The Roles and Interactions of the Red Clover Necrotic Mosiac Virus Capsid and Movement Proteins in Cell-to-Cell and Long Distance Movement.

MCB-9418700摘要 植物病毒如何从最初接种的细胞转移到邻近的细胞，并最终系统地引起疾病还不清楚。 红三叶草坏死花叶病毒（RCNMV）是研究植物病毒运动的一个模式系统。 为了了解细胞间和长距离运动的机制，设计了一系列遗传、生化和细胞生物学实验来解决以下问题：运动蛋白（MP）如何促进病毒在细胞间的传播？ MP在细胞内的什么地方起作用？ MP中的功能域可以区分吗？ MP和衣壳蛋白（CP）在病毒长距离移动中的作用是什么？长距离移动需要病毒粒子的形成吗？运动和衣壳蛋白相互作用以实现长距离运动吗？ 如果有，宿主因素与MP和/或CP相互作用导致全身感染？ 回答这些问题的方法概括在以下三个目标中：1）产生和表征RCNMV CP的丙氨酸扫描突变体。2)分析野生型和突变体RCNMV MP在转基因植物中的行为。3)鉴定促进病毒细胞间和/或长距离移动的宿主因子，并表征MP、CP和宿主因子之间的相互作用。 将构建一系列CP丙氨酸扫描突变体并分析CP表达、病毒体形成及其促进长距离移动的能力。 将产生表达野生型和突变MP的转基因植物。 这些将用于确定MP的亚细胞定位以及用于互补实验。 大肠杆菌中过量产生的野生型和突变型CP和MP将被显微注射到各种植物细胞类型中，并测定修饰胞间连丝和将大分子从一种细胞类型转运到另一种细胞类型的能力。 MP和CP之间的假设相互作用将通过遗传学、生物化学、凝胶迁移试验、酵母双杂交系统和/或突变分析进行评估。 将使用酵母双杂交系统、lgt 11菌落杂交或亲和色谱法筛选来自RCNMV系统性宿主植物的cDNA表达文库中与MP特异性相互作用的宿主因子。 这些实验结果将有助于我们更好地理解植物病毒的传播和致病机制。 它们还将作为研究植物病毒如何篡夺正常宿主功能以实现感染以及植物如何在细胞与细胞之间进行通信的基础。 参与病毒运动的宿主因子的鉴定将提供对蛋白质-蛋白质相互作用、宿主特异性以及胞间连丝的结构和功能的深入了解。 该提案旨在了解植物RNA病毒如何从细胞转移到细胞。 在这个过程中有几个参与者，一种称为运动蛋白（MP）的蛋白质，这种蛋白质包括病毒的外壳以构成衣壳（CP），以及植物细胞之间穿过相邻细胞的细胞壁的精致组织的通道，即胞间连丝。 洛梅尔实验室以前的工作已经表明，运动蛋白的氨基酸序列的变化（通过定点诱变）使病毒在细胞间的传播失能。 还表明CP突变改变了病毒长距离移动的能力，例如，从叶子到叶子。 存在CP与MP的假定相互作用，并且现在将通过CP的受控诱变来探索该相互作用的性质，随后通过分析细胞到细胞的运动以及通过确定CP与突变体和野生型MP的相互作用的性质来探索。 还将确定这些蛋白质与宿主蛋白质相互作用的性质，因为假设一些宿主蛋白质参与长距离运输。植物细胞间运输的性质是植物细胞生物学家目前面临的主要问题之一，并代表了我们基础知识的逐步增加将在基础（胞间连丝作用）和应用（植物抗病性）领域带来潜在的巨大回报。 ***

MCB-9418700 Abstract How plant viruses move from the initially inoculated cell to neighboring cells and eventually systemically to cause disease is not understood. Red clover necrotic mosaic virus (RCNMV) has been developed as a model system to study plant virus movement. To understand the mechanisms of both cell-to-cell and long distance movement, a series of genetic, biochemical and cell biology experiments are designed to address the following questions: How does the movement protein (MP) facilitate viral spread from cell to cell? Where within the cell does the MP appear to act? Can fuctional domains be distinguished in the MP? What are the roles of the MP and capsid protein (CP) in long distance movement of the virus? I s virion formation requried for long distance movement? Do the movement and capsid protiens interact with one another to achieve long distance movement? What if any, host factors interact with the MP and/or CP to cause a systemic infection? The approaches to answer these questions are outlined in the following three objectives: 1) Generate and characterize alanine scanning mutants of the RCNMV CP. 2) Analyze wild-type and mutant RCNMV MP behavior in transgenic plants. 3) Identify host factors that facilitate viral cell-to-cell and/or long distance movement and characterize the interactions between MP, CP, and host factors. A series of CP alanine scanning mutants will be constructed and analyzed for CP expression, virion formation, and their ability to facilitate long distance movement. Transgenic plants will be generated expressing the wild-type and mutant MPs. These will be used to determine the subcellular localization of MPs as well as for complementation experiments. Wild-type and mutant CPs and MPs over-produced in E.coli will be microinjected into various plant cell types and assayed for the ability to modify plasmodesmata and transport macromoleucles from one cell type to another. The hypothesized interactions betw een MP and CP will be assessed genetically, biochemically, in gel shift assays, by the yeast two hybrid system, and/or by mutational analysis. cDNA expression libraries from RCNMV systemic host plants will be screened for a host factor(s) that specifically interacts with the MP using the yeast two hybrid system, lgt11 colony hybridization, or affinity chromatography. Results from the proposed expeirments will afford a better understanding of the mechanisms by which plant viruses move and ultimately cause disease. They will also serve as a basis for studying how plant viruses usurp normal host functions to achieve infection and how plants communicate from cell to cell. The identification of host factors involved in viral movement will provide insights into protein-protein interactions, host specificity, and the structure and function of plasmodesmata. %%% This proposal seeks to understand how plant RNA viruses move from cell to cell. There are several players in the process, a protein called movement protein (MP) , the protein which comprises the coat of the virus to make up the capsid (CP), and the exquisitely organized channels between plant cells which traverse the cell wall of adjacent cells, the plasmodesmata. Previous work in the Lommel lab has shown that changes in the amino acid sequence (through site- directed mutagenesis) of the movement protein incapacitate viral spread from cell to cell. It has also been shown that CP mutations change the ability of the virus to move over long distances, e.g., from leaf to leaf. There is a postulated interaction of CP with MP and the nature of that interaction will now be explored through controlled mutagenesis of CP, followed by analyzing cell to cell movement and by determining the nature of the interaction of CP with mutant and wild type MP. The nature of the interaction of these proteins with host proteins will also be determined, since it is hypothesized that some host protein is involved in lo ng distance transport. The nature of cell-to-cell transport in plants is one of the main questions confronting plant cell biologists currently and typifies the field in that incremental increases in our basic knowledge will lead to potentially large pay-offs in both fundamental (plasmodesmatal action) and applied (plant disease resistance) areas. ***