权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Studies on interaction between potyvirus VPg and plant eIF(iso)4E

马铃薯Y病毒属VPg与植物eIF(iso)4E相互作用的研究

基本信息

批准号：
16580031
负责人：
MIYOSHI Hiroshi
金额：
$ 2.37万
依托单位：
St.Marianna-University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16580031/
关键词：
Arabidopsis thaliana Potyvirus translation initiation factor genome-linked protein 翻訳開始因子

项目摘要

I.Expression, purification and crystallization of Arabidopsis thaliana translation initiation factor eIF(iso)4E and turnip mosaic virus (TuMV) genome-linked protein (VPg).Expression and purification of A.thaliana eIF(iso)4E and TuMV VPg were established by using E.coli - pET vector system. Crystallization of A.thaliana eIF(iso)4E and TuMV VPg have been still investigating and three-dimensional of A.thaliana eIF(iso)4E was generated by using the X-ray structure of human eIF4E (Tomoo, K.et al., Biochim Biophys Acta, 2005) as the template.II.Binding analyses for the interaction between plant virus VPg and plant translational initiation factors.Affinity chromatography on m^7GTP-Sepharose showed that bound A.thaliana eIF(iso)4E was eluted with crude TuMV VPg. Further column studies with purified VPg and other A.thaliana eIF4E isoforms showed that VPg preferentially bound eIF(iso)4E. Structural data implicate Trp-46 and Trp-92 in eIF(iso)4E in cap recognition. When Trp-46 or Trp-92 were chan … More ged to Leu, eIF(iso)4E lost the ability to form a complex with both VPg and m^7GTP-Sepharose. This suggests that the VPg-binding site is located in or near the cap-recognition pocket on eIF(iso)4E. Affinity constants for the interactions with eIF(iso)4E of VPg and capped RNA oligomer were determined using surface plasmon resonance (SPR). The K_D values showed that the binging affinity of VPg for eIF(iso)4E is stronger than that of capped RNA. This suggests that viral VPg can interfere with formation of a translational initiation complex on host plant cellular mRNA by sequestering eIF(iso)4E. Further experiments with affinity chromatography showed that VPg forms a ternary complex with eIF(iso)4E and eIF(iso)4G. Thus, VPg may participate in viral translational initiation by functioning as an alternative cap-like structure (Miyoshi, H.et al., Biochimie, 2006).Furthermore, kinetic analysis for the interaction between TuMV VPg and wheat eIF(iso)4E was investigated by fluorescence titration collaborating with Prof.D.J Goss (The City University of New York). Scatchard analysis revealed the binding affinity (K_a) and average binding sites (n) for VPg were 25.14x10^6 M^<-1> and 0.85,respectively. Addition of eIFiso4G to the eIFiso4E increases 1.5 times the binding affinity with VPg as compared to the eIFiso4E alone. However, eIFiso4G alone did not bind with VPg. Addition of m^7G cap to the eIFiso4E-VPg or eIFiso4E-iso4G-VPg complex further increased the binding affinity. The van't Hoff analyses showed that VPg binding is enthalpy-driven and entropy favorable with a large negative ΔH°(-28.34 kJmole^<-1>), and positive ΔS°(38.40 Jmole^<-1>K^<-1>). These results suggest that the VPg binding site is located on or near the cap recognition pocket on eIFiso4E. Further, the formation of a cap binding complex containing VPg suggests the possibility of direct participation of VPg in the translation of the viral genome (ASBMB Annual Meeting 2006). Less

1 .拟南芥翻译起始因子eIF(iso)4E和芜菁花叶病毒（TuMV）基因组连锁蛋白（VPg）的表达、纯化和结晶。利用大肠杆菌- pET载体体系，建立了拟菌eIF(iso)4E和TuMV VPg的表达和纯化。利用人eIF4E的x射线结构（Tomoo， K.et al., biochem Biophys Acta, 2005）为模板，构建了拟沙菌eIF(iso)4E的三维结构（植物病毒VPg与植物翻译启动因子相互作用的结合分析），并对拟沙菌eIF(iso)4E和TuMV VPg进行了研究。m^7GTP-Sepharose亲和层析表明，结合的A.thaliana eIF(iso)4E被粗TuMV VPg洗脱。进一步用纯化的VPg和其他拟菌eIF4E异构体进行柱状研究，发现VPg优先结合eIF(iso)4E。结构数据表明，eIF(iso)4E中的Trp-46和Trp-92在帽识别中起作用。当Trp-46或Trp-92与Leu更紧密结合时，eIF(iso)4E失去了与VPg和m^7GTP-Sepharose形成复合物的能力。这表明vpg结合位点位于eIF(iso)4E上的帽状识别口袋内或附近。利用表面等离子体共振（SPR）测定了VPg和被盖RNA寡聚物与eIF(iso)4E相互作用的亲和常数。K_D值表明VPg对eIF(iso)4E的绑定亲和力比封帽RNA强。这表明病毒VPg可以通过隔离eIF(iso)4E来干扰宿主植物细胞mRNA上翻译起始复合物的形成。进一步的亲和层析实验表明VPg与eIF(iso)4E和eIF(iso)4G形成三元配合物。因此，VPg可能通过替代帽状结构参与病毒翻译起始（Miyoshi， H.et al., Biochimie, 2006）。此外，与d.j. Goss教授（纽约城市大学）合作，利用荧光滴定法研究了TuMV VPg与小麦eIF(iso)4E相互作用的动力学分析。Scatchard分析显示VPg的结合亲和力（K_a）和平均结合位点(n)分别为25.14x10^6 M^<-1>和0.85。在eIFiso4E中加入eIFiso4G与VPg的结合亲和力是单独加入eIFiso4E的1.5倍。然而，eIFiso4G单独不与VPg结合。在eIFiso4E-VPg或eIFiso4E-iso4G-VPg复合物上添加m^7G帽进一步提高了结合亲和力。van't Hoff分析表明VPg结合是焓驱动的，熵有利，负ΔH°（-28.34 kmol ^<-1>）和正ΔS°（38.40 kmol ^<-1>K^<-1>）。这些结果表明VPg结合位点位于eIFiso4E的帽识别口袋上或附近。此外，含有VPg的帽结合复合体的形成表明VPg可能直接参与病毒基因组的翻译（ASBMB年会2006）。少