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

描述（由申请方提供）：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后退机制来制备特异性融合蛋白。在大麦黄矮病毒（BYDV）中，通读是通过UAG终止子近端的序列元件和下游数百个核苷酸的远端元件的组合来编程的。对于BYDV和大多数其他RNA病毒，宿主因子参与程序性终止密码子通读是未知的。长期的目标是鉴定和表征参与BYDV程序化通读的宿主基因。为了便于遗传方法，研究中使用的模式宿主生物是酵母。本研究的三个具体目标是：（1）建立BYDV程序通读酵母实验系统。设计包括通读编程序列元件的单和双报告构建体以证明通读发生在酵母中。设计具有烟草花叶病毒（TMV）通读序列的类似双报告基因构建体以测试影响BYDV通读的基因是否也影响TMV通读。(2)野生型酵母中BYDV和TMV序列的终止密码子通读的定量。使用（1）的双报告基因构建体，将量化通读效率。研究结果表明酵母是否是研究BYDV通读遗传学的合适模式宿主。(3)参与BYDV程序化通读的酵母基因的鉴定和表征。使用基于酵母的通读系统，将筛选酵母过表达文库以鉴定显著提高通读效率的基因。将详细描述如此鉴定的基因，包括测试它们的过表达是否也影响TMV通读。同时，将直接使用通读报告系统检测已知影响翻译终止、终止密码子通读或翻译保真度的酵母基因的作用。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-宿主相互作用遗传学的理解。许多致病性RNA病毒感染人类。从宿主方面了解更多关于病毒-宿主相互作用的信息，通常有可能为旨在了解和控制致病性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.