Simon MCB-9315948 Abstract Turnip crinkle virus is an ideal system to study the mechanism of RNA recombination. The genome is among the smallest viral RNA genomes and is associated with satellite RNAs and numerous small, subviral RNAs (Sat-RNA) which are the products of recombination events within the genomic RNA or between the genomic and subviral RNAs. The development of an in vitro recombination as well as a protoplast recombination system has allowed a more complete characterization of the elements and factors which mediate recombination in this system. A model of recombination has resulted from our work which suggests that coat protein helps to mediate the recombination event by possibly positioning the templates in proximity. In order to test this model and to continue characterizing the event, we will: 1. Establish protoplast and in vitro assays for recombination. 2. Conduct structural studies on motif I (the sequences found at the right sides of all junctions of recombinant RNA) using both mutagenesis and chemical and enzymatic probes. 3. Determine the role of coat protein in recombination between sat-RNA D and Sat-RNA C using gel-shift assays and 4. Assay for binding of the RNA-dependent RNA polymerase to motif I. %%% Viruses which depend on RNA-dependent RNA polymerases for replication of their genomic and sub-genomic RNAs are subject to high inherent mis-incorporation of nucleotides that can lead to heterogeneous mixtures of related species. Thus, RNA recombination is now thought to have been, and perhaps still is, a major factor in the evolution of viruses. The discovery that the turnip crinkle virus contained nearly the entire sequence of a satellite RNA joined to two regions of genomic RNA by this laboratory suggested that non-homologous recombination occurs. That fact, as well as small genome size and the generation of recombinant molecules in a non-random manner suggested that a detailed examination of the sequences, structures and mechanisms inv olved in the recombinational event would be productive. It is critical in many ways to have a better understanding of the mechanisms used by viruses in the evolutionary process and the information resulting from the specific goals pursued in this work will enhance that understanding.
