苜蓿银纹夜蛾核多角体病毒E25序列突变对其入核定位和芽殖型病毒体增殖的影响  

Effects of Mutations in the Autographa Californica Multiple Nucleopolyhedrovirus E25 on Its Trafficking to Nucleus and Budded Virus Production

作　　者：罗晓春[1] 岳秀丽[1] 黎路林[1] 

机构地区：[1]华中师范大学生命科学学院,遗传调控与整合生物学湖北省重点实验室,武汉430079

出　　处：《病毒学报》2013年第5期535-543,共9页Chinese Journal of Virology

基　　金：国家自然科学基金项目(31170145)

摘　　要：为了认识杆状病毒E25不同区段序列对其入核运输和定位及对病毒复制的作用,用egfp依次替代苜蓿银纹夜蛾核多角体病毒e25不同区段序列或插入其不同位点构建7个e25突变体,分别插入e25缺失型或野生型bacmid构建了14个e25突变型bacmids,用于对Sf9细胞的转染-感染实验。免疫荧光和共聚焦显微镜分析显示,由EGFP替代E25 2～45aa构成的融合蛋白在被转染细胞质中弥散分布;EGFP插入1aa/2aa处构成的融合蛋白沿核膜外侧分布,未入核;EGFP替代46～118aa或插入118aa/119aa处形成的E25-EGFP在细胞核膜内、外侧和核质中呈稀疏点块状分布。EGFP替代119～228aa或插入45/46aa或C-末端的融合蛋白的定位与野生型bacmid转染细胞中的E25相似,呈环状或弥散分布。这些结果显示虽然E25N-端序列对其定向入核运输和膜定位是必需的;但其中间段序列对其入核运输和定位也有一定影响。在正常E25和E25-EGFP突变子同时存在的情况下,不同的E25-EGFP突变子与正常E25呈现共定位特征,显示E25可能以二聚体或多聚体形式存在。在转染-感染实验中,只有兼含正常e25和2～45aa编码序列被替代的e25突变体的bacmid能够产生高感染性芽殖型病毒体(Budded virus,BV);其它e25突变型bacmids只能产生少量或完全不能产生感染性BV,显示E25结构严谨,所有替代和插入突变都导致其功能丧失或异常。This study was performed to investigate the effects of different regions of the Autographa califor- nica multiple nucleopolyhedrovirus envelope protein E25 on its trafficking into nucleus and nuclear localiza- tion in host cells and on virus replication. Fourteen recombinant bacmids, each containing an e25 mutant with substitution or insertion of egfp, in the absence or presence of the native e25, were constructed and used to transfect S f9 cells. The E25-EGFP fusion proteins and native E25 expressed in the cells transfect- ed with individual recombinant bacmid were traced by autofluorescence from EGFP or by immuno-fluores- cence assays. Confocal microscopy revealed that the E25-EGFP fusion protein with the N-domain （2-45aa） of E25 substituted by EGFP only distributed in the cytoplasm in transfected cells~ and the fusion protein with EGFP inserted at the laa/2aa site of E25 completely remained outside of the nucleus and resided along the nuclear membrane. The E25 EGFPs with 46-118aa of E25 substituted by EGFP or with EGFP inserted at the 118aa/119aa site were present outside, across from the nuclear membrane or in nuclear plasm in dot- like shapes. The fusion proteins with the C-domain substituted by EGFP or with EGFP inserted at the site of 45/46aa or at the C-terminal formed a condensed ring or spread throughout the nucleus, in a similar manner to the E25 distributed in the ceils transfected by the e25-knockout repair bacmid. These results prove that the N-terminal domain is critical for nuclear transportation of E25 and possibly to its position on the cytoplasm membrane as well~ and the sequence downstream of the N-terminal domain also affects traf- ficking and nuclear localization of the protein. In cells transfected with bacmids containing both the native e25 and individual e25-egfp mutants, the E25-EGFP fusion proteins co-localized with E25 individually, showing similar patterns of subcellular localization as E25 mutants in the absence of native E25 in most ca- ses, suggesting that the E25 likely exists

关 键 词：杆状病毒 苜蓿银纹夜蛾核多角体病毒 被膜蛋白E25 入核定位 

分 类 号：R373.9[医药卫生—病原生物学]