Analysis of the novel modalities of gene expression of the smallest (220 nucleotides) replicating virusoid

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Viroids and virusoids (viroid-like satellite RNAs) are small single-stranded (220-450 nucleotide) covalently closed circular (CCC) RNAs with no coding capacity for proteins (with the exception of the virusoid associated with the rice yellow mottle sobemovirus (scRYMV) which is the subject of this proposal) and are the smallest replicating circular RNA pathogens. They usually replicate through a "rolling circle" model producing larger concatemers which are then processed into monomeric forms either with a self-splicing hammerhead ribozyme (virusoids and viroids in the Avsunviroidae family) or by cellular enzymes. We reported (Collins et al, 1998.Virology 241:269-275) the characterization and sequence of this smallest scRYMV virusoid (220 nt) which depends on RYMV helper virus for replication and packaging. However, this is the smallest amongst all known viroids and virusoids and the only one to have an open reading (ORF) and codes for a basic 146/166 amino acid protein (16/18kD). Our goal is to unravel and comprehend the entire life cycle of this scRYMV. The objectives are: 1. Confirm the existence of 16/18 kD protein in RYMV infected rice by immunological assays and by mass spectroscopy. 2. Study the translation of this CCCRNA and in particular to demonstrate the internal initiation of translation of a Eukaryotic mRNA. 3. Determine the function (s) of this 16/18 kD protein and identify host proteins involved in the replication of a virusoid. 4. Study the initiation of replication of the CCCRNA and develop an in vitro functional system (RYMV RNA polymerase). Viral and host proteins involved in replication will be expressed in E. coli for replication of this virusoid. 5. Analyze the structure-function, pathogenesis and other novel properties of this virusoid. 6. Continue the investigation of degradation of E. coli RNAs by short double-stranded sequences from the scRYMV. Development of an E. coli model system to study the effects of double-stranded (DS) sequences on the degradation and regulation of genes (dicer-like effect and interference with micro RNAs (miRNAs)). Application of this E. coli system to elucidate the pathogenesis seen in infected plants with viruses, viroids and virusoids.