FIG. 4.

C/EBPβ represses HPV-18 activity. (A) C/EBPβ-induced repression of the HPV-18 URR is independent of the switch region OL22 in HeLa cells. CAT assays were carried out with extracts from HeLa cells transfected with a CAT plasmid containing the wild-type HPV-18 URR (p18URR) (upper panel) or with a construct containing mutations in the switch region of the HPV-18 URR (p18URR-22M1) (lower panel) (4). HeLa cells were transfected with 3 μg of p18URR or p18URR-22M1 together with increasing amounts of CMV-C/EBPβ (0.1 to 0.8 μg) and 0.6 μg RSV/L (18) as an internal control. Relative CAT activities were quantified relative to the activities obtained with p18URR and p18URR-22M1 cotransfected with 0.8 μg of pcDNAI, which were set at 1, and the results of representative CAT assays are shown. Relative CAT activities were as follows. Upper panel (p18URR): 0 μg of C/EBPβ, 1.0; 0.1 μg of C/EBPβ, 0.51; 0.2 μg of C/EBPβ, 0.36; 0.3 μg of C/EBPβ, 0.24; 0.4 μg of C/EBPβ, 0.20; 0.5 μg of C/EBPβ, 0.18; 0.6 μg of C/EBPβ, 0.12; 0.7 μg of C/EBPβ, 0.08; 0.8 μg of C/EBPβ, 0.06. Lower panel (p18URR-22M1): 0 μg of C/EBPβ, 1.0; 0.1 μg of C/EBPβ, 0.47; 0.2 μg of C/EBPβ, 0.25; 0.3 μg of C/EBPβ, 0.19; 0.4 μg of C/EBPβ, 0.15; 0.5 μg of C/EBPβ, 0.13; 0.6 μg of C/EBPβ, 0.09; 0.7 μg of C/EBPβ, 0.06; 0.8 μg of C/EBPβ, 0.05. (B) C/EBPβ represses E6-E7 mRNA expression in HeLa cells. Cells were transfected with increasing amounts of CMV-C/EBPβ or pcDNAI, as indicated in the figure, and 0.6 μg of RSV/L (18) to monitor for transfection efficiency. Cytoplasmic RNA was extracted and separated in a 1.2% agarose gel (upper panel). The filter was hybridized with a ³²P-labeled HPV-18 E6-E7 DNA probe and was exposed to X-ray film for 3 days. Beside controls with transfected pcDNAI, RNA from HeLa cells transfected only with 0.6 μg of RSV/L only (18) (lane 13) and RNA from nontransfected HeLa cells (/) (lane 14) were used. The arrowheads indicate the positions of the 28S and 18S RNAs.