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

摘要植物病毒如何从最初接种的细胞传播到邻近细胞，并最终系统性地导致疾病，目前尚不清楚。红三叶坏死花叶病毒(RCNMV)是研究植物病毒运动的模型系统。为了了解细胞间和远距离运动的机制，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之间的传播？下院议员似乎在细胞内的什么地方行动？在MP中可以区分功能域吗？MP和衣壳蛋白(CP)在病毒的长距离移动中起什么作用？我是S长距离运动所需的病毒粒子吗？运动和衣壳蛋白是否相互作用以实现远距离运动？如果有的话，宿主因素与MP和/或CP相互作用而引起全身性感染怎么办？回答这些问题的方法概述在以下三个目标中：1)产生和鉴定RCNMV CP的丙氨酸扫描突变体。2)分析野生型和突变型RCNMV MP在转基因植株中的行为。3)确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的表达、病毒粒子的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型MPS的转基因植株。这些将被用来确定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将被显微注射到各种类型的植物细胞中，并检测其修饰胞间连丝和将大分子从一种细胞类型转移到另一种细胞类型的能力。MP和CP之间的假想相互作用将通过遗传、生化、凝胶迁移分析、酵母双杂交系统和/或突变分析进行评估。将利用酵母双杂交系统、lgt11菌落杂交或亲和层析从RCNMV系统寄主植物中筛选出与MP特异相互作用的寄主因子(S)。拟议中的考察结果将使人们更好地了解植物病毒移动并最终导致疾病的机制。它们还将作为研究植物病毒如何篡夺正常寄主功能以实现感染以及植物如何在细胞之间进行通信的基础。识别参与病毒运动的宿主因子将有助于深入了解蛋白质-蛋白质相互作用、宿主特异性以及胞间连丝的结构和功能。这项建议旨在了解植物RNA病毒是如何在细胞间传播的。在这个过程中有几个参与者，一种被称为运动蛋白(MP)的蛋白质，这种蛋白质组成病毒外壳(CP)，以及穿越相邻细胞细胞壁的植物细胞之间的精心组织的通道，胞间连丝。Lommel实验室之前的工作表明，运动蛋白氨基酸序列的变化(通过定点突变)使病毒无法在细胞间传播。研究还表明，CP突变改变了病毒长距离传播的能力，例如从树叶到树叶。存在CP与MP相互作用的假设，现在将通过CP的受控突变，然后分析细胞到细胞的运动，并通过确定CP与突变体和野生型MP相互作用的性质来探索这种相互作用的性质。这些蛋白质与宿主蛋白相互作用的性质也将被确定，因为它假设某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，因为我们基础知识的增加将在基础(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. ***