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.

项目摘要/摘要 病毒已经发展出多种机制，以劫持宿主的蜂窝机械，以获得自己的优势。 RNA病毒基因组无处不在的是离散的结构化RNA元素，在逃避中起着至关重要的作用 宿主细胞免疫力并促进病毒传播。因此，病毒RNA结构复杂地与病毒联系在一起 感染和疾病。重要的是，生物医学研究的主要目标是了解病毒如何操纵 主机机械。这是理解结构化RNA元素的结构功能关系 在病毒中，基因组对于疫苗和治疗的未来开发至关重要。 许多RNA病毒基因组都包含结构或功能模拟转移RNA（tRNA）的区域 他们与宿主细胞机械互动和操纵策略的一部分。这些tRNA模拟物被发现 通过病毒基因组和感染多种宿主的病毒。具有tRNA模拟的重要例子 用作这种类型的病毒机制的模型系统是3'中发现的结构（TLS）的tRNA 未翻译的植物感染病毒区域。这些TLS是几十年前首次确定的，已经 以三种方式模仿TRNA，并增强无细胞提取物中的翻译。 尽管研究了数十年和一些TLS的高分辨率结构信息，但它们如何增强 翻译仍然未知。该建议旨在了解TLS在翻译中的贡献 增强病毒基因组。第一步是识别TLS相互作用的其他宿主细胞组件 与Cognate trnas相比，这是如何比较的。确定完整的互动合作伙伴将允许建立 可测试的模型以阐明TLS翻译增强功能背后的机制。 此外，重要的是要确定TLSs是否通过额外的tRNA靶向酶识别 由于tRNA修饰酶作为最近的证据表明对RNA的化学修饰对于 结构和功能。目标2提案，以研究TLS的假设模型为伪poly（a） 尾巴。使用荧光共振能量传递（FRET），然后进行翻译测定将确定是否是否 RNA转录本的固有折叠对于将3'TLS连接到5'盖很重要。 确定tRNA模拟在某些病毒的3端如何绕过poly（a）尾巴的必要性 将增强对主机翻译的强制性和潜在替代要求的理解 机械。这项工作将洞悉病毒如何使用tRNA模仿其益处，tRNA模拟方式 它可以产生宿主细胞机械，然后增强对tRNA模仿及其的一般理解 对基因表达的影响。