Extant hybrids of RNA viruses and viroid-like elements

Earth’s life may have originated as self-replicating RNA. Some of the simplest current RNA replicators are RNA viruses, defined by linear RNA genomes encoding an RNA-dependent RNA polymerase (RdRP), and subviral agents with single-stranded, circular RNA genomes, such as viroids encoding paired self-cleaving ribozymes. Amongst a massive expansion of candidate viroid and viroid-like elements, we report that fungal pathogens, ambiviruses, are viroid-like elements which undergo rolling circle replication and encode their own viral RdRP, thus they are a distinct hybrid infectious agent. These findings point to a deep evolutionary history between modern RNA viruses and sub-viral elements and offer new perspectives on the evolution of primordial infectious agents, and RNA life. One-Sentence Summary Novel infectious agents resembling self-cleaving viroid-like RNAs whilst encoding a viral RNA-dependent RNA polymerase.


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Serving dual functions as encoding for genetic information, and as a biological catalyst, RNA has 45 been proposed to predate DNA and protein at the origin of life in an "RNA World" (1,2). Further, extant sub-viral and viral agents have been suggested to be "living fossils" and can help illuminate the molecular biology of Earth's first life (3)(4)(5).
Viruses having RNA genomes (realm Riboviria) are infectious agents defined by a linear RNA genome encoding one of their hallmark replication polymerases (replicases), either an RNA 50 dependent RNA polymerase (RdRP) for RNA viruses, or a Reverse Transcriptase (RT) for retroviruses. These replicases, and thus Riboviria, are monophyletic in nature as based on alignment of the conserved palm domain of RdRP and RT (6).
Viroids and viroid-like entities (such as the human satellite virus, Hepatitis Delta Virus), are subviral RNA infectious agents of plants and animals ( Fig 1A). These sub-viral agents are typified by 55 single-stranded circular RNA (circRNA) genomes which form extensive secondary structures, typically rod or branched-rod folding, and most encode hallmark paired ribozymes, one in each strand polarity, necessary for their rolling-circle replication. So far, the self-cleaving RNA ribozymes reported in infectious circRNA (7,8) are hammerhead (HHRz), hairpin (HPRz) and hepatitis Delta (DVRz) ribozymes (9,10). In contrast to RNA viruses, infectious circRNAs 60 replicate by RNA polymerases encoded in trans by the host or helper viruses. Additionally, while viroids are non-coding RNAs (8), Delta and Delta-like viruses have been assigned the realm Ribozyviria, and encode for the Delta Antigen proteins (11). Overall, the simple genomes and small ribozymes of infectious circRNAs are why these elements are regarded as molecular fossils of the prebiotic world (3). 65 Ambiviruses are a recently characterized and widespread group of fungal single-stranded RNA infectious agents with unusual genomic features (12,13) (Fig. 1A). An ambivirus (~5 kb genome) encodes for two conserved open read frames (ORFs) A and B, one on each strand -a so-called bicistronic, ambisense genome. ORF-A has a remote similarity to RdRP leading to these agents being reported as RNA viruses. However, Northern blotting, RT-PCR, and de novo transcript 70 assembly indicated contiguity between 3' and 5' terminal ends, inconsistent with the assumed typical linear RNA virus genome (12,13).
