杆状病毒Ac78 C末端保守区域的功能初步分析

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Group I NPVs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(vAc78: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(budded virion,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24 h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.

Functional Analysis of the C-terminal Conserved Region in Baculovirus Ac78

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:del105-108), 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 Ie1 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:del105-108 exhibited the typical characteristics of baculovirus infection, which were similar to those of vAc78:HA-transfected cells. Immunofluorescence microscopy showed that, in the nuclei of vAc78:del105-108-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.