PHYSICAL INTERACTION BETWEEN THE HERPES-SIMPLEX VIRUS TYPE-1 IMMEDIATE-EARLY REGULATORY PROTEINS ICPO AND ICP4

The herpes simplex virus type 1 immediate-early protein ICPO enhances expression of a spectrum of viral genes alone and synergistically with ICP4. To test whether ICPO and ICP4 interact physically, we performed far-Western blotting analysis of proteins from mock-, wild-type, and ICP4 mutant virus infected cells with in vitro-synthesized [S-35]Met-labeled ICPO and ICP4 as probes. The ICP I and ICPO polypeptides synthesized in vitro exhibited molecular weights similar to those of their counterparts in herpes simplex virus type 1-infected cells, and the in vitro-synthesized ICP 1 was able to bind to a probe containing the ICP4 consensus binding site. Far-Western blotting experiments demonstrated that ICPO interacts directly and specifically with ICP I and with itself. To further define the interaction between ICPO and ICP4 we generated a set of glutathione S transferase (GST)-ICPO fusion proteins that contain GST and either ICPO N-terminal amino acids 1 to 244 or 1 to 394 or C-terminal amino acids 395 to 616 or 395 to 775. Using GST-ICPO fusion protein affinity chromatography and in vitro-synthesized [S-35]Met-labeled ICPO and ICP4, ICP4 was shown to interact preferentially with the fusion protein containing ICPO C-terminal amino acids 395 to 775, whereas ICPO interacted efficiently with both the N-terminal GST-ICPO fusion proteins and the C-terminal GST-ICPO fusion proteins containing amino acids 395 to 775. Fusion protein affinity chromatography also demonstrated that the C-terminal 235 amino acid residues of ICP4 are important for efficient interaction with ICPO. Collectively, these results reveal a direct and specific physical interaction between ICPO and ICP4.