BACKGROUND: This study investigates elements of herpes simplex virus type 1 (HSV-1) which influence transgene expression in tetracycline-regulated expression systems. METHODS: Different HSV-1 mutants were used to infect Vero cells that had been transfected with plasmids containing the luciferase gene under the control of tet-off or tet-on tetracycline-regulation systems. RESULTS: The baseline level of luciferase expression was elevated after infection with HSV-1 mutants lacking one or more immediate early genes encoding transactivating factors: ICP27, ICP4 and ICP0. With the tet-off system, not only was baseline expression elevated, but there was a complete loss of induction upon removal of tet when this regulatory system was brought into the cell by infection with helper virus-free amplicon vectors. Elevation of luciferase expression was also observed upon infection with the same HSV-1 mutants following transfection with a plasmid containing only a CMV minimal promoter driving luciferase (pUHC13-3). Only one HSV mutant (14Hdelta3), which bears a disruption in the transactivation domain of VP16 and is deleted for both ICP4 genes, did not increase baseline luciferase expression after transfection of pUHC13-3. The disregulating effects were dependent on virus dose and were not influenced by treatment with interferon (IFN)-alpha, which suppresses viral gene expression. Additional assays involving cotransfection of pUHC13-3 with a plasmid encoding of the HSV-1 transactivating factor ICP4 revealed that ICP4 was the most potent inducer of gene expression from the tetO/CMV minimal promoter. CONCLUSION: These results indicate that proteins encoded in the HSV-1 genome, especially the transactivating immediate early gene products (ICP4, ICP27 and ICP0) and the VP16 tegument protein can activate the tetO/ minimal CMV promoter and thereby interfere with the integrity of tetracycline-regulated transgene expression.
BACKGROUND: This study investigates elements of herpes simplex virus type 1 (HSV-1) which influence transgene expression in tetracycline-regulated expression systems. METHODS: Different HSV-1 mutants were used to infect Vero cells that had been transfected with plasmids containing the luciferase gene under the control of tet-off or tet-on tetracycline-regulation systems. RESULTS: The baseline level of luciferase expression was elevated after infection with HSV-1 mutants lacking one or more immediate early genes encoding transactivating factors: ICP27, ICP4 and ICP0. With the tet-off system, not only was baseline expression elevated, but there was a complete loss of induction upon removal of tet when this regulatory system was brought into the cell by infection with helper virus-free amplicon vectors. Elevation of luciferase expression was also observed upon infection with the same HSV-1 mutants following transfection with a plasmid containing only a CMV minimal promoter driving luciferase (pUHC13-3). Only one HSV mutant (14Hdelta3), which bears a disruption in the transactivation domain of VP16 and is deleted for both ICP4 genes, did not increase baseline luciferase expression after transfection of pUHC13-3. The disregulating effects were dependent on virus dose and were not influenced by treatment with interferon (IFN)-alpha, which suppresses viral gene expression. Additional assays involving cotransfection of pUHC13-3 with a plasmid encoding of the HSV-1 transactivating factor ICP4 revealed that ICP4 was the most potent inducer of gene expression from the tetO/CMV minimal promoter. CONCLUSION: These results indicate that proteins encoded in the HSV-1 genome, especially the transactivating immediate early gene products (ICP4, ICP27 and ICP0) and the VP16 tegument protein can activate the tetO/ minimal CMV promoter and thereby interfere with the integrity of tetracycline-regulated transgene expression.
Authors: S Goverdhana; M Puntel; W Xiong; J M Zirger; C Barcia; J F Curtin; E B Soffer; S Mondkar; G D King; J Hu; S A Sciascia; M Candolfi; D S Greengold; P R Lowenstein; M G Castro Journal: Mol Ther Date: 2005-08 Impact factor: 11.454
Authors: E Scott Halstead; Todd M Umstead; Michael L Davies; Yuka Imamura Kawasawa; Patricia Silveyra; Judie Howyrlak; Linlin Yang; Weichao Guo; Sanmei Hu; Eranda Kurundu Hewage; Zissis C Chroneos Journal: Respir Res Date: 2018-01-05