FIG. 5.

The inhibitory effect of the NS1 plasmid is ablated by a frameshift in the NS1 ORF. (A) The NS1 cDNA was mutagenized to delete 2 nt from codon eight, which shifts the reading frame and terminates the ORF three codons later. This is illustrated in the sequence of codons 1 to 11 of the NS1 ORF. This mutation would preclude translation from either the first or eighth codons, which are AUG. The next AUG in the 139-codon ORF is at codon 80. (B) Western blot analysis showing the expression of the NS1 protein from the wild-type (lanes 1 to 5) or mutant (lanes 6 to 9) plasmid. The indicated amounts of plasmid were transfected into 293 cells infected with vTF7-3. Cell lysates were prepared 40 h later and analyzed with a Western blot probed with an antiserum against a synthetic peptide of the NS2 protein. (C) Expression of CAT by the C2 minigenome complemented by the N, P, L, and M2 (both ORFs) plasmids together with the indicated amount of wild-type or mutant NS1 plasmid. Cells were harvested 40 h postinfection, and lysates were assayed for the ability to acetylate [¹⁴C]chloramphenicol as visualized by thin-layer chromatography and autoradiography (the unreacted chloramphenicol comprises the bottom row of spots, and the acetylated forms comprise the two higher rows).