REGULATION OF CAP-INDEPENDENT TRANSLATION IN PLUS-STRAND RNA VIRUSES

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This project will benefit society by having college undergraduates, PhD level graduate students and postdoctoral researchers participate in research designed to address how viruses engage in one of their most important activities, synthesis of their required proteins. This research will be presented at scientific conferences, used in the teaching of introduction to biology for biology majors, and used to introduce undergraduates at the University of Maryland and Bowie State University to cutting edge procedures designed to study how the virus manipulates its genome in ways that were not previously thought to occur. In addition, this research adds a new dimension of understanding to a procedure (protein synthesis) that was thought to be mostly solved by exploring the possible regulation of a key aspect of virus biology: how they switch from being a template for making proteins to having that template replicated by changing how the template interacts with specific proteins. By studying three different viruses that all appear to use the same process to control how proteins are made, results should have a broad impact on virus research in general. Thus the results of this research should be applicable not only to plant viruses, but also animal and human viruses, including ones that cause serious disease in animals and plants. Recent results have led to the hypothesis that translation enhancers located in 3' UTRs of plant viruses need to be cleared of bound translation factors/ribosomes for replication of the template to proceed. PEMV contains three 3'cap-independent translation enhancers (3'CITEs): kl-TSS, PTE, and TSS, only two of which are used by the genomic RNA (kl-TSS/PTE) while all three are used by the subgenomic RNA. In Aim 1, SHAPE RNA structure probing and mutagenesis will be used to determine whether a sequence exclusive to the gRNA alters the structure of the TSS to keep it from functioning. In Aim 2, compensatory mutations and SHAPE will be used to examine a newly discovered element (the CAS) for its ability to block translation factor binding to downstream 3' CITEs in three viruses. The newly identified replication element DUO will also be investigated for interaction with CAS during translation, allowing the critical kl-H hairpin to bind CAS, keeping CAS from binding to the downstream 3'CITE and interfering with translation factor binding. Aim 3 will employ mutagenesis, SHAPE and EMSA to determine if CAS interacts with, and destabilizes its downstream 3'CITE, and whether this interferes with binding of translation factors and RdRp transcription. Aim 4 will investigate the relationship among DUO, kl-H and CAS to determine how CAS is blocked from binding its 3'CITE.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.