Bipartite Geminvirus SqLCV Movement Protein Function

这项研究的目的是了解植物病毒与其宿主之间发生的细胞相互作用，这种相互作用决定了病毒的容许宿主范围，并导致全身性感染的发展。研究的病毒是南瓜卷叶病毒（SqLCV），与大多数植物病毒不同，它具有DNA基因组。由病毒DNA编码的两种蛋白质，BR1和BL1，在感染期间对病毒的细胞间运动很重要。BR1主要存在于细胞核中，BL1主要存在于细胞壁/质膜中。Lazarowitz提出了一个模型，其中BL1和BR1协同作用，指导病毒基因组的短距离细胞间运动，而BR1促进病毒在韧皮部的长距离运动。该模型预测，BL1增加细胞壁间连丝的大小排斥极限，以促进病毒gemone向相邻未感染的韧皮部细胞移动。它进一步预测BR1是一种结合病毒ssDNA并将其移动到细胞外周的核穿梭蛋白。在细胞外围，复合物与BL1相互作用，使其能够局部移动到邻近的未感染细胞和通过韧皮部提供长距离运输的筛元件。对该模型进行了以下测试：1)使用直接测定和组织特异性启动子来证明BL1和BR1在细胞间和系统运动、宿主范围和组织特异性中起作用；2)在感染和转基因植物中将BL1和BR1定位到特定的组织和亚细胞区室，并确定它们对疟原虫大小排除极限的影响；3)表征BL1和BR1的生化特性。4)鉴定和纯化与BL1相互作用的宿主蛋白，并克隆和表征编码这些蛋白的基因。在后一种情况下，预计参与病毒运输的蛋白质将与通常参与细胞间通信和调节韧皮部运输的蛋白质相同。本研究的目的是了解植物病毒与其宿主之间发生的细胞相互作用，这种相互作用决定了病毒的容许宿主范围，并导致全身性感染的发展。研究的病毒是南瓜卷叶病毒（SqLCV），与大多数植物病毒不同，它具有DNA基因组。由病毒DNA编码的两种蛋白质，BR1和BL1，在感染期间对病毒的细胞间运动很重要。BR1主要存在于细胞核中，BL1主要存在于细胞壁/质膜中。这些研究验证了BR1作为穿梭器将DNA从细胞核带到质膜的模型。一旦到达质膜，该模型预测BR1与BL1协同工作，允许病毒成分通过植物细胞之间的连接，即胞间连丝运输。显微镜研究将确定BR1和BL1在感染和转基因植物中的组织和亚细胞位置。显微注射研究将直接检查每种蛋白质对BR1-DNA复合物和分级大小的非特异性分子的细胞间运动的影响。组织培养模型系统的研究将确定BR1和BL1正确靶向质膜或细胞核以及彼此直接相互作用所需的结构域。这些研究的最终目的是鉴定与BL1相互作用的宿主细胞蛋白。这些研究表明，病毒的短距离或长距离移动是否使用了不同的机制，韧皮部限制性病毒的移动机制是否与其他病毒不同。了解这些现象背后的机制是制定抗病植物工程策略的必要条件。＊＊＊

9417664 Lazarowitz The aim of the research is to understand the cellular interactions that occur between a plant virus and its host that define the permissive host range of the virus and which lead to the development of systemic infection. The virus examined is the squash leaf curl virus (SqLCV) which, unlike most plant viruses, has a DNA genome. Two proteins encoded by the viral DNA, BR1 and BL1, are important for the cell-tocell movement of the virus during infection. BR1 is predominantly found in the nucleus and BL1 is found predominantly at the cell wall/plasma membrane. Lazarowitz has proposed a model in which BL1 and BR1 act in concert to direct the short distance cell-to-cell movement of the viral genome, and BR1 promotes viral long distance movement in the phloem. This model predicts that BL1 increases the size exclusion limit of plasmodesmata in the cell wall to facilitate viral gemone movement to adjacent uninfected phloem cells. It further predicts that BR1 is a nuclear shuttle protein that binds viral ssDNA and moves it to the cell periphery. At the cell periphery the complex interacts with BL1 which allows it to move locally to adjacent uninfected cells and sieve elements which provide long distance transport through the phloem. The model is tested by 1) using direct assays and tissue-specific promoters to demonstrate that BL1 and BR1 function in cell-to-cell and systemic movement, host range and tissue specificity, 2) localizing BL1 and BR1 in infected and transgenic plants to specific tissues and subcellular compartments and determining their effects on plasmodemal size-exclusion limit, 3) characterizing the biochemical properties of BL1 and BR1, and their interactions that lead to transport of viral nucleic acids, and 4) identifying and purifying host proteins interacting with BL1, and cloning and characterizing the genes encoding these proteins. In the latter situation, it is expected that the proteins involved in viral transport wil l be the same as those normally involved in cell-to-cell communication and in the regulation of phloem transport. %%% The aim of the research is to understand the cellular interactions that occur between a plant virus and its host that define the permissive host range of the virus and which lead to the development of systemic infection. The virus examined is the squash leaf curl virus (SqLCV) which, unlike most plant viruses, has a DNA genome. Two proteins encoded by the viral DNA, BR1 and BL1, are important for the cell-tocell movement of the virus during infection. BR1 is predominantly found in the nucleus and BL1 is found predominantly at the cell wall/plasma membrane. The studies test the model that BR1 acts as a shuttle to bring the DNA to the plasma membrane from the nucleus. Once at the plasma membrane, the model predicts that BR1 works in concert with BL1 to allow viral component transport through the connections between plant cells, the plasmodesmata. Microscopy studies will define the tissue and subcellular location of BR1 and BL1 in infected and transgenic plants. Microinjection studies will directly examine the effects of each protein on the cell-to-cell movement of the BR1-DNA complexes and non- specific molecules of graded size. Studies in tissue culture model systems will define the domains of BR1 and BL1 required for their correct targeting to the plasma membrane or nucleus and for direct interaction with each other. The studies are ultimately directed at identifying the host cell proteins that interact with BL1. The studies show whether different mechanisms are used for virus short or long distance movment, and if phloemrestricted viruses move by a different mechanism from other viruses. Understanding the mechanism underlying these phenenomena is required in order to develop strategies for engineering virus- resistant plants. ***