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

一、拟南芥翻译起始因子eIF（iso）4 E和芜菁花叶病毒（TuMV）基因组连接蛋白（VPg）的表达、纯化和结晶利用大肠杆菌- pET载体系统，建立了拟南芥eIF（iso）4 E和TuMV VPg的表达和纯化体系。拟南芥eIF（iso）4 E和TuMV VPg的结晶仍在研究中，并且通过使用人eIF（iso）4 E的X射线结构产生拟南芥eIF（iso）4 E的三维结构（Tomoo，K.et al.，II.植物病毒VPg和植物翻译起始因子之间相互作用的结合分析.在m^7 GTP-Sepharose上的亲和层析显示结合的拟南芥eIF（iso）4 E被TuMV VPg粗品洗脱。用纯化的VPg和其他拟南芥eIF 4 E同种型进行的进一步柱研究显示VPg优先结合eIF（iso）4 E。结构数据暗示Trp-46和Trp-92在cap识别中的eIF（iso）4 E中。当Trp-46或Trp-92改变时， ...更多信息 eIF（iso）4 E与Leu结合后，失去了与VPg和m^7 GTP-Sepharose形成复合物的能力。这表明VPg结合位点位于eIF（iso）4 E上的帽识别口袋中或附近。使用表面等离子体共振（SPR）测定VPg和加帽RNA寡聚体与eIF（iso）4 E相互作用的亲和力常数。KD值表明，VPg与eIF（iso）4 E的结合亲和力强于加帽RNA。这表明病毒VPg可以通过隔离eIF（iso）4 E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）4 E和eIF（iso）4G形成三元复合物。因此，VPg可通过作为替代帽样结构起作用而参与病毒翻译起始（Miyoshi，H.et al.，此外，通过与D.J Goss教授（纽约）合作的荧光滴定研究了TuMV VPg和小麦eIF（iso）4 E之间相互作用的动力学分析。Scatchard分析表明，VPg的结合亲和力（K_a）为25.14 × 10 ~（^6）M^，平均结合位点数（n）<-1>为0.85。与单独的eIFiso 4 E相比，向eIFiso 4 E中添加eIFiso 4G使与VPg的结合亲和力增加1.5倍。然而，单独的eIFiso 4G不与VPg结合。向eIFiso 4 E-VPg或eIFiso 4 E-iso 4G-VPg复合物中添加m^7G帽进一步增加了结合亲和力。范特霍夫分析表明，VPg结合是熵驱动的，具有大的负ΔH°（-28.34 kJ mole ^<-1>）和正ΔS°（38.40 Jmole^<-1>K^<-1>）。这些结果表明VPg结合位点位于eIFiso 4 E上的帽识别口袋上或附近。此外，含有VPg的帽结合复合物的形成表明VPg直接参与病毒基因组翻译的可能性（ASBMB Annual Meeting 2006）。少

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