We have previously proposed a new virus lifestyle or yadokari/yadonushi nature exhibited by a positive-sense ssRNA virus, yadokari virus 1 (YkV1), and an unrelated 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 co-packages 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 on the YkV1 polyprotein, abolished its replication competency. Mutations were also introduced in the conserved 2A-like peptide motif, hypothesized to cleave the YkV1 polyprotein co-translationally. 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 capsid solely packaging YkV1 dsRNA and RdRp. These results provide proof-of-concept that a capsidless (+)ssRNA virus is hosted by an unrelated dsRNA virus.
Importance
Viruses typically encode their own capsids that encase their genomes. However, a capsidless (+)ssRNA virus, YkV1, depends on an unrelated dsRNA virus, YnV1, for encapsidation and replication. We have previously shown that YkV1 highjacks the capsid of YnV1 for trans-encapsidation of its own RNA and RdRp. YkV1 was hypothesized to divert the hetero-capsid as the replication site, as is commonly observed for dsRNA viruses. Herein, mutational analyses showed that the RdRp and 2A-like domains on 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 capsid 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.
Purification of the major sialoglycoproteins of 13762 MAT-B1 and MAT-C1 rat ascites mammary adenocarcinoma cells by density gradient centrifugation in cesium chloride and guanidine hydrochloride.