Bm59基因缺失对家蚕核型多角体病毒(BmNPV)复制、转录及组装的影响
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摘要
Bm59是家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,Bm NPV)的核心基因之一。利用λRed重组技术和Bac-to-Bac系统构建野生型病毒wt Bacmid-polh-egfp、缺失型病毒Bm59ko-Bacmid-polh-egfp以及补回型病毒Bm59re-Bacmidpolh-egfp,并分别转染家蚕卵巢培养细胞Bm N,研究Bm59基因在病毒侵染、增殖和组装中的功能。对病毒滴度的检测结果表明3种类型病毒都能产生有活力的子代病毒并使细胞感染发病,但Bm59ko-Bacmid-polh-egfp在各个时相的滴度值均显著低于其他2种类型病毒(P<0.05)。电子显微镜下观察Bm59ko-Bacmid-polh-egfp转染的细胞中只有少量细长的杆状病毒粒子,而其他2种类型病毒转染细胞后则能产生大量具有囊膜包裹的成熟病毒粒子。qRT-PCR检测结果显示,Bm59缺失型病毒基因组DNA的复制能力显著降低(P<0.05),早期基因lef-3、晚期基因vp39和极晚期基因p10的转录水平显著低于野生型病毒(P<0.05)。综上所述,Bm59基因虽然是家蚕核型多角体病毒复制的非必需基因,但会显著影响病毒的增殖速度和病毒粒子的组装,对病毒各个时期的基因转录水平具有下调作用。
Bm59 gene is one of the core genes of Bombyx mori nucleopolyhedrovirus( Bm NPV). In this study,the λRed recombination system and the Bac-to-Bac system were used to create wild type virus wt Bacmid-polh-egfp,knockout type virus Bm59ko-Bacmid-polh-egfp and repair type virus Bm59re-Bacmid-polh-egfp,all of which were transfected into Bm N cells for exploring function of Bm59 gene in viral infection,replication and assembly. Test results of viral titer indicated that all three type viruses were able to produce viable progeny viruses capable of infecting new cells. However,titer of Bm59ko-Bacmid-polh-egfp was significantly lower than the other two viruses in all phases( P < 0. 05). Observation under transmission electron microscope showed that there were very few thin and long baculoviral particles in cells transfected by Bm59ko-Bacmid-polh-egfp, whereas a great many mature viral particles wrapped in envelops were produced in cells transfected by the other two type viruses. Test result of qRT-PCR revealed that Bm59 deletion had a strong down-regulatory effect on viral DNA replication( P < 0. 05) and significantly reduced the tran-scription levels of early gene lef-3,late gene vp39,and very late gene p10 in comparison to wild type virus( P < 0. 05).These results indicate that Bm59 is a non-essential gene for viral replication of Bm NPV. However,it significantly influences viral reproduction and assembly and has a down-regulatory effect on gene transcription levels in all phases.
Bm59 gene is one of the core genes of Bombyx mori nucleopolyhedrovirus( Bm NPV). In this study,the λRed recombination system and the Bac-to-Bac system were used to create wild type virus wt Bacmid-polh-egfp,knockout type virus Bm59ko-Bacmid-polh-egfp and repair type virus Bm59re-Bacmid-polh-egfp,all of which were transfected into Bm N cells for exploring function of Bm59 gene in viral infection,replication and assembly. Test results of viral titer indicated that all three type viruses were able to produce viable progeny viruses capable of infecting new cells. However,titer of Bm59ko-Bacmid-polh-egfp was significantly lower than the other two viruses in all phases( P < 0. 05). Observation under transmission electron microscope showed that there were very few thin and long baculoviral particles in cells transfected by Bm59ko-Bacmid-polh-egfp, whereas a great many mature viral particles wrapped in envelops were produced in cells transfected by the other two type viruses. Test result of qRT-PCR revealed that Bm59 deletion had a strong down-regulatory effect on viral DNA replication( P < 0. 05) and significantly reduced the tran-scription levels of early gene lef-3,late gene vp39,and very late gene p10 in comparison to wild type virus( P < 0. 05).These results indicate that Bm59 is a non-essential gene for viral replication of Bm NPV. However,it significantly influences viral reproduction and assembly and has a down-regulatory effect on gene transcription levels in all phases.
