Characterization of the DNA-binding properties of herpes simplex virus regulatory protein ICP4

ICP4 is a herpesvirus-encoded protein that is expressed during the immediate-early phase of productive infection and is required for efficient transcription of viral genes during the early and late phases of infection. Previous studies have shown that ICP4 is a component of specific protein-DNA complexes but have not revealed whether native ICP4 directly recognizes specific nucleotide sequences. Using DNA affinity chromatography, we have purified ICP4 to near homogeneity. The purified preparation consists primarily of dimeric and tetrameric forms of ICP4. As shown by DNase protection experiments, purified ICP4 binds directly to specific sequences in viral DNA. The stability of ICP4-DNA complexes is increased over 100-fold by shifting the temperature from 23 to 4 degrees C. The equilibrium dissociation constant of ICP4-DNA complexes formed at 4 degrees C in the presence of 100 mM NaCl was determined to be approximately 1.1 nM.