Intellectual Merit: Plant viruses cause diseases that affect a significant number of food crops world-wide with potentially severe consequences on food supplies and economic conditions. Most viruses 'commandeer' the host cell's protein synthesis machinery to make viral proteins. Barley yellow dwarf virus (BYDV) is one of the two most widespread and economically significant plant viruses, affecting wheat, barley and oat crops worldwide. The aims of this project will advance the basic science of plant viruses, while enabling technologies for the improved control of harmful plant viruses and the engineering of modified viruses for production of high-value proteins using plants. BYDV contains an unusual RNA structure, a translational enhancer (BTE), in the 3' untranslated region (UTR) of the mRNA that interacts with a 5' UTR stem loop. This long-range interaction promotes efficient translation of the viral mRNA by a mechanism that is not yet fully understood. The project will employ the 3' UTR of BYDV as a model system to elucidate mechanisms of long-range RNA interactions involving 3'UTR elements. The overall goal of the project is to identify the molecular interactions that enable viral mRNA to compete efficiently with the much higher concentrations of cellular RNA for access to ribosomes and translation factors. The three critical steps in the assembly of viral protein synthesis initiation complexes will be investigated: 1) The binding of eIFs (eukaryotic initiation factors) to the viral 3'-BTE; 2) the interaction of ribosomes with viral 3'-BTE and 5'-UTR sequences; and 3) the interaction between viral 3'-BTE and 5'-UTR regions. A library of BYDV mRNAs with varying translational efficiency will be used to determine the correlation between eIF binding and translation, the binding affinity of ribosomes for viral RNA sequences, and the role of the interaction between 3' and 5'-UTRs in each of these steps. The project will employ a range of biochemical and biophysical techniques to achieve these aims, including ribosome 'toeprint' analysis, direct fluorescence assays, and RNA binding assays analyzed by mass spectroscopic analysis.
Broader Impacts: The significance of the research and the range of experimental approaches employed will provide excellent training opportunities for students, ranging from the high school to the graduate school level, in both biochemical assays and quantitative biophysical measurements. Students at each level will receive appropriate training in molecular biology techniques including expression of cloned proteins, translational assays, and other important biochemical techniques. Fluorescence spectroscopy and mass spectrometry used in this project will provide quantitative and biophysical training for more advanced students. Hunter College recruits and enrolls significant numbers of minority students under-represented in the sciences, and also sponsors a summer research program for them. Students will be recruited from this program to participate in the research. Thus, the project will contribute to training a diverse workforce in science and technology. High school teachers will carry out research in the lab during the summers, using quantitative fluorescence measurements to determine equilibrium and thermodynamic properties and enhance their content knowledge of applications of thermodynamics to biological systems. The Principal Investigator will work with faculty from the Borough of Manhattan Community College (an Hispanic serving institution) to increase research opportunities for their faculty and students, providing access to state-of-the-art equipment and research guidance when appropriate. By participating in the research project, high school teachers and community college faculty will increase their knowledge of current research techniques and their abilities to convey to their students the excitement of science.
