A capsidless (+)RNA yadokarivirus hosted by a dsRNA virus is infectious as particles, cDNA, and dsRNA

Capsidless yadokariviruses (members of the order <i>Yadokarivirales</i>) with (+)RNA genomes divert the capsid of their partner icosahedral double-stranded RNA (dsRNA) viruses in different families of the order <i>Ghabrivirales</i> into the replication site. A yadokarivirus, AfSV2, has been reported from a German strain of the ascomycete fungus <i>Aspergillus foetidus</i> coinfected by two dsRNA viruses, a victorivirus (AfSV1<i>,</i> family <i>Pseudototiviridae</i>) and an alternavirus (AfFV, family <i>Alternaviridae</i>). Here, we identified AfSV1 as the partner of AfSV2 in a Japanese <i>A. foetidus</i> strain after showing the infectiousness of AfSV2 in three forms: virus particles (heterocapsid), transforming full-length complementary DNA (cDNA), and purified replicated form (RF) dsRNA that is believed to be inactive as a translational template. Virion transfection of virus-free <i>A. foetidus</i> protoplasts resulted in the generation of two strains infected either by AfSV1 alone or by both AfSV1 and AfSV2. Transformants with AfSV2 full-length cDNA launched AfSV2 infection only in the presence of AfSV1, but not those with AfSV2 RNA-directed RNA polymerase mutant cDNA. The purified fractions containing AfSV2 RF dsRNA also launched infection when transfected into protoplasts infected by AfSV1. Treatment with dsRNA-specific RNase III, but not with proteinase K, S1 nuclease, or DNase I, abolished the infectivity of AfSV2 RF dsRNA. Furthermore, we confirmed the infectiousness of gel-purified AfSV2 RF dsRNA in the presence of AfSV1. Taken together, our results show the unique infectious entity of AfSV2 and the expansion of yadokarivirus partners in the family <i>Pseudototiviridae</i> and provide interesting evolutionary insights.IMPORTANCEThe viral phylum <i>Pisuviricota</i> accommodates members with both double-stranded RNA (dsRNA) and (+)RNA genomes. Some members of the second group display peculiar virus lifestyles. These include (+)RNA yadkariviruses, which are capsidless and highjack the capsid of their partner dsRNA viruses in the order <i>Ghabrivirales</i> of a different phylum <i>Duplornaviricota</i>. We identified the partner dsRNA virus (AfSV1, a victorivirus) of a yadokarivirus (AfSV2) from the ascomycete <i>Aspergillus foetidus</i>. AfSV2 is infectious in the presence of AfSV1 in three forms: purified particles, transforming full-length complementary DNA, and, surprisingly, the purified replicative form dsRNA. These combined results expand yadokarivirus partner viruses to the family <i>Pseudototiviridae</i> and provide evidence for AfSV2 as a unique infectious entity as well as interesting evolutionary insights.