Analysis of Viroid Replication

Viroids, as a consequence of not encoding any protein, are extremely dependent on their hosts. Replication of these minimal genomes, composed exclusively by a circular RNA of 246–401 nt, occurs in the nucleus (family Pospiviroidae) or in the chloroplast (family Avsunviroidae) by an RNA-based rolling-circle mechanism with three steps: (1 ) synthesis of longer-than-unit strands catalyzed by host DNA-dependent RNA polymerases recruited and redirected to transcribe RNA templates, (2 ) cleavage to unit-length, which in family Avsunviroidae is mediated by hammerhead ribozymes, and (3 ) circularization through an RNA ligase or autocatalytically. This consistent but still fragmentary picture has emerged from a combination of studies with in vitro systems (analysis of RNA preparations from infected plants, transcription assays with nuclear and chloroplastic fractions, characterization of enzymes and ribozymes mediating cleavage and ligation of viroid strands, dissection of 5′ terminal groups of viroid strands, and in situ hybridization and microscopy of subcellular fractions and tissues), and in vivo systems (tissue infiltration studies, protoplasts, studies in planta and use of transgenic plants expressing viroid RNAs).