymerizationofactininSf9cells重组杆状病毒AcMNPV-BmKIT对Sf9细胞肌动蛋白早期电缆形成和的核聚合反应的作用AbstractAutographacalifornicanuclearpolyhedrosisvirus(AcMNPV)isoneofthemostimportantbaculoviridae.However,theapplicationofAcMNPVasabiocontrolagenthasbeenlimited.Previously,weengineeredButhusmartensiiKarschinsecttoxin(BmKIT)geneintothegenomeofAcMNPV.ThebioassaydataindicatedthattherecombinantbaculovirusAcMNPV-BmKITsignificantlyenhancedtheanti-insectefficacyofthevirus.Theactincytoskeletonisthemajorcomponentbeneaththesurfaceofeukaryoticcells.Inthisreport,theeffectsofAcMNPV-BmKITontheformationofearlycablesofactinandnuclearfilamentous-actin(F-actin)werestudied.Theresultsindicatedthatthesebaculovirusinducedrearrangementoftheactincytoskeletonofhostcellsduringinfectionandactinmightparticipateinthetransportationofbaculovirusfromcytoplasmtothenuclei.AcMNPV-BmKITdelayedtheformationofearlycablesofactinandnuclearF-actinandacceleratedtheclearanceofactininthenuclei.IntroductionTheactincytoskeletonisthemajorcomponentbeneaththesurfaceofeukaryoticcells.Manybacterialandviralpathogensinducerearrangementoftheactincytoskeletonofhostcellsduringinfection.Thismaybeassociatedwithviralgenomereplication,transcription,virusassembly,mobility,orbudding(Cudmoreetal.1997).Severalviruseshavebeenshowntointeractwiththecytoskeletonornuclearmatrixintheirreplication,suchvariedactivitiesasgenomereplication,proteinsynthesisandtransport,andassembly,transport,andreleaseof摘要苜蓿银蚊夜蛾多角体病毒(AcMNPV)是一种最重要的杆状病毒。但是，将AcMNPV用作一种生物介质却很有限。之前，我们建立了一种钳蝎属病毒(BmKIT)基因插入到AcMNPV基因组中。活体鉴定数据表明杆状病毒重组体AcMNPV-BmKIT显著增强了昆虫的抗病毒性。肌动蛋白是细胞骨架下层主要组分。本文研究了AcMNPV-BmKIT对肌纤蛋白和核内肌动蛋白微丝早期电缆形成的作用。结果显示感染后，这些重组杆状病毒诱导了宿主细胞肌动蛋白骨架发生重排，而肌动蛋白可能参与转运杆状病毒进入细胞核。AcMNPV-BmKIT延缓了肌动蛋白和核内肌动蛋白微丝早期电缆的形成，加速了核内肌动蛋白的清除。引言肌动蛋白细胞骨架是真核细胞下层主要组分。许多杆状病毒和病毒病原体感染宿主细胞，诱导肌动蛋白细胞骨架发生重排。这可能与病毒基因组复制，转录，病毒装配，迁移，芽殖有关。已经发现多种病毒复制与细胞骨架或核基质发生相互作用，其他多种活动如基因组复制，蛋白质合成和运输，装配，装运，病毒粒子释放都与这种结构有关。virionshavebeenassociatedwiththesestructures.Aseriesofpreciselycontrolledactincytoskeletonrearrangementshavebeenreportedtooccurduringthebaculovirusreplicationcycle:shortlyafterthereleaseofviralnucleocapsidsintothecytoplasmfromendosomes,filamentous-actin(F-actin)cablesareformedinthecytoplasm;then,afternucleocapsidpenetrationintothenucleus,F-actincablesarereorganized;later,granular-actin(G-actin)monomersaredriventoaccumulateinthenucleusandarepolymerizedintoF-actin(KasmanandVolkman2000;OhkawaandVolkman1999).ThecytoplasmicF-actincablesarepossiblyinvolvedinfacilitatingnucleocapsidpenetrationintothenucleus,whereasnuclearactinpolymerizationisessentialforviralnucleocapsidmorphogenesis(Lietal.2010).Scorpionsusetheirvenomasaweapontohuntandforself-defense.Theeffectivecomponentsofthevenomareagroupofproteinscomposedof30–80aminoacidswhichpossessneurotoxicity(Moskowitzetal.1998).Scorpionvenomcontainsphopholipase,mucopolysaccharides,hyaluronidase,lowrelativemolecularmassmoleculeslikehistamineandserotonin,proteaseinhibitors,histaminereleasersandpolypeptidylcompoundsandionchannelsarethemaintargetsforvariousneurotoxins(CesteleandCatterall2000).Thetoxinsclasscanbeseparatedintotwoclasses:toxinsactiveonmammals(MaTx)andtoxinsactiveoninsects(ITx;Gordonetal.1998).Excitatoryinsecttoxinsactspecificallyoninsectsandinduceafastexcitatorycontractionparalysisuponinjection.Depressantinsecttoxinsinduceaslowdepressantandflaccidparalysisprecededbyashorttransientphaseofcontracture(Gurevitzetal.1998).ButhusmartensiiKarsch(BmK)isakindofscorpionswhichvenomcontainsexcitatoryinsecttoxins(BmKIT).BmKITarepolypeptidesof69–72aminoacidresidues,actingspecificallyoninsectsandinduceafastexcitatorycontractionparalysisuponinjection(Goudetetal.2002).TheexpressedBmKITkillscottonbollwormsatveryhighratesandconsiderablyretardslarvaldevel-已有报道，杆状病毒复制循环时，一系列肌动蛋白细胞骨架重排受精确调控：病毒核衣壳从内涵体释放到细胞质后不久，胞质内肌动蛋白微丝电缆形成；之后，核衣壳渗透入胞核，肌动蛋白微丝电缆重新组织；最后，核内粒状肌动蛋白单体逐渐堆积，聚合形成肌动蛋白微丝。细胞质丝状肌动蛋白微丝很可能参与促进病毒核衣壳侵入宿主细胞核，鉴于原子核的肌动蛋白聚合作用对病毒核衣壳形态形成成蝎子用毒液当武器来捕获猎物和保护自身。毒液的有效成分是一组含有30-80个氨基酸组成的，具有神经毒的蛋白质。蝎毒含有磷脂酶，粘多糖，玻璃酸酶，几乎不含相对分子量类似组织胺，血清素，蛋白酶抑制药，组胺释放者和聚合化合物，以及离子通道等，多种神经毒素的主要底物。这种毒素可以分成两类：哺乳动物类(MaTx)和昆虫类(ITx;Gordonetal.1998)。兴奋性昆虫毒素特异性作用于昆虫，注射后引起快速兴奋、收缩、瘫痪。抑制性昆虫毒素在短暂的痉挛之后引起镇静、不完全麻痹。东亚钳蝎(BmK)是蝎子的一种，它的毒液含有兴奋性昆虫毒素(BmKIT)。BmKIT是含69–72个氨基酸残基的聚合物，特异性作用于昆虫，注射后引起快速兴奋、收缩、瘫痪。表达BmKIT杀灭棉铃虫的效率非常高，能有效抑制幼虫发育，毒素含量高，以及蝎毒对昆虫严格的选择性，使之成为理想的生物杀虫剂。opment(Haoetal.2005)andthehightoxicityandstrictinsectselectivityofthisscorpiontoxinmakeitapromisingbiologicalinsecticide.Thebaculoviridaeisalargefamilyofenveloped,rod-shapedviruseswithcircular,doublestranded,supercoiledDNAgenomesrangingfrom100to180kb(Volkmanetal.1995).Autographacalifornicanuclearpolyhedrosisvirus(AcMNPV)isoneofthemostimportantbaculoviridae.Butasforbiocontrolagents,theuseofAcMNPVhasbeenlimited.Theiruseaspesticideshasbeenlesssuccessful,onereasonisthetimerequiredforaninfectedinsecttoceasefeeding.Previously,weengineeredBmKITgeneintothegenomeofAcMNPV.ThebioassaydataindicatedthattherecombinantbaculovirusAcMNPV-BmKITsignificantlyenhancedtheanti-insectefficacyofthevi