[目的]家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,BmNPV)是生产上危害最严重的病原之一。BmNPV感染BmN-SWU1细胞将细胞周期阻滞于G2/M期。CyclinB是调控细胞周期G2期向M期转换的重要细胞周期蛋白。因此,研究BmNPV感染后CyclinB变化对解析病毒调控细胞周期的机制具有重要意义,同时探究这个过程中与CyclinB互作的病毒蛋白,可为构建家蚕转基因品系提供分子靶标。[方法]qRT-PCR检测BmNPV感染后BmCyclinB的表达变化;免疫荧光观察病毒感染前后BmCyclinB的定位变化,通过细胞质细胞核蛋白分离实验验证。免疫共沉淀钓取与BmCyclinB互作的病毒蛋白。BmNPV感染期间敲除BmNPV IAP1观察BmCyclinB的入核比例。[结果]BmNPV感染后BmCyclinB转录水平下调。BmNPV感染前BmCyclinB主要定位于细胞质,而感染后主要定位于细胞核。BmNPV感染BmN-SWU1细胞后促进BmCyclinB在核内积累。共钓取了7个与BmCyclinB互作的病毒蛋白,免疫共沉淀和细胞共定位证明BmNPV IAP1与BmCyclinB之间存在相互作用。敲除BmNPV IAP1后BmCyclinB进入细胞核的数量显著减少。[结论]BmNPV IAP1可通过与BmCyclinB互作,促进BmCyclinB在核内积累。
[Objective]Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the most serious pathogens in sericulture.BmNPV infection blocked the cell cycle at G2/M phase in BmN-SWU1 cells.CyclinB is an important cyclin that regulates the transition from G2 phase to M phase of cell cycle.Therefore,studying the changes of CyclinB after BmNPV infection is of great significance to elucidate the mechanism of viral regulation of cell cycle and meanwhile,exploring the viral proteins interacting with CyclinB during this process can provide molecular targets for the construction of transgenic strains of Bombyx mori.[Methods] Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of BmCyclinB after BmNPV infection.The localization change of BmCyclinB after viral infection was observed by immunofluorescence and verified by cytoplasmic and nuclear protein separation experiment.Co-immunoprecipitation was employed to catch the viral proteins that interacted with BmCyclinB.The proportion of BmCyclinB into the nucleus was observed by knocking down BmNPV IAP1 after BmNPV infection.[Results]The transcription level of BmCyclinB was down-regulated after BmNPV infection and BmCyclinB was mainly located in cytoplasm before BmNPV infection and in nucleus after infection.BmNPV infection of BmN-SWU1 cells promoted the nuclear accumulation of BmCyclinB.A total of 7 viral proteins interacting with BmCyclinB were identified.The interaction between BmNPV IAP1 and BmCyclinB was confirmed by co-immunoprecipitation and cell co-localization.The amount of BmCyclinB entering the nucleus was significantly reduced after BmNPVIAP1 was knocked out during BmNPV infection. [Conclusion]BmNPV IAP1 can promote the accumulation of BmCyclinB in the nucleus by interacting with BmCyclinB.