Studies on roles of potyviral protein on plant viral disease

1.TuMV的VPG与小麦胚芽翻译起始因子eIF(Iso)4E和eIFiso4F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)4E和eIF(Iso)4F的相互作用。荧光猝灭数据表明，VPG在eIF(Iso)4E上存在一个结合位点。Line weaver-Burk图表明，EIF(Iso)4E的VPG和邻氨基甲酰基-m^7GTP之间以混合型竞争配体结合。荧光停流研究表明，EIF(Iso)4E和EIF(Iso)4F与VPG快速结合，表明VPG与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。TEV结构RNA的加入增强了EIF(Iso)4F-VPG的结合，表明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复合体的形成表明vpg可能直接参与t…的翻译。马铃薯Y病毒VPG直接影响小麦胚芽的体外翻译。我们发现，在小麦胚芽提取液中加入VPG可以促进未封顶病毒mRNA的翻译，而抑制封顶病毒mRNA的翻译。这为未封顶的病毒mRNA提供了显著的竞争优势。我们证明了eIF(Iso)4F可以与eIF4F竞争VPG结合。这里提供的定量数据表明，eIF4F-VPG相互作用通过增加eIF4F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。III.TuMV VPG与拟南芥EIF(Iso)4E相互作用并抑制体外翻译。体外翻译分析表明，VPG的加入显著抑制了eIF(Iso)4E-重组小麦胚芽提取物中封顶RNA的翻译。这一结果表明，VPG通过与eIF(Iso)4E结合来抑制帽子依赖的翻译起始。我们目前的结果可能表明，在包埋阶段产生的过量VPG通过抑制eIF(Iso)4E与细胞mRNAs之间的复合体的形成，关闭了宿主细胞中依赖帽子的翻译启动。较少

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