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.
