The peach latent mosaic viroid replication initiation site is located at a universal position that appears to be defined by a conserved sequence

Viroids replicate through a rolling circle mechanism that is exclusively RNA dependent. In this study, we initially revisited the determination of the replication initiation sites of peach latent mosaic viroid (PLMVd). A universal initiation site for each of the PLMVd polarities (position A50/C51 and U284 for the plus and minus strands, respectively) that is shared by a relatively wide repertoire of viroid variants was identified, in agreement with a previous report based on a different methodology. Subsequently, an in vitro selection procedure based on a model rolling circle replication assay was developed. This latter experiment led to the identification of a highly conserved CAGACG box which is reminiscent of the sequence found in the vicinity of the PLMVd initiation sites. The conserved sequence contributes to delineating the initiation site and provides an explanation for the presence of a specific universal initiation site on the PLMVd molecule.