CAREER: Structural and mechanistic studies of RNA-mediated enteroviral genome replication

肠道病毒具有单链(+)正义RNA基因组，含有复杂的RNA元件，通过招募病毒和宿主蛋白形成病毒复制所需的核糖核蛋白(RNP)复合体来指导基因组复制。了解这些RNA元件的3D结构和相应的RNPs对于理解基本的病毒学过程是必不可少的。该项目将研究RNA介导的肠道病毒基因组复制的结构和机制基础，并确定RNA结构如何利用病毒和宿主蛋白质在病毒感染期间促进复制。这些发现可以为未来的治疗或预防战略提供参考，以对抗导致人类疾病和全球大流行的病原体。该项目还将为不同的高中生、本科生和研究生提供以RNA为中心的研究机会，鼓励他们在RNA生物化学、结构生物学或相关的STEM领域从事更高的学习和职业生涯。该研究将利用抗原结合抗体片段(Fabs)作为伴侣，确定与肠道病毒复制相关的RNA和RNP的晶体结构，并使用生物化学和分子生物学工具来阐明RNA-RNA、RNA-蛋白质和蛋白质-蛋白质的相互作用。本研究的目的是：1)确定7种主要肠道病毒(肠道病毒A-C和鼻病毒A-D)5‘端顺式复制元件(CRs)的晶体结构；2)阐明这些5’Cres与病毒3C、3CD融合和人PCBP蛋白的相互作用；3)研究3‘Cres的晶体结构及其在(-)正义RNA复制中间体3’端形成的RNPs。预计结果将揭示结构-功能关系，为这一主要类别的病毒病原体的复制机制提供新的见解。该项目由生物科学局分子和细胞生物科学部的遗传机制和分子生物物理学项目联合资助。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Enteroviruses have a single-strand (+)-sense RNA genome containing complex RNA elements that direct genome replication by recruiting viral and host proteins to form ribonucleoprotein (RNP) complexes required for viral replication. Knowledge of the 3D structures of these RNA elements and corresponding RNPs is essential for understanding fundamental virological processes. The project will investigate the structural and mechanistic underpinnings of RNA-mediated enteroviral genome replication and determine how RNA structures exploit viral and host proteins to promote replication during viral infections. The findings can inform future therapeutic or preventive strategies to combat pathogens that cause human diseases and global pandemics. The project will also provide opportunities for RNA-centric research to a diverse group of high school, undergraduate, and graduate students, encouraging them to pursue higher studies and careers in RNA biochemistry, structural biology, or related STEM fields.The research will utilize antigen-binding antibody fragments (Fabs) as chaperones for determining the crystal structures of enteroviral replication-linked RNAs and RNPs and use biochemistry and molecular biology tools to elucidate RNA-RNA, RNA-protein, and protein-protein interactions. The objectives are to 1) determine the crystal structures of 5′ cis-acting replication elements (CREs) from seven major enteroviral genotypes (enterovirus A-C and rhinovirus A-D), 2) elucidate the interactions of these 5′CREs with viral 3C, 3CD fusion, and human PCBP proteins, and 3) investigate the crystal structures of 3′CREs and their RNPs formed within the 3′-end of the (-)-sense RNA replication intermediate. The outcomes are expected to reveal structure-function relationships that will provide new insights into the replication mechanism of this major class of viral pathogens.This project is jointly funded by the Genetic Mechanisms and Molecular Biophysics programs of the Molecular and Cellular Biosciences Division in the Biological Sciences Directorate.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.