A combination of HSV-TK expression and pTP-mi5 expression leads to an additive inhibitory effect on adenoviral DNA replication upon treatment with GCV. A549 cells were transduced with the adenoviral HSV-TK/pTP-mi5 expression vector, AdTO-TK-pTP-mi5, or the respective control vector, AdTO-TK-mi-, expressing a negative control amiRNA at an MOI of 100 TCID50/cell. At 24 h post-transduction, cells were infected with wt Ad5 at an MOI of 0.01 TCID50/cell, and GCV was or was not added at a concentration of 1.2 μM. A. Wt Ad5 genome copy numbers were determined at 48 h post-infection with wt Ad5 by qPCR using a primer/probe set specific for the E1A gene present only on wt Ad5. B. The infection kinetics for wt Ad5 was monitored over a time span of 6 days by determining genome copy numbers in the same way as described for (A). Data represent the means ± SD of 3 independent transductions/infections. *p < 0.05, **p < 0.01, ***p < 0.001.