Here we report that ambiviruses have circular RNA genomes, encoding paired self-cleaving ribozymes, enabling a viroid-like rolling circle mechanism of replication, however, we further show that ORF-A is indeed a functional RdRP like that of a classical RNA virus. Thus, ambiviruses 75 appear to have arisen from the hybridization of a viroid-like genomic backbone with the hallmark RNA virus gene RdRP, and as such are a distinct class of infectious agent. This finding bridges the RNA virospheres of viruses and viroids, posing deep questions regarding their origins and (co-)evolution. 80 Starting with the hint that ambiviruses may have non-standard linear RNA genomes (Supplementary text and fig. S1), we analyzed published ambiviruses for sequence homology to structured RNA covariance models with INFERNAL (14). This uncovered head-to-tail oriented, ambisense HHRz or HPRz motifs in 28/30 (93%) of the GenBank sequences, reminiscent of subviral circRNAs (table S1). To expand the known sequence diversity of ribozyme-bearing 85 subviral agents and ambiviruses, we adapted the Serratus ultra-high throughput computing architecture (15) to search for ribozymes using INFERNAL across 198,194 raw metagenome/metatranscriptomes freely-available in the Sequence Read Archive (16,17). Combining the top 5,000 ribozyme-hit libraries with our previous RNA virus assemblage and the Transcriptome Shotgun Assembly (TSA), we created the "RNA Deep Virome Assemblage" 90 (RDVA) of 58,557 libraries. From the 12.5 billion assembled contigs, 34 million contained 5'-3' kmer overlaps suggestive of a circular molecule. Filtering the potential circular contigs further for those encoding two paired-ambisense ribozymes resulted in a discovery set of 32,393 putative viroid and viroid-like elements, which clustered into 20,364 novel species-like operational taxonomic units (sOTU) at 90% nucleotide sequence identity. These datasets included up to 863 95 unique RdRP-encoding ambiviruses, which clustered into 378 sOTU at 90% palmprint identity (18).
The initially reported homology between ambivirus ORF-A and RdRP was marginal owing to their deep divergence (12,13). Advances in in silico structural prediction have extended the capacity to assess for deep protein homology from sequences alone (19). AlphaFold2 prediction 100 of ORF-A strongly supports (pLDDT confidence up to 93.5 for several models) that this protein takes on the classic right-handed palm domain architecture, with highest similarity to negarnavirus RdRPs (Influenza A, Z-score >19, RDSM 3.8A) ( fig. S2). Critically, the expanded multiple sequence alignment shows the strong sequence conservation across the essential polymerase catalytic motifs A, B, and C (18) (Fig. 1B). Altogether this lends strong support that 105 the ambivirus ORF-A encodes for a catalytically active RdRP.
Further screening of public databases (see Material and Methods) extended the number of ambiviral sOTUs up to 439 species. Prediction of the secondary structures of minimal free energy for the ambiviral potential circular genomes revealed than the majority (90%) of these RNAs adopt a perfect rod or quasi-rod like structure of very high stability, akin to most plant 110 viroids and Delta-like viruses. However, about 10% of the ambivirus genomes adopt a highly branched RNA conformation, still stable and somehow keeping a main rod-like architecture (Fig. 1C), which suggests that extensive genomic base-pairing structural constraints are preserved across ambiviruses. ORF-A shows a classic polymerase palm fold structure and strong sequence conservation of catalytic residues in motif A, B, and C is seen across all ambiviruses. (C) Circular plots of the predicted RNA secondary structures of two representative ambiviruses. On the left, an example of the characteristic rod-like structure predicted for most ambiviruses. On the right, an example of a highly branched architecture predicted for some ambivirus RNA genomes, usually carrying a mixed pair of HHRz/HPRz 125 motifs.
