Experimental characterization of zeta viruses and discovery of betaviroid-like RNAs expand the diversity of coding circular RNA replicons

Viroid-like RNAs (vdlRNAs) are infectious agents characterized by single-stranded circular genomes folding into compact secondary structures, often containing self-cleaving ribozymes involved in replication. Recent metatranscriptomic studies have expanded their diversity across multiple ecological niches and revealed novel vdlRNAs groups with protein-coding capacity, including zeta viruses, characterized by the potential to encode an endless tandem-repeat protein in each polarity strand. However, besides their in silico identification, no experimental evidence supported their existence and molecular features. Here, three new zeta viruses were identified and one of them was molecularly characterized from soil. In addition, a novel type of coding vdlRNAs, named betaviroid-like RNAs was discovered. Similar to zeta viruses, betaviroid-like RNAs are circular RNAs containing, in each polarity strand, one ribozyme and one open reading frame (ORF) encompassing the full-length genome. Unlike zeta viruses, betaviroid-like ORFs are not endless but longer than the unit-length genome. Existence of both circular RNA polarity strands, absence of DNA forms, and self-cleavage activity of ribozymes were experimentally confirmed for both zeta viruses and betaviroid-like RNAs, providing data consistent with symmetric rolling-circle replication. Additional zeta viruses and betaviroid-like RNAs were identified in publicly available metatranscriptomic datasets from wetlands of different geographic areas, supporting their broad distribution. Sixteen zeta viruses contain diverse self-cleaving ribozymes, including delta, type II and III hammerhead, twister, and hairpin ribozymes, expanding the known ribozyme diversity of this group. Altogether, our results provide the first experimental characterization of zeta viruses and betaviroid-like RNAs expanding the diversity of circular RNA replicons.