HSV-1 infected cell proteins influence 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.


CONCLUSIONS
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.
Herrlinger
U
U
Neurology Service, Massachusetts General Hospital and Harvard Medical School, Charlestown 02129, USA. ulrich.herrlinger@uni-tuebingen.de
Pechan
P A
PA
Jacobs
A H
AH
Woiciechowski
C
C
Rainov
N G
NG
Fraefel
C
C
Paulus
W
W
Reeves
S A
SA
D000818:Animals; D002522:Chlorocebus aethiops; D015870:Gene Expression / Q000187:drug effects*; D018014:Gene Transfer Techniques; D017781:Genes, Immediate-Early; D018259:Herpesvirus 1, Human / Q000235:genetics*; D017874:Immediate-Early Proteins / Q000235:genetics; D008156:Luciferases / Q000235:genetics; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D013752:Tetracycline / Q000494:pharmacology*; D014158:Transcription, Genetic; D014162:Transfection; D014709:Vero Cells; D014764:Viral Proteins / Q000378:metabolism
D017874:Immediate-Early Proteins; D014764:Viral Proteins; C048925:herpes simplex virus, type 1 protein ICP4; D008156:Luciferases; D013752:Tetracycline