Fig. 4: Double inducible chemogenetic switches overcome major challenges with single molecule systems.

a Schematic illustration of the ST7-Tet double inducible expression cassette inserted into TK locus. The TetR and split T7 fragment proteins were expressed under continuous vaccinia virus promoters and a GFPLuc fusion protein was incorporated under T7 promoter preceding a TetO element which binds TetR protein. The binding of split T7 fragments in the presence of Rapalogs and dissociation of the TetR from TetO upon Dox administration leads to initiation of transcription and gene expression. b, c Representative fluorescent images and quantitation of luciferase activity from U2OS cells 24 h following infection with VV-ST7-TetR-iGFPLuc in the presence or absence of Dox and rapamycin. mCherry signal indicates virally infected cells. d Schematic illustration of the ST7-Cumate double inducible expression cassette inserted into the same locus as described in A. The CymR and split T7 fragment proteins were expressed under continuous vaccinia virus promoters and GFPLuc was incorporated under T7 promoter preceding a CuO element which binds CymR protein. The binding of split T7 fragments in the presence of Rapalogs and dissociation of the CymR from CuO upon cumate administration leads to initiation of transcription and gene expression. e, f Representative fluorescent images and quantitation of luciferase activity from U2OS cells 24 h following infection with VV-ST7-CymR-iGFPLuc in the presence or absence of cumate and rapamycin. g Schematic illustration of the Cu-Tet double inducible expression cassette inserted into the same locus as described in A. The CymR and TetR proteins were expressed under continuous vaccinia virus promoters and GFPLuc was incorporated under different vaccinia virus promoters preceding a CuO element which binds CymR protein, and a TetO element which binds TetR protein. Dissociation of the CymR from CuO and TetR from TetO upon cumate and Dox administration leads to initiation of transcription and gene expression. h Luciferase signal (in RLU) was quantified 24 h after infection of U2OS cells with viruses expressing GFPLuc under the control of CuTet and various native and synthetic vaccinia promoters in the presence or absence of 100 µg/ml cumate and 100 ng/ml Dox. i, j Representative fluorescent images and quantitation of luciferase activity from HEK293T, Hela, Vero, HT-29, 786-O cells infected with VV-CymR-TetR-iGFPLuc in the presence or absence of cumate and Dox after 24 h. IRF signal indicates virally infected cells. k Schematic illustration of the CuTet double inducible expression cassette inserted in the intergenic ___location between UL26 and UL27 open reading frames of the HSV-1 genome. The CymR-T2A-rtTA3 fusion protein was expressed under the UBC promoter. For expression of the GFPLuc fusion protein, TetO followed by a minimal CMV promoter and CuO was used. In the presence of Dox, rtTA3 binds to the TetO and enhances CMV promoter activity which will be at maximum activity if CymR is also dissociated from CuO upon cumate administration. IRF is continuously expressed from the virus, to monitor viral infection. l, m Representative fluorescent images and quantitation of luciferase activity from U2OS cells infected with VV-CymR-TetR-iGFPLuc in the presence or absence of cumate and Dox after 24 h. IRF signal indicates virally infected cells. Scale bars = 40 μm in (c, f, i, m). Data indicate means ± SD of three (b, h, j, l) or four (e) 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.