VIRULENCE DETERMINANTS IN PICORNAVIRUS GENOMIC RNA

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心机构，不一定是研究者机构。柯萨奇病毒B3（CVB 3）是病毒性心肌炎的主要原因，导致胰腺炎，并在I型糖尿病中发挥作用。与其他小核糖核酸病毒一样，CVB 3使用内部核糖体进入位点（IRES）翻译其RNA基因组，由此通过识别基因组5'非翻译区（5' UTR）中的高度结构化RNA元件，将核糖体直接募集到起始密码子。本研究的总体目标是了解5 'UTR及其相关IRES的结构和功能。本计划的具体目标是探索柯萨奇病毒B3（CVB 3）中发现的5 'UTR和IRES。IRES的结构完整性对于病毒增殖和毒力至关重要。鉴于IRES在病毒感染中的核心作用，了解IRES结构和功能的细节至关重要。对于小核糖核酸病毒IRES，已经提出了一个二级结构模型的基础上系统发育比较，但这个模型还没有经过实验测试。本研究的目的是确定CVB 3 IRES的溶液结构，从两个毒性野生型和减毒突变病毒。两个具体的目标将测试的假设，IRES折叠成一个稳定的结构，需要适当的功能。在具体目的1中，将确定野生型CVB 3 IRES的结构。碱基特异性化学修饰剂如硫酸二甲酯和酮醛将用于探测折叠IRES RNA中核苷酸的可及性。化学探测的结果将通过实验确定IRES的结构。在特定目标2中，将定位介导CVB 3毒力表型的结构差异。将化学探测方法应用于病毒的减毒突变株，将揭示构成毒力基础的关键结构变化。理解CVB 3 IRES的结构和结构动力学将提供对灭活该病毒的靶点的深入了解。这些结果将有助于寻找有效的抗病毒药物和疫苗，不仅对CVB 3，而且对许多其他引起疾病的小核糖核酸病毒。

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.