Translational Enhancement by tRNA Mimics

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This project addresses the question of how viruses, which depend on the infected host cells for making virus proteins, use the host cell protein-producing machinery, the ribosome, in a manner that is highly efficient. The research will exploit and extend the novel discovery that the genetic material of a virus contains specific structures that can bind to the ribosomes and that thereby allow them to be quickly and efficiently reused. Key outcomes of this project will be broadly applicable in many fields, including in biotechnology to enhance the efficiency of protein production. The outcomes may also lead to novel approaches to control a variety of viruses that infect plants and animals. This project will provide educational and research training opportunities for high school students, college undergraduates, graduate students and postdoctoral researchers. The outcomes of the research will be presented at scientific conferences, taught in introductory courses to biology students, and used to introduce undergraduates and high school students to the life cycle of viruses. The principal investigator will work with Advanced Placement students at DuVal High School (98% under-represented minority students) to prepare them for college, serve on the board of the National Academy of Sciences partnership with the entertainment industry and will also serve as a scientific consultant for programming by the Discovery and National Geographic Channels.The plant virus "Pea enation mosaic umbravirus" has a bipartite positive-strand RNA genome containing two 3' proximal T-shaped structures (kl-TSS and 3'TSS). These structures are critical for translation of the uncapped viral RNAs into proteins. 3'TSS is an example of the long-known, better-studied tRNA-like structures found in viral RNAs, but the existence of the kl-TSS was unsuspected and came to light during previous research. The newly found, internal kl-TSS, with its three-way branched secondary structure, binds to 40S, 60S and 80S ribosomes and can simultaneously engage in a long-distance interaction with 5H2, a coding region hairpin located near the 5'Untranslated Region (UTR). The 3'TSS is also likely to connect with 5H2, and the whole system will be exploited to learn how different tRNA-mimics interact with ribosomes to enhance translation. Specifically, a novel "pick-up and delivery" hypothesis will be evaluated, i.e., the idea that multiple TSS may be connected via RNA:RNA interactions with the ends of virus-encoded Open Reading Frames (ORF), to gather ribosomal subunits downstream of an ORF at the end of translation and relay them back to the 5'UTR for another round of translation. In addition, it will be tested whether the kl-TSS can enhance translation of capped mRNAs produced from expression constructs. These experiments will inform our understanding of how this novel and possibly widespread use of tRNA mimicry operates to enhance translation and to control plant virus life cycles. The project will also lead to the development of expression cassettes with enhanced translation of encoded proteins with potentially extensive agricultural as well as laboratory applications.