Figure 3

Enhanced production of progeny virus by introduction of the precursor terminal protein (pTP) gene. (A) Scheme of pTP-overexpressing E1B55K-deleted oncolytic adenovirus. (B) Expressions of pTP protein in DU145 cells after Ad-3484-pTP infection at indicated MOIs for 48 h. (C) Western blot analysis for detection of protein levels, including Adenovirus type 5, in A549 and Hep3B cells infected using various MOIs of Ad-3484-pTP for 48 h. (D) Western blot analysis for detection of protein levels, including adenovirus type 5, in various cancer cells as well as in 293A cells infected with 10 MOIs of Ad-3484-pTP for 48 h. The numbers indicate the relative band intensity of Ad-3484-pTP to control (Ad-3484-NC) after band intensities were measured with a densitometer. (E) Estimation of viral DNA copy number after pTP overexpression. After infection with Ad-3484-pTP at 10 MOI for 24 h in 293A cells, cells and media were harvested. Purified DNA after CsCl density gradient ultracentrifugation and Tris-dialysis of viral soup was measured at OD260 and OD280 for copy-number calculations. Error bars represent standard errors from three independent experiments. (F) After infections with the same amount of each oncolytic adenovirus (MOI 10 with Ad-3484-NC or Ad-3484-pTP), and purification of virus following CsCl density gradient ultracentrifugation and Tris-dialysis of viral soup from 293A cells, total virus particles (VP/ml) were calculated by measuring absorbance at OD260 and plaque-forming units (PFU/ml), which indicates that the amounts of infectious virus particles were successfully calculated by TCID50 titration assays in 293A cells. Error bars represent standard errors from three independent experiments. P values less than 0.05 were considered statistically significant (*P < 0.05). (G) PFU titration assays of adenovirus produced by each human cancer cell line following Ad-3484-NC or Ad-3484-pTP infections at MOI 50 for 4 h, and incubations for 48 h with media exchanged after PBS washing. Adenovirus packaging cells (293A) were infected at MOI 0.1. Error bars represent standard errors from three independent experiments. P values less than 0.05 were considered statistically significant (**P < 0.01; ***P < 0.001).