FIG. 4.

In vitro transcription with the Sendai virus L mutants. VVT7-infected A549 cells were transfected with no plasmids (Mock) or with the Sendai virus P (1.5 μg) and the indicated wt or mutant L (0.5 μg) plasmids. (A) Cytoplasmic cell extracts were incubated with polymerase-free wt Sendai virus RNA-NP (1 μg) and [α-³²P]CTP (200 μCi/ml) as described previously (7, 8). Total RNA was isolated and separated on an agarose-urea gel. The position of the NP and P transcripts which comigrate is indicated. (B) Samples of the extracts were immunoblotted with α-P antibody. The position of the P protein is indicated. (C) For leader RNA synthesis the extracts were incubated with polymerase-free wt RNA-NP and unlabeled nucleotides, and the leader RNA was detected by Northern analysis with a ³²P-labeled oligonucleotide probe complementary to le+ RNA as described previously (7). The position of the le+ product is indicated.