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Journal of Virology, July 2001, p. 6042-6051, Vol. 75, No. 13
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.13.6042-6051.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Oligomerization Mediated by a Helix-Loop-Helix-Like Domain of Baculovirus IE1 Is Required for Early Promoter Transactivation

Victoria A. Olson, Justin A. Wetter, and Paul D. Friesen*

Institute for Molecular Virology and Department of Biochemistry, Graduate School and College of Agricultural and Life Sciences, University of Wisconsin---Madison, Madison, Wisconsin 53706

Received 17 January 2001/Accepted 9 April 2001

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.

* Corresponding author. Mailing address: Institute for Molecular Virology, Bock Laboratories, University of Wisconsin---Madison, 1525 Linden Dr., Madison, WI 53706-1596. Phone: (608) 262-7774. Fax: (608) 262-7414. E-mail: pfriesen{at}facstaff.wisc.edu.

Journal of Virology, July 2001, p. 6042-6051, Vol. 75, No. 13
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.13.6042-6051.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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Copyright © 2001 by the American Society for Microbiology. All rights reserved.