Bipartite Geminivirus Movement Protein Function

该项目的目标是阐明植物病毒编码的运动蛋白（MP）如何协调病毒基因组的复制及其在植物细胞中和植物细胞之间的定向运动，并探索MP在赋予植物病毒感染组织限制中的作用。了解病毒与宿主在这一水平上的相互作用对于对抗病毒性疾病非常重要，因为只有从这些知识中才能开发出合理的抗病毒策略。遗传学研究表明，MP决定了植物病毒的寄主范围和致病特性。因此，了解它们的功能是开发策略，工程植物，将是一个广泛的geminiviruses.bipartite双生病毒南瓜曲叶病毒（SqLCV）和卷心菜曲叶病毒（CLCV）编码两个MP，NSP和MPB。它们协同作用，在细胞内和细胞之间移动病毒单链DNA基因组。NSP是一种核穿梭蛋白，其在病毒基因组在核中的复制位点处结合病毒基因组，并在核和细胞质之间移动这些基因组。MPB将NSP-基因组复合物捕获在细胞质中，并使它们沿着独特的内质网（ER）衍生的小管移动到并穿过发育中的韧皮部细胞的壁。通过定点诱变，已经在每个MP内鉴定了功能结构域。NSP和MPB之间的相互作用受到植物细胞特异性活性的调节，突变研究表明这种调节可能涉及MP的磷酸化。绿色荧光蛋白（GFP）的使用进一步表明SqLCV感染是韧皮部限制的。为了了解NSP和MPB的功能，它们作用的信号通路，以及MPB在病毒感染的韧皮部限制中的作用，现在需要进行生物化学和遗传学研究。我们还希望了解和开发新的体内和体外方法来研究它们的功能。该项目的具体目标是研究MPB-ER相互作用及其在病毒感染的韧皮部限制中的作用，研究AtNSI的功能，AtNSI是一种与NSP相互作用的功能未知的拟南芥蛋白，并鉴定与MPB相互作用的宿主细胞蛋白。这些方法将利用葫芦科植物和拟南芥的不同特征。将研究表达游离GFP的CLCV和CLCV-SqLCV重组体以确定CLCV感染在拟南芥中是否是韧皮部限制的以及MPB在感染拟南芥的能力中的作用。MPB与GFP的融合将被用于确定MPB在韧皮部限制病毒感染中的作用以及韧皮部发育对病毒运动的影响。确定AtNSI的亚细胞位置和组织特异性和时间表达模式将提供关于其功能的信息。将使用体外NSP直接结合试验和酵母反向双杂交筛选来鉴定AtNSI相互作用缺陷的NSP突变体;这些突变体将通过基于PCR的技术进行筛选。鉴定与MPB相互作用的宿主蛋白将定义其功能以及其如何靶向ER，并且将通过使用酵母SOS募集系统、相互作用克隆和与MPB共纯化来完成。

The goal of the project is to elucidate how plant virus-encoded movement proteins (MPs) act to coordinate replication of the viral genome with its directional movement through and between plant cells, and to explore the role of MPs in conferring tissue restriction to plant virus infection. Understanding virus-host interactions at this level is important for combating viral diseases, for only from such knowledge can rational anti-viral strategies be developed. Genetic studies show that MPs determine the host range and pathogenic properties of plant viruses. Thus, understanding their functions is essential for developing strategies to engineer plants that will be resistant to a broad range of geminiviruses.The bipartite geminiviruses squash leaf curl virus (SqLCV) and cabbage leaf curl virus (CLCV) encode two MPs, NSP and MPB. These act cooperatively to move the viral single strand DNA genome within and between cells. NSP is a nuclear shuttle protein that binds viral genomes at their site of replication in the nucleus, and moves these between the nucleus and the cytoplasm. MPB traps NSP-genome complexes in the cytoplasm and moves them along unique endoplasmic reticulum (ER)-derived tubules to and across the walls of developing phloem cells. By site-directed mutagenesis, functional domains have been identified within each MP. The interactions between NSP and MPB are regulated by an activity that is specific to plant cells, and mutational studies suggest that this regulation may involve phosphorylation of the MPs . Use of the green fluorescent protein (GFP) has further shown that SqLCV infection is phloem-limited. To understand the functions of NSP and MPB, the signaling pathways through which they act, and the role of MPB in the phloem restriction of virus infection, now requires biochemical and genetic studies. We also want to understand, and develop new in vivo and in vitro approaches to investigate their functions. The specific goals of the project are to investigate MPB-ER interactions and their roles in the phloem-restriction of virus infection, to investigate the function of AtNSI, an Arabidopsis protein of unknown function that interacts with NSP and to identify host cell proteins that interact with MPB. These approaches will take advantage of different features of cucurbits and Arabidopsis. CLCV expressing free GFP and CLCV-SqLCV recombinants will be studied to determine if CLCV infection is phloem-restricted in Arabidopsis and the role of MPB in the ability to infect Arabidopsis. Fusions of MPB with GFP expressed from tissue-specific promoters in transgenic Arabidopsis wll be used to determine the role of MPB in the phloem limitation to virus infection and the effects of phloem development on virus movement. Determining the subcellular location and tissue-specific and temporal expression patterns of AtNSI will provide information on its function. Direct binding assays with NSP in vitro and a yeast reverse two-hybrid screen will be used to to identify NSP mutants with defects in AtNSI interaction; these will be screened by a PCR-based technique. Identifying host proteins that interact with MPB will define its function and how it targets to ER, and will be accomplished by using the yeast SOS-recruitment system, interaction cloning, and co-purification with MPB.