引文
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[16]史利利,蒋彩英,于威,等.Bm NPV orf98对家蚕核型多角体杆状病毒复制、转录及包装的影响[J].浙江大学学报(农业与生命科学版),2015,41(6):623-630
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[18]袁佳,解纯刚,汪凤梅,等.lef-12基因缺失对家蚕核型多角体病毒(Bm NPV)复制和基因转录的影响[J].蚕业科学,2014,40(3):468-474
[19]ONO C,KAMAGATA T,TAKA H,et al.Phenotypic grouping of141 Bm NPVs lacking viral gene sequences[J].Virus Res,2012,165(2):197-206
[20]KE J H,WANG J W,DENG R Q,et al.Autographa californica multiple nucleopolyhedrovirus ac66 is required for the efficient egress of nucleocapsids from the nucleus,general synthesis of preoccluded virions and occlusion body formation[J].Virology,2008,374(2):421-431
[2]HERNIOU E A,OLSZEWSKI J A,CORY J S,et al.The genome sequence and evolution of baculoviruses[J].Annu Rev Entomol,2003,48:211-234
[3]YU M,CARSTENS E B.Identification of a domain of the baculovirus Autographa californica multiple nucleopolyhedrovirus singlestrand DNA-binding protein LEF-3 essential for viral DNA replication[J].J Virol,2010,84(12):6153-6162
[4]MIKHAILOV V S,ROHRMANN G F.Baculovirus replication factor LEF-1 is a DNA primase[J].J Virol,2002,76(5):2287-2297
[5]FAULKNER P,KUZIO J,WILLIAMS G V,et al.Analysis of p74,a PDV envelope protein of Autographa californica nucleopolyhedrovirus required for occlusion body infectivity in vivo[J].J Gen Virol,1997,78(Pt 12):3091-3100
[6]PENG K,VAN OERS M M,HU Z,et al.Baculovirus per os infectivity factors form a complex on the surface of occlusion-derived virus[J].J Virol,2010,84(18):9497-9504
[7]ZHANG J,DONG Z Q,ZHANG C D,et al.Identification of a novel nuclear localization signal of baculovirus late expression factor 11[J].Virus Res,2014,184(5):111-119
[8]SPARKS W O,HARRISON R L,BONNING B C.Autographa californica multiple nucleopolyhedrovirus ODV-E56 is a per os infectivity factor,but is not essential for binding and fusion of occlusionderived virus to the host midgut[J].Virology,2011,409(1):69-76
[9]GUO Z J,QIU L H,AN S H,et al.Open reading frame 60 of the Bombyx mori nucleopolyhedrovirus plays a role in budded virus production[J].Virus Res,2010,151(2):185-191
[10]TANG Q,LI G,YAO Q,et al.Bm91 is an envelope component of ODV but is dispensable for the propagation of Bombyx mori nucleopolyhedrovirus[J].J Invertebr Pathol,2013,113(1):70-77
[11]GE J Q,YANG Z N,TANG X D,et al.Characterization of a nucleopolyhedrovirus with a deletion of the baculovirus core gene Bm67[J].J Gen Virol,2008,89(Pt 3):766-774
[12]HARRISON R L,LYNN D E.Genomic sequence analysis of a nucleopolyhedrovirus isolated from the diamondback moth,Plutella xylostella[J].Virus Genes,2007,35(3):857-873
[13]WANG R,DENG F,HOU D,et al.Proteomics of the Autographa californica nucleopolyhedrovirus budded virions[J].J Virol,2010,84(14):7233-7242
[14]LI Y,WANG J,DENG R,et al.vlf-1 deletion brought Ac MNPV to defect in nucleocapsid formation[J].Virus Genes,2005,31(3):275-284
[15]BIDESHI D K,FEDERICI B A.The Trichoplusia ni granulovirus helicase is unable to support replication of Autographa californica multicapsid nucleopolyhedrovirus in cells and larvae of T.ni[J].J Gen Virol,2000,81(Pt 6):1593-1599
[16]史利利,蒋彩英,于威,等.Bm NPV orf98对家蚕核型多角体杆状病毒复制、转录及包装的影响[J].浙江大学学报(农业与生命科学版),2015,41(6):623-630
[17]WU W,LIN T,PAN L,et al.Autographa californica multiple nucleopolyhedrovirus nucleocapsid assembly is interrupted upon deletion of the 38K gene[J].J Virol,2006,80(23):11475-11485
[18]袁佳,解纯刚,汪凤梅,等.lef-12基因缺失对家蚕核型多角体病毒(Bm NPV)复制和基因转录的影响[J].蚕业科学,2014,40(3):468-474
[19]ONO C,KAMAGATA T,TAKA H,et al.Phenotypic grouping of141 Bm NPVs lacking viral gene sequences[J].Virus Res,2012,165(2):197-206
[20]KE J H,WANG J W,DENG R Q,et al.Autographa californica multiple nucleopolyhedrovirus ac66 is required for the efficient egress of nucleocapsids from the nucleus,general synthesis of preoccluded virions and occlusion body formation[J].Virology,2008,374(2):421-431