Three new clades of putative viral RNA-dependent RNA polymerases with rare or unique catalytic triads discovered in libraries of ORFans from powdery mildews and the yeast of oenological interest Starmerella bacillaris

Abstract

High throughput sequencing allowed the discovery of many new viruses and viral organizations increasing our comprehension of virus origin and evolution. Most RNA viruses are currently characterized through similarity searches of annotated virus databases. This approach limits the possibility to detect completely new virus-encoded proteins with no detectable similarities to existing ones, i.e., ORFan proteins. A strong indication of the ORFan viral origin in a metatranscriptome is the lack of DNA corresponding to an assembled RNA sequence in the biological sample. Furthermore, sequence homology among ORFans and evidence of co-occurrence of these ORFans in specific host individuals, provides further indication of a viral origin. Here we use this theoretical framework to report the finding of three conserved clades of protein-coding RNA segments without a corresponding DNA in fungi. Protein sequence and structural alignment suggest these proteins are distantly related to viral RNA dependent RNA polymerases (RdRP). In these new putative viral RdRP clades no GDD catalytic triad is present, but the most common putative catalytic triad is NDD, and a clade with GDQ, a triad previously unreported at that site. SDD, HDD ADD are also represented. For most members of these three clades, we were able to associate a second genomic segment, coding for a protein of unknown function. We provisionally named this new group of viruses ormycovirus. Interestingly, all the members of one of these sub-clades (gammaormycovirus) accumulate more minus sense RNA than plus sense RNA during infection.

Identification of RNA virus-derived RdRp sequences in publicly available transcriptomic datasets

“…We took all classified sequences within each pHMM group, aligned them, and manually located the conserved motif C. Consistent with previous work, we saw six possible variations of the core amino acid triplet: GDD, SDD, GDN, IDD, ADN and ADD (in order of frequency) (Figure 7). Interestingly, Forgia et al (2022a) identified additional variations (NDD, GDQ and HDD; besides SDD and ADD) in the ormycoviruses – a recently discovered group of fungi-infecting viruses that are highly divergent from other known RNA viruses.…”

“…Callanan et al, 2020). Approaches based on predicted secondary or tertiary protein structure such as HHpred (Zimmermann et al, 2018), Phyre2 (Kelley et al, 2015) or AlphaFold (Jumper et al, 2021) could also be useful to find more divergent RdRp sequences (Wolf et al, 2020; Charon et al, 2022; Forgia et al, 2022a; Lee et al, 2022). For example, homology of the quenyavirus RdRp to previously known RNA virus RdRps was detectable with HHpred but not with BLASTP (Obbard et al, 2020) or our approach.…”

“…The ∼5,500 RNA virus (kingdom Orthornavirae ) species currently represented in GenBank constitute just a tiny fraction of the estimated millions of RNA virus species on Earth (Geoghegan & Holmes, 2017; Kuhn et al, 2019; Dance, 2021; Harvey & Holmes, 2022). Recent high-throughput RNA sequencing (RNA-Seq) studies – performed with the express purpose of identifying RNA viruses – have revealed vast numbers of novel RNA viruses, and many new family-level virus clades in diverse eukaryotic host organisms (Cook et al, 2013; Li et al, 2015; Shi et al, 2016, 2018; Olendraite et al, 2017; Charon et al, 2020; Chiapello et al, 2020; Sutela et al, 2020; Wolf et al, 2020; Batson et al, 2021; Chen et al, 2022; Forgia et al, 2022a; Kinsella et al, 2022; Rosario et al, 2022; reviewed in Dolja & Koonin, 2018; Greninger, 2018; Obbard, 2018; Zhang et al, 2019; Cobbin et al, 2021; Harvey & Holmes, 2022). However, a much larger number of RNA-Seq studies are performed for projects that are unrelated to virus discovery, but instead aim to study the transcriptomes of the targeted cellular organisms.…”

A collection ofTrichodermaisolates from natural environments in Sardinia, a biodiversity hotspot, reveals a complex virome that includes negative-stranded mycoviruses with unprecedented genome organizations

“…The term "Ormycoviruses" has been recently coined to describe a novel taxonomic group including three conserved clades of protein-coding RNA segments of viral origin, typically associated with a second RNA segment with unknown function (34). Using in-silico structural prediction approaches, we were able to demonstrate clear structural conservation of these RNA segments with previously characterized viral RdRps, but with very limited protein sequence conservation, which initially prevented their detection through similarity searches.…”

“…Additionally, another 47% of the identified viral sequences were predicted to possess a double-stranded RNA genome and have been assigned to the Durnavirales order (three new and two already known sequences) or to the Ghabrivirales order (three novel sequences). Lastly, one sequence appeared to belong to a recently proposed group of allegedly positive single-stranded RNA viruses named 'ormycoviruses' (34).…”