A combination of HSV-TK expression/GCV treatment and siRNA-mediated silencing of adenoviral gene expression leads to an additive inhibitory effect on adenoviral DNA replication. A549 cells were transfected with 30 nM siRNA directed against the hexon (hex), protease (prot), IVa2, pTP, and viral DNA polymerase (pol) transcripts. A nontargeting siRNA served as a negative control. At 24 h after transfection, cells were transduced with either the adenoviral HSV-TK expression vector, AdEE4-TK (right panels), or the analogous EGFP expression vector, AdEE4 (left panels), at an MOI of 100 TCID50/cell. Another 24 h later, 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. At 48 h after infection with wt Ad5, the cultures were analyzed for wt Ad5 multiplication. A. Genome copy numbers were determined by qPCR using a primer/probe set specific for the E1A gene present only on wt Ad5. B. The output of infectious virus progeny was determined by TCID50 assays. Data represent the means ± SD of 3 independent transfections/infections.