FIG 3.

The RdRp activity of VP1, the dsRNA binding activity of VP3, and both the 5′ and 3′ UTRs are required for VP1/VP3-mediated translation initiation of the uncapped IBDV RNA. (A) HEK 293T cells were cotransfected with the indicated plasmids, and the amount (in micrograms) of each plasmid for transfection is indicated above the lanes. Western blot analysis was performed to assess the expression of VP1, VP3, and VP4 at 72 h posttransfection. The transcription of viral mRNA from the transfected constructs was assessed by RT-PCR assay using primer pairs specific for vp1, vp3, and vp4 or for the plasmid backbone as a control. (B) HEK 293T cells were cotransfected with the indicated plasmids, and the amount (in micrograms) of each plasmid for transfection is indicated above the lanes. Western blot analysis of the cell lysate of the transfected cells was performed to assess the expression of VP1, VP3, and VP4 at 72 h posttransfection. (C) The supernatant of HEK 293T cells cotransfected with the indicated plasmids was passaged at 72 h posttransfection, and the images were taken under a phase-contrast microscope at 12 h postinfection. Scale bar, 100 μm. (D) Schematic representation of the constructs for the generation of the uncapped plus-sense RNA of segment A with deletion of the 5′ UTR (p1-mAΔ5) or 3′ UTR (p1-mAΔ3). (E) HEK 293T cells were transfected with the indicated plasmids, and the amount (in micrograms) of each plasmid for transfection is indicated above the lanes. Western blot analysis was performed to assess the expression of VP1, VP3, and VP4. The transcript level of vp4 under each condition was analyzed by RT-PCR.