Studies on roles of potyviral protein on plant viral disease

I. TuMV VPg与小麦胚翻译起始因子eIF（iso）4 E和eIF（iso）4F的相互作用测定并比较了TuMV VPg与小麦胚真核翻译起始因子eIF（iso）4 E和eIF（iso）4F的相互作用。荧光猝灭数据显示VPg在eIF（iso）4 E上存在一个结合位点。线性韦弗-伯克图显示了VPg和邻氨基苯甲酰-m^7 GTP对eIF（iso）4 E的混合型竞争性配体结合。eIF（iso）4 E和eIF（iso）4F与VPg的荧光停流研究显示了快速结合，表明VPg和m^7G帽之间存在动力学竞争。VPg蛋白的结合速度比cap类似物快得多。增强eIF（iso）4F-VPg结合与另外的结构化RNA来自TEV表明，涉及VPg的翻译起始发生在内部核糖体进入位点。此外，含有VPg的蛋白质-RNA复合物的形成表明VPg可能直接参与蛋白质的翻译。 关于我们 病毒基因组。马铃薯Y病毒VPg直接影响小麦胚的体外翻译，我们发现，在小麦胚提取物中加入VPg会导致未加帽病毒mRNA翻译的增强和加帽病毒mRNA翻译的抑制。这为未加帽的病毒mRNA提供了显著的竞争优势。我们证明了eIF（iso）4F可以与eIF 4F动力学竞争VPg结合。本文提供的定量数据表明了一种模型，其中eIF 4F-VPg相互作用通过增加eIF 4F对TEV RNA的亲和力来增强帽非依赖性翻译。这是VPg直接参与翻译启动的第一个证据。TuMV VPg与拟南芥eIF（iso）4 E相互作用并抑制其体外翻译，本研究通过研究TuMV VPg与拟南芥eIF（iso）4 E之间的相互作用来探讨马铃薯Y病毒VPg对宿主细胞翻译起始的影响。体外翻译分析表明，VPg的加入显着抑制加帽RNA在eIF（iso）4 E-重构小麦胚芽提取物的翻译。该结果表明VPg通过与eIF（iso）4 E结合抑制帽依赖性翻译起始。我们目前的研究结果可能表明，过量的VPg产生的在pH4.0阶段关闭帽依赖性的翻译起始在宿主细胞中通过抑制复合物的形成eIF（iso）4 E和细胞的mRNA。少

I. Interaction of VPg of TuMV with Wheat Germ Translation Initiation Factors eIF(iso)4E and eIFiso4F.The interaction between VPg of TuMV and wheat germ eukaryotic translation initiation factors eIF(iso)4E and eIF(iso)4F were measured and compared. The fluorescence quenching data showed the presence of one binding site on eIF(iso)4E for VPg. A Line weaver-Burk plot indicates mixed-type competitive ligand binding between VPg and anthraniloyl-m^7GTP for eIF(iso)4E. Fluorescence stopped-flow studies of eIF(iso)4E and eIF(iso)4F with VPg show rapid binding, suggesting kinetic competition between VPg and m^7G cap. The VPg protein binds much faster than cap analogs. Enhancement of eIF(iso)4F-VPg binding with the addition of a structured RNA derived from TEV suggests that translation initiation involving VPg occurs at internal ribosomal entry sites. Furthermore, the formation of a protein-RNA complex containing VPg suggests the possibility of direct participation of VPg in the translation of t … More he viral genome.II. Potyvirus VPg directly affects wheat germ in vitro translation.We show that addition of VPg to wheat germ extract leads to enhancement of uncapped viral mRNA translation and inhibition of capped viral mRNA translation. This provides a significant competitive advantage to the uncapped viral mRNA. We demonstrate that eIF(iso)4F can kinetically compete with eIF4F for VPg binding. The quantitative data presented here suggest a model where eIF4F-VPg interaction enhances cap-independent translation by increasing the affinity of eIF4F for TEV RNA. This is the first evidence of direct participation of VPg in translation initiation.III. TuMV VPg interacts with Arabidopsis thaliana eIF(iso)4E and inhibits in vitro translation.The interaction between TuMV VPg and Arabidopsis thaliana eIF(iso)4E was investigated to address the influence of potyviral VPg on host cellular translational initiation. In vitro translation analysis showed that the addition of VPg significantly inhibited translation of capped RNA in eIF(iso)4E-reconstituted wheat germ extract. This result indicates that VPg inhibits cap-dependent translational initiation via binding to eIF(iso)4E. Our present results may indicate that excess VPg produced at the encapsidation stage shuts off cap-dependent translational initiation in host cells by inhibiting complex formation between eIF(iso)4E and cellular mRNAs. Less