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INVESTIGATOR: Kraft, J. J.; Kraft, J. J.

PERFORMING INSTITUTION:
IOWA STATE UNIVERSITY
2229 Lincoln Way
AMES, IOWA 50011

RNA STRUCTURE-MEDIATED REGULATION OF CAP-INDEPENDENT TRANSLATION BY PLANT VIRUSES

NON-TECHNICAL SUMMARY: Plant viruses in the Carmovirus, Panicovirus and Umbravirus genera are economically important viruses of soybean, legumes and lentils in US and other parts of the world causing up to 100% crop loss. These viruses occur and cause economic impact wherever these and similar crops are grown. In addition to its economical importance, viruses described here use an array of unconventional tools to express encoded genes. Little is known how carmo-, panico-, and umbraviruses are able to recruit translation machinery in the absence of canonical modifications in their genome. Even less is known of how they are able to break resistance encoded by natural recessive resistance genes. The success of plant breeding for improved avirulence determinants depends on how much we know about virus gene replication and expression. The purpose of this study is to determine the roles of RNA sequences from these three different viral genera in cap-independent translation initiation, their interaction with the protein synthesis machinery and how these interactions modify RNA structure using combination of structural and functional methods such as X-ray crystallography and site-directed mutagenesis. A detailed understanding of mechanism of cap-independent translation employed by these plant viruses will make it possible to 1. identify key regulatory steps and find common themes for viral translation shared with other economically important plant viruses that will ultimately suggest new ways to control viral disease and reduce crop losses in US and world; and 2. use the RNA translation signals to control expression of nonviral genes in transgenic plants expressing any gene of agricultural interest.

OBJECTIVES: Unlike host mRNAs, many viruses lack a cap or poly(A) tail and yet they are efficiently translated. Many plant viruses harbor sequences in their genome that allow them to escape the cellular control of translation initiation and express encoded genes. My goal is to understand how viruses in the Carmovirus, Panicovirus and Umbravirus genera accomplish this to effectively parasitize the plant host. Objectives are: 1. Determine the structure and sequences required by a novel RNA called the Panicum mosaic virus-like cap-independent Translation Element (PTE) by conducting mutagenesis, translation assays, structure probing and X-ray crystallography. 2. Understanding the molecular mechanism of PTE RNA-mediated translation by conducting structural analysis of the high affinity interaction of the PTE with translation initiation factor eIF4E. The following outcomes are expected: in year 1 mapping the core PTE secondary structure, purification of large quantities of PTE and PTE/eIF4E crystal targets and obtain single PTE and PTE-eIF4E crystals is planned. Plans for year 2 include improvement of resolution limit of obtained crystals to less than 3 angstroms by optimizing crystal growing conditions, and then solving the PTE and PTE-eIF4E crystal structure. Structure interpretation in conjunction with mutagenesis and structure probing data will relate the PTE structure to its function to 1. model how PTE recruits eIF4E in the absence of 5' cap to initiate translation; 2. provide insight into translation mechanisms used by related plant viruses. (this in turn could help in design of antiviral agents targeting viral translation mechanisms); and 3. explain how these and related viruses are able to break plant-mediated resistance for more successful breeding with improved avirulence determinants. Acquired results will be published in peer reviewed scientific journals and presented at scientific conferences.