Understanding viroids, endogenous circular RNAs, and viroid-like RNAs in the context of biogenesis

Viroids are a group of noncoding subviral RNAs that infect plant hosts.Currently, there are 44 formal viroid species grouped into 2 families, 39 members in Pospiviroidae, and 5 members in Avsunviroidae [1].All viroids share the following characteristics: (1) possessing highly structured circular RNA genomes; (2) lacking protein-coding capacity; (3) lacking DNA intermediates/templates; (4) replicating autonomously without helper virus; (5) using host RNA polymerases that normally recognize DNA templates; and (6) exhibiting transmissibility (Fig 1A and 1B).Interestingly, members of Avsunviroidae possess ribozyme activity, which is in contrast to members of Pospiviroidae.Viroids were originally considered as pathogens causing crop diseases, but now there are many examples where their infections are symptomless or latent [2].In general, symptom development depends on the combinations of viroid strains and hosts.With the rapid development of RNA-Seq technology and bioinformatic tools, an explosive number of endogenous circular RNAs have been identified in nearly all examined organisms across kingdoms.These circular RNAs are encoded by genomes, mostly derived from splicing.Some circular RNAs have been characterized to perform diverse functions [3,4].More recently, metatranscriptomic analyses have uncovered thousands of RNAs that are predicted to contain the consensus sequence of the hammerhead ribozyme and adopt a circular conformation [5,6].Due to these similarities to chloroplastic viroids, those sequences are often termed "viroid-like RNAs."In addition to the well-known viroid analog hepatitis delta virus (HDV) that infects humans [7], some viroid-like RNAs were shown to infect fungal hosts [8].Therefore, more viroid-like RNA pathogens infecting diverse hosts across kingdoms can be expected.Here, we revisit the establishment of the viroid concept and compare the diverse circular RNA biogenesis mechanisms, which can help to understand novel viroid-like RNA pathogens. Question 1: What is the definition of viroid?In the early 20th century, a "spindling-tuber disease" was observed in Irish potato varieties.It was identified as a symptom of infection, but the causal agent was not revealed until the late 1960s [9].The pathogenic agent (potato spindle tuber viroid (PSTVd)) was characterized as a free RNA that is much smaller than any viral genome [9].Given its small size, PSTVd was