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

所有病毒都必须接管宿主的蛋白质合成(翻译)机制。细胞信使核糖核酸需要5‘ 帽和聚(A)尾招募核糖体，并以一种受调控的方式启动翻译。许多病毒RNA 避免这一控制步骤，并避免宿主防御，通过缺乏5‘帽或聚(A)尾巴。相反，许多人像病毒一样 MRNAs在非翻译区(UTRs)中含有促进高效帽非依赖性的序列 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开发，特别是 以独特的病毒翻译机制为目标。这种知识还可以将病毒作为基因加以利用。 在人类中的治疗载体，或作为表达载体在 植物。这项建议的重点是新奇的大小写无关的翻译元素(BTE)在3‘UTR. 大麦黄矮病(BYDV)和其他病毒RNA，促进RNA 5‘端的翻译启动。 这一过程需要5‘和3’非编码区之间的远距离碱基配对。我们的目标是确定如何 BTE招募翻译机器。在目标一中，我们将确定顺序和结构 无细胞小麦大批量诱变和翻译对BTE高分辨率的要求 胚芽提取物和植物原生质体中。在AIM II中，我们将剖析 翻译起始因子elF4G和elF4E，以及可能是BTE所需的其他因素 翻译。我们将通过滤膜结合、表面等离子激元观察突变因子与BTE RNA的结合 共振和RNA足迹分析。突变因子的功能将通过重组而被识别 去除因子的无细胞提取物，以及通过病毒诱导的基因沉默来消耗细胞中的因子。在AIM ，核糖体进入RNA的机制将通过蔗糖梯度离心法进行研究 RNA-核糖体复合体、脚趾印和其他方法。在整个项目中，BTE的作用 并将评估其在病毒复制中的相互作用。一种模式病毒及主要植物的研究 病原体可能有助于理解也使用帽非依赖性的小核糖核酸病毒(例如脊髓灰质炎) 翻译受非典型肺炎病毒(如SARS)和黄病毒(如肺炎)之间的相互作用调节。 登革热，西尼罗河)，通过远距离RNA碱基配对调节基因表达和复制。 最后，本研究将为真核生物的翻译启动机制提供基础性的见解。

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.