Bacteriophages of the family cystoviridae are unique in that they have genomes of 3 segments of double-stranded RNA packaged precisely within a preformed procapsid. Phi6 is the first member of this family and has been studied extensively. A model has been proposed for the mechanism of precise genomic packaging in phi6. The model is strongly supported by the confirmation of predicted results in both in vitro and in vivo packaging. The packaging involves the serially dependent transport of plus strands s, m and I into preformed procapsids with specificity determined by changes in the binding sites on the outside of P1, the major structural protein of the procapsid. The goal of this project is to determine the interaction of the RNA molecules with specific parts of protein P1; to identify the specific elements involved in the changing of the RNA binding sites as the packaging program procedes. This proposal is for the investigation of the location of the binding sites, their amino acid sequences and the determination of the interaction between the RNA and the binding sites. A major question posed is whether the binding sites for the different RNA molecules are physically separate from each other or whether they are overlapping and involve the same regions of protein P1. Approaches utilized in this project include the cross-linking of specific RNA molecules to their binding sites and the use of phage display libraries to locate binding sites. Collaborative structural studies are also proposed in which cores that have packaged different amounts of RNA will be examined by cryoelectron microscopy to determine their changes in conformation. This study also includes a comparative investigation of genomic packaging in the distantly related bacteriophage phi8, which seems to follow the general rules determined for phi6 but with less stringency and with a number of additional features, particularly the participation of host factors and viral genes not found in other members of the cystoviridae. The cystoviridae are the only segmented dsRNA viruses whose mode of packaging is understood. There is no current understanding of the genomic packaging of the reoviridae, segmented dsRNA viruses that cause disease in plants and animals, including humans.
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