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 [(1)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 [(1)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 (1)ssRNA virus snatches capsids from an unrelated dsRNA virus to replicate with its own RdRp, thereby mimicking the typical dsRNA virus lifestyle.