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

项目摘要 我们的主要目标是了解内部核糖体进入位点(IRESS)和3‘帽非依赖性翻译 增强子(3‘CITES)促进基因组在(+)链RNA病毒中的帽子非依赖性翻译。尽管高度 进化遥远病毒的多样性序列和预测的二级结构、IRESS和3个ʹ 从宿主中招募相同的组件来启动基因组翻译。病毒IRESS的结构信息 直接与核糖体结合的能力有限，而且对三维(3D)结构和相互作用的了解也很有限 通过其他机制促进翻译的IRESS和3‘CITES在很大程度上是未知的。我们将使用X光 结晶学，以确定这些RNA的高分辨率晶体结构，重点是类型I和类型 II小核糖核酸病毒IRESS和番茄病毒3‘端因其独特的募集翻译起始机制而被引用 因子和核糖体亚基通过使用模块化RNA结构域的多步骤动态组装过程。 我们的策略使用Fab片段作为伴侣来结晶和确定RNA和RNPs的结构， 这是派作为博士后帮助开发的一项技术的扩展。自从搬到UMBC以来，我们已经获得了 柯萨奇病毒IRES结构域V的晶体(I型IRES的一例)与Fab BL3-6形成的复合体中的衍射物 到3.36？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 与心脏病毒J-K和甲型肝炎病毒DV结构中观察到的类似的三向连接(PI以前的 工作)。优化结晶条件，获得高分辨率的衍射谱数据，分析SAXS数据 以获取溶液中的结构信息，并纯化用于结合的人eIF4G热-1结构域 研究正在进行中。最近，我们解决了Saguaro的T形结构域的2.9？分辨率的晶体结构 仙人掌病毒3型ꞌ与Fab BL3-6形成一个复合体，并鉴定其与人eIF4E的结合。许多病毒 在番茄病毒科中包含3个具有相似结构域的ꞌCITE，这表明这些RNAs采用一个共享的 模拟mRNA5ꞌ-帽的拓扑结构，用于结合eif4E。因此，我们可以确定其晶体结构。 不同种类的病毒IRESS，3‘CITES，以及一些细胞IRESS。FAB方法成功地解决了 几种RNA的晶体结构，但尚未证明RNP复合体的晶体结构；第二个目标是 整合这种FAB辅助技术来结晶和确定RNP络合物的结构。第三 目标是创建抗RNA单链可变片段(ScFv)作为RNA可视化的独特探针 促进宿主细胞中病毒RNA的定位、跟踪和量化。我们取得了很有希望的成果 这方面的初步数据，包括三种抗RNA单链抗体及其ScFv-GFP融合的发展 基于现有的抗RNA Fabs。带有和不带有GFP标签的scFv与同源RNA靶标结合 亲和力类似于它们的父代Fabs。综合起来，拟议的研究将提供对以下方面的更深入的见解 (+)-正义RNA病毒中帽非依赖性翻译启动的机制和解锁机会 开发针对这些导致人类、动物和植物疾病的病原体的RNA靶向疗法。

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.