LOCALIZATION OF A 34-AMINO-ACID SEGMENT IMPLICATED IN DIMERIZATION OFTHE HERPES-SIMPLEX VIRUS TYPE-1 ICP4 POLYPEPTIDE BY A DIMERIZATION TRAP

The herpes simplex virus type 1 immediate-early protein ICP4 plays an essential role in the regulation of the expression of all viral genes. It is the major trans activator of early and late genes and also has a negative regulatory effect on immediate-early gene transcription. IC P4 is a sequence-specific DNA-binding protein and has always been puri fied in a dimeric form. The part of the protein that consists of the e ntire highly conserved region 2 and of the distal portion of region 1 retains the ability to specifically associate with DNA and to form hom odimers in solution. In an attempt to map the dimerization domain of I CP4, we used a dimerization trap assay, in which we screened deletion fragments of this 217-amino-acid stretch for sequences that could conf er dimerization properties on a heterologous cellular transcription fa ctor (LFB1), which binds to its cognate DNA sequence only as a dimer. The analysis of these chimeric proteins expressed in vitro ultimately identified a stretch of 34 amino acids (343 to 376) that could still c onfer DNA-binding activity on the LFB1 reporter protein and thus appar ently contained the ICP4 dimerization motif. Consistent with this resu lt, a truncated ICP4 protein containing amino acids 343 to 490, in spi te of the complete loss of DNA-binding activity, appeared to retain th e capacity to form a heterodimer with a longer ICP4 peptide after coex pression in an in vitro translation system.