VIRULENCE DETERMINANTS IN THE COXSACKIEVIRUS B3 GENOME

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 柯萨奇病毒B3(CVB3)是病毒性心肌炎、胰腺炎和I型糖尿病的主要病因。与所有微小核糖核酸病毒一样，CVB3利用内部核糖体进入位点(IRES)翻译其RNA基因组，通过识别5‘非翻译区(5’NTR)中的高结构RNA元件，将核糖体直接招募到起始密码子。这项研究的总体目标是了解5‘NTR及其相关IRES的结构和功能。这项建议的具体目标是探索决定CVB3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折叠成稳定结构的假设，这是正常功能所必需的。根据特定目标1的建议，确定了野生型CVB3 IRES的结构。正如在特定目标2中提出的，调节毒力表型的差异已经被定位。目前研究的目标是定义决定毒力的最小RNA元件，并探索支撑IRES功能的RNA-蛋白质相互作用。有两个特定的目标将检验这一假设，即5‘NTR中独立折叠的小RNA结构为细胞和病毒蛋白提供识别特征，这些特征共同决定病毒的毒力。在特定的目标1中，以前被确定为毒力决定因素的RNA结构域的溶液结构将在基因组的毒力和非毒力变体中确定。这些变体将包括自然发生的序列、定点突变和嵌合结构。我们建立的碱基特异性化学修饰剂的方法将探索核苷酸在折叠的RNA结构域中的可及性。在具体目标2中，将通过研究RNA-蛋白质相互作用来探索IRES功能的分子机制。利用我们已建立的化学修饰技术对RNA-蛋白质复合体进行探索性研究将有助于深入了解5‘NTR的构象动力学和功能状态。这些结果将有助于寻找有效的抗病毒药物和疫苗，不仅针对CVB3，而且还针对其他导致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. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.