Fig 4.

Production of positive-sense RNA by LACV polymerase variants. Comparison of wt L and the D79A mutant. Cells were transfected with constructs for the minigenome system (Fig. 1). Untransfected cells (UT) were used as a reference, and plasmid mixes without L (no L) were used as a negative control. At 72 h after transfection, total RNA was isolated and levels of positive-sense minigenome RNA were determined. (a) Primer extension analysis. RNA was reverse transcribed using radiolabeled primers specific for positive-sense (+) Ren-Luc transcripts or negative-sense (−) Ren-Luc transcripts and for cellular 5S rRNA transcripts. (b) Strand-specific real-time RT-PCR. Note that the amount of the negative-sense vRen minigenome plasmid was lowered to 5 ng per well to the reduce background signal. rel., relative. Mean values and standard deviations from three independent experiments are shown.