Quantitative Analysis of Viral mRNA Translation

植物病毒性疾病影响世界范围内大量粮食作物，对经济状况和粮食供应都可能产生严重影响。蛋白质合成是病毒感染的关键步骤，但尚未得到很好的理解。这项研究将增加我们对植物病毒蛋白合成以及这一重要生化过程如何被调控的理解。关于IRES和起始因子的结构信息提供了关于形成的配合物的信息。然而，定量数据对于确定起始因子如何竞争不同的mRNA位点、中间体的形成以及复合物的动力学和热力学稳定性是必要的。本研究将利用荧光光谱、停流动力学和圆二色性来确定单个起始因子在植物病毒mRNA内部起始中的作用，这些因子之间的相互作用以及已知IRES元素，以及这些因子如何改变mRNA结构。这些数据将用于确定在各种生理条件下mRNA选择的模型。定量信息和相互作用协同性的确定将获得48S起始复合物组装和与病毒IRES结合的详细信息。这些信息可以潜在地用于通过基因工程或生化制剂来关闭病毒蛋白质的合成。此外，病毒蛋白质的合成通常比宿主细胞蛋白质的合成更简单，因此有可能利用这项研究的信息来开发系统，以生产有益营养的所需蛋白质、抗体或具有其他经济用途。

Plant viral diseases affect a significant number of food crops world-wide and can have severe impact both on economic conditions and food supply. Protein synthesis is a key step in viral infection yet is not well understood. This research will increase our understanding of plant viral protein synthesis and how this important biochemical process can be regulated. Structural information about IRES and initiation factors is providing information about the complexes formed. However, quantitative data are necessary to determine how initiation factors compete for different mRNA sites, the formation of intermediates, and the kinetic and thermodynamic stability of complexes. This research will utilize fluorescence spectroscopy, stopped-flow kinetics and circular dichroism to determine the role of individual initiation factors in plant viral mRNA internal initiation, the interaction of these factors with each other and known IRES elements, and how these factors modify mRNA structure. These data will be used to determine a model for selection of mRNA under various physiologic conditions. Quantitative information and determination of the cooperativity of interactions will yield detailed information on the assembly of the 48S initiation complex and binding to viral IRES. This information can potentially be used to shut off viral protein synthesis either through genetic engineering or biochemical agents. Further, viral protein synthesis in general is simpler than host cell protein synthesis so there is potential to use the information from this research to develop systems to produce desired proteins which are nutritionally beneficial, antibodies, or have other economic uses.