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

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需要一个5'帽和poly(a)尾巴来招募核糖体并以一种受调节的方式启动翻译。许多病毒rna通过缺乏5'帽或聚(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（UTRs）中含有序列，这有助于高效的帽无关翻译。了解病毒是如何做到这一点的，可以开发出专门针对独特病毒转译机制的抗病毒药物。这一知识也允许利用病毒作为人类基因治疗载体，或作为在植物中生产定制药物多肽的表达载体。本研究的重点是大麦黄矮（BYDV）和其他病毒RNA的3‘ UTR中的新型帽独立翻译元件（BTE），该元件促进了RNA 5’端的翻译起始。这个过程需要在5‘和3’ utr之间进行长距离碱基配对。我们的目标是确定BTE如何招募翻译机制。在Aim I中，我们将通过大规模诱变和在无细胞小麦胚芽提取物和植物原生质体中的翻译，以高分辨率确定BTE的序列和结构要求。在Aim II中，我们将剖析翻译起始因子eIF4G和eIF4E的作用和结构要求，以及bte介导的翻译可能需要的其他因素。我们将通过各种RNA-蛋白相互作用实验观察突变因子与BTE RNA的结合。突变因子的功能将通过重组耗尽因子的无细胞提取物和通过病毒诱导的基因沉默耗尽因子的细胞来识别。在Aim 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、足印和其他方法来研究。在整个项目中，将评估BTE及其相互作用者在病毒复制方面的作用。这项对模式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（如脊髓灰质炎），这些病毒也采用由utr之间相互作用调节的cap-independent翻译，以及通过长距离RNA碱基配对调节基因表达和复制的尼多病毒（如SARS）和黄病毒（如登革热、西尼罗河病毒）。最后，该研究将为真核生物翻译机制提供基础见解。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转译机制。我们正在使用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理解脊髓灰质炎病毒、普通感冒鼻病毒、SARS病毒、登革热病毒和西尼罗河病毒等医学上重要的病毒调控基因表达和复制的机制。最后，该研究将为所有高等生物细胞的翻译机制提供基本的见解。

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.