INRAE, Biologie du Fruit et Pathologie, University of Bordeaux, UMR 1332, F-33140 Villenave d'Ornon, France.
Faculty of Sciences, University of Sydney, Charles Perkins Center D17, Camperdown Campus, Sydney, NSW 2006, Australia.
Int J Mol Sci. 2020 Aug 5;21(16):5618. doi: 10.3390/ijms21165618.
The infectious cycle of potyviruses requires the formation of a complex between the viral genome-linked protein VPg and the host eukaryotic translation initiation factor 4E, eIF4E. Mutations associated with plant resistance to potyviruses were previously mapped at the eIF4E surface, while on the virus side, mutations leading to plant resistance breaking were identified within the VPg. In the present study, fluorescence spectroscopy was used to probe the contribution of the VPg intrinsically disordered region bearing amino acids determinant of the resistance breaking, to the VPg-eIF4E binding mechanism. Synthetic peptides encompassing the VPg central region were found to tightly bind to eIF4E. Fluorescence energy transfer experiments show that, upon binding to eIF4E, the N and C termini of the VPg fragment move closer to one another, at a distance compatible with a α-helix folding. When the VPg region, which contains amino acids associated with resistance breakdown, is appended to VPg, the complex formation with eIF4E switches from a single-step to a two-step kinetic model. This study revisits a recent investigation of the VPg-eIF4E complex by specifying the contribution of the VPg central helix and its appended disordered region to VPg association with eIF4E.
马铃薯 Y 病毒属的感染周期需要病毒基因组连接蛋白 VPg 与宿主真核翻译起始因子 4E（eIF4E）之间形成复合物。先前已将与植物抗马铃薯 Y 病毒属相关的突变映射到 eIF4E 表面，而在病毒方面，已确定导致抗药性丧失的突变位于 VPg 内。在本研究中，荧光光谱法被用于探测具有抗性断裂决定因素的 VPg 固有无序区对 VPg-eIF4E 结合机制的贡献。包含 VPg 中心区域的合成肽被发现与 eIF4E 紧密结合。荧光能量转移实验表明，与 eIF4E 结合后，VPg 片段的 N 端和 C 端彼此靠近，距离与α-螺旋折叠兼容。当包含与抗性断裂相关的氨基酸的 VPg 区域附加到 VPg 上时，与 eIF4E 的复合物形成从单步动力学模型转变为两步动力学模型。这项研究通过指定 VPg 中心螺旋及其附加的无序区域对 VPg 与 eIF4E 结合的贡献，重新考察了最近对 VPg-eIF4E 复合物的研究。
