Quantitative Analysis of Viral mRNA Translation

植物病毒病影响世界各地的大量粮食作物，并可对经济条件和粮食供应产生严重影响。蛋白质合成是病毒感染的关键步骤，但尚不清楚。这项研究将增加我们对植物病毒蛋白质合成以及如何调控这一重要生化过程的理解。有关IRES和启动因子的结构信息提供了有关形成的络合物的信息。然而，定量数据对于确定起始因子如何竞争不同的mRNA位点、中间体的形成以及络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谱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的IRES元件，以及这些因子是如何改变mRNA结构的。这些数据将被用来确定在不同生理条件下选择mRNA的模型。定量信息和相互作用协同性的确定将产生关于48S起始复合体的组装和与病毒IRES结合的详细信息。这些信息可能被用来通过基因工程或生化试剂来切断病毒蛋白质的合成。此外，一般来说，病毒蛋白质合成比宿主细胞蛋白质合成更简单，因此有可能利用这项研究的信息开发系统来生产所需的蛋白质，这些蛋白质对营养有益、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.