FIG. 5.

R2 RNA encodes VP1 and VP2. (A) CMV-driven AMDV-G capsid constructs transfected into CRFK cells (as described in Results). The predicted RNA products they generate, their relevant ORFs (black, frame 1; striped, frame 2; gray, frame 3), and their protein products are indicated. Within the nonstructural region, the black bar indicates NS1-encoding ORF1, the striped box indicates NS3-encoding ORF2, and the gray box indicates NS2-encoding ORF3. The large capsid protein-encoding ORF shared by VP1 and VP2 in the right-hand end of the genome is read in ORF3, indicated by an open box. (B) Two days later, total cell lysates were separated on SDS-10% PAGE gels and analyzed by Western blotting, and total RNA was analyzed by RNase protection assay using probe D3 (D). Expression of HA-tagged GFP following transfection of an expression plasmid (C1GFPHA) was used as an internal control and is shown at the bottom of panel B and as the GFP-HA band in panel D. (C) Proteins generated from HA-tagged NS1 and NS2 constructs were separated on SDS-12% PAGE gels and analyzed by Western blotting using a monoclonal antibody to the HA tag (Sigma). (D) RNase protection assay using probe PD3, which spans the D3 donor at nt 2214 and thus detects all RNAs generated by these constructs, showing that they all make equivalent amounts of RNA. The band marked by a star was likely the undigested probe. Unspl, unspliced; Spl, spliced.