VIRULENCE DETERMINANTS IN PICORNAVIRUS GENOMIC RNA

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。柯萨奇病毒B3(CVB3)是病毒性心肌炎的主要原因，可引起胰腺炎，显然在I型糖尿病中起作用。与其他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，通过识别基因组5‘非翻译区(5’UTR)中的高结构RNA元件，将核糖体直接招募到起始密码子。本研究的总体目标是了解5‘非编码区及其相关IRES的结构和功能。这项建议的具体目的是探索在柯萨奇病毒B3(CVB3)中发现的5‘非编码区和IRES。IRES的结构完整性对于病毒增殖和毒力也是至关重要的。鉴于IRES在病毒感染中的核心作用，必须了解IRES的结构和功能的细节。对于微小核糖核酸病毒IRES，已经提出了一个基于系统发育比较的二级结构模型，但该模型还没有得到实验验证。本研究的目的是确定CVB3IRES的溶液结构，包括强毒野毒和弱毒突变病毒。两个特定的目标将检验IRES折叠成正常功能所需的稳定结构的假设。在具体目标1中，将确定野生型CVB3 IRES的结构。碱基特异的化学修饰剂，如硫酸二甲酯和酮氧醛，将被用来探测折叠的IRES RNA中核苷酸的可及性。化学探测的结果将在实验上确定IRES的结构。在特定的目标2中，将定位介导CVB3毒力表型的结构差异。将化学探测方法应用于该病毒的减毒突变株，将揭示毒力背后的关键结构变化。了解CVB3IRES的结构和结构动力学将为灭活该病毒的靶点提供洞察力。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，而且还针对引起小核糖核酸病毒的其他疾病。

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.