Control of cap-independent translation by a viral 3' UTR

通过病毒 3 UTR 控制帽独立翻译

• 批准号: 8667461
• 负责人: Wyatt ALLEN MILLER
• 金额: $ 27.59万
• 依托单位: IOWA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2017-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8667461
• 关键词: 3' Untranslated Regions; 5' Untranslated Regions; Affinity; Amino Acids; Animal Experimentation; Animals; Anisotropy; Antiviral Agents; Base Pairing; Behavior; Beryllium; Binding; Binding Sites; Biological Assay; Bypass; Cell Extracts; Cells; Complex; Crystallization; Data Collection; Dengue; Elements; Enhancers; Experimental Designs; Funding; Gene Expression; Genome; Goals; Hepatitis C virus; Human; Human Virus; Hydroxyl Radical; In Vitro; Indium; Infection; Ions; Knowledge; Light; Luteovirus; Malignant Neoplasms; Mammalian Cell; Maps; Measures; Medicine; Messenger RNA; Methods; Modeling; Molecular; Mosaic Viruses; Nucleotides; Oncogenes; Panicum; Peptide Initiation Factors; Pharmacologic Substance; Phase; Plant Viruses; Plants; Poly(A) Tail; Polyacrylamide Gel Electrophoresis; Production; Protein Biosynthesis; Proteins; Protoplasts; RNA; RNA Binding; RNA Folding; RNA Recognition Motif; RNA Viruses; Race; Recruitment Activity; Research; Resolution; Ribosomes; Scaffolding Protein; Scanning; Structure; System; Testing; Therapeutic; Translations; Twin Multiple Birth; United States National Institutes of Health; Viral; Virus; Virus Replication; Work; X-Ray Crystallography; arm; cancer type; design; feeding; in vivo; interest; mutant; novel; protein purification; public health relevance; translation factor; tumor; viral RNA

DESCRIPTION (provided by applicant): All viruses depend on the host's translation (protein synthesis) machinery. For this reason, host cells have evolved numerous antiviral mechanisms that shut down or otherwise regulate translation. In the molecular arms race, viruses, in turn, have evolved ways to bypass host translational control. It is this essential step in virus replication that is the focus of this proposal. Host mRNAs contain a 5' "cap" structure and a 3' poly(A) tail that interact with translation initiation factors which recruit the ribosome to the mRNA. In contrast, the RNAs of most RNA viruses are uncapped, so they have evolved RNA structures that recruit the ribosome by noncanonical, cap-independent mechanisms. Many uncapped plant viral RNAs contain a cap-independent translation element (CITE) in the 3' untranslated region that facilitates efficient ribosome entry at the 5' end of the genome. In NIH-funded research the PI's lab showed that this is facilitated by long-distance base pairing between the 3' CITE, which binds a translation initiation factor, and the 5' untranslated region. Unanswered is how the CITE RNA structure causes it to bind a translation initiation factor with high affinity, leading to recruitment of the ribosome. Here, a variety of approaches will be applied to determine the structural requirements of two unrelated 3' CITEs, and the translation factors with which they interact. These include (i) the Barley yellow dwarf virus-like translation element (BTE) which binds and requires initiation factor eIF4G and not eIF4E; and (ii) the Panicum mosaic virus-like translation element (PTE), which binds and requires eIF4E - a protein known previously to bind only to the 5' cap structure. The three aims all can be performed independently, but the knowledge gained from each will feed into the other two aims. Aim I uses multiple, factor-depletable translation systems of mutant CITEs and mutant cognate translation factors with which they interact. This will reveal the key nucleotides and amino acids required for interaction and translation function. The second aim uses a variety of methods to measure the interactions of the mutant CITEs with mutant translation factors. The third aim will determine CITE structure at high resolution by ion-dependent RNA folding and X-ray crystallography methods. This project will provide a new understanding of the way in which viruses take over the cell, which may, in turn, suggest potential targets for antiviral drugs. Although this work focuses on model plant viruses, many growing human viruses such as dengue and hepatitis C viruses use similar mechanisms. Also, this work will shed new light on how the translational machinery works, and the translation system is extremely highly conserved between plants and animals. For example, the PTE functions in mammalian cells and we will use human cells and extracts to study how it uses eIF4E to usurp the ribosomes. Over-active eIF4E causes tumors and restriction of its function inhibits many types of cancers. The tightly binding PTE RNA may provide structural knowledge for design of eIF4E-inhibiting cancer therapeutics.

描述（由申请人提供）：所有病毒都依赖于宿主的翻译（蛋白质合成）机制。因此，宿主细胞进化出了许多抗病毒机制，可以关闭或以其他方式调节翻译。在分子军备竞赛中，病毒反过来又进化出了绕过宿主翻译控制的方法。病毒复制的这一重要步骤是本提案的重点。宿主 mRNA 包含 5'“帽”结构和 3'poly(A) 尾，可与翻译起始因子相互作用，将核糖体募集至 mRNA。相比之下，大多数 RNA 病毒的 RNA 是无帽的，因此它们进化出了通过非规范的、不依赖帽子的机制招募核糖体的 RNA 结构。许多无帽植物病毒 RNA 在 3' 非翻译区含有帽独立翻译元件 (CITE)，有助于核糖体有效进入基因组 5' 端。在 NIH 资助的研究中，PI 实验室表明，结合翻译起始因子的 3' CITE 和 5' 非翻译区域之间的长距离碱基配对促进了这一点。尚未解答的是 CITE RNA 结构如何使其以高亲和力结合翻译起始因子，从而导致核糖体的募集。在这里，将应用多种方法来确定两个不相关的 3' CITE 的结构要求以及它们相互作用的翻译因子。这些包括 (i) 大麦黄矮病毒样翻译元件 (BTE)，它结合并需要起始因子 eIF4G 而不是 eIF4E； (ii) 黍花叶病毒样翻译元件 (PTE)，它结合并需要 eIF4E——一种先前已知仅结合 5' 帽结构的蛋白质。这三个目标都可以独立执行，但从每个目标中获得的知识将反馈到其他两个目标中。目标 I 使用突变型 CITE 和与其相互作用的突变型同源翻译因子的多个因子耗尽翻译系统。这将揭示所需的关键核苷酸和氨基酸 交互和翻译功能。第二个目标使用多种方法来测量突变体 CITE 与突变体翻译因子的相互作用。第三个目标是通过离子依赖性 RNA 折叠和 X 射线晶体学方法确定高分辨率的 CITE 结构。该项目将对病毒接管细胞的方式提供新的认识，这反过来可能会提出抗病毒药物的潜在靶标。尽管这项工作的重点是模型植物病毒，但许多正在生长的人类病毒（例如登革热和丙型肝炎病毒）也使用类似的机制。此外，这项工作将为翻译机制的工作原理提供新的线索，并且翻译系统在植物和动物之间极其高度保守。例如，PTE 在哺乳动物细胞中发挥作用，我们将使用人类细胞和提取物来研究它如何利用 eIF4E 来侵占核糖体。过度活跃的 eIF4E 会导致肿瘤，限制其功能可以抑制多种癌症。紧密结合的 PTE RNA 可以为 eIF4E 抑制癌症疗法的设计提供结构知识。