VIRULENCE DETERMINANTS IN PICORNAVIRUS GENOMIC RNA

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Coxsackievirus B3 (CVB3) is the leading cause of viral myocarditis, causes pancreatitis and clearly plays a role in type I diabetes. Like other picornaviruses, CVB3 translates its RNA genome using an internal ribosome entry site (IRES), whereby ribosomes are recruited directly to an initiation codon by recognizing a highly structured RNA element in the 5' nontranslated region (5'UTR) of the genome. The overall goal of this research is to understand the structure and function of the 5'UTR and its associated IRES. The specific objective of this proposal is to explore the 5'UTR and IRES found in coxsackievirus B3 (CVB3). Structural integrity of the IRES is critically important for viral multiplication and also for virulence. Given the central role of the IRES to viral infection it is essential to understand the details of IRES structure and function. For the picornavirus IRES, a secondary structure model has been proposed based upon phylogenetic comparison, but this model has not been tested experimentally. The goal of this research is to determine the solution structure of the CVB3 IRES from both virulent wild type and attenuated mutant viruses. Two specific aims will test the hypothesis that the IRES folds into a stable structure that is required for proper function. In specific aim 1 the structure of a wild type CVB3 IRES will be determined. Base-specific chemical modifying agents such as dimethyl sulfate and kethoxal will be used to probe the accessibility of nucleotides in the folded IRES RNA. Results of the chemical probing will experimentally determine the structure of the IRES. In specific aim 2 structural differences that mediate the virulence phenotype in CVB3 will be mapped. Applying chemical probing methods to attenuated mutant strains of the virus will reveal the critical structural changes that underlie virulence. Understanding the structure, and structural dynamics of the CVB3 IRES will provide insight into targets for inactivating this virus. These results will aid in the search for effective antivirals and vaccines, not only for CVB3 but also for a host of other disease causing picornaviruses.