Physical interaction between the herpes simplex virus type 1 immediate-early regulatory proteins ICP0 and ICP4.
The herpes simplex virus type 1 immediate-early protein ICP0 enhances expression of a spectrum of viral genes alone and synergistically with ICP4. To test whether ICP0 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 ICP0 and ICP4 as probes. The ICP4 and ICP0 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 ICP0 interacts directly and specifically with ICP4 and with itself. To further define the interaction between ICP0 and ICP4, we generated a set of glutathione S-transferase (GST)-ICP0 fusion proteins that contain GST and either ICP0 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-ICP0 fusion protein affinity chromatography and in vitro-synthesized [35S]Met-labeled ICP0 and ICP4, ICP4 was shown to interact preferentially with the fusion protein containing ICP0 C-terminal amino acids 395 to 775, whereas ICP0 interacted efficiently with both the N-terminal GST-ICP0 fusion proteins and the C-terminal GST-ICP0 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 ICP0. Collectively, these results reveal a direct and specific physical interaction between ICP0 and ICP4.
Division of Molecular Genetics, Dana-Farber Cancer Institute, Boston, Massachusetts.
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002522:Chlorocebus aethiops; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D017781:Genes, Immediate-Early; D016679:Genome, Viral; D018259:Herpesvirus 1, Human / Q000235:genetics / Q000378:metabolism*; D017874:Immediate-Early Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D014176:Protein Biosynthesis; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012156:Reticulocytes / Q000378:metabolism; D017384:Sequence Deletion; D014158:Transcription, Genetic; D044767:Ubiquitin-Protein Ligases; D014709:Vero Cells
D004268:DNA-Binding Proteins; D017874:Immediate-Early Proteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; C048925:herpes simplex virus, type 1 protein ICP4; D008715:Methionine; D044767:Ubiquitin-Protein Ligases; C051804:Vmw110 protein, Human herpesvirus 1