Functional Role of BYDV 3' RNA Translational Enhancer Element

Cuny Hunter College, New York NY

Investigators

Abstract

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.