Mutational analysis of the RNA-binding domain of the Prunus necrotic ringspot virus (PNRSV) movement protein reveals its requirement for cell-to-cell movement
Mutational analysis of the RNA-binding domain of the Prunus necrotic ringspot virus (PNRSV) movement protein reveals its requirement for cell-to-cell movement
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DOI：
10.1016/j.virol.2005.05.020
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发表时间：
2005-08-15
期刊：
影响因子：
3.7
通讯作者：
Pallás, V
中科院分区：
文献类型：
--
作者：
Herranz, MC;Sanchez-Navarro, JA;Pallás, V
李坏死环斑病毒 (PNRSV) 的运动蛋白 (MP) 是细胞间运动所必需的。使用 GFP 融合蛋白进行的 MP 亚细胞定位研究揭示了相邻细胞之间的高度点状结构，据信代表胞间连丝。删除 PNRSV MP 的 RNA 结合域 (RBD) 可消除细胞间的运动。为了确定体内控制病毒运输的特征，对该 RBD 进行了突变分析。尽管所有突变体在原生质体中的积累水平与野生型（wt）MP 观察到的相似，但正电荷的丢失阻止了细胞间的运动。使用代表突变体和野生型 RBD 的合成肽通过 EMSA 分析来研究 RNA 结合亲和力，突变体中的 RNA 结合亲和力大约低 20 倍。圆二色性分析表明，肽的二级结构并未受到突变的显着影响。讨论了病毒 RNA 和 MP 之间的亲和力变化在病毒细胞间运动中的作用。 (C) 2005 Elsevier Inc. 保留所有权利。
The movement protein (MP) of Prunus necrotic ringspot virus (PNRSV) is required for cell-to-cell movement. MP subcellular localization studies using a GFP fusion protein revealed highly punctate structures between neighboring cells, believed to represent plasmodesmata. Deletion of the RNA-binding domain (RBD) of PNRSV MP abolishes the cell-to-cell movement. A mutational analysis on this RBD was performed in order to identify in vivo the features that govern viral transport. Loss of positive charges prevented the cell-to-cell movement even though all mutants showed a similar accumulation level in protoplasts to those observed with the wild-type (wt) MP. Synthetic peptides representing the mutants and wild-type RBDs were used to study RNA-binding affinities by EMSA assays being approximately 20-fold lower in the mutants. Circular dichroism analyses revealed that the secondary structure of the peptides was not significantly affected by mutations. The involvement of the affinity changes between the viral RNA and the MP in the viral cell-to-cell movement is discussed. (C) 2005 Elsevier Inc. All rights reserved.