FIG. 3.

The large Rep proteins are required and sufficient to enhance the ratio of spliced to unspliced AAV RNA. 293 cells were transfected with the minimal P5P40VP reporter plasmid in the presence of adenovirus, and different Rep proteins were provided in trans by cotransfection. For the sample shown in lane 11, Rep was supplied by the plasmid RepSM. pSK45mP5ATG, a full-length AAV construct in which the initiating AUG for the large Rep proteins has been mutated, is described in the text. Total RNA was protected by the RP probe, and bands specifically protected by P40-initiated spliced and unspliced RNAs from a representative experiment are designated. Quantitations of the ratio of spliced to unspliced P40-initiated RNAs are shown and are averages of the results of at least three separate experiments, with standard deviations in parentheses. All of the Rep-supplying plasmids contained silent mutations in the region protected by the RP probe and are described in Materials and Methods. The arrow and arrowhead denote the unspliced and spliced RNAs, respectively, from the P5+P19 promoters (lanes 10 and 11) and from the P5 promoter (lanes 1 to 9), with some low-level contribution from plasmid read-around (lanes 1 to 9).