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 multicapsid nucleopolyhedrovirus (AcMNPV). Transactivation by IE1 is stimulated when early viral promoters are cis linked to homologous-region (hr) enhancer 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 homophilic interactions in IE1 transactivation, we have mapped the IE1 domains required for oligomerization. We report here that IE1 oligomerizes by a mechanism independent of enhancer binding, as demonstrated by in vitro pull-down assays using fusions of IE1 (582 residues) to the C terminus of glutathione S-transferase. In vivo oligomerization of IE1 was verified by immunoprecipitation of IE1 complexes from extracts of plasmid-transfected SF21 cells. Analyses of a series of site-directed IE1 insertion mutations indicated that a helix-loop-helix (HLH)-like domain extending from residue 543 to residue 568 is the primary determinant of oligomerization. Replacement of residues within the hydrophobic face of the putative dimerization domain disrupted IE1 homophilic interactions and caused loss of IE1 transactivation of hr-dependent promoters in plasmid transfection assays. Thus, oligomerization is required for IE1 transcriptional stimulation. HLH mutations also reduced IE1 stability and abrogated transactivation of non-hr-dependent promoters. These data support a model wherein IE1 oligomerizes prior to DNA binding to facilitate proper interaction with the symmetrical recognition sites within the hr enhancer and thereby promote the transcription of early viral genes.
Olson
V A
VA
Institute for Molecular Virology, Graduate School and College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, WI 53706, USA.
Wetter
J A
JA
Friesen
P D
PD
D001665:Binding Sites; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins; D018257:Helix-Loop-Helix Motifs; D017874:Immediate-Early Proteins / Q000737:chemistry / Q000502:physiology*; D011401:Promoter Regions, Genetic; D015534:Trans-Activators / Q000737:chemistry / Q000502:physiology*; D015533:Transcriptional Activation
C069183:Ac-IE-1 protein, Autographa californica nucleopolyhedrovirus; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D017874:Immediate-Early Proteins; D015534:Trans-Activators