Fig. 3: Cumate-inducible system controls viral gene expression in vitro and in vivo.

a Schematic illustration of the cumate-inducible expression cassette inserted into TK locus. The CymR protein was expressed under a continuous vaccinia virus promoter and a GFPLuc fusion protein incorporated under various vaccinia virus promoters preceding a CuO element which binds CymR protein. The dissociation of the CymR from CuO upon cumate administration leads to initiation of transcription and gene expression. Blue fluorescence protein is expressed from the virus to monitor its presence in infected cells. b, c Representative fluorescent images and quantitation of luciferase signal (RLU) from U2OS cells 24 h after infection at MOI 0.1 with viruses expressing the GFPLuc fusion protein (VV-CymR-iGFPLuc) under the control of various native and synthetic vaccinia promoters. Expression of GFPLuc was induced with 100 µg/ml cumate. d Quantitation of viral titers within HT-29 tumors from CD-1 nude mice seven days following infection with VV-CymR-iGFPLuc (1E7 PFU/tumor) and treatment with varying amounts of cumate. e, f C57BL/6 mice were fed with varying amounts of cumate in their diet for 25 days and weighed at regular intervals for up to 55 days to measure cumate toxicity. Serum was collected at days 25 and 55 to measure different toxicity indicators. g–j HT-29 tumors from CD-1 nude mice were infected with VV-CymR-iGFPLuc (1E7 PFU/ml) and mice fed cumate diets according to the schedule and amounts shown. Bioluminescence images were taken, and luciferase activity quantified 1 and 2 days following initial virus treatment. After day 2, cumate diets were switched to a normal rodent diet and the control group received a diet containing 6000 mg/kg of cumate. Additional images were acquired following the diet switch, at day 4 post virus infection. Bar graphs show the total flux signal measured in the tumor area at the indicated days. Scale bars = 40 μm in (b). Data indicate means ± SD of three (c), twenty (d), and five (e–j) five biological replicates. ns P > 0.05, *P < 0.05 **P < 0.003841, ***P < 0.000125, ****P < 0.001 in unpaired two-samples t-test. Source data are provided as a Source Data file.