CAA: Identification of Host Factors Regulating the Intra- and Intercellular Movement of Bipartite Geminiviruses

9707580 Lazarowitz病毒编码运动蛋白的作用是允许植物病毒穿过植物细胞壁的屏障，系统地感染寄主植物。了解运动蛋白的功能对于确定植物病毒感染的发病阶段和设计抗病植物的策略至关重要。运动蛋白还提供了一种独特的方法来定义植物细胞内和细胞间运输的途径。本实验室对两节双病毒南瓜卷叶病毒（SqLCV）编码的两个运动蛋白BL1和BR1的研究表明，这两个蛋白具有不同的功能，并以合作的方式移动病毒单链（ss）DNA基因组。BR1是一种核穿梭蛋白，它结合病毒的ssDNA基因组并将其移进移出细胞核。BL1是BR1的细胞质陷阱，通过在细胞质中捕获BR1基因组复合物并引导它们穿过细胞壁，为运动提供方向性，这显然与内质网衍生的小管有关。本实验室的研究进一步表明，BL1和BR1的相互作用可能通过两种运动蛋白的翻译后磷酸化来调节。该研究将在斯克里普斯研究所（Scripps Research Institute）的休假期间进行，旨在直接研究翻译后磷酸化在BL1和BR1相互作用中的作用，并探索以BR1为模型核穿梭蛋白开发可渗透烟草细胞系统用于核出口体外研究的可行性。磷酸化研究将在杰夫·哈珀博士的实验室进行；开发用于核出口调查的渗透性植物细胞系统的尝试将在拉里·杰雷斯博士的建议和指导下进行。具体目标是：(1)通过电喷雾质谱测序确定体内BL1和BR1的磷酸化位点；(2)检测BL1和BR1是植物中普遍存在的钙依赖性钙调素不依赖性激酶的体外底物，还是其他激酶的底物；(3)体外验证BL1与BR1是否直接结合，以及磷酸化对这种相互作用的影响；(4)以BR1为模型核穿梭蛋白，探索构建烟草植物核输出渗透细胞系统的可行性。许多植物病毒从植物表面的一些点进入植物，然后通过植物细胞壁上的通道进行细胞间运动，在植物中系统地从一个细胞传播到另一个细胞。病毒本身含有由病毒基因组编码的“运动蛋白”，这些蛋白促进病毒颗粒或遗传物质在细胞内从细胞核到细胞质以及在植物内从一个细胞到另一个细胞的运动。Lazarowitz博士一直在研究南瓜卷叶病毒的运动蛋白。拟议的研究将为Lazarowitz博士提供必要的新生化和生物物理技能，以研究蛋白质功能和蛋白质：蛋白质相互作用的调节，并将允许她进行初步研究，以开发一种渗透性植物细胞系统，以研究体外核输出。这些技术对于确定BL1和BR1的功能至关重要。重要的是，这些新的技能和系统将使她的研究计划转向两个令人兴奋的新领域：(1)生物化学研究，以确定植物细胞将分子引导到细胞壁的途径，并调节细胞壁通道的形成和功能；(2)核出口机制的基础研究。她将在这次休假期间获得的技术和知识视角将提高她自己的研究能力，并为她的学生和博士后提供培训。＊＊＊

9707580 Lazarowitz Viral-encoded movement proteins act to allow a plant virus to cross the barrier of the plant cell wall and systemically infect the host plant. Understanding the function of movement proteins is essential for defining the stages of pathogenesis in plant virus infections and devising strategies to engineer disease-resistant plants. Movement proteins also provide a unique approach to defining pathways of intracellular and intercellular transport in plants. This laboratory's investigation of the two movement proteins, BL1 and BR1, encoded by the bipartite geminivirus squash leaf curl virus (SqLCV) have shown that the two proteins have distinct functions and act in a cooperative manner to move the viral single strand (ss)DNA genome. BR1 is a nuclear shuttle protein that binds the viral ssDNA genome and moves it into and out of the nucleus. BL1 is a cytoplasmic trap for BR1, providing directionality to movement by trapping BR1-genome complexes in the cytoplasm and directing these to across the cell wall, apparently in association with tubules derived from the endoplasmic reticulum. The studies from this laboratory further suggest that the interaction of BL1 and BR1 may be regulated through posttranslational phosphorylation of both movement proteins. The proposed studies, to be done during a sabbatical leave at the Scripps Research Institute, are designed to directly investigate the role of posttranslational phosphorylation in the interaction of BL1 and BR1, and to explore the feasibility of developing a permeabilized tobacco cell system for in vitro studies on nuclear export using BR1 as a model nuclear shuttling protein. The phosphorylation studies will be done in the laboratory of Dr. Jeff Harper; the attempt to develop a permeabilized plant cell system for investigating nuclear export will be done with the advice and guidance of Dr. Larry Gerace. The specific goals are to: (1) identify in vivo sites of phosphorylation in BL1 and BR1 by electrospray mass spectrometry sequencing; (2) test whether BL1 and BR1 are in vitro substrates for calcium-dependent calmodulin-independent kinases prevalent in plants, or for other kinases; (3) demonstrate in vitro whether BL1 and BR1 directly bind each other and the effects of phosphorylation on this interaction; and (4) explore the feasibility of developing a permeabilized tobacco plant cell system for investigating nuclear export using BR1 as a model nuclear shuttle protein. Many plant viruses gain entry to the plant at points on the surface of the plant, and are subsequently spread systemically from cell to cell through the plant by intercellular movement through channels in the plant cell walls. The viruses contain their own "movement proteins" encoded by their viral genomes which facilitate the movement of viral particles or genetic material from the nucleus to the cytoplasm within a cell and from cell to cell within a plant. Dr. Lazarowitz has been studying the movement proteins of the Squash Leaf Curl Virus. The proposed studies will provide Dr. Lazarowitz with essential new biochemical and biophysical skills to study the regulation of protein function and protein:protein interactions, and will allow her to do pilot studies to develop a permeabilized plant cell system to investigate nuclear export in vitro. These techniques are essential for defining the functions of BL1 and BR1. Importantly, these new skills and systems will allow her to redirect her research program into two exciting new areas: (1) biochemical studies to define the pathways by which plant cells direct molecules to the wall, and regulate the formation and functioning of channels in the cell wall; and (2) basic studies on the mechanism of nuclear export. The techniques and intellectual perspective that she will acquire during this sabbatical leave will enhance both her own research capabilities and the training she can provide to her students and postdoctoral fellows. ***