Elucidating structure-function relationships of viral tRNA-like structures

项目摘要/摘要 病毒进化出不同的机制，为了自己的利益劫持宿主的细胞机器。 RNA病毒基因组中普遍存在的是离散的结构化RNA元件，它们在躲避病毒的过程中起着至关重要的作用 宿主细胞免疫和促进病毒繁殖。因此，病毒的RNA结构与病毒有着千丝万缕的联系 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 主机机器。因此，了解结构RNA元件的结构-功能关系发现 对病毒基因组的研究是疫苗和疗法未来发展的基础。 许多rna病毒基因组包含在结构或功能上模仿转移rna(Trna)为 它们与宿主细胞机器相互作用和操纵的策略的一部分。我们发现了这些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 作为这种类型病毒机制的模型系统的是在3‘端发现的tRNA样结构(TLS) 植物感染病毒的非翻译区。这些TLS在几十年前首次被发现，并已被 显示以三种方式模拟tRNA，并在无细胞提取物中增强翻译。 尽管经过几十年的研究和几个TLS的高分辨率结构信息，它们如何增强 翻译仍不得而知。这项建议旨在了解翻译服务在翻译中的贡献 病毒基因组的增强。第一步是确定TLS相互作用的其他宿主细胞组件 以及这与同源tRNA相比有什么不同。确定一整套互动合作伙伴将允许建立 可测试的模型，以阐明翻译促进功能背后的机制。 此外，重要的是确定TLSS是否被额外的tRNA靶向酶识别，如 最近的证据表明，tRNA修饰酶对RNA的化学修饰对 结构和功能。目的2研究作为伪聚(A)的TLS假想模型 尾巴。使用荧光共振能量转移(FRET)和翻译分析将确定 RNA转录本的内在折叠对于3‘TLS到5’帽的通讯是重要的。 确定tRNA如何在某些病毒的3‘端模拟可以绕过多聚(A)尾巴的必要性 将加强对主办方翻译的强制性和潜在替代要求的理解 机械设备。这项工作将深入了解病毒如何利用tRNA模仿来造福自己，tRNA模仿是如何 能够迷惑宿主细胞机制，并进一步增强对tRNA模拟和其 对基因表达的影响。

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.