Interaction of cell and virus proteins with DNA sequences encompassing the promoter/regulatory and leader regions of the herpes simplex virus thymidine kinase gene.
During the course of a productive infection with herpes simplex virus (HSV), gene expression is coordinately regulated in a cascade fashion. Three major kinetic classes of genes, termed alpha, beta, and gamma, are sequentially activated. The mechanism responsible for repression and subsequent activation of beta and gamma genes is not known. A mobility-shift electrophoresis assay was used to examine DNA fragments containing the promoter/regulatory and the mRNA leader regions of the thymidine kinase gene (TK, a model beta gene) for their ability to bind proteins present in nuclear extracts prepared from uninfected and infected cells. Specific complexes unique to each extract were formed. Using a monoclonal antibody specific for ICP4 (the major regulatory protein of HSV) we demonstrated that this protein is present in the complexes formed between probes encompassing either the promoter/regulatory or leader sequence DNAs and proteins in infected-cell extracts. These complexes formed despite the lack of a high affinity binding site for ICP4 in either of these regions. The stability of complexes formed in infected-cell extracts with DNA probes containing the promoter/regulatory, leader region, and a high affinity ICP4-binding site were compared by dissociation analysis. The relative kd(obs) for these DNA-protein complexes was in the order: TK-leader region much greater than TK-promoter/regulatory region greater than or equal to high affinity ICP4-binding site. Cu+/1,10-phenanthroline footprinting revealed that infected-cell complexes which form on a probe containing a high affinity ICP4-binding site generate a protection pattern, whereas those formed on a probe containing the TK-leader sequence do not. In contrast, complexes formed with the latter probe in extracts from uninfected cells are kinetically stable and refractile to cleavage. A model for activation of the TK gene which incorporates these results is presented.
Papavassiliou
A G
AG
Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
Silverstein
S J
SJ
D001665:Binding Sites; D001667:Binding, Competitive; D003001:Cloning, Molecular; D015342:DNA Probes; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D017874:Immediate-Early Proteins; D010618:Phenanthrolines / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D021382:Protein Sorting Signals / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D018139:Simplexvirus / Q000201:enzymology* / Q000235:genetics; D013937:Thymidine Kinase / Q000235:genetics*; D054334:Viral Regulatory and Accessory Proteins / Q000378:metabolism
D015342:DNA Probes; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D017874:Immediate-Early Proteins; D010618:Phenanthrolines; D021382:Protein Sorting Signals; D054334:Viral Regulatory and Accessory Proteins; C048925:herpes simplex virus, type 1 protein ICP4; D013937:Thymidine Kinase; C025205:1,10-phenanthroline