LEF-6乙酰化修饰在家蚕核型多角体病毒侵染过程中的调控作用

Study on Regulatory Function of LEF-6 Acetylation Modification During the Infection Process of Bombyx mori Nucleopolyhedrovirus

晚期表达因子6(late expression factor 6,LEF-6)是家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,BmNPV)复制的非必需因子,能促进病毒晚期基因的转录,提高病毒粒子的产量。BmNPV感染家蚕细胞前后的蛋白质乙酰化修饰差异组学结果显示,在病毒侵染过程中,LEF-6有2个赖氨酸残基(K85和K94)发生了乙酰化修饰。为了深入探究LEF-6乙酰化修饰在病毒侵染过程中的调控机制,利用Red重组技术和Bac-to-Bac系统分别构建lef-6敲除型(lef-6-KO-Bacmid)和补回型(lef-6-RE-Bacmid)的病毒,再利用定点突变技术将赖氨酸(K)突变为谷氨酰胺(Q)模拟乙酰化修饰,将赖氨酸(K)突变为精氨酸(R)模拟去乙酰化修饰,利用实时荧光定量PCR(qPCR)技术分析病毒基因组的复制情况。结果发现lef-6-KO-Bacmid型病毒基因组拷贝数显著下降,2个赖氨酸位点的乙酰化修饰对病毒基因组复制也具有显著抑制作用(P<0.01),而去乙酰化修饰没有显著影响;病毒增殖能力检测和滴度分析结果也显示乙酰化修饰可以显著抑制病毒活力,降低子代病毒的产量;LEF-6乙酰化修饰对病毒晚期基因vp39转录具有显著抑制作用(P<0.01);利用激光共聚焦显微镜技术分析LEF-6的亚细胞定位,发现乙酰化修饰后该蛋白主要定位在细胞质中。研究结果将为深入了解BmNPV与家蚕细胞之间的互作调控机制奠定基础。

Late expression factor 6(LEF-6)is a non-essential factor for the replication of Bombyx mori nucleopolyhedrovirus(BmNPV),which can promote the transcription of late viral genes and increase the production of virus particles.Results from protein acetylation modification differential omics before and after the BmNPV infection showed that two lysine(K)residues(K85 and K94)of LEF-6 can be acetylated during viral infection.To explore the regulatory mechanism of LEF-6 acetylation modification in the process of virus infection,lef-6 knock-out(lef-6-KO-Bacmid)and repaired(lef-6-RE-Bacmid)Bacmid were constructed using Red recombination technology and Bac-to-Bac system.Then,lysine(K)was mutated into glutamine(Q)to stimulate acetylation modification,and lysine(K)was mutated into arginine(R)to simulate deacetylation modification by means of site-directed mutagenesis.Afterwards,real-time PCR(qPCR)was used to analyze the replication of viral genome.It was found that copy number of viral genome of lef-6-KO-Bacmid was significantly reduced,and acetylation modifications of two lysine sites also had significant inhibitory effects on the viral genome replication(P<0.01),while deacetylation modification has no significant effect.In addition,test on virus proliferation ability and titer analysis showed that acetylation modification can significantly inhibit virus viability and reduce the yield of progeny viruses.Furthermore,LEF-6 acetylation modification was found to have a significant inhibitory effect on the transcription of late viral gene vp39(P<0.01).According to analysis on LEF-6 subcellular localization by laser confocal microscopy,it was found that LEF-6 protein was mainly located in the cytoplasm after acetylation modification.These results will lay the foundation for in-depth understanding of the regulatory mechanism of interaction between BmNPV and silkworm cells.