Figure 1.

Effect of PAP on the synthesis of RNA3 by the viral replicase. (A) PAP-treated and untreated BMV RNA3 in vitro transcripts were used as template for the BMV replicase assay in the presence of radiolabeled CTP. Product RNA3 was separated in a 7 M urea/12% acrylamide gel and visualized by autoradiography. Control is positive-strand, radiolabeled BMV RNA3 in vitro transcript to serve as a size marker. (B) PAP-treated and untreated BMV RNA3 in vitro transcripts were incubated with BMV replicase in the presence of radiolabeled CTP and aliquots were removed at the indicated times. The RNA products were precipitated and the amount synthesized over time was quantified by scintillation counting. Points are means ± SE for three separate experiments. Circles represent PAP-treated template RNA3 and squares represent untreated RNA3. (C) A time-course analysis of the status of PAP-treated BMV RNA3 in vitro transcripts used as template for the replicase assay and primer extension. PAP-treated and untreated BMV RNA3 transcripts were incubated with the replicase (RdRp) or in buffer alone, and aliquots were removed at the indicated times. RNA was analyzed by northern blot for positive-strand BMV RNA3.