Reconstruction of the ambivirus RdRP phylogeny shows a complex natural history of the ambivirus RdRP and their ribozymes. Four out of the ten known self-cleaving ribozymes (20) were found in ambivirus genomes thereby suggesting that multiple recombination and/or 130 horizontal transfer events of ribozymes have occurred (Fig. 2). Intriguingly, there is also extensive mixture of two different ribozymes on a single genome. Similar ribozyme mixtures are observed in the 5% of our discovery set of small viroid-like genomes, supporting that different ribozyme classes may coexist within a genome.  S3). To determine cleavage occurred in vivo, 5' rapid amplification of cDNA ends (RACE) was performed from infected Tulasnella spp. and Cryphonectria parasitica extracts, and again showed perfect 155 agreement with in silico and in vitro results (Fig. 3 A, B and fig. S4). Such functional self-cleaving ribozymes on both polarities are only known to occur in diverse viroid-and Delta-like agents, all of which possess circRNA genomes which replicate through a symmetric rolling circle mechanism. Another hallmark of symmetric rolling circle replication is the in vivo accumulation of circRNAs of both polarities (23). To evaluate if ambiviruses have 175 circular RNA genomes, northern blotting against CpAV1 was done under native and denaturing conditions (Fig. 3 C, D). Under native conditions, the CpAV1 genomic RNA migrated as a monomer (around the expected 4,623 nt), while under denaturing conditions, two bands were resolved. The retarded band would correspond to a circular molecule, and indeed the upper band shows preferential resistance to RNase R exonuclease treatment ( fig. S5). Together this 180 demonstrates that in vivo CpAV1, and by extension ambiviruses, replicate through a symmetric rolling circle mechanism.
Finally, to associate a phenotype to ambivirus infection, we obtained ambivirus-infected and ambivirus-free isogenic conidial isolate and show that CpAV1 causes hypovirulence in its fungal host ( fig. S6), a feature that is useful for biocontrol of this important chestnut tree disease; this 185 is the first example of a biotechnologically exploitable property linked to this new group of infectious agents. Furthermore, these agents could develop into circular RNA expression vectors, a further step into protein expression stabilization from RNA templates.
Motivated to see if other "RNA viruses" share a viroid-like genomic backbone, we searched the RDVA set for contigs with dual polarity paired-ribozymes and identified up to 16 circular contigs 190 of approximately 3 kb encoding RdRP-like ORFs (table S2 and Fig. 4). These sequences show homology to grapevine-associated mitoviruses genomes in GenBank. Among them, we detected 7 novel circular genomes similar to Fusarium asiaticum mitoviruses which all carry dual-polar variants of a rare and complex self-cleaving motif, the Varkud Satellite ribozyme (VSRz), so far only described in the mitochondrial VS plasmid of some Neurospora isolates (24). 195 The self-cleaving activity of mitovirus VSRzs was experimentally confirmed, whereas the predicted secondary structure of these agents was found not to be rod-like but highly branched (Fig. 4). These findings extend the presence of circular genomes with paired ribozymes to a different group of RNA mycoviruses and suggests that the hybridization of viroid-like agents and RNA viruses has occurred multiple times in evolutionary history. Infectious circular RNAs are evidently more widespread than previously appreciated, ranging from small viroid/viroid-like, medium sized delta-like, to large virus-like elements, which are hybrid forms of infectious RNAs with circular genomes encoding autocatalytic ribozymes and viral RdRPs. While computational advances will drive the exponential discovery of these elements (similar to RNA viruses) (15,25), emphasis should be placed on collaborative 215 molecular characterization of this new universe of infectious agents. While commonly we associate these entities as infectious agents of mitochondria (some mitoviruses), fungi (ambiviruses), plants (viroids), or animals (delta-like viruses), these host-distributions likely reflect our ignorance of the full diversity of infectious circular RNAs across all kingdoms.
Given the rapid mutation rate of viroids (26), the disposition of viroid-like elements to swap 220 ribozymes (as shown here), and combined with the limitations of phylogenetics, the deep evolutionary relationships between infectious circRNA (such as the genomic origin of ambiviruses) is likely unknowable. Further, structurally simple ribozymes, such as HHRz can arise spontaneously in evolution (27), and thus embedding in relatively simple head-to-head ligated RNAs supports the notion that the simplest classes of infectious circRNA may be 225 evolving de novo continuously. As viroid-like circRNA can acquire genes, such as a delta-antigen, or viral RdRP and give rise to more complex entities, it follows that host RNA polymerases may have been exapted to give rise to Earth's primordial "viruses". Perhaps most tantalizingly, the acquisition of an early RNA replicase ribozyme would serve as a blueprint for simplest replicator of the RNA world, and thus the origin of life.