Construction and characterization of an aureusvirus defective RNA

•A defective RNA of CLSV was constructed.•This replicon was used to study CLSV RNA replication.•RNA sequences and structure important for virus replication were identified.•The results revealed similarities and differences between CLSV and TBSV.

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.