TheBarley stripe mosaic virusγb protein promotes viral cell-to-cell movement by enhancing ATPase-mediated assembly of ribonucleoprotein movement complexes
TheBarley stripe mosaic virusγb protein promotes viral cell-to-cell movement by enhancing ATPase-mediated assembly of ribonucleoprotein movement complexes
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大麦条纹花叶病毒 γb 蛋白通过增强 ATP 酶介导的核糖核蛋白运动复合物的组装来促进病毒细胞间运动
DOI：
10.1371/journal.ppat.1008709
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发表时间：
2020-07-01
期刊：
影响因子：
6.7
通讯作者：
Zhang, Yongliang
中科院分区：
文献类型：
--
作者：
Jiang, Zhihao;Zhang, Kun;Zhang, Yongliang
五个不同科的九个属的病毒使用三重基因阻断（TGB）蛋白进行病毒移动。TGB模块分为两类：hordei-like和potex-like。虽然TGB介导的病毒运动已被广泛研究，病毒核糖核蛋白（vRNP）运动复合物的成分和它们参与vRNP介导的运动的机制的确定还远未完成。在本研究中，TGB 1蛋白复合物形成过程中大麦条纹花叶病毒（BSMV）感染的免疫沉淀揭示了γ B蛋白的存在下，在产品。进一步的实验表明，TGB 1与γ bin在体外和体内相互作用，并且γ b-TGB 1定位于叶绿体和胞间连丝（PD）的外周。对本氏烟草（Nicotiana benthamiana）表皮细胞中γ B蛋白的亚细胞定位分析表明，在病毒感染的不同阶段，γ B蛋白除了定位于叶绿体外，还靶向ER、肌动蛋白丝和PD。通过追踪BSMV感染过程中γ B的定位，我们证明了γ B是有效的细胞间运动所必需的。γ B的N-末端与TGB 1 ATP酶/解旋酶结构域相互作用并增强该结构域的ATP酶活性。体外翻译和免疫共沉淀（co-IP）分析表明，ATP水解可促进TGB 1-TGB 3-TGB 2复合物的形成。γ B蛋白在ATP存在下正调节复合物的形成，表明γ B通过直接促进TGB 1 ATP酶介导的vRNP运动复合物组装在BSMV细胞间运动中具有新的作用。我们进一步证明了ATP酶活性的消除消除了马铃薯X病毒（PVX）和甜菜坏死黄脉病毒（BNYVV）TGB 1蛋白的PD和肌动蛋白靶向作用。这些结果扩展了我们对γ B的多功能作用的理解，并为TGB 1 ATP酶结构域在编码TGB的病毒的运动中的功能提供了新的见解。病毒核糖核蛋白（vRNP）运动复合物包括大麦或马铃薯样三重基因块（TGB）和病毒RNA代表植物病毒运动的重要模型。然而，病毒核糖核蛋白（vRNP）运动复合物的成分以及它们在vRNP组装和随后的相互作用中的生物学意义远未完成。此外，高度保守的TGB 1 ATP酶结构域在vRNP介导的运动中的机制作用仍然是一个谜。在这里，我们证明，γ B蛋白作为一种新的正调节BSMV细胞到细胞的运动，通过直接与TGB 1蛋白相互作用，在vRNP运动复合物的组装，一个过程，可以进一步增强γ B蛋白的TGB 1 ATP酶介导的ATP水解的体外生化测定验证了一个重要的作用。我们还扩展了我们的研究BSMV TGB 1 ATP酶的PVX和BNYVV，并提出了一个模型的进化保守模式的能量耦合vRNP运动复杂的装配之间的不同TGB编码病毒。我们的研究结果解决了TGB 1 ATP酶活性和vRNP运动复合体组装之间的知识差距，并扩大了我们对γ B在BSMV感染中的多方面作用的理解。
Nine genera of viruses in five different families use triple gene block (TGB) proteins for virus movement. The TGB modules fall into two classes: hordei-like and potex-like. Although TGB-mediated viral movement has been extensively studied, determination of the constituents of the viral ribonucleoprotein (vRNP) movement complexes and the mechanisms underlying their involvement in vRNP-mediated movement are far from complete. In the current study, immunoprecipitation of TGB1 protein complexes formed duringBarley stripe mosaic virus(BSMV) infection revealed the presence of the gamma b protein in the products. Further experiments demonstrated that TGB1 interacts with gamma bin vitroandin vivo, and that gamma b-TGB1 localizes at the periphery of chloroplasts and plasmodesmata (PD). Subcellular localization analyses of the gamma b protein inNicotiana benthamianaepidermal cells indicated that in addition to chloroplast localization, gamma b also targets the ER, actin filaments and PD at different stages of viral infection. By tracking gamma b localization during BSMV infection, we demonstrated that gamma b is required for efficient cell-to-cell movement. The N-terminus of gamma b interacts with the TGB1 ATPase/helicase domain and enhances ATPase activity of the domain. Inactivation of the TGB1 ATPase activity also significantly impaired PD targeting.In vitrotranslation together with co-immunoprecipitation (co-IP) analyses revealed that TGB1-TGB3-TGB2 complex formation is enhanced by ATP hydrolysis. The gamma b protein positively regulates complex formation in the presence of ATP, suggesting that gamma b has a novel role in BSMV cell-to-cell movement by directly promoting TGB1 ATPase-mediated vRNP movement complex assembly. We further demonstrated that elimination of ATPase activity abrogates PD and actin targeting ofPotato virus X(PVX) andBeet necrotic yellow vein virus(BNYVV) TGB1 proteins. These results expand our understanding of the multifunctional roles of gamma b and provide new insight into the functions of TGB1 ATPase domains in the movement of TGB-encoding viruses.Author summary Plant viruses employ varied movement strategies to mediate local and systemic infections. Viral ribonucleoprotein (vRNP) movement complexes comprising either the hordei- or potex-like triple gene block (TGB) and viral RNAs represent important models for plant virus movement. However, the constituents of viral ribonucleoprotein (vRNP) movement complexes as well as their biological significance in vRNP assembly and subsequent interactions are far from complete. Additionally, the mechanistic roles of the highly conserved TGB1 ATPase domain in vRNP-mediated movement remain an enigma. Here, we demonstrate that the gamma b protein acts as a novel positive regulator of BSMV cell-to-cell movement by directly interacting with the TGB1 protein.In vitrobiochemical assays verified an essential role of TGB1 ATPase-mediated ATP hydrolysis in assembly of vRNP movement complexes, a process that can be further enhanced by the gamma b protein. We also extend our studies of BSMV TGB1 ATPase to those of PVX and BNYVV, and suggest a model for an evolutionally conserved mode of energy-coupled vRNP movement complex assembly among different TGB-encoding viruses. Our results address the knowledge gap between TGB1 ATPase activity and vRNP movement complex assembly and expand our understanding of the multifaceted roles of gamma b in BSMV infection.