Structural and mechanistic studies of cap-independent genome translation in (+)-strand RNA viruses

( )链RNA病毒中帽独立基因组翻译的结构和机制研究

• 批准号: 10713117
• 负责人: Deepak Koirala
• 金额: $ 34.34万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE COUNTY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-10 至 2028-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10713117
• 关键词: 3-Dimensional; Adopted; Affinity; Animal Diseases; Binding; Binding Sites; Cactaceae; Cardiovirus; Cells; Complex; Coxsackie Viruses; Crystallization; Data; Development; Distant; Enhancers; Etiology; Fab Immunoglobulins; Family; Genome; Goals; Hepatitis A Virus; Human; Internal Ribosome Entry Site; Knowledge; Messenger RNA; Molecular Chaperones; Outcomes Research; Parents; Peptide Initiation Factors; Plant Diseases; Postdoctoral Fellow; Process; RNA; RNA Probes; RNA Viruses; RNA-targeting therapy; Resolution; Ribosomes; Shapes; Structure; Technology; Tombusviridae; Tombusvirus; Translation Initiation; Translations; Viral; Virus; Visualization; Work; X-Ray Crystallography; global health; human disease; insight; pandemic disease; pathogen; recruit; three dimensional structure; viral RNA

Project Summary Our main goal is to understand how internal ribosome entry sites (IRESs) and 3' cap-independent translation enhancers (3'CITEs) promote cap-independent translation of genomes in (+)-strand RNA viruses. Despite highly diverse sequences and predicted secondary structures, IRESs and 3ʹCITEs from evolutionarily distant viruses recruit the same components from the host to initiate genome translation. Structural information for viral IRESs that bind directly to the ribosome is limited and understanding of 3-dimensional (3D) structures and interactions of IRESs and 3'CITEs that promote translation by other mechanisms is largely unknown. We will use X-ray crystallography to determine the high-resolution crystal structures of these RNAs, focusing on the type I and type II picornaviral IRESs and tombusvirus 3'CITEs due to their unique mechanisms of recruiting translation initiation factors and the ribosomal subunits through a multistep, dynamic assembly process using modular RNA domains. Our strategy employs Fab fragments as chaperones to crystallize and determine structures of RNAs and RNPs, an extension of a technology that PI helped develop as a postdoc. Since moving to UMBC, we have obtained crystals of coxsackievirus IRES domain V (an example of type I IRES) in a complex with Fab BL3-6 that diffracted to 3.36 Å resolution (the first 3D structural information for type I IRESs). A partial structure of the dV contains a 3-way junction analogous to that observed in cardiovirus J-K and hepatitis A virus dV structures (PI's previous work). Optimization of crystallization conditions to obtain high-resolution diffraction data, analysis of SAXS data to access in-solution structural information, and purification of the human eIF4G HEAT-1 domain for binding studies are underway. Recently, we solved the 2.9 Å resolution crystal structure of a T-shaped domain of saguaro cactus virus 3ꞌCITE in a complex with Fab BL3-6 and characterized its binding with human eIF4E. Many viruses within the tombusviridae family contain 3ꞌCITEs with similar domains, suggesting that these RNAs adopt a shared topology to mimic mRNA 5ꞌ-cap for binding eIF4E. We are thus poised to determine the crystal structures of different kinds of viral IRESs, 3'CITEs, and some cellular IRESs. The Fab approach has successfully solved the crystal structures of several RNAs, but it has not been demonstrated for RNP complexes; the second goal is to integrate this Fab-assisted technology to crystallize and determine the structures of RNP complexes. The third goal is to create anti-RNA single-chain variable fragments (scFvs) as unique probes for RNA visualization to facilitate the localization, tracking, and quantification of viral RNAs in host cells. We have obtained promising preliminary data in this direction, including the development of three anti-RNA scFvs and their scFv-GFP fusions based on the existing anti-RNA Fabs. The scFvs with and without GFP tags bind the cognate RNA targets with affinities similar to their parent Fabs. When taken together, the proposed studies will provide deeper insights into the mechanisms of cap-independent translation initiation in (+)-sense RNA viruses and unlock opportunities for developing RNA-targeted therapeutics against these pathogens that cause human, animal, and plant diseases.

项目摘要 我们的主要目标是了解内部核糖体入口站点（IRESS）和3'无关的翻译 增强子（3'CITES）促进基因组中基因组的帽单位翻译（+） - 链RNA病毒。尽管很高 潜水序列和预测的二级结构，IRESS和3次来自进化的病毒 从宿主募集相同的组件以启动基因组翻译。病毒IRESS的结构信息 直接与核糖体结合的是有限的，并且了解3维（3D）结构和相互作用 通过其他机制促进翻译的IRESS和3'cites是未知的。我们将使用X射线 晶体学以确定这些RNA的高分辨率晶体结构，重点是I型和类型 II picornaviral iress和tombusvirus 3'cites，由于其独特的招募翻译启动机制 因子和核糖体亚基通过模块化RNA域的多步动组装过程。 我们的战略员工fab碎片作为伴侣，以结晶并确定RNA和RNP的结构， PI帮助发展后的技术的扩展。自移居UMBC以来，我们已经获得了 coxsackievivirus ires域的晶体V（I型IRES的示例）与Fab Bl3-6的复合体中 到3.36Å分辨率（I型IRESS的第一个3D结构信息）。 DV的部分结构包含一个 3向连接类似于在心脏病毒J-K和肝炎A病毒DV结构中观察到的连接（PI的先前 工作）。优化结晶条件以获得高分辨率衍射数据，分析SAXS数据 访问解决方案的结构信息，并纯化人EIF4G热1结构域以结合 研究正在进行中。最近，我们解决了Saguaro T形域的2.9Å分辨率晶体结构 仙人掌病毒3游戏中引用了fab bl3-6的综合体，并将其与人EIF4E的结合进行了特征。许多病毒 在Tombusviridae家族中包含3个带有相似域的引用，这表明这些RNA采用了共享 拓扑模拟MRNA 5-CAP，用于结合EIF4E。因此，我们被中毒以确定 不同种类的病毒率，3'cites和一些细胞IRESS。工厂方法已成功解决了 几种RNA的晶体结构，但尚未证明RNP复合物。第二个目标是 集成了这种Fab辅助技术，以结晶并确定RNP复合物的结构。第三 目标是创建抗RNA单链可变片段（SCFV）作为RNA可视化的独特问题 促进宿主细胞中病毒RNA的定位，跟踪和定量。我们得到了诺言 在这个方向上的初步数据，包括开发三个抗RNA SCFV及其SCFV-GFP融合 基于现有的抗RNA Fabs。带有和不带GFP标签的SCFV与 类似于父母晶圆厂的亲和力。当一起，拟议的研究将为您提供更深入的见解 （+）中无关转换启动的机制 - 感知RNA病毒和解锁机会 针对引起人，动物和植物性疾病的这些病原体开发针对RNA靶向的疗法。