Va. Olson et al., Oligomerization mediated by a helix-loop-helix-like domain of baculovirus IE1 is required for early promoter transactivation, J VIROLOGY, 75(13), 2001, pp. 6042-6051
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.