FIG. 3.

RNA present in the cytoplasm of infected cells is viral dsRNA. (A) MA104 cells were transfected for 1 h with 10 μg/ml of purified rotavirus dsRNA or 10 μg/ml of rotavirus gene 10 mRNA or (B) were infected with rotavirus RRV at an MOI of 3. Eight hours posttransfection or postinfection, the cells were fixed, permeabilized, and treated with 2 μg/ml of RNase III or 1 μg/ml of RNase A for 2 h at 37°C. After extensive washes, the cells were stained with MAb J2 to dsRNA (α-dsRNA) (green) and with a polyclonal antibody to NSP5 (α-NSP5) (red) as primary antibodies, followed by incubation with goat anti-mouse immunoglobulin G coupled to Alexa 488 and goat anti-rabbit immunoglobulin G coupled to Alexa 568 as secondary antibodies, respectively. The cell nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI; in blue). (C) CsCl-purified TLPs and DLPs or rotavirus dsRNA were incubated with RNase III or RNase A as indicated above for 2 h at 37°C. The genomic dsRNA was phenol extracted and analyzed by PAGE and silver staining as described in Materials and Methods.