9505829 Bruenn Many of the double-stranded RNA viruses of lower eucaryotes are non-infectious viruses that persist indefinitely by replicating in tandem with their host cells, their spread mediated entirely by sexual and asexual reproduction. They have adapted to this lifestyle in some cases by packaging extra RNA not essential to the virus but encoding products of selective value to their host cells. These products are cellular toxins that kill cells not harboring the virus (the killer phenotype). Several such systems are known among the fungal dsRNA viruses, the best studied of which is the Saccharomyces cerevisiae virus (ScV). A number of characteristics make this system very attractive for studying nucleic acid-protein interactions. First, the host cells are readily amenable to molecular and classical genetics. This makes it possible to use the killer phenotype for screening and selection of viral mutants. Second, the virus separately encapsidates its RNAs, making many experiments simpler than those in the dsRNA viruses of higher eucaryotes, many of which have as many as 12 unique dsRNAs encapsidated in each viral particle. Third, extensive analysis of the single essential large viral dsRNA (4580 bp) has mapped many viral functions. There are only two viral proteins, one the major capsid polypeptide and product of the cap gene, and the second a multifunctional protein the result of frameshifting between the two open reading frames (cap and pol) to produce a cap-pol fusion protein. The cap-pol fusion protein has the enzymatic and packaging activities of the virion. Among the cis-acting sites is the viral binding site (packaging site) which has (for RNA) a very high affinity for its recognition protein (the cap-pol fusion protein). This research will define the interaction between cap-pol and this binding site by mapping all the elements of specificity in both the RNA and the protein. Analysis will include in vitro mutagenesis of both cap-pol and the binding site, domain swapping experiment s between related RNA binding sites, domain swapping experiments between related cap-pol proteins, optimization of the RNA binding site, and optimization of the cap-pol binding domain. The minimal size of the optimal RNA biding site is known (19 nucleotides) as is the region of the viral protein necessary for binding (73-200 amino acids). This is one of the few systems in which both in vivo and in vitro systems for selection and analysis of altered binding sites and binding protein exist. It promises to contribute significantly to understanding RNA-protein interactions. %%% Many of the double-stranded RNA viruses of lower eucaryotes are non-infectious viruses that persist indefinitely by replicating in tandem with their host cells, their spread mediated entirely by sexual and asexual reproduction. They have adapted to this lifestyle in some cases by packaging extra RNAs not essential to the virus but encoding products of selective value to their host cells. These products are cellular toxins that kill cells not harboring the virus (the killer phenotype). Several such systems are known among the fungal dsRNA viruses, the best studied of which is the Saccharomyces cerevisiae virus (ScV). A number of characteristics make this system very attractive for studying nucleic acid-protein interactions. First, the host cells are readily amenable to molecular and classical genetics. This makes it possible to use the killer phenotype for screening and selection of viral mutants. Second, the virus separately encapsidates its RNAs, making many experiments simpler than those in the dsRNA viruses of higher eucaryotes. Third, extensive analysis of the single essential large viral dsRNA (4580 bp) has mapped many viral functions. There are only two viral proteins, the major capsid polypeptide and the cap-pol fusion protein, which has the enzymatic and packaging activities of the virion. This research seeks to understand the nature of the interaction between cappol and its viral RNA binding site by mapping all th e elements of specificity in both the RNA and the protein. This is one of the few systems in which both in vivo and in vitro systems for selection and analysis of altered binding sites and binding protein exist. It promises to contribute significantly to understanding RNA-protein interactions. ***
