杨扇舟蛾颗粒体病毒经口侵染因子PIF0～PIF6间分子互作的酵母双杂交验证
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摘要
为阐明杨扇舟蛾颗粒体病毒(Clostera anachoreta granulovirus,ClanGV)的口服侵染机制,利用酵母双杂交技术,通过双向互作方法研究了ClanGV的经口侵染因子PIF0、PIF1、PIF2、PIF3、PIF4、PIF5和PIF6之间的分子互作情况。自激活试验证明,PIF5的重组诱饵载体(B5)有自激活现象,其它重组诱饵载体均无自激活现象。酵母双杂交试验结果表明,ClanGV的4个经口侵染因子PIF1～PIF4之间存在双向互作现象,而且这4个蛋白均能同时与自身发生互作,暗示这4个经口侵染因子有可能是以二聚体或多聚体的形式存在并发挥功能。在ClanGV经口侵染因子间的6组单向互作中,PIF5作为捕获载体时分别能与PIF1、PIF3和PIF4发生互作,PIF6作为诱饵载体时分别能与PIF3和PIF4发生互作,PIF0作为捕获载体时能与PIF4发生互作。表明ClanGV的经口侵染因子像核型多角体病毒(nucleopolyhedrovirus,NPV)一样,在病毒粒子表面通过分子互作形成复合体并在病毒的经口侵染过程中发挥功能。
In order to elucidate the mechanism of oral infection of Clostera anachoreta granulovirus(ClanGV), the protein interactions of per os infectivity factors(PIFs) of ClanGV(PIF0, PIF1, PIF2,PIF3, PIF4, PIF5 and PIF6) were studied by yeast two-hybrid technique. Self-activation experiments demonstrated that only the recombinant bait vector(B5) of PIF5 had self-activation in this study. The results of yeast two-hybrid indicated that PIF1, PIF2, PIF3 and PIF4 of ClanGV had two-way interactions between any two proteins; these proteins could interact with itself, which suggested that these PIFs might be present with dimers or polymers in complex. In addition, this study also explained six groups of unidirectional interactions between ClanGV PIFs: as a trap vector, PIF5 could interact with PIF1,PIF3 and PIF4, respectively; as a bait vector, PIF6 had interactions with PIF3 and PIF4, respectively,and as a trap vector, PIF0 could interact with PIF4. This study indicated that the PIFs formed a complex on the surface of ClanGV virions by molecular interactions and played an important role in the course of oral infection, which was similar to nucleopolyhedrovirus(NPV).
In order to elucidate the mechanism of oral infection of Clostera anachoreta granulovirus(ClanGV), the protein interactions of per os infectivity factors(PIFs) of ClanGV(PIF0, PIF1, PIF2,PIF3, PIF4, PIF5 and PIF6) were studied by yeast two-hybrid technique. Self-activation experiments demonstrated that only the recombinant bait vector(B5) of PIF5 had self-activation in this study. The results of yeast two-hybrid indicated that PIF1, PIF2, PIF3 and PIF4 of ClanGV had two-way interactions between any two proteins; these proteins could interact with itself, which suggested that these PIFs might be present with dimers or polymers in complex. In addition, this study also explained six groups of unidirectional interactions between ClanGV PIFs: as a trap vector, PIF5 could interact with PIF1,PIF3 and PIF4, respectively; as a bait vector, PIF6 had interactions with PIF3 and PIF4, respectively,and as a trap vector, PIF0 could interact with PIF4. This study indicated that the PIFs formed a complex on the surface of ClanGV virions by molecular interactions and played an important role in the course of oral infection, which was similar to nucleopolyhedrovirus(NPV).
