Regulation of transcription in vitro from herpes simplex virus genes

Self Cite

On the basis of experiments with mutant virus and transfection with isolated genes, the herpes simplex virus immediate-early gene product ICP4 is known to positively regulate the transcription of viral early and late genes and negatively regulate expression from its own promoter. Binding of ICP4 to DNA sequences in several viral genes has been reported, yet the significance of ICP4-DNA interaction in transcriptional activation remains unclear. We have studied this problem by using the early glycoprotein D (gD) gene, which possesses a binding site at approximately-100 relative to the RNA initiation site. We linked this promoter and various mutant constructs to the chloramphenicol acetyltransferase gene in order to measure promoter activity in transient transfections both in the presence and in the absence of an ICP4-encoding plasmid. The natural promoter was activated 3.3-fold, and a deletion construct lacking the binding site was activated minimally (1.7-fold). Constructs containing multiple tandem repeats of the binding site (three or five inserts) demonstrated higher expression in the presence of ICP4 than did the natural promoter while retaining low levels of expression when unstimulated. Gel mobility shift assays and DNase I footprinting analyses indicated that ICP4 associated with multiple binding sites. In vitro transcription from a gD promoter construct containing multiple binding sites showed increased RNA synthesis in the presence of partially purified ICP4. These data provide the first direct evidence that binding of ICP4 to a specific DNA sequence in the gD gene contributes to activation of transcription.

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Role for DNA-protein interaction in activation of the herpes simplex virus glycoprotein D gene

Tedder

Pizer

1988

Self Cite

“…Consequently, the significance of the binding site in the DE promoter for the gD gene is of some interest. Pizer et al (47) have suggested that it has a positive effect from in vitro transcription studies, whereas Arsenakis et al (1) argue that there may be negative effects as well. However, in transient cotransfection assays the IE175-binding site in the gD promoter is not required for IE175 activation of gD-CAT activity (P. O'Hare and A. Haig, unpublished data).…”

Section: Discussionmentioning

confidence: 99%

Direct correlation between a negative autoregulatory response element at the cap site of the herpes simplex virus type 1 IE175 (alpha 4) promoter and a specific binding site for the IE175 (ICP4) protein

Roberts

Boundy

O’Hare

et al. 1988

135

copies of a consensus response element commonly referred to as the 29,33,51,63), plus, in some cases, an overlapping consensus octamer element, ATGCTAAT (39; C. apRhys, D. M. Ciufo, E. A. O'Neill, T. J. Kelly, and G. S. Hayward, submitted for publication). In addition, 11 CCCGCCC elements, which appear to be associated with basal expression properties of the IE175-IE68 region, have been shown by DNA footprinting studies to bind to the purified cellular Sp-1 factor (25).During HSV infection, the IE175 gene product is essential for progression of the lytic cycle (8, 50, 60) and for activation of the transcription of the delayed-early (DE) class of viral genes (30,31,46,50). The isolated viral DE and late promoters, whether associated with their own genes (11,55) or as hybrid HSV promoter-driven reporter genes, also respond to virus superinfection in an IE175-dependent fashion, both when in an integrated state in permanent DNAtransfected cell lines (6,9,11,12,35,53) and in transientexpression assays with superinfecting virus (14,40). In DNA cotransfection assays, the isolated IE175 gene encodes a trans-acting factor that stimulates expression of HSV DE promoter targets (13,21,41,52) and inhibits expression from its own promoter in IE175- CAT (9,22,42,43). Although the HSV IE110 gene product was shown to stimulate expression 4307 on July 6, 2020 by guest http://jvi.asm.org/ Downloaded from with polynucleotide kinase and [-y-32P]ATP. The two com-J. VIROL.on July 6, 2020 by guest http://jvi.asm.org/ Downloaded from DNA TARGET SITES FOR IE175 BINDING AND AUTOREGULATION plementary oligonucleotides were annealed by heating to 65°C in 50 mM NaCI-10 mM MgCl2-25 mM Tris hydrochloride (pH 8.0)-100 ,ug of bovine serum albumin per ml-2 mM P-mercaptoethanol followed by slow cooling. For cloning, the double-stranded oligonucleotides were ligated after phosphorylation with polynucleotide kinase to BamHI-plus-BglII-cleaved pGH59 DNA. The resulting plasmids, pGH123a and pGH123b, contained sequences from -8 to

“…It binds to sequences in the promoter regions and transcribed noncoding regions of various HSV-1 genes as part of a protein complex (1,20,21,50,51,65), and recent evidence suggests that it may bind directly to DNA on its own (64). Partly purified preparations of ICP4 have also been found to stimulate transcription of HSV-1 early and late genes in vitro (1,73). However, it is not known whether direct binding of ICP4 is necessary for this transcriptional stimulation to occur.…”

mentioning

confidence: 99%

The herpes simplex virus type 1 alpha protein ICP27 can act as a trans-repressor or a trans-activator in combination with ICP4 and ICP0

Sekulovich

Leary

Sandri-Goldin

1988

182

147

The herpes simplex virus type 1 (HSV-1) a proteins ICP4, ICPO, and ICP27 are transacting proteins which affect HSV-1 gene expression. To investigate potential interactions between these a products and to determine the specificity of action of the a proteins in combination with each other compared with their activities individually, we performed a series of transient-expression assays. In these assays we used plasmids containing the a genes encoding ICP4, ICPO, and ICP27 either singly or in combination as effectors and HSV-1 genes of different kinetic classes and heterologous genes as targets. The HSV-1 targets consisted of promoter-regulatory domains from a (ICPO and ICP27), 0 (thymidine kinase and alkaline exonuclease), P-y (glycoprotein D, glycoprotein B, and VP5), and y (glycoprotein C) genes, each fused to the chloramphenicol acetyltransferase (CAT) gene. The heterologous target genes consisted of the simian virus 40 early promoter with enhancer and the Rous sarcoma virus long terminal repeat promoter and enhancer each fused to the CAT gene. Target