Functional Role of BYDV 3' RNA Translational Enhancer Element

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。该项目的研究将探索一种特定植物病毒的遗传物质如何形成一种不寻常的结构，这种结构能够在宿主细胞中启动蛋白质的生产（一种称为翻译的过程）。这项研究的信息不仅有可能用于控制病毒，而且有可能用于开发生产有益营养的蛋白质、蛋白质抗体或具有经济用途的蛋白质的系统。在更基本的层面上，由于翻译的相似性，这种知识可能与其他主要病毒性疾病有关，如丙型肝炎、艾滋病毒和西尼罗河病毒。由于其相对较小的尺寸和明确的成分，植物病毒作为一个模型系统，以增加对更复杂的病毒和细胞翻译的理解。该项目将为从高中到研究生的学生提供生物化学和定量测量方面的优秀培训。过去参与研究的许多学生都来自代表性不足的少数群体，这种情况未来可能会继续下去。这些学生将为科学和技术领域的多元化和训练有素的劳动力做出贡献。该项目还将涉及高中教师和社区大学教师。当RNA病毒感染细胞时，它必须与宿主细胞mRNA竞争以产生病毒蛋白。病毒通过许多策略来实现这一点，其中许多涉及独特的病毒RNA结构。大麦黄矮病毒（BYDV）在mRNA的3'非翻译区（UTR）含有一个不寻常的RNA结构（BTE）。初步结果表明，BTE与核糖体、真核起始因子（eIFs）结合，并与5' UTR干环相互作用。这种新的蛋白质合成起始机制的细节尚不清楚。该项目将研究起始复合物组装的三个关键步骤。这些工作包括：1)确定BYDV的40S/43S核糖体亚基与3' BTE结合的限速步骤；2)核糖体向5' UTR转移的分析；3)3' BTE与5' UTR相互作用的分析。荧光光谱将用于确定每个分离的蛋白质对核糖体亚基结合的影响。核糖体在5' UTR上的转移位点和扫描速率以及eif对这些速率的影响将通过FRET技术测量。在单分子荧光的新应用中，将确定40S核糖体亚基从3' UTR结合位点转移到5' UTR的速率。BYDV 5'-3' RNA相互作用似乎是短暂的，因此很难阐明该复合物对核糖体亚基转移的影响。为了进一步量化这些相互作用，将使用与康乃馨意大利环斑病毒相关但更稳定的相互作用的5'-3' utr。这里描述的实验将增加对分子相互作用的理解，这种相互作用使病毒mRNA能够有效地与更高浓度的细胞RNA竞争进行翻译。

Plant viral diseases affect a significant number of food crops world-wide and can have severe impact both on economic conditions and food supply. The research in this project will probe how the genetic material of a specific plant virus form an unusual structure that is able to initiation the production of proteins (a process called translation) in the host cell. There is a potential to use information from this research not only to control viruses but also to develop systems for production of proteins that are nutritionally beneficial, protein antibodies or proteins that have economic uses. At a more fundamental level, because of similarities in translation, this knowledge may be relevant to other major viral diseases such as hepatitis C, HIV and West Nile. Because of their relatively small size and well-defined components, plant viruses serve as a model system to increase the understanding of more complex viral and cellular translation. This project will provide excellent training for students from high school through graduate school in both biochemical and quantitative measurements. Many of the students involved in research in the past have been from underrepresented minority groups and this is likely to continue in the future. These students will contribute to a diverse and well trained workforce in science and technology. This project will also involve high school teachers and community college faculty.When an RNA virus infects a cell, it must out-compete host cell mRNA to produce viral proteins. Viruses accomplish this by a number of strategies, many involving unique viral RNA structures. Barley yellow dwarf virus (BYDV) contains an unusual RNA structure (a BTE) in the 3' untranslated region (UTR) of the mRNA. In preliminary results, it was shown that the BTE binds ribosomes, eukaryotic initiation factors (eIFs) and interacts with a 5' UTR stem loop. The details of this novel mechanism of protein synthesis initiation are unknown. This project will investigate three critical steps in assembly of an initiation complex. These include 1) determining the rate-limiting steps for 40S/43S ribosomal subunit binding to the 3' BTE of BYDV; 2) analysis of ribosome transfer to the 5' UTR and 3) 3' BTE and 5' UTR interactions. Fluorescence spectroscopy will be used to determine the effects of each of the isolated proteins on ribosomal subunit binding. The site of ribosome transfer at the 5' UTR and the rates of scanning and influence of eIFs on those rates will be measured by FRET techniques. In a novel application of single molecule fluorescence, the rate of transfer of the 40S ribosomal subunit from the 3' UTR binding site to the 5' UTR will be determined. The BYDV 5'-3' RNA interaction appears to be transient and it is therefore difficult to elucidate the effects of the complex on ribosomal subunit transfer. To further quantitate these interactions, the related, but much more stable interaction of the carnation Italian ringspot virus 5'-3' UTRs will be used. The experiments described here will increase understanding of the molecular interactions that allow viral mRNA to compete efficiently with the much higher concentration of cellular RNA for translation.