Role for DNA-protein interaction in activation of the herpes simplex virus glycoprotein D gene.
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.
Tedder
D G
DG
Department of Microbiology and Immunology, University of Colorado School of Medicine, Denver 80262.
Pizer
L I
LI
D000818:Animals; D001665:Binding Sites; D004262:DNA Restriction Enzymes; D004279:DNA, Viral / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D017874:Immediate-Early Proteins; D007700:Kinetics; D010957:Plasmids; D011401:Promoter Regions, Genetic; D018139:Simplexvirus / Q000235:genetics*; D014158:Transcription, Genetic; D014709:Vero Cells; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
D004279:DNA, Viral; D017874:Immediate-Early Proteins; D014764:Viral Proteins; C048925:herpes simplex virus, type 1 protein ICP4; D004262:DNA Restriction Enzymes