Analysis of Processes Associated with Replication of Subviral RNAs in the TCV System

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The processes by which plant viral and subviral RNAs replicate and produce disease symptoms are not understood. One particularly intriguing question concerns the origin of linear subviral RNA multimers. A proposed model suggests that multimers are formed by the same process as the generation of discontinuous RNAs in the same system; the replicase reinitiates synthesis before releasing the newly synthesized strand. This model will be tested by following the formation of multimers and monomers in a protoplast replication system as well as analyzing different satellite RNA mutations which affect the ratio of monomers and multimers produced in vivo. A second question involves the recent finding that reiterative copying of redundant nucleotide can occur under specific circumstances in a subviral RNA accumulating in plants. In certain animal viruses, this process, known as replicase stuttering, results in the exposure of cryptic reading frames. A model involving a stable stem loop structure in the region of the nucleotide additions will be tested using deletion analysis and in vitro mutagenesis techniques. The possibility of a physiological role for reiterative nucleotide copying will be addressed by transferring a "stuttering cassette" to a heterologous viral RNA and testing for the ability to insert additional, non- encoded nucleotides. //