FIG. 1.
5′ and 3′ proximal regions 1, 4, and 5 are required for RNA1 replication. (A) Schematic of known RNA1 and RNA3 cis elements and the deletions made within RNA1. Insertion of a 4-nt sequence causes a frameshift (fs) in RNA1 (F1fs) that prevents full-length protein A translation. Interaction between the distal subgenomic control element (DSCE) and the proximal subgenomic control element (PSCE) is required for RNA3 synthesis in yeast (31). RNA3 translates the RNA silencing inhibitor protein B2 (FB2). The internal replication element (intRE) and 3′ replication element (3′ RE) are required for RNA1 replication (31). Five RNA1 deletions were made and analyzed: 1, nt 1 to 377; 2, nt 377 to 1373; 3, nt 1377 to 2276; 4, nt 2287 to 2843; and 5, nt 2847 to 3107. (B and C) Total RNA was extracted from Drosophila cells (B) or yeast (C) expressing protein A and wild-type (wt) or deletions of RNA1fs. Total RNA (2 μg) was analyzed by Northern blotting with 32P-labeled cRNA probes for positive- or negative-strand RNA1 (nt 1 to 377 or nt 2718 to 3064) or 18S rRNA. The diamond in panel B represents a B2-specific, protein A-independent background band. The star in panel C represents a background band. The arrowheads in panels B and C mark the locations of negative-strand RNA1 bands. The band labeled dsRNA3 likely corresponds to both double-stranded RNA3 and RNA3 dimers (1, 31). Levels of negative-strand RNA1 were graphed as the percentage of wild-type RNA1fs levels. The measurements in panel B were made in the absence of B2.