FIG. 1.

(A) Inhibitory effect of AAV on HPV-16 LCR promoter activity at different molar ratios. SiHa cells were cotransfected with pBL-16LCR-CAT6 reporter plasmid (10 μg) and either an AAV plasmid (pAV1, which carries WT AAV DNA within the pBR322 vector) (2, 4, 10, or 20 μg) or a control plasmid (pBR322; 1, 2, 5, or 10 μg) to yield HPV/AAV molar ratios of 10:1, 5:1, 2:1, and 1:1. Transfected cells were cultured for 48 h and harvested, and the cell extracts were prepared for CAT assay as described previously (25). Acetylated and nonacetylated spots were quantified by electronic autography with an instant imager (Packard, Meriden, Conn.). The promoter activity in extracts of cells cotransfected with pBL-16LCR-CAT6 and pBR322 was set as 100%. Calculation of the relative activity is described in text. The data are the means ± standard errors of four individual experiments. (B) Representative thin-layer chromatography analysis of the effect of AAV on the HPV-16 LCR in SiHa (HPV-positive) and C-33A (HPV-free) cells. The cells were cotransfected with reporter pBL-16LCR-CAT6 and either effector plasmid pAV1 or vector pBR3224 at molar ratios of 2.5:1 and 1:1. The subsequent steps were the same as described above. The chromatography results presented are from one of three individual experiments. −, pBLCAT6 (negative control); V, pBR322; A, pAV1; RCA, relative CAT activity.