An aromatic amino acid and associated helix in the C-terminus of the potato leafroll virus minor capsid protein regulate systemic infection and symptom expression.
An aromatic amino acid and associated helix in the C-terminus of the potato leafroll virus minor capsid protein regulate systemic infection and symptom expression.
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DOI：
10.1371/journal.ppat.1007451
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发表时间：
2018-11
期刊：
影响因子：
6.7
通讯作者：
Gray SM
中科院分区：
文献类型：
--
作者：
Xu Y;Da Silva WL;Qian Y;Gray SM
马铃薯卷叶病毒（PLRV）次要结构蛋白的C末端区域，即通读蛋白（RTP），参与病毒的高效移动、组织嗜性和症状产生。对众多C末端缺失的分析确定了一个对RTP功能必需的五氨基酸基序。一种表达缺失这五个氨基酸的RTP（Δ5aa - RTP）的PLRV突变体在系统感染和症状表达方面受到损害。尽管Δ5aa - RTP突变体能够进行长距离移动，但在系统感染的叶片中观察到有限的感染病灶，这表明这五个氨基酸调节接种叶片中病毒的韧皮部装载和/或向系统感染组织的卸载。5aa缺失没有改变RTP翻译的效率，也没有损害RTP的自身相互作用或它与病毒移动蛋白P17的相互作用。然而，该缺失确实改变了RTP的亚细胞定位。当与PLRV感染性克隆共表达时，带有绿色荧光蛋白（GFP）标签的野生型RTP定位在细胞周边不连续的点状斑点处，并与胞间连丝相关联，尽管其定位取决于植物组织的发育阶段。相比之下，Δ5aa - RTP - GFP在细胞质中聚集。结构建模表明，5aa缺失预计会扰乱一个α - 螺旋基序。在接种后十周，30株感染Δ5aa - RTP的植株中有2株出现了野生型病毒感染表型。通过深度测序对这些植株中的病毒群体进行分析，发现缺失位点附近的序列发生了重复，预计这会恢复α - 螺旋基序。RTP的亚细胞分布受5 - 氨基酸基序的调节，该基序承受着强大的选择压力，进而有助于病毒的高效长距离移动和系统症状的诱导。
蛋白质的功能通常取决于其由氨基酸组成和化学性质所决定的结构。PLRV RTP的C末端约200个氨基酸具有无序的特征，但这个蛋白质结构域参与病毒移动、组织嗜性和症状产生。对病毒突变体、病毒群体及其细胞生物学的分析使我们能够在无序的RTP C末端鉴定出一段包含一个保守芳香族氨基酸的短有序氨基酸序列。这种结构特征以宿主依赖的方式决定病毒的高效长距离移动和症状表达。缺乏此特征的病毒突变体会通过自身序列的重复产生补偿性突变，从而恢复芳香族氨基酸和相关的α - 螺旋结构基序，进而恢复野生型症状和移动表型。了解病毒的适应能力应有助于设计稳定的病毒构建体和可持续的宿主抗性策略。
The C-terminal region of the minor structural protein of potato leafroll virus (PLRV), known as the readthrough protein (RTP), is involved in efficient virus movement, tissue tropism and symptom development. Analysis of numerous C-terminal deletions identified a five-amino acid motif that is required for RTP function. A PLRV mutant expressing RTP with these five amino acids deleted (Δ5aa-RTP) was compromised in systemic infection and symptom expression. Although the Δ5aa-RTP mutant was able to move long distance, limited infection foci were observed in systemically infected leaves suggesting that these five amino acids regulate virus phloem loading in the inoculated leaves and/or unloading into the systemically infected tissues. The 5aa deletion did not alter the efficiency of RTP translation, nor impair RTP self-interaction or its interaction with P17, the virus movement protein. However, the deletion did alter the subcellular localization of RTP. When co-expressed with a PLRV infectious clone, a GFP tagged wild-type RTP was localized to discontinuous punctate spots along the cell periphery and was associated with plasmodesmata, although localization was dependent upon the developmental stage of the plant tissue. In contrast, the Δ5aa-RTP-GFP aggregated in the cytoplasm. Structural modeling indicated that the 5aa deletion would be expected to perturb an α-helix motif. Two of 30 plants infected with Δ5aa-RTP developed a wild-type virus infection phenotype ten weeks post-inoculation. Analysis of the virus population in these plants by deep sequencing identified a duplication of sequences adjacent to the deletion that were predicted to restore the α-helix motif. The subcellular distribution of the RTP is regulated by the 5-aa motif which is under strong selection pressure and in turn contributes to the efficient long distance movement of the virus and the induction of systemic symptoms. Protein function is often dependent on its structure that is determined by the composition and chemistry of its amino acids. The C-terminal ~200 amino acids of the PLRV RTP are characteristically disordered yet this protein domain is involved in virus movement, tissue tropism and symptom development. Analysis of virus mutants, virus populations, and their cell biology allowed us to identify a short-ordered stretch of amino acids containing a conserved aromatic amino acid in the disordered RTP C-terminus. This structural feature determines efficient long-distance virus movement and symptom expression in a host-dependent manner. Virus mutants deficient in this feature generate compensatory mutations by duplication of their own sequence that restore the aromatic amino acid and the associated α-helix structural motif and in turn restore the wild-type symptom and movement phenotypes. Understanding the adaptive abilities of the virus should help to design stable virus constructs and sustainable host resistance strategies.
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