Elucidating structure-function relationships of viral tRNA-like structures

阐明病毒 tRNA 样结构的结构与功能关系

• 批准号: 10462144
• 负责人: Jillian Ramos
• 金额: $ 4.8万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2023-03-26
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10462144
• 关键词: 3' Untranslated Regions; Amino Acyl-tRNA Synthetases; Awareness; Biological; Biological Models; Biomedical Research; Bypass; Cell physiology; Cells; Cellular Structures; Chemicals; Communication; Complex; Disease; Elements; Elongation Factor; Enzymes; Fluorescence Resonance Energy Transfer; Future; Gene Expression; Goals; Guanosine Triphosphate; Hepatitis C virus; Human Herpesvirus 4; Immunity; In Vitro; Infection; Lead; Link; Malignant Neoplasms; Mass Spectrum Analysis; Methods; Modeling; Modification; Organism; Plants; Play; Poly(A) Tail; Process; Property; Proteins; Proteome; Proteomics; Publishing; RNA; RNA Folding; RNA Viruses; RNA-Protein Interaction; Reporter; Resolution; Role; Structure; Structure-Activity Relationship; System; Testing; Therapeutic; Transcript; Transfer RNA; Translations; Variant; Viral; Viral Genome; Viral Physiology; Viral Proteins; Virus; Virus Diseases; Work; base; functional mimics; insight; mimicry; translation assay; vaccine development; viral RNA

Project Summary/Abstract Viruses have evolved diverse mechanisms to hijack the host’s cellular machinery for their own advantage. Ubiquitous in RNA viral genomes are discrete structured RNA elements that play an essential role in evading host cell immunity and promoting viral propagation. Therefore, viral RNA structure is intricately linked to viral infection and disease. Importantly, a major goal of biomedical research is to understand how viruses manipulate the host machinery. Thus, understanding the structure-function relationship of structured RNA elements found in viral genomes is fundamental for future development of vaccines and therapeutics. Many RNA viral genomes contain regions that structurally or functionally mimic transfer RNA (tRNA) as part of their strategy to interact with and manipulate the host cell machinery. These tRNA mimics are found throughout viral genomes and in viruses that infect diverse hosts. Important examples of tRNA mimicry that have served as a model system for this type of viral mechanism are tRNA Like Structures (TLS) found in the 3’ untranslated region of plant-infecting viruses. These TLSs were first identified decades ago and have been shown to mimic tRNAs in three ways and enhance translation in cell-free extracts. Despite decades of study and high-resolution structural information of a few TLSs, how they enhance translation remains unknown. This proposal aims to understand the contribution of the TLS in translation enhancement of the viral genome. The first step is to identify additional host cell components the TLS interacts with and how this compares to cognate tRNAs. Identifying the full set of interacting partners will allow for building testable models to elucidate the mechanism behind the translation enhancement function of the TLS. Furthermore, it is important to determine if the TLSs are recognized by additional tRNA-targeted enzymes, such as tRNA modification enzymes as recent evidence has shown chemical modifications to RNA are important for structure and function. Aim 2 proposes to investigate the hypothesized model of the TLS as a pseudo-poly(A) tail. Using fluorescence resonance energy transfer (FRET) followed by translation assays will determine if intrinsic folding of the RNA transcript is important for communication of the 3’ TLS to the 5’ cap. Identifying how tRNA mimics at the 3’ end of certain viruses can bypass the necessity of a poly(A) tail will enhance the understanding of the mandatory and potential alternative requirements of the host translation machinery. This work will yield insight into how viruses use tRNA mimicry for their benefit, how tRNA mimics are able to delude host cell machinery, and furthermore enhance the general understanding of tRNA mimicry and its influence on gene expression.

项目总结/摘要 病毒已经进化出各种各样的机制来劫持宿主的细胞机器以获得自身的优势。 在RNA病毒基因组中普遍存在的是离散结构的RNA元件，其在逃避病毒感染中起着重要作用。 宿主细胞免疫和促进病毒繁殖。因此，病毒RNA的结构与病毒 感染和疾病。重要的是，生物医学研究的一个主要目标是了解病毒如何操纵 主机机械。因此，理解结构化RNA元件的结构-功能关系， 在病毒基因组中是未来疫苗和治疗药物开发的基础。 许多RNA病毒基因组含有在结构上或功能上模拟转运RNA（tRNA）的区域， 这是它们与宿主细胞相互作用和操纵宿主细胞机制的策略的一部分。这些tRNA模拟物 在病毒基因组和感染不同宿主的病毒中。tRNA模拟的重要例子， 作为这类病毒机制的模型系统的是在3'端发现的tRNA样结构（TLS）。 感染植物的病毒的非翻译区。这些TLS在几十年前首次被发现， 显示以三种方式模拟tRNA并增强无细胞提取物中的翻译。 尽管几十年的研究和一些TLS的高分辨率结构信息，它们如何增强 翻译仍然未知。本文旨在了解TLS在翻译中的作用 增强病毒基因组。第一步是确定TLS相互作用的其他宿主细胞成分 以及与同源tRNA的比较。确定全套相互作用的合作伙伴将允许建立 可测试的模型来阐明TLS的翻译增强功能背后的机制。 此外，重要的是确定TLS是否被另外的tRNA靶向酶识别，如 作为tRNA修饰酶，最近的证据表明，对RNA的化学修饰对于 结构和功能。目的2提出研究TLS作为伪聚（A）的假设模型 尾巴使用荧光共振能量转移（FRET），然后进行翻译测定，将确定 RNA转录本的内在折叠对于3 ′ TLS与5 ′帽的通讯是重要的。 确定某些病毒3'端的tRNA模拟物如何绕过poly（A）尾的必要性 将加强对主机翻译的强制性和潜在替代性要求的理解 机械.这项工作将深入了解病毒如何利用tRNA模拟来获得利益，tRNA模拟物如何 能够欺骗宿主细胞机制，并进一步增强对tRNA模拟及其 影响基因表达。