Plant Responses to Virus Replication

本研究探讨了植物病毒，特别是烟草花叶病毒（TMV）的复制和发病机制。研究涉及TMV外壳蛋白泛素化对病毒感染过程的意义。这包括观察胞质外壳蛋白自由池中泛素外壳蛋白的外观，定位泛素外壳蛋白在TMV病毒上和感染细胞内的位置，观察转基因植物中泛素外壳蛋白的泛素化，并通过监测细胞中泛素的产生来表征植物细胞对病毒感染的反应。第二个实验领域涉及在病毒感染过程中病毒核酸和蛋白质在叶绿体中建立的机制。我们设计了专门的实验，以确定成熟的病毒外壳蛋白是由细胞质进入叶绿体，还是由病毒RNA在叶绿体内合成，以及2)研究病毒RNA进入叶绿体的机制。这项工作的长期目标是增加对植物病毒在受感染的植物细胞中引起致病反应的机制的理解。重点是烟草花叶病毒（TMV）。第一个目标涉及调查TMV（和许多其他病毒）含有附着在完整病毒颗粒上的蛋白泛素这一发现的意义。泛素是一种存在于所有高等植物和动物细胞中的小蛋白质。已知它在许多细胞中与选定的蛋白质形成共价附着物，并在细胞调节，蛋白质降解和应激反应中发挥作用。本研究的第二个目标是观察到TMV RNA能够在感染期间进入叶绿体，这与叶绿体是TMV感染期间细胞破坏的主要部位这一事实相结合。研究将集中在病毒RNA如何进入叶绿体上。这个问题将首先通过开发一种无细胞系统来观察分离叶绿体对病毒RNA的摄取来解决。一旦这个系统开始运作，RNA摄取的更具体方面就可以通过实验来解决。

This research investigates the mechanisms of replication and pathogenesis of plant viruses, particularly the tobacco mosaic virus (TMV). Studies involve the significance of ubiquitination of the TMV coat protein to the viral infection process. This includes looking at the appearance of ubiquinated coat protein in the free pool of cytosolic coat proteins, localizing the site of the ubiquinated protein both on the TMV viruon and within infected cells, looking at the ubiquination of coat protein in transgenic plants and characterizing the response of plant cells to viral infection by monitoring the cell's production of ubiquitin. A second area of experimentation involves the mechanism whereby viral nucleic acids and proteins become established in the chloroplast during the process of viral infection. Specific experiments are designed 1) to ascertain whether mature viral coat protein enters the chloroplast from the cytosol or is synthesized within the chloroplast from viral RNA and 2) to investigate the mechanism by which viral RNA is incorporated into the chloroplast. The long-term goal of this work is an increased understanding of the mechanisms by which plant viruses bring about pathogenic responses in infected plant cells. Emphasis will be placed on the tobacco mosaic virus (TMV). The first goal involves investigating the significance of the finding that TMV (and numerous other viruses) contain the protein ubiquitin attached to intact virus particles. Ubiquitin is a small protein found in the cells of all higher plants and animals. It is known to form covalent attachments to selected proteins in numerous cells and plays a role in cell regulation, protein degradation and stress responses. A second goal of this research addresses the observation that TMV RNA is able to enter the chloroplast during infection which is coupled with the fact that the chloroplast is the major site of cellular disruption during TMV infection. Studies will focus on how the viral RNA gets into the chloroplast. This problem will be approached initially by developing a cell-free system for looking at the uptake of viral RNA by isolated chloroplasts. Once this system is operating, more specific aspects of the RNA uptake can be addressed experimentally.