Proof of Concept of the Yadokari Nature: a Capsidless Replicase-Encoding but Replication-Dependent Positive-Sense Single-Stranded RNA Virus Hosted by an Unrelated Double-Stranded RNA Virus.

We previously proposed a new virus lifestyle or yadokari/yadonushi nature exhibited by a positive-sense single-stranded RNA (ssRNA) virus, yadokari virus 1 (YkV1), and an unrelated double-stranded RNA (dsRNA) virus, yadonushi virus 1 (YnV1) in a phytopathogenic ascomycete, Rosellinia necatrix. We have proposed that YkV1 diverts the YnV1 capsid to trans-encapsidate YkV1 RNA and RNA-dependent RNA polymerase (RdRp) and replicate in the heterocapsid. However, it remains uncertain whether YkV1 replicates using its own RdRp and whether YnV1 capsid copackages both YkV1 and YnV1 components. To address these questions, we first took advantage of the reverse genetics tools available for YkV1. Mutations in the GDD RdRp motif, one of the two identifiable functional motifs in the YkV1 polyprotein, abolished its replication competency. Mutations were also introduced in the conserved 2A-like peptide motif, hypothesized to cleave the YkV1 polyprotein cotranslationally. Interestingly, the replication proficiency of YkV1 mutants in the host fungus agreed with the cleavage activity of the 2A-like peptide tested using a baculovirus expression system. Cesium chloride equilibrium density gradient centrifugation allowed for the separation of particles, with a subset of YnV1 capsids solely packaging YkV1 dsRNA and RdRp. These results provide proof of concept that a capsidless positive-sense ssRNA [(+)ssRNA] virus is hosted by an unrelated dsRNA virus. IMPORTANCE Viruses typically encode their own capsids that encase their genomes. However, a capsidless positive-sense single-stranded RNA [(+)ssRNA] virus, YkV1, depends on an unrelated double-stranded RNA (dsRNA) virus, YnV1, for encapsidation and replication. We previously showed that YkV1 highjacks the capsid of YnV1 for trans-encapsidation of its own RNA and RdRp. YkV1 was hypothesized to divert the heterocapsid as the replication site, as is commonly observed for dsRNA viruses. Herein, mutational analyses showed that the RdRp and 2A-like domains of the YkV1 polyprotein are important for its replication. The active RdRp must be cleaved by a 2A-like peptide from the C-proximal protein. Cesium chloride equilibrium density gradient centrifugation allowed for the separation of particles, with YnV1 capsids solely packaging YkV1 dsRNA and RdRp. This study provides proof of concept of a virus neo-lifestyle where a (+)ssRNA virus snatches capsids from an unrelated dsRNA virus to replicate with its own RdRp, thereby mimicking the typical dsRNA virus lifestyle.

An exploration of ambigrammatic sequences in narnaviruses.

Narnaviruses have been described as positive-sense RNA viruses with a remarkably simple genome of ~3 kb, encoding only a highly conserved RNA-dependent RNA polymerase (RdRp). Many narnaviruses, however, are 'ambigrammatic' and harbour an additional uninterrupted open reading frame (ORF) covering almost the entire length of the reverse complement strand. No function has been described for this ORF, yet the absence of stops is conserved across diverse narnaviruses, and in every case the codons in the reverse ORF and the RdRp are aligned. The >3 kb ORF overlap on opposite strands, unprecedented among RNA viruses, motivates an exploration of the constraints imposed or alleviated by the codon alignment. Here, we show that only when the codon frames are aligned can all stop codons be eliminated from the reverse strand by synonymous single-nucleotide substitutions in the RdRp gene, suggesting a mechanism for de novo gene creation within a strongly conserved amino-acid sequence. It will be fascinating to explore what implications this coding strategy has for other aspects of narnavirus biology. Beyond narnaviruses, our rapidly expanding catalogue of viral diversity may yet reveal additional examples of this broadly-extensible principle for ambigrammatic-sequence development.

A novel monopartite dsRNA virus isolated from the phytopathogenic fungus Ustilaginoidea virens strain GZ-2.

In this study, we describe a novel mycovirus isolated from Ustilaginoidea virens strain GZ-2, which was designated "Ustilaginoidea virens nonsegmented virus 2" (UvNV-2). The genome of UvNV-2 contains two overlapping open reading frames (ORFs). ORF1 encodes an unknown protein, and ORF2 encodes a putative RNA-dependent RNA polymerase (RdRp), which is most closely related to that of Purpureocillium lilacinum nonsegmented virus 1 (PINV-1) and is likely to be expressed by a + 1 ribosomal frameshift within the sequence CCC_UUU_UAG. A phylogenetic analysis of the RdRp of UvNV-2 showed that UvNV-2 is an unclassified mycovirus.

Molecular characterization of a novel double-stranded RNA mycovirus of Trichoderma asperellum strain JLM45-3.

In this study, we describe a novel mycovirus isolated from Trichoderma asperellum, which was designated Trichoderma asperellum dsRNA Virus 1 (TaRV1). The sequence analysis revealed that TaRV1 has two discontinuous open reading frames (ORF), ORF1 and ORF2. A hypothetical protein and an RNA-dependent RNA polymerase are encoded by ORF1 and ORF2, respectively. Phylogenetic analysis based on RdRp sequences clearly places TaRV1 in a taxonomically unassigned dsRNA mycovirus group.