FIG 3.

Overexpression of PUBS inhibits HPV DNA replication. (A) HPV DNA replication levels in C33A cells transfected with increasing amounts of PUBS-YFP or E1 UBS-YFP expression vector (25, 50, and 75 ng). The empty YFP vector (75 ng) was used as a negative control (−). Assays were performed with two different amounts of E1 expression vector (5 ng and 1.25 ng), as indicated, and without E1 as a baseline (i.e., no DNA replication) control (×). DNA replication activity is reported as a percentage of the signal obtained with the YFP-negative control. Each value represents the average from two independent experiments, each performed in triplicates, with the standard deviation indicated by an error bar. (B) Same as panel A but using the E1Δ protein instead of wild-type E1. (C) Western blot showing the relative expression of PUBS-YFP and E1 UBS-YFP in transfected C33A cells. Proteins were detected with anti-GFP antibodies. (D) Cell cycle analysis. C33A cells were transfected with a plasmid encoding PUBS-YFP or YFP alone as a control, and their DNA was stained with Hoechst 48 h posttransfection. The cell cycle distribution of the YFP-positive cell population was analyzed by flow cytometry. (E) Colony formation assay. C33A cells were either mock transfected or transfected with an expression vector for PUBS-YFP or E1 UBS-YFP and selected for approximately 3 weeks in bleomycin-containing medium. Drug-resistant colonies were fixed in methanol and stained with methylene blue.