引文
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Faulkner P,Kuzio J,Williams GV,Wilson JA.1997.Analysis of p74,a PDV envelope protein of Autographa californica nucleopolyhedrovirus required for occlusion body infectivity in vivo.Journal of General Virology,78(12):3091-3100
Haas-Stapleton EJ,Washburn JO,Volkman LE.2004.P74 mediates specific binding of Autographa californica M nucleopolyhedrovirus occlusion-derived virus to primary cellular targets in the midgut epithelia of Heliothis virescens larvae.Journal of Virology,78(13):6786-6791
Horton HM,Burand JP.1993.Saturable attachment sites for polyhedron derived baculovirus on insect cells and evidence for entry via direct membrane fusion.Journal of Virology,67(4):1860-1868
Huang ZH,PanMJ,Zhu SL,Zhang H,Wu WB,Yuan MJ,Yang K.2017.The Autographa californica multiple nucleopolyhedrovirus ac83 gene contains a cis-acting element that is essential for nucleocapsid assembly.Journal of Virology,91(5):e02110-16
Kikhno I,Gutiérrez S,Croizier L,Croizier G,Ferber ML.2002.Characterization of pif,a gene required for the per os infectivity of Spodoptera littoralis nucleopolyhedrovirus.Journal of General Virology,83(12):3013-3022
Kuzio J,Jaques R,Faulkner P.1989.Identification of p74,a gene essential for virulence of baculovirus occlusion bodies.Virology,173(2):759-763
Lepore LS,Roelvink PR,Granados RR.1996.Enhancin,the granulosis virus protein that facilitates nucleopolyhedrovirus(NPV)infections,is a metalloprotease.Journal of Invertebrate Pathology,68(2):131-140
Li X,Song J,Jiang T,Liang C,Chen X.2007.The N-terminal hydrophobic sequence of Autographa californica nucleopolyhedrovirus PIF-3 is essential for oral infection.Archives of Virology,152(10):1851-1855
Luo SJ,Zhang YF,Xu XS,Westenberg M,Vlak JM,Wang HL,Hu ZH,Deng F.2011.Helicoverpa armigera nucleopolyhedrovirus occlusion-derived virus-associated protein,HA100,affects oral infectivity in vivo but not virus replication in vitro.Journal of General Virology,92(6):1324-1331
Ohkawa T,Washburn JO,Sitapara R,Sid E,Volkman LE.2005.Specific binding of Autographa californica M nucleopolyhedrovirus occlusion-derived virus to midgut cells of Heliothis virescens larvae is mediated by products of pif genes Ac119 and Ac022 but not by Ac115.Journal of Virology,79(24):15258-15264
Peng K,van Lent JW,Boeren S,Fang M,Theilmann DA,Erlandson MA,Vlak JM,van Oers MM.2012.Characterization of novel components of the baculovirus per os infectivity factor complex.Journal of Virology,86(9):4981-4988
Peng K,van Oers MM,Hu Z.2010.Baculovirus per os infectivity factors form a complex on the surface of occlusion-derived virus.Journal of Virology,84(18):9497-9504
Szewczyk B,Hoyos-Carvajal L,Paluszek M,Skrzecz I,de Souza ML.2006.Baculoviruses:re-emerging biopesticides.Biotechnology Advances,24(2):143-160
Volkman LE.2007.Baculovirus infectivity and the actin cytoskeleton.Current Drug Targets,8(10):1075-1083
Wang RR,Deng F,Hou DH,Zhao Y,Guo L,Wang HL,Hu ZH.2010.Proteomics of the Autographa californica nucleopolyhedrovirus budded virions.Journal of Virology,84(14):7233-7242
Xiang XW,Chen L,Guo AQ,Yu SF,Yang R,Wu XF.2011.The Bombyx mori nucleopolyhedrovirus(BmNPV)ODV-E56 envelope protein is also a per os infectivity factor.Virus Research,155(1):69-75
Yang L,Zhou GY,Liang J,Ni Y,LüQ,Zhang XY.2014.Isolation,screening and identification of the endogenous microorganisms antagonizing poplar canker.Journal of Plant Protection,41(4):438-446(in Chinese)[杨蕾,周国英,梁军,倪杨,吕全,张星耀.2014.防治杨树溃疡病内生菌的分离筛选及鉴定.植物保护学报,41(4):438-446]
Zhang CL,Ji XM,Pan SQ,Che QM.1992.Experiment on large-scale control of Clostera anachoreta by granulovirus.Journal of Jilin Forestry Science and Technology,(6):33-34,40(in Chinese)[张春龙,纪晓明,潘淑琴,车秋明.1992.颗粒体病毒大面积防治杨扇舟蛾试验.吉林林业科技,(6):33-34,40]
Zhang XX,Liang ZP,Yin XM,Shao XF.2015.Proteomic analysis of the occlusion derived virus of Clostera anachoreta granulovirus.Journal of General Virology,96(8):2394-404
Zhang ZX.2012.Some changes about the virus classification system in ICTV ninth report.Chinese Journal of Virology,28(5):595-599(in Chinese)[张忠信.2012.ICTV第九次报告对病毒分类系统的一些修改.病毒学报,28(5):595-599]
