VIRULENCE DETERMINANTS IN THE COXSACKIEVIRUS B3 GENOME

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。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 研究中心，而研究中心不一定是研究者所在的机构。 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。 与所有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，由此核糖体通过识别5'非翻译区（5' NTR）中的高度结构化RNA元件而直接募集至起始密码子。 本研究的总体目标是了解5 'NTR及其相关IRES的结构和功能。 本提案的具体目标是探索决定CVB 3毒力的RNA元件和RNA-蛋白质相互作用。 上一笔赠款的两个具体目标已经实现，证实了IRES折叠成正常功能所需的稳定结构的假设。 如具体目的1中所提出的，确定了野生型CVB 3 IRES的结构。 如具体目标2所述，已绘制了介导毒力表型的差异。 目前研究的目标是确定决定毒力的最小RNA元件，并探索IRES功能的基础RNA-蛋白质相互作用。 两个具体的目标将测试的假设，小，独立折叠的RNA结构在5 'NTR提供识别功能的细胞和病毒蛋白，共同决定病毒的毒力。 在具体目标1中，将在基因组的毒性和非毒性变体中确定先前鉴定为毒力决定簇的RNA结构域的溶液结构。 这些变体将包括天然存在的序列、定点突变体和嵌合构建体。 我们建立的碱基特异性化学修饰剂的方法将探测折叠RNA结构域中核苷酸的可及性。 在具体目标2中，IRES功能的分子机制将通过RNA-蛋白质相互作用的研究来探索。 利用我们建立的化学修饰技术对RNA-蛋白质复合物进行探测研究将提供对5 'NTR的构象动力学和功能状态的深入了解。 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，而且对许多其他致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 plays a role in type I diabetes. Like all 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'NTR). The overall goal of this research is to understand the structure and function of the 5'NTR and its associated IRES. The specific objective of this proposal is to explore the RNA elements and the RNA-protein interactions that determine virulence in CVB3. The two specific aims of the previous grant were accomplished, confirming the hypothesis that the IRES folds into a stable structure that is required for proper function. As proposed in specific aim 1, the structure of a wild type CVB3 IRES was determined. As proposed in specific aim 2, differences that mediate the virulence phenotype have been mapped. The goal of the current research is to define the minimal RNA elements that determine virulence and explore the RNA-protein interactions that underlie IRES function. Two specific aims will test the hypothesis that small, independently folding RNA structures in the 5'NTR provide recognition features for cellular and viral proteins that together determine viral virulence. In specific aim 1, the solution structure of RNA domains previously identified to be virulence determinants will be determined in virulent and non-virulent variants of the genome. These variants will include naturally occurring sequences, site-directed mutants and chimeric constructs. Our established methods of base-specific chemical modifying agents will probe the accessibility of nucleotides in the folded RNA domains. In specific aim 2 the molecular mechanism of IRES function will be explored by studies of RNA-protein interactions. Probing studies of RNA-protein complexes using our established chemical modification techniques will provide insight into the conformational dynamics and functional states of the 5'NTR. 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.