The Highly Conserved Basic Domain I of Baculovirus IE1 Is Required for hr Enhancer DNA Binding and hr -Dependent Transactivation

Abstract

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 K 152 and K 154 or K 160 and K 161 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.The major transcriptional regulator of Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) is the immediate-early protein IE1. Highly conserved among members of Baculoviridae, this 67-kDa DNA-binding protein is postulated to participate in critical processes during baculovirus infection, including the initiation of DNA replication and transregulation of transcription from early and late viral promoters (1, 13, 15, 17-20, 22, 23, 26, 28, 30, 34, 35, 39-41). Current evidence suggests that IE1's regulatory functions are mediated through its interaction with homologous region (hr) sequences that are repeated throughout the circular DNA genome of AcMNPV (reviewed in references 9 and 27). The baculovirus hrs are transcription enhancers and possible origins of DNA replication (11,12,14,20,22,30,37,41,42). In transfection assays, cis linkage of the hr element to baculovirus promoters can boost IE1-mediated transactivation as much as 200-fold (30, 39, 41). During infection, IE1 also colocalizes with viral DNA replication factories in the nucleus, where it may function as a DNA origin (hr)-binding protein (31,35,40). Despite these roles in transcription and DNA replication, the molecular mechanisms by which IE1 functions through DNA binding are unknown.AcMNPV IE1 is a 582-residue nuclear phosphoprotein wit...

Transactivation, Dimerization, and DNA-Binding Activity of White Spot Syndrome Virus Immediate-Early Protein IE1

“…Like many other virus immediate-early proteins that bind DNA in a dimeric form (8,16,40,57), our evidence suggests that WSSV IE1 also has this FIG. 5.…”

“…IE1 has a strong affinity for self-interaction. Many virus immediate-early proteins are in dimeric form when they bind to DNA (8,16,40,57). We therefore performed an in vitro biochemical binding assay to determine whether IE1 can also self-interact directly.…”

Focal Distribution of Baculovirus IE1 Triggered by Its Binding to the hr DNA Elements

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An inducible system for highly efficient production of recombinant adeno-associated virus (rAAV) vectors in insect Sf9 cells

“…The mechanism by which DNA binding promotes IE-1 transactivation is unknown. Olson et al (13) hypothesized that DNA binding is required for conformational changes in IE-1, a prerequisite to subsequent interaction with other transcription factors and trans stimulation. However, binding to hr alone is insufficient for IE-1-mediated enhancer activity (20)(21)(22).…”