FIG. 4.

Transient viral DNA replication assay and transcriptional transactivation assay. (A) A total of 1 μg of either pCMV₄E2 wild type or pCMV₄E2S206T-expressing vector was cotransfected with 0.5 μg of pHPV16E1E2TTL and various amounts of pCMV₄E1 into 293T cells. After 72 h, low-molecular-weight Hirt DNAs were isolated and subjected to DpnI and BamHI digestion. DpnI-resistant DNAs were detected by Southern blotting with a radiolabeled probe. The amounts of pCMV₄E2 or pCMV₄E1 in each transfection are indicated above the panel. DpnI-resistant pHPV₁₆E₁E₂TTL DNA were detected only in the presence of both E1 and E2 (lanes 4 to 6 and 8 to 10). A DpnI-digested fragment of pHPV16E1E2TTL (indicated by an arrowhead) was detected equally in all samples, indicating a similar efficiency of transfection for each sample. (B) A total of 0.5 μg of either wild-type pCMV₄E2 or pCMV₄E2S206T was cotransfected with 0.25 μg of a luciferase reporter plasmid, pBS1073 (black columns) or pBS1013 (white columns), into 293T cells. After 48 h, cells were harvested and subjected to analysis to measure luciferase activity as instructed by the manufacturer (Promega, Madison, WI). The y axis indicates chemiluminescence per second (CPS). Three independent transfections were performed.