FIG. 2.

The 2a ORF-dependent pathway recruits RNA2 derivatives to functional RNA replication complexes. Negative-strand RNA2 accumulation was assayed by a two-cycle RNase protection assay (29) with equal amounts of total RNA extracted from yeast expressing each of the RNA2 derivatives shown in Fig. 1B in the presence (+1a) or absence (−1a) of 1a as indicated. After initial hybridization and RNase treatment to remove excess positive-strand RNA2, the remaining double-stranded RNA was denatured, hybridized with a ³²P-labeled RNA probe corresponding to nt 1441 to 1685 of positive-strand RNA2, and treated with RNases A and T₁. The reaction products were electrophoresed and autoradiographed. The relative strength of the negative-strand signal for each RNA2 derivative, averaged over three independent experiments, is shown at the bottom.