摘要
杆状病毒模式种苜蓿丫纹夜蛾核多角体病毒（Autographa californica multiple nucleopolyhedrovirus，AcMNPV）的orf78（即Ac78）是最近被发现的杆状病毒核心基因，在杆状病毒的生活周期中具有重要功能．氨基酸序列分析表明，Ac78的C末端105～108位氨基酸区域在GroupINPVs旁系同源物中高度保守．为研究该保守区域在Ac78功能中的作用，利用Bac—to-Bac杆状病毒表达载体系统成功构建了缺失该保守区域，并且携带绿色荧光蛋白基因和多角体蛋白基因的Ac78截短补回型重组病毒（vAe78：del105．108）．荧光显微镜分析和病毒生长曲线测定结果表明，在vAc78：del105．108转染的Sf9细胞中，感染性的芽生型病毒粒子（buddedvirion，BV）产生量与Ac78全长补回型重组病毒（vAc78：HA）基本一致；电镜观察发现，在vAc78：del105．108转染的细胞中，呈现与vAc78：HA的现象一致的典型的杆状病毒感染特征，多粒包埋型病毒粒子（multiple nucleocapsid—enveloped occlusion-derived virion，M—ODV）以及包埋有M-ODV的包涵体均能正常形成；免疫荧光实验表明，在vAc78：del105．108感染的Sf9细胞中，从病毒感染细胞24h时开始，Ac78专一定位于感染细胞的内核膜附近，与vAc78：HA的现象一致．上述结果表明，Ac78的C末端105-108位氨基酸保守区域对于BV和M—ODV的有效产生以及Ac78的亚细胞定位非必需．
Ac78 of the archetype of the Baculoviridae, Autographa californica multiple nucleopolyhedrovirus （AcMNPV） , is a recently identified baculovirus core gene. It plays an important role in the baculovirus life cycle. An amino acid （aa） alignment of the Ac78 homologs showed that Ac78 C-terminal aa 105 ~ 108 are highly conserved in group I NPVs. To investigate the role of Ac78 aa 105 - 108 in the baculovirus life cycle, an Ac78 gene truncated AcMNPV recombinant （ vAc78 ： de1105-108） , in which the Ac78 cassette without residues 105 - 108, was constructed successfully with Bac-to-Bac system. To examine the effect on occlusion body morphogenesis and to facilitate examination of virus infection, the AcMNPV polyhedrin （Polh） gene and the enhanced green fluorescence protein （Egfp; referred to as Gfp in the present study） gene whose expression was driven by its own promoter and the AcMNPV immediate-early gene Iel promoter, respectively, were also inserted into the recombinant virus. Fluorescence microscopy and titration assay demonstrated that Sf9 cells transfected with vAc78：del105- 108 or vAc78：HA showed a normal and steady increase in virus production and similar virus growth kinetics. Electron microscopy showed that the cells transfected with vAc78：dell05-108 exhibited the typical characteristics of baculovirus infection, which were similar to those of vAc78 ： HA- transfected ceils. Immunofluorescence microscopy showed that, in the nuclei of vAc7$： dell05-10g-infected Sf9 cells, discrete foci of red fluorescence appeared near the inner nuclear membrane at 24 hour post infection （h p. i. ） , and they were condensed at 48 h p. i. , by 72 h p. i. , Ac78 was concentrated within the ring zone and formed larger foci. In summary, we provided the evidence demonstrating that removal of the Ac78 aa 105 - 108 did not affect the productions of budded virions and M-ODVs and the subcellular localization pattern of Ac78 in Sf9 cells. That is, Ac78 aa 105 - 108 is not essential for its role during the baculovirus life cycle.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2015年第7期748-756,共9页
Chinese Journal of Biochemistry and Molecular Biology
基金
广东省自然科学基金资助项目(No.2014A030313664)~~
关键词
苜蓿丫纹夜蛾核多角体病毒
C末端保守区域
芽生型病毒粒子
多粒包埋型病毒粒子
Autographa californica multiple nucleopolyhedrovirus （AcMNPV）
conserved C-terminal region
budded virion （BV）
multiple nucleocapsid-enveloped occlusion-derived virion （M-ODV）
