Construction and characterization of an aureusvirus defective RNA

Defective RNAs (D RNAs) are small RNA replicons derived from viral RNA genomes. No D RNAs have been found associated with members of the plus-strand RNA virus genus Aureusvirus (family Tombusviridae). Accordingly, we sought to construct a D RNA for the aureusvirus Cucumber leaf spot virus (CLSV) using the known structure of tombusvirus defective interfering RNAs as a guide. An efficiently accumulating CLSV D RNA was generated that contained four non-contiguous regions of the viral genome and this replicon was used as a tool to studying viral cis-acting RNA elements. The results of structural and functional analyses indicated that CLSV contains counterparts for several of the major RNA elements found in tombusviruses. However, although similar, the CLSV D RNA and its components are distinct and provide insights into RNA-based specificity and mechanisms of function.