Fang MG,Nie YC,Wang Q,Deng F,Wang RR,Wang HZ,Wang HL,Vlak JM,Chen XW,Hu ZH.2006.Open reading frame 132 of Heliocoverpa armigera nucleopolyhedrovirus encodes a functional per os infectivity factor(PIF-2).Journal of General Virology,87(9):2563-2569
Faulkner P,Kuzio J,Williams GV,Wilson JA.1997.Analysis of p74,a PDV envelope protein of Autographa californica nucleopolyhedrovirus required for occlusion body infectivity in vivo.Journal of General Virology,78(12):3091-3100
Haas-Stapleton EJ,Washburn JO,Volkman LE.2004.P74 mediates specific binding of Autographa californica M nucleopolyhedrovirus occlusion-derived virus to primary cellular targets in the midgut epithelia of Heliothis virescens larvae.Journal of Virology,78(13):6786-6791
Horton HM,Burand JP.1993.Saturable attachment sites for polyhedron derived baculovirus on insect cells and evidence for entry via direct membrane fusion.Journal of Virology,67(4):1860-1868
Huang ZH,PanMJ,Zhu SL,Zhang H,Wu WB,Yuan MJ,Yang K.2017.The Autographa californica multiple nucleopolyhedrovirus ac83 gene contains a cis-acting element that is essential for nucleocapsid assembly.Journal of Virology,91(5):e02110-16
Kikhno I,Gutiérrez S,Croizier L,Croizier G,Ferber ML.2002.Characterization of pif,a gene required for the per os infectivity of Spodoptera littoralis nucleopolyhedrovirus.Journal of General Virology,83(12):3013-3022
Kuzio J,Jaques R,Faulkner P.1989.Identification of p74,a gene essential for virulence of baculovirus occlusion bodies.Virology,173(2):759-763
Lepore LS,Roelvink PR,Granados RR.1996.Enhancin,the granulosis virus protein that facilitates nucleopolyhedrovirus(NPV)infections,is a metalloprotease.Journal of Invertebrate Pathology,68(2):131-140
Li X,Song J,Jiang T,Liang C,Chen X.2007.The N-terminal hydrophobic sequence of Autographa californica nucleopolyhedrovirus PIF-3 is essential for oral infection.Archives of Virology,152(10):1851-1855
Luo SJ,Zhang YF,Xu XS,Westenberg M,Vlak JM,Wang HL,Hu ZH,Deng F.2011.Helicoverpa armigera nucleopolyhedrovirus occlusion-derived virus-associated protein,HA100,affects oral infectivity in vivo but not virus replication in vitro.Journal of General Virology,92(6):1324-1331
Ohkawa T,Washburn JO,Sitapara R,Sid E,Volkman LE.2005.Specific binding of Autographa californica M nucleopolyhedrovirus occlusion-derived virus to midgut cells of Heliothis virescens larvae is mediated by products of pif genes Ac119 and Ac022 but not by Ac115.Journal of Virology,79(24):15258-15264
Peng K,van Lent JW,Boeren S,Fang M,Theilmann DA,Erlandson MA,Vlak JM,van Oers MM.2012.Characterization of novel components of the baculovirus per os infectivity factor complex.Journal of Virology,86(9):4981-4988
Peng K,van Oers MM,Hu Z.2010.Baculovirus per os infectivity factors form a complex on the surface of occlusion-derived virus.Journal of Virology,84(18):9497-9504
Szewczyk B,Hoyos-Carvajal L,Paluszek M,Skrzecz I,de Souza ML.2006.Baculoviruses:re-emerging biopesticides.Biotechnology Advances,24(2):143-160
Volkman LE.2007.Baculovirus infectivity and the actin cytoskeleton.Current Drug Targets,8(10):1075-1083
Wang RR,Deng F,Hou DH,Zhao Y,Guo L,Wang HL,Hu ZH.2010.Proteomics of the Autographa californica nucleopolyhedrovirus budded virions.Journal of Virology,84(14):7233-7242
Xiang XW,Chen L,Guo AQ,Yu SF,Yang R,Wu XF.2011.The Bombyx mori nucleopolyhedrovirus(BmNPV)ODV-E56 envelope protein is also a per os infectivity factor.Virus Research,155(1):69-75
Yang L,Zhou GY,Liang J,Ni Y,LüQ,Zhang XY.2014.Isolation,screening and identification of the endogenous microorganisms antagonizing poplar canker.Journal of Plant Protection,41(4):438-446(in Chinese)[杨蕾,周国英,梁军,倪杨,吕全,张星耀.2014.防治杨树溃疡病内生菌的分离筛选及鉴定.植物保护学报,41(4):438-446]
Zhang CL,Ji XM,Pan SQ,Che QM.1992.Experiment on large-scale control of Clostera anachoreta by granulovirus.Journal of Jilin Forestry Science and Technology,(6):33-34,40(in Chinese)[张春龙,纪晓明,潘淑琴,车秋明.1992.颗粒体病毒大面积防治杨扇舟蛾试验.吉林林业科技,(6):33-34,40]
Zhang XX,Liang ZP,Yin XM,Shao XF.2015.Proteomic analysis of the occlusion derived virus of Clostera anachoreta granulovirus.Journal of General Virology,96(8):2394-404
Zhang ZX.2012.Some changes about the virus classification system in ICTV ninth report.Chinese Journal of Virology,28(5):595-599(in Chinese)[张忠信.2012.ICTV第九次报告对病毒分类系统的一些修改.病毒学报,28(5):595-599]