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Tetracycline repressor-regulated gene repression in recombinant human cytomegalovirus.

ABSTRACT: The tetracycline repressor (TetR)-regulated gene expression system from Escherichia coli was used to control gene expression in recombinant human cytomegalovirus (HCMV). To adapt the TetR system in HCMV, derivatives of the viral US11 (early) gene promoter, which controls the beta-glucuronidase reporter gene, were constructed by systematic insertion of the tetracycline operator (tetO) sequences. Gene expression from constructs containing two or three appropriately placed tetO sequences adjacent to the TATA box were efficiently repressed by a TetR-VP16 fusion protein (tTA) in a transient expression system. Efficient repression (50- to 120-fold) also occurred in tTA-expressing stably transfected human cells which were infected with recombinant HCMV containing a US11 promoter surrounded by three tetO sequences. The tTA-mediated gene repression was relieved in the presence of 1 microgram of tetracycline per ml. The results of this study are significant in three respects. (i) This is the first demonstration that a TetR-derived protein can be used to efficiently repress gene expression in a mammalian system. (ii) Efficient repression was dependent on the presence of the transcriptional activation domain from the herpes simplex virus type 1 VP16 protein. (iii) The ability to regulate gene expression in a controlled fashion in order to elucidate viral gene function is an important development in the HCMV field. The tTA-mediated gene repression system may be extremely useful for creating host-range mutants in essential genes in order to study their role in the HCMV replicative cycle, a system that is otherwise exceedingly difficult to genetically dissect.

SUBMITTER: Kim HJ

PROVIDER: S-EPMC188934 | biostudies-other | 1995 Apr

REPOSITORIES: biostudies-other

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Tetracycline repressor-regulated gene repression in recombinant human cytomegalovirus.

Kim H J HJ Gatz C C Hillen W W Jones T R TR

Journal of virology 19950401 4

The tetracycline repressor (TetR)-regulated gene expression system from Escherichia coli was used to control gene expression in recombinant human cytomegalovirus (HCMV). To adapt the TetR system in HCMV, derivatives of the viral US11 (early) gene promoter, which controls the beta-glucuronidase reporter gene, were constructed by systematic insertion of the tetracycline operator (tetO) sequences. Gene expression from constructs containing two or three appropriately placed tetO sequences adjacent t ...[more]

PMID: 7884907

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Project description:The region of the human cytomegalovirus (HCMV) genome between the UL127 promoter and the major immediate-early (MIE) enhancer is referred to as the unique region. The role of this region during a viral infection is not known. In wild-type HCMV-infected permissive fibroblasts, there is no transcription from the UL127 promoter at any time during productive infection. Our investigators previously reported that the region upstream of the UL127 TATA box repressed expression from the UL127 promoter (C. A. Lundquist et al., J. Virol. 73:9039-9052, 1999). The region was reported to contain functional NF1 DNA binding sites (L. Hennighausen and B. Fleckenstein, EMBO J. 5:1367-1371, 1986). Sequence analysis of this region detected additional consensus binding sites for three transcriptional regulatory proteins, FoxA (HNF-3), suppressor of Hairy wing, and CAAT displacement protein. The cis-acting elements in the unique region prevented activation of the early UL127 promoter by the HCMV MIE proteins. In contrast, deletion of the region permitted very high activation of the UL127 promoter by the viral MIE proteins. Mutation of the NF1 sites had no effect on the basal activity of the promoter. To determine the role of the other sites in the context of the viral genome, recombinant viruses were generated in which each putative repressor site was mutated and the effect on the UL127 promoter was analyzed. Mutation of the putative Fox-like site resulted in a significant increase in expression from the viral early UL127 promoter. Insertion of wild-type Fox-like sites between the HCMV immediate-early (IE) US3 TATA box and the upstream NF-kappaB-responsive enhancer (R2) also significantly decreased gene expression, but mutated Fox-like sites did not. The wild-type Fox-like site inhibits activation of a viral IE enhancer-containing promoter. Cellular protein, which is present in uninfected or infected permissive cell nuclear extracts, binds to the wild-type Fox-like site but not to mutated sites. Reasons for repression of UL127 gene transcription during productive infection are discussed.

Dataset Information

Tetracycline repressor-regulated gene repression in recombinant human cytomegalovirus.

Publications

Tetracycline repressor-regulated gene repression in recombinant human cytomegalovirus.

Similar Datasets

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