Background This study investigates elements of herpes simplex virus type 1
(HSV-1) which influence transgene expression in tetracycline-regulated expr
ession systems.
Methods Different HSV-1 mutants were used to infect Vero cells that had bee
n transfected with plasmids containing the luciferase gene under the contro
l of tet-off or tet-on tetracycline-regulation systems.
Results The baseline level of luciferase expression was elevated after infe
ction with HSV-1 mutants lacking one or more immediate early genes encoding
transactivating factors: ICP27, ICP4 and ICP0. With the tet-off system, no
t only was baseline expression elevated, but there was a complete loss of i
nduction upon removal of tet when this regulatory system was brought into t
he 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 (14H Delta 3),
which bears a disruption in the transactivation domain of VP16 and is dele
ted for both ICP4 genes, did not increase baseline luciferase expression af
ter transfection of pUHC13-3. The disregulating effects were dependent on v
irus dose and were not influenced by treatment with interferon (IFN)-alpha,
which suppresses viral gene expression. Additional assays involving cotran
sfection of pUHC13-3 with a plasmid encoding of the HSV-1 transactivating f
actor ICP4 revealed that ICP4 was the most potent inducer of gene expressio
n 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. Copyright (C) 2000 John Wiley & Sons, Ltd.