FIG 5.
SsYkV1 is hosted in virions of SsBV3. (A) Dual culture of strains SCH941R115 and SCH941A1 (or SCH941A1hyg, a hygromycin B-resistant strain) on PDA plates for 7 days. The new isolates were picked from the colony margin of the virus-free strain. RT-PCR was performed to detect the mycovirus content in the new isolates with specific primers for six mycoviruses infecting strain SCH941. Lane NC: negative control (double distilled water instead of cDNA). (B) Agarose gel electrophoresis profile of viral nucleic acid released from virions isolated from strains AT3 and PT18 on a 0.7% agarose gel. (C) Ten percent SDS-PAGE analysis of the protein components of viral particles purified from the SsBV3- and SsYkV1-coinfected strain (lane AT3), the SsBV3-infected strain (lane PT18), and the virus-free strain (lane SCH941A1). The molecular weight of the protein bands was estimated by the protein markers. Each protein band marked by a red line was identified by LC-MS/MS, and the corresponding peptide information is shown in Table S2 in the supplemental material. (D) Western blot analysis of virions purified from a 40% CsCl layer using anti-SsYkV1 RdRp polyclonal antibodies. The position of the 75-kDa protein is indicated on the right. Amino acids 1 to 160 of the SsYkV1 ORF were used to produce polyclonal antibodies. (E) RT-PCR and 0.7% agarose gel electrophoresis analysis of the viral nucleic acid released from virions in strain AT3. The left lane was not treated with S1 nuclease, and the right lane was treated with S1 nuclease. (F) Schematic representation of the fractionation of virus particles based on a CsCl density gradient. The darker the blue, the higher the CsCl concentration. The dotted line indicates that 1-mL fractions were taken for nucleic acid extraction. Fractions 24 and 25 (highlighted in red) represent the fractions containing encapsidated forms of SsBV3 only and SsBV3 plus SsYkV1, respectively. (G) Electrophoretic profile of viral RNA extracted from different fractions after CsCl density gradient centrifugation.