Fig. 1.

Construction and characterization of the RK-G8-A1-A2Flag cell line. (A) Diagram depicting the assay to determine dual late promoter activity of intermediate promoters. A cell that expresses the three late transcription factors G8, A1 and A2, is infected with a VACV mutant with a deletion of the A23 intermediate transcription factor and is transfected with a plasmid containing LUC regulated by a strict intermediate (I) promoter or by a dual intermediate/late (I/L) promoter. Black circle indicates no LUC expression; yellow circle indicates LUC expression. (B) Diagram of a tricistronic cassette regulated by a CMV promoter. The picornavirus 2A-like sequences F2A and T2A separate the G8R and A1L ORFs and the A1L and A2L ORFs, respectively. The A2L ORF has a C-terminal Flag tag. (C) Western blots showing G8, A1 and A2 expression. A2 was detected with antibody to the Flag epitope; G8 and A1 were detected with antibody to the 2A peptide. The positions of protein markers in kDa are shown on the left and the A2, G8 and A1 bands on the right. (D) Expression of a late and intermediate gene. RK-13, RK-G8-A1-A2Flag, or RK13-A8/A23 cells were infected with vA23Δ and transfected with plasmids encoding the late F17R and intermediate G8R ORFs with 3XFlag tags regulated by their natural promoters. After 18 h, the cells were lysed and analyzed by Western blotting with anti-Flag antibody. The single flag epitope on the A2 protein was not detected in the small amount of extract used for analysis of the triple flag on the F17 and G8 proteins.