Genetics of Barley yellow dwarf virus stop-codon readthrough in yeast

描述(申请人提供)：多种正义RNA病毒，包括一些哺乳动物逆转录病毒，使用终止密码子通读的翻译消退机制来制造特定的融合蛋白。在大麦黄矮病毒(BYDV)中，通读是由UAG末端近端的序列元件和下游数百个核苷酸的远端元件的组合编程的。对于黄矮病病毒和大多数其他RNA病毒来说，宿主因素在程序性终止密码子通读中的参与是未知的。长期的目标是鉴定和鉴定与黄矮病病毒程序化通读有关的宿主基因。为了便于遗传方法，研究中使用的模型宿主生物体是酵母。本研究的三个具体目的是：(1)建立研究酵母中黄矮病病毒程序通读的实验系统。包括通读编程序列元素的单报告和双报告结构被设计来证明在酵母中发生了通读。类似的带有烟草花叶病毒(TMV)通读序列的双报告结构被设计来测试影响BYDV通读的基因是否也影响TMV通透。(2)野生型酵母中BYDV和TMV序列终止密码子通读量的测定。使用(1)的双报告结构，通读效率将被量化。这些结果将表明酵母是否是研究BYDV通读的遗传学的合适的模式宿主。(3)BYDV程序化通读相关酵母基因的鉴定与鉴定。使用基于报告的通读系统，将筛选酵母过表达文库，以识别显著提高通读效率的基因。这样确定的基因将被详细描述，包括测试它们的过度表达是否也影响TMV的通读。同时，已知的影响翻译终止、终止密码子通读或翻译保真度的酵母基因的影响将直接用通读报告系统进行测试。公共卫生相关性：该项目涉及基础研究，旨在促进我们对RNA病毒与宿主相互作用的遗传学的理解。许多致病的RNA病毒感染人类。从宿主端了解更多关于病毒-宿主相互作用的信息，通常有可能为旨在了解和控制致病RNA病毒感染的研究提供信息。

DESCRIPTION (provided by applicant): A variety of positive-sense RNA viruses, including some mammalian retroviruses, use the translation receding mechanism of stop-codon readthrough to make specific fusion proteins. In Barley yellow dwarf virus (BYDV), readthrough is programmed by a combination of a sequence element proximal to the UAG stop, and a distal element several hundred nucleotides downstream. The involvement of host factors in programmed stop codon readthrough is unknown for BYDV and for most other RNA viruses. The objective in the long term is to identify and characterize host genes involved in BYDV programmed readthrough. To facilitate a genetic approach, the model host organism used in the study is yeast. The three specific aims of the study are: (1) Development of the experimental system for studying BYDV programmed readthrough in yeast. Single- and double-reporter constructs including the readthrough programming sequence elements are designed to demonstrate that readthrough occurs in yeast. Similar double-reporter constructs with a Tobacco mosaic virus (TMV) readthrough sequence are designed to test whether genes that affect BYDV readthrough also affect TMV readthrough. (2) Quantification of stop codon readthrough of BYDV and TMV sequences in wild-type yeast. Using the double-reporter constructs of (1), readthrough efficiencies will be quantified. The results will indicate whether yeast is an suitable model host for studying the genetics of BYDV readthrough. (3) Identification and characterization of yeast genes involved with BYDV programmed readthrough. Using the reporter-based readthrough system, a yeast overexpression library will be screened to identify genes that increase readthrough efficiency significantly. Genes so identified will be characterized in detail, including testing whether their overexpression also influences TMV readthrough. In parallel, the effect of yeast genes known to affect translation termination, stop codon readthrough, or translation fidelity will be tested directly with the readthrough reporter system. Public health relevance: The project involves basic research designed to advance our understanding of the genetics of RNA virus-host interactions. A number of pathogenic RNA viruses infect humans. Knowing more about virus-host interactions from the host side has the potential, in general, of informing research designed to understand and control infections by pathogenic RNA viruses.