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Journal of Virology, May 2003, p. 5668-5677, Vol. 77, No. 10
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.10.5668-5677.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

The Highly Conserved Basic Domain I of Baculovirus IE1 Is Required for hr Enhancer DNA Binding and hr-Dependent 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 6 January 2003/ Accepted 24 February 2003

The immediate-early protein IE1 is the principal transcriptional regulator of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV). Transactivation by IE1 is dramatically stimulated by cis linkage of the affected promoter to AcMNPV homologous region (hr) elements that contain palindromic 28-bp repeats (28-mers) with enhancer activity. This hr-dependent transcriptional enhancement requires binding of the 28-mer by dimeric IE1. Here, we have defined IE1 domains required for this DNA binding in order to investigate the mechanism of IE1 function. Analysis of a panel of IE1 insertion mutations indicated that disruption of a highly conserved domain (residues 152 to 161) consisting of mostly positive-charged residues (basic domain I) abolished hr-dependent transactivation. Targeted mutagenesis of basic residues within basic domain I caused loss of hr-dependent transactivation but had no effect on IE1 oligomerization, nuclear localization, or hr-independent transactivation of viral promoters. Alanine substitutions of K152 and K154 or K160 and K161 impaired IE1 binding to 28-mer DNA as a homodimer, indicating that these basic residues are required for enhancer binding. Consistent with a DNA-binding defect, 28-mer interaction was improved by heterodimerization with wild-type IE1 or by increasing mutated IE1 concentrations. DNA binding mediated by basic domain I was also required for IE1 transactivation that occurred through physically separated, unlinked hr elements. We concluded that basic domain I is the enhancer-binding domain for IE1. Our data also suggest that DNA binding activates IE1 for transcriptional enhancement, possibly through a conformational change involving basic domain I.

* 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}wisc.edu.

Journal of Virology, May 2003, p. 5668-5677, Vol. 77, No. 10
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.10.5668-5677.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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