Structure and Function of Plant Virus 3' RNA Translational Enhancer Elements

植物病毒影响许多粮食作物，导致严重的经济和粮食供应问题。大麦黄矮病毒（BYDV）和玉米坏死条纹病毒（MNeSV）是世界范围内对谷类作物的蚜虫传播威胁。这些正链RNA病毒是已知翻译最有效的mRNA之一，因此可以在生物技术中用于低成本生产蛋白质;然而，新兴病毒仍然是影响作物的严重问题。该项目将通过确定病毒蛋白合成的机制细节来确定干预的目标步骤，从而解决开发新的抗病毒策略的主要障碍。该项目还为从高中到研究生阶段的学生提供了良好的培训。一个结构化的夏季研究计划将被开发为一个模型，提供研究机会，在该机构的本科生人数更多。这些学生中有许多来自代表性不足的少数民族，他们的参与将有助于建立一支多样化和训练有素的科学队伍。本项目将研究选择mRNA翻译和组装起始复合物的关键方面。蛋白质合成起始因子3（eIF 3）的作用将通过使用荧光光谱法来测定与翻译增强子元件结合的亲和力和动力学，并通过结构探测来确定它如何重塑RNA。将通过单分子荧光研究核糖体募集的机制，包括对BYDV和结构更简单的MNeSV的比较分析，以了解病毒蛋白质合成中的共同主题。冷冻电镜实验和化学探测将用于确定蛋白质-RNA相互作用的结构细节。这种全面的方法有望提供一个更好的理解植物病毒如何在翻译中胜过宿主mRNA。由于这些机制可能发生在其他真核生物系统中，并在压力条件下调节翻译，因此该项目的结果预计将广泛适用。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的知识价值和更广泛的影响审查标准进行评估来支持。

Plant viruses affect a number of food crops leading to severe economic and food supply problems. Barley yellow dwarf virus (BYDV) and maize necrotic streak virus (MNeSV) are aphid-borne threats to cereal crops worldwide. These positive strand RNA viruses are some of the most efficiently translated mRNA known, and can therefore be co-opted in biotechnology for low-cost production of proteins; however, emerging viruses remain a severe problem affecting crops. This project will address major roadblocks in developing new anti-viral strategies by determining mechanistic details of viral protein synthesis that identify target steps for intervention. The project also provides excellent training for students from high school through graduate levels. A structured summer research program will be developed as a model for offering research opportunities to larger numbers of undergraduates at the institution. Many of these students are from under-represented minorities and their participation will contribute to a diverse and well-trained scientific workforce. This project will investigate critical aspects in the selection of mRNA for translation and assembly of an initiation complex. The role of protein synthesis initiation factor 3 (eIF3) will be determined by using fluorescence spectroscopy to measure affinity and kinetics of binding to the translation enhancer element, and structural probing to determine how it remodels RNA. The mechanism of ribosome recruitment will be investigated by single molecule fluorescence, including comparative analysis of BYDV and the structurally simpler MNeSV to understand common themes in viral protein synthesis. Cryo-EM experiments and chemical probing will be employed to determine structural details about the protein-RNA interactions. This comprehensive approach is expected to provide a better understanding of how plant viruses outcompete host mRNA in translation. Since these mechanisms likely occur in other eukaryotic systems and regulate translation under stress conditions, the results of this project are expected to be broadly applicable.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.