Jeremy A Bruenn MCB 9727630 RNA-Protein Interactions in a Double-Stranded RNA Virus of Sacchromyces Cerevisiae 1. Technical Abstract - 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 inessential 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), with a Kd of less than 1 nM. The present proposal study seeks to understand the nature of the interaction between Cap-Pol and this b inding site. This will involve further in vitro selection of binding sites, cross-linking and mutagenesis experiments to map active site residues in the Cap-Pol protein, structural determination of the 20-base RNA binding site, and optimization of the Cap-Pol binding domain. 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. 2. Non-technical Abstract - There is a group of small viruses of fungi and protozoans in which the genome is a single large double-stranded RNA. These viruses express their genomes by transcribing the RNA in the viral particles and extruding one strand (the plus strand) into the cytoplasm. Their RNAs may either be translated by the cell?s machinery or packaged in newly assembled viral particles. Consequently, the viral particles must choose from among all the RNAs in the cell the one RNA that is viral for packaging. This is accomplished in the yeast virus by the specific recognition of a small sequence in the RNA by a domain of 75-150 amino acids in the viral polymerase. This interaction is examined in this study by a number of molecular biological, biochemical, and biophysical techniques. The very high affinity of the RNA for its viral protein and the very small RNA sequence recognized only 20 bases) and the simplicity of the packaging process in this group of viruses makes this system an attractive one for understanding how RNA viruses construct their particles.
