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

植物病毒如何从最初接种的细胞转移到邻近细胞并最终系统性地引起疾病尚不清楚。红三叶草坏死花叶病毒（RCNMV）是研究植物病毒运动的一个模型系统。为了了解细胞间和长距离运动的机制，我们设计了一系列的遗传、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？在MP中可以区分功能域吗？MP和衣壳蛋白（CP）在病毒长距离运动中的作用是什么？长距离移动需要病毒粒子的形成吗？运动蛋白和衣壳蛋白是否相互作用以实现长距离运动？如果有的话，宿主因子与MP和/或CP相互作用会导致全身性感染吗？回答这些问题的方法概括为以下三个目标：1)生成和表征RCNMV CP的丙氨酸扫描突变体；2)分析野生型和突变型RCNMV MP在转基因植物中的行为。3)识别促进病毒细胞间和/或长距离运动的宿主因子，并表征MP， CP和宿主因子之间的相互作用。构建一系列CP丙氨酸扫描突变体，分析其CP表达、病毒粒子形成和远距离移动能力。将产生表达野生型和突变型MPs的转基因植株。这些将用于确定MPs的亚细胞定位以及互补实验。在大肠杆菌中过量生产的野生型和突变型CPs和MPs将被微注射到各种植物细胞类型中，并检测其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之间假设的相互作用将通过遗传学、生物化学、凝胶移位试验、酵母双杂交系统和/或突变分析进行评估。来自RCNMV系统寄主植物的cDNA表达文库将使用酵母双杂交系统、lgt11集落杂交或亲和层析筛选特异性与MP相互作用的寄主因子。拟议实验的结果将有助于更好地理解植物病毒移动和最终致病的机制。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间进行交流的基础。识别参与病毒运动的宿主因子将为蛋白质-蛋白质相互作用、宿主特异性以及间连丝的结构和功能提供见解。这一建议旨在了解植物RNA病毒如何在细胞间传播。在这个过程中有几个参与者，一种叫做运动蛋白（MP）的蛋白质，这种蛋白质组成了病毒的外壳，构成了衣壳（CP），以及植物细胞之间组织精细的通道，穿过相邻细胞的细胞壁，即胞间连丝。Lommel实验室先前的工作表明，运动蛋白的氨基酸序列的变化（通过定点突变）使病毒在细胞间传播失能。研究还表明，CP突变改变了病毒长距离传播的能力，例如在叶子到叶子之间传播。假设CP与MP之间存在相互作用，现在将通过CP的受控诱变来探索这种相互作用的性质，随后分析细胞间的运动，并确定CP与突变型和野生型MP之间相互作用的性质。这些蛋白与宿主蛋白相互作用的性质也将被确定，因为假设一些宿主蛋白参与了长距离运输。植物细胞间运输的性质是目前植物细胞生物学家面临的主要问题之一，也是该领域的典型问题之一，因为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）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. ***