IDENTIFICATION AND MAPPING OF DIMERIZATION AND DNA-BINDING DOMAINS INTHE C-TERMINUS OF THE IE2 REGULATORY PROTEIN OF HUMAN CYTOMEGALOVIRUS

The 80-kDa IE2 nuclear phosphoprotein encoded by the human cytomegalov irus (HCMV) major immediate-early (MIE) gene behaves both as a nonspec ific transactivator of heterologous reporter genes and as a specific r epressor of its own promoter-enhancer region. To begin to examine the biochemical properties of the IE2 protein, we prepared panels of N-ter minal and C-terminal truncation mutants by in vitro translation proced ures. In cross-linking experiments, the C-terminal half of IE2 (which is sufficient for down-regulation) formed dimers but N-terminal segmen ts did not do so. Cotranslated Oct2/IE2 fusion proteins containing the same IE2 C-terminal region from codons 266 to 579 also formed mixed-s ubunit DNA-bound oligomeric complexes in gel mobility shift assays. Fu rthermore, an IE2 domain bounded by codons 388 to 542 proved to immuno precipitate as heterodimers with cotranslated subunits containing know n epitopes for specific antibodies. Deletion up to codon 428 or trunca tion back to codon 504 prevented this interaction. In direct gel shift DNA-binding assays, a bacterial GST/IE2(346-579) fusion protein bound to a 30-mer oligonucleotide probe encompassing the major immediate-ea rly gene negative cis-regulatory target DNA sequence but failed to bin d to a single-base-pair insertion mutant probe (DELTACRS). This specif ic DNA-binding activity was abolished by further deletion up to codon 388 on the N-terminal side or by truncation at codon 542 on the C-term inal side. Therefore, the minimal DNA-binding domain requires addition al amino acid motifs on both sides of the dimerization domain. This se gment of IE2 is functionally important for both transactivation and do wn-regulation and contains several highly conserved amino acid motifs that are shared amongst the equivalent HCMV, simian CMV, mouse CMV, ra t CMV, and human herpesvirus 6 proteins from other betaherpesviruses.