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: 7464806
• 负责人: Wyatt ALLEN MILLER
• 金额: $ 22.74万
• 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 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7464806
• 关键词: 3' Untranslated Regions; 5' Untranslated Regions; Affinity; Antiviral Agents; Barley; Base Pairing; Binding; Biological Assay; Bypass; Cells; Centrifugation; Class; Communication; Complex; Conserved Sequence; Custom; Dengue; Development; Elements; Enhancers; Family Picornaviridae; Flavivirus; Gene Expression; Gene Silencing; Gene Transduction Agent; Genetic Translation; Genome; Genomics; Germ; Goals; Host Defense; Human; Human Virus; Indium; Initiator Codon; Internal Ribosome Entry Site; Knowledge; Lead; Light; Luteovirus; Maps; Mediating; Medical; Messenger RNA; Modeling; Mutagenesis; Parasites; Peptide Initiation Factors; Pharmacologic Substance; Plant Viruses; Plants; Poliomyelitis; Poly(A) Tail; Process; Protein Biosynthesis; Proteins; Protoplasts; RNA; RNA I; Range; Recruitment Activity; Regulation; Research; Resolution; Ribosomes; Role; Scanning; Sequence Analysis; Severe Acute Respiratory Syndrome; Structure; Sucrose; Surface Plasmon Resonance; System; Translation Initiation; Translations; Untranslated Regions; Viral; Viral Proteins; Virus; Virus Replication; West Nile virus; Wheat; base; expression vector; insight; mutant; novel; pathogen; polypeptide; reconstitution; translation factor; viral RNA

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 elF4G and elF4E, and possibly other factors that are required for BTE-mediated translation. We will observe binding of mutant factors with the BTE RNA by filter binding, surface plasmon resonance, and RNA footprinting assays. The functions of mutant factors will be discerned by reconstituting factor-depleted cell-free extracts, and by depleting cells of factors via virus-induced gene silencing. In Aim , 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 initiation mechanisms.

所有病毒都必须接管宿主的蛋白质合成（翻译）机器。细胞的mRNA需要5'端 帽和poly（A）尾以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）尾来避免该控制步骤并避免宿主防御。相反，许多病毒 mRNA在非翻译区（UTR）中含有序列，这些序列有助于高效的帽非依赖性 翻译.了解病毒是如何做到这一点的，可能会导致抗病毒药物的发展， 靶向独特的病毒翻译机制。这一知识也可以利用病毒作为基因 治疗载体，或作为表达载体以产生定制的药物多肽， 植物该建议集中在3' UTR中的新的帽独立翻译元件（BTE）， 大麦黄矮病病毒（BYDV）和其它病毒RNA，其促进RNA 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招募翻译机器。在目标I中，我们将确定序列和结构 通过高容量诱变和在无细胞小麦中的翻译以高分辨率获得BTE的要求 胚芽提取物和植物原生质体中。在目标II中，我们将剖析 翻译起始因子eIF 4G和eIF 4 E，以及可能的其他BTE介导的转录所需的因子。 翻译.我们将观察突变因子与BTE RNA通过滤膜结合、表面等离子体激元 共振和RNA足迹分析。突变因子的功能将通过重组 因子耗尽的无细胞提取物，以及通过病毒诱导的基因沉默耗尽细胞的因子。在Aim中 ，核糖体进入RNA的机制将通过蔗糖梯度离心来研究， RNA-核糖体复合物、足印和其他方法。在整个项目中，BTE的作用 并评估其在病毒复制中的相互作用。本研究以一种模式病毒和主要植物为对象， 病原体可能有助于理解小核糖核酸病毒（如脊髓灰质炎），也采用帽非依赖性 翻译受UTR与巢状病毒（例如SARS）和黄病毒（例如CMV）之间的相互作用调节。 登革热，西尼罗河），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起始机制提供基础性的见解。