Physical interactions between the herpes simplex virus type 1 immediate-early regulatory proteins ICP0

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 [35S]Met-labeled ICPO and ICP4 as probes. The ICP4 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 ICP4 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 ICP4 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 24