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: 7655506
• 负责人: Wyatt ALLEN MILLER
• 金额: $ 22.71万
• 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 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7655506
• 关键词: 3' Untranslated Regions; 5' Untranslated Regions; Affinity; Animals; Antiviral Agents; Barley; Base Pairing; Binding; Biological Assay; Bypass; Cells; Centrifugation; Common Cold; Communication; Complex; Conserved Sequence; Custom; Dengue; Dengue Virus; Development; Elements; Enhancers; Family Picornaviridae; Flavivirus; Gene Expression; Gene Silencing; Gene Transduction Agent; Genetic Translation; Genome; Germ; Goals; Host Defense; Human; Human Virus; Human poliovirus; Indium; Initiator Codon; Internal Ribosome Entry Site; Knowledge; Lead; Light; Luteovirus; Maps; Mediating; Medical; Messenger RNA; Modeling; Mutagenesis; Organism; Parasites; Peptide Initiation Factors; Pharmacologic Substance; Plant Viruses; Plants; Poliomyelitis; Polioviruses; Poly(A) Tail; Process; Protein Biosynthesis; Proteins; Protoplasts; RNA; RNA Viruses; RNA-Protein Interaction; Recruitment Activity; Regulation; Research; Resolution; Rhinovirus; Ribosomes; Role; SARS coronavirus; Scanning; Sequence Analysis; Severe Acute Respiratory Syndrome; Structure; Sucrose; Translation Initiation; Translations; Untranslated Regions; Viral; Viral Proteins; Virus; Virus Replication; West Nile virus; Wheat; base; expression vector; insight; mutant; novel; pathogen; polypeptide; public health relevance; reconstitution; translation factor; viral RNA

DESCRIPTION (provided by applicant): All viruses must take over the host's protein synthesis (translation) machinery. Cellular mRNAs require a 5' cap and poly(A) tail to recruit the ribosome and initiate translation in a regulated manner. Many viral RNAs avoid this control step, and avoid host defenses, by lacking a 5' cap or poly(A) tail. Instead, many viral mRNAs harbor sequences in the untranslated regions (UTRs) that facilitate highly efficient cap-independent translation. Understanding how viruses do this could lead to development of antiviral agents that specifically target unique viral translation mechanisms. This knowledge could also allow exploitation of viruses as gene therapy vectors in humans, or as expression vectors to produce custom pharmaceutical polypeptides in plants. This proposal focuses on the novel cap-independent translation element (BTE) in the 3' UTR of barley yellow dwarf (BYDV) and other viral RNAs that facilitates translation initiation at the 5' end of the RNA. This process requires long-distance base pairing between the 5' and 3' UTRs. Our goal is to determine how the BTE recruits the translational machinery. In Aim I we will determine the sequence and structural requirements of the BTE at high resolution by high volume mutagenesis, and translation in cell-free wheat germ extracts and in plant protoplasts. In Aim II we will dissect the role and structural requirements of translation initiation factors eIF4G and eIF4E, and possibly other factors that are required for BTE-mediated translation. We will observe binding of mutant factors with the BTE RNA by a variety of RNA-protein interaction assays. The functions of mutant factors will be discerned by reconstituting factor-depleted cell-free extracts, and in cells depleted of factors by virus-induced gene silencing. In Aim III, the mechanism of ribosome entry on the RNA will be investigated by sucrose gradient centrifugation of RNA-ribosome complexes, toeprinting, and other approaches. Throughout the project, the role of the BTE and its interactors in virus replication will be assessed. This research on a model virus and major plant pathogen may contribute to understanding picornaviruses (e.g. polio) that also employ cap-independent translation regulated by interactions between the UTRs, and nidoviruses (e.g. SARS) and flaviviruses (e.g. dengue, West Nile) that regulate gene expression and replication by long-distance RNA base pairing. Finally, the research will provide fundamental insight on eukaryotic translation mechanisms. PUBLIC HEALTH RELEVANCE: All viruses must take over the host's protein synthesis (translation) machinery. Viruses of plants and animals share many common mechanisms for translation. We are using a plant virus as a small, easy-to-use model to investigate the mechanisms by which an RNA virus interacts with the host translational apparatus. This research may contribute to understanding mechanisms by which medically important viruses such as poliovirus, common cold rhinoviruses, the SARS virus, dengue virus, and West Nile viruses regulate gene expression and replication. Finally, the research will provide fundamental insight on translation mechanisms in cells of all higher organisms.

描述（由申请人提供）：所有病毒必须接管宿主的蛋白质合成（翻译）机制。细胞mRNA需要5'帽和poly（A）尾来募集核糖体并以受调控的方式启动翻译。许多病毒RNA通过缺少5'端帽或poly（A）尾来避免这一控制步骤，并避免宿主防御。相反，许多病毒mRNA在非翻译区（UTR）中含有促进高效帽非依赖性翻译的序列。了解病毒如何做到这一点可能会导致开发专门针对独特病毒翻译机制的抗病毒剂。这一知识也可以允许利用病毒作为人类的基因治疗载体，或作为表达载体在植物中产生定制的药物多肽。本研究的重点是大麦黄矮病毒（BYDV）和其他病毒RNA的3'非翻译区中的新的帽独立翻译元件（BTE），其促进RNA的5'末端的翻译起始。该过程需要5'和3' UTR之间的长距离碱基配对。我们的目标是确定BTE如何招募翻译机器。在目的I中，我们将通过高容量诱变以高分辨率确定BTE的序列和结构要求，以及在无细胞小麦胚芽提取物和植物原生质体中的翻译。在目的II中，我们将剖析翻译起始因子eIF 4G和eIF 4 E的作用和结构要求，以及可能需要BTE介导的翻译的其他因子。我们将通过各种RNA-蛋白质相互作用试验观察突变因子与BTE RNA的结合。突变因子的功能将通过重构因子耗尽的无细胞提取物来辨别，并且在因子耗尽的细胞中通过病毒诱导的基因沉默来辨别。在目标III中，核糖体进入RNA的机制将通过RNA-核糖体复合物的蔗糖梯度离心、脚趾印法和其他方法来研究。在整个项目中，将评估BTE及其相互作用因子在病毒复制中的作用。对模型病毒和主要植物病原体的研究可能有助于理解小核糖核酸病毒（例如脊髓灰质炎），这些病毒也采用由UTR之间的相互作用调节的帽非依赖性翻译，以及巢状病毒（例如SARS）和黄病毒（例如登革热，西尼罗河病毒）通过长距离RNA碱基配对调节基因表达和复制。最后，该研究将为真核生物翻译机制提供基础性的见解。 公共卫生相关性：所有病毒都必须接管宿主的蛋白质合成（翻译）机制。植物和动物的病毒有许多共同的翻译机制。我们正在使用一种植物病毒作为一个小的，易于使用的模型来研究RNA病毒与宿主翻译装置相互作用的机制。这项研究可能有助于了解医学上重要的病毒，如脊髓灰质炎病毒，普通感冒鼻病毒，SARS病毒，登革热病毒和西尼罗河病毒调节基因表达和复制的机制。最后，这项研究将为所有高等生物细胞中的翻译机制提供基本的见解。