9728277 Simon The TCV RdRp, after synthesizing full-length or near full-length plus-strands of the associated RNA sat-RNA D, switches to using minus-strands of either a second associated RNA, sat-RNA C, or the TCV genomic RNA with transcription reinitiating just 3' of stable hairpins. This system has been reconstructed for in vitro studies by creating a chimeric RNA consisting of the recombination hot-spot from sat-RNA D plus-strands at the 3' end connected by a short loop to sat-RNA C minus-strands including the sat-RNA C recombination hot-spot hairpin (motif1-hairpin). Using partially purified TCV RdRp, recombination in vitro is detected as an intramolecular extension product from the 3' end of the sat-RNA D sequence using the sat-RNA C-derived region as template. The motif1-hairpin and a short intrastrand base-paired region just upstream of the motif1-hairpin are important for primer extension both in vitro and in vivo. Recombination between sat-RNA D and TCV genomic RNA in vivo does not apparently involve a base-paired region 3' of the TCV motif3-hairpin recombination hot-spot. In addition, transcription start sites for subgenomic (sg) RNA promoters are hot-spots for recombination in vivo. This research will: ( 1 ) establish an in vitro primer extension system for the TCV motif3-hairpin region and (2) sgRNA promoter region analogous to the system established for the sat-RNA C motif1-hairpin. Using a mutagenesis strategy, the roles of the hairpins and single-stranded regions surrounding the motif'3-hairpin or sgRNA promoter hairpin will be determined. In addition, the in vivo effects of adding sequences capable of forming heteroduplexes with sat-RNA D plus-strands upstream and downstream of the TCV motif3-hairpin and sgRNA promoter will be established. (3) The mechanism of RdRp processing of the 3' end of the nascent strand will be determined using the in vitro primer extension constructs labeled at the 3' end. By examining small labeled products of the primer-extension reaction, a determ ination of whether processing involves endonucleolytic or exonucleolytic activity and also the length of the 3' end sequence that is removed will be made. RNA recombination is one of the major processes involved in the evolution of RNA virus genomes. In addition, analyses of sequences and structures that are important for recombination have provided major insights into a poorly characterized enzyme, the enzyme encoded by all RNA viruses that replicate through RNA intermediates, called RNA-dependent RNA polymerase (RdRp). The turnip crinkle virus (TCV) system is an unusually powerful system for studying RNA recombination since recombination can be studied using the whole organism (plant), cell culture (protoplast) and in vitro systems. In addition, recombination in the TCV system is between subviral (satellite) RNAs (such as sat-RNAs C and D) that are non-coding and are substantially smaller than the viral genomic RNA, or between sat-RNAs and the genomic RNA. This research will establish the sequences and structures on the RNA molecules that are important in the recombination event. In addition, the ability of the RdRp to repair truncations at the ends of virus RNAs using a process similar to recombination will be analyzed.
