Characterization of late gene expression factor LEF-11 form Bombyx mori nucleopolyhedrovirus

来源 :中国蚕学会第八届青年学术研讨会 | 被引量 : 0次 | 上传用户:yinleish
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  Baculovirus late expression factors (LEFs) are involved in viral DNA replication and viral gene regulation.Baculovirus lef-11 gene encodes a putative protein with predicted molecular weight about 13KDa.Currently its function remains unclear.Studies have reported that Autographa californica multiple nucleopolyhedrovirus (AcMNPV) LEF-11 localized within the nucleus of Sf9 cells after infection,and genomic knockout experiments have revealed that this protein is involved in viral replication and late/very late gene activation.However,the mechanism by which baculovirus LEF-11 exact function is not yet fully understood.To investigate the role of lef-11 in the BmNPV life cycle,a lef-11 knockout virus (vBmlef11KO) was constructed through homologous recombination in Escherichia coli.To determine the effect of lef-11 knockout on viral propagation,enhanced green fluorescent protein (egfp) genes were inserted into the polyhedrin locus of lef11KO Bacmid by Tn7-mediated trans-position in the Bac-to-Bac system.After transfection these recombinant Bacmids into BmN-SWU1 cells,the lef-11 Knockout bacmid leads to a defect in infectious budded virus production while the lef-11 repair bacmid can rescue this defect and the budded virus titers can reach the wild type levels.RT-qPCR analysis indicated that lef-11 is required for amplification but not initiation of viral DNA replication.To screen and investigate host proteins which interact with BmNPV LEF-11,Coimmunoprecipitation (Co-IP) assay was used to characterize protein candidates interacting with LEF-11 bait in BmN-SWU1 cells infected with BmNPV.Eleven protein candidates were identified by Liquid chromatography tandem-mass spectrometry (LC-MS/MS),including five baculoviral proteins (LEF3,LEF-8,BM21,V-CATH and Chitinase),and six proteins from B.mori (XPD,PP5,CDC48/P97,PP2A,ROCK and Actin).Further bimolecular fluorescence protein complementation assays (BiFC) confirmed the interaction of BmNPV LEF-11 with host PP2A.BmNPV replication was blocked in infected BmN-SWU1 cells in the presence of Okadaic acid,an effective inhibitor of PP2A enzyme activity.These results suggest that BmNPV LEF-11 may regulate PP2A to facilitate virus multiplication.
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