FIG. 2.

In vitro mRNA synthesis with SeV L mutants in A549 cytoplasmic extracts. To express the SeV P and L proteins, A549 cells were infected with VV-T7 at an MOI of 2.5 PFU/cell and transfected with wt P and wt or mutant L plasmids (the mock sample was VV-T7 infected but had no plasmids). (Top) Cytoplasmic extracts were prepared and incubated at 30°C with polymerase-free SeV RNA-N template in the presence of [α-³²P]CTP. Labeled RNA products were purified and analyzed by agarose-urea gel electrophoresis and visualized by autoradiography. The position of the SeV NP mRNA is indicated. “% Txn” shows mRNA levels relative to those of wt SeV L protein (100%) using a PhosphorImager and represents the average of two or three experiments where variation was less than 15%. (Bottom) Immunoblot analysis of a portion of the A549 extracts used for in vitro transcription using a mixture of SeV P and L antibodies. The positions of the P and L proteins are indicated.