甜菜夜蛾核多角体病毒VP39蛋白在苜蓿丫纹夜蛾核多角体病毒中的功能研究

Functional analysis of Spodoptera exigua multiple nucleopo lyhedrovirus VP39 in Autographa californica MNPV

【目的】构建表达甜菜夜蛾核多角体病毒(Spodoptera exigua multiple nucleopolyhedrovirus,SeMNPV)VP39蛋白的vp39假型苜蓿丫纹夜蛾核多角体病毒(Autographa californica MNPV,AcMNPV),明确SeMNPV VP39是否能取代AcMNPV VP39在AcMNPV中行使功能,为深入探究杆状病毒的核衣壳装配机理打下理论基础。【方法】利用Bac-to-Bac杆状病毒表达载体系统在AcMNPV vp39缺失型重组bacmid(bAcvp39KO)的基础上构建携带SeMNPV vp39基因、绿色荧光蛋白基因(Enhanced green fluorescence protein,egfp)和多角体蛋白基因(Polyhedrin,polh)的重组病毒(vAcSevp39:FLAG)。利用Western blotting检测分析SeMNPV vp39在vAcSevp39:FLAG转染的草地贪夜蛾(Spodoptera frugiperda)IPLB-Sf21-AE clonal isolate 9(Sf9)细胞中的表达情况,通过荧光显微镜观察vAcSevp39:FLAG在Sf9中的感染和扩散情况,采用病毒滴度测定并绘制病毒生长曲线检测vAcSevp39:FLAG的感染性芽生型病毒粒子产量,并以电子显微镜观察vAcSevp39:FLAG转染的Sf9细胞中的形态发生情况。【结果】Western blotting检测结果表明,SeMNPV VP39能在vAcSevp39:FLAG转染的Sf9细胞中获得表达;荧光显微镜观察和病毒生长曲线测定结果表明,在vAcSevp39:FLAG转染的Sf9细胞中无感染性的芽生型病毒粒子产生,与AcMNPV vp39缺失型重组病毒(vAcvp39KO)的现象一致;电子显微镜观察发现,与vAcvp39KO不同的是,在vAcSevp39:FLAG转染的细胞中虽然未产生核衣壳,但在感染细胞的核中存在大量空的透明长管状衣壳结构,表明SeMNPV VP39能挽救vAcvp39KO的衣壳结构装配,但在AcMNPV中不具备装配核衣壳的能力。【结论】SeMNPV VP39在AcMNPV中虽能形成衣壳结构,但不能有效装配核衣壳,导致无芽生型病毒粒子和包埋型病毒粒子产生。

【Objective】A vp39 pseudotyped Autographa californica multiple nucleopolyhedrovirus(AcMNPV)in which AcMNPV vp39 was replaced with Spodoptera exigua MNPV(SeMNPV)VP39 was constructed to determine whether SeMNPV VP39 could replace AcMNPV VP39 in AcMNPV.This study laid a theoretical foundation for further exploring the nucleocapsid assembly mechanism of baculovirus.【Method】Via Tn7-mediated transposition,the SeMNPV vp39 gene under the control of the AcMNPV vp39 promoter with a FLAG tag prior to the SeMNPV vp39 stop codon,to-gether with the enhanced green fluorescence protein(egfp)and polyhedrin(polh)genes,was inserted into the AcMNPV vp39 knockout bacmid(bAcvp39KO)polh locus to construct vp39 pseudotyped AcMNPV(vAcSevp39:FLAG).Western blotting was performed to determine whether SeMNPV vp39 was expressed in Spodoptera frugiperda IPLB-Sf21-AE clonal isolate 9(Sf9)cells transfected with vAcSevp39:FLAG.The viral replication and infection in Sf9 cells transfected with vAcSevp39:FLAG were monitored using a fluorescence microscope.Viral growth curve analysis was performed by 50%tissue culture infective dose(TCID50)endpoint dilution assay to further evaluate the ability of vAcSevp39:FLAG to pro-duce infectious budded virion(BV).Thin section of vAcSevp39:FLAG-transfected Sf9 cells was observed using an elec-tron microscope to investigate the virus morphogenesis.【Result】Western blotting showed that SeMNPV VP39 was detected in Sf9 cells transfected with vAcSevp39:FLAG.Fluorescence microscopy and viral growth curve analysis showed that no infectious BVs were produced in vAcSevp39:FLAG-transfected Sf9 cells,which was consistent to AcMNPV vp39 knockout recombinant virus(vAcvp39KO).Electron microscopy demonstrated that,different from vAcvp39KO,in Sf9 cells transfected with vAcSevp39:FLAG,masses of abnormally elongated empty capsid structures were observed inside the nuclei,but no nucleocapsids formed.【Conclusion】SeMNPV VP39 has the ability to assemble tubular capsid-like structures but not nucleocapsids in AcMNPV,resulting in no BV and occlusion-derived virion produced.