Oligomerization mediated by a helix-loop-helix-like domain of baculovirus IE1 is required for early promoter transactivation

IE1 is a principal transcriptional regulator of Autographa californica mult icapsid nucleopolyhedrovirus (AcMNPV). Transactivation by IE1 is stimulated when early viral promoters are cis linked to homologous-region (hr) enhanc er sequences of AcMNPV. This transcriptional enhancement is correlated with the binding of IE1 as a dimer to the 28-bp palindromic repeats comprising the hr enhancer. To define the role of hemophilic interactions in IE1 trans activation, we have mapped the IE1 domains required for oligomerization. We report here that IE1 oligomerizes by a mechanism independent of enhancer b inding, as demonstrated by in vitro pull-down assays using fusions of IE1 ( 582 residues) to the C terminus of glutathione S-transferase. In vivo oligo merization of IE1 was verified by immunoprecipitation of IE1 complexes from extracts of plasmid-transfected SF21 cells. Analyses of a series of site-d irected IE1 insertion mutations indicated that a helix-loop-helix (HLH)-lik e domain extending from residue 543 to residue 568 is the primary determina nt of oligomerization. Replacement of residues within the hydrophobic face of the putative dimerization domain disrupted IE1 hemophilic interactions a nd caused loss of IE1 transactivation of hr-dependent promoters in plasmid transfection assays. Thus, oligomerization is required for IE1 transcriptio nal stimulation. HLH mutations also reduced IE1 stability and abrogated tra nsactivation of non-ltr-dependent promoters. These data support a model whe rein IE1 oligomerizes prior to DNA binding to facilitate proper interaction with the symmetrical recognition sites within the hl enhancer and thereby promote the transcription of early viral genes.