FIG. 4.

Both the external and internal promoter elements are necessary for efficient RNA synthesis. (A) Schematic diagram illustrating the sites of point mutations in the +56(13) minigenome. The first gene start sequence is illustrated with a hatched rectangle, Le-specific sequence is shown as a thin black rectangle, and nonspecific sequence is shown as a thin white rectangle. The position of the single nucleotide change to introduce an NsiI restriction site is indicated as NsiI, and the positions of the three C-to-A substitutions in the promoter elements are indicated with lightning symbols. (B and C) Northern blot analysis of intracellular positive-sense RNA produced from the wild-type (lane 2) and mutant (lanes 3 to 7) minigenomes, as indicated. Lane 1 is a negative control of RNA from cells transfected with plasmid encoding a minigenome with a wild-type Le but no L polymerase. Panel B shows total intracellular RNA and panel C shows RNA from cell lysates that were treated with MCN prior to RNA purification. (D and E) Quantitation of antigenome and mRNA 1 generated from the mutant minigenomes by phosphorimage analysis. Each RNA value was calculated as a percentage of the wild-type value (100%) and each error bar represents the standard error of the mean. It should be noted that the quantitation of antigenome and mRNA 1 includes quantitation of the more slowly migrating RNA species, in addition to the transcripts that migrated similarly to those from the wild-type minigenome. Panel D shows analysis of antigenome RNA (black bars) and mRNA 1 (white bars) measured in the total RNA samples, and panel E shows analysis of encapsidated antigenome measured in the MCN-treated RNA samples.