DNA-dependent transregulation by IE1 of Autographa californica nuclear polyhedrosis virus: IE1 domains required for transactivation and DNA binding

Abstract

IE1 is the principal early transregulator of Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV). The 582-residue protein stimulates viral transcription and binds as a dimer to 28-bp palindromic repeats (28-mers) comprising the AcMNPV homologous region (hr) transcription enhancers. To define IE1 domains responsible for hr-dependent transactivation, we first constructed a series of IE1 fusions to the DNA binding domain of the yeast GAL4 transactivator. In transfection assays, GAL4-IE1 fusions stimulated transcription from a TATA-containing AcMNPV promoter only upon cis linkage to GAL4 DNA binding sites. IE1 N-terminal residues 8 to 118 were sufficient for GAL4-binding-site-dependent transactivation. To identify IE1 residues required for hr interaction, we tested a series of IE1 mutations for 28-mer binding by using electrophoretic mobility shift assays. Deletion of IE1 residues other than the N-terminal transactivation domain eliminated 28-mer binding. Of 14 insertion mutations, only IE1 I425 and IE1 I553 failed to bind the 28-mer either as homodimers or as heterodimers with functional IE1. In contrast to insertion IE1 I425 , IE1 I553 also failed to compete with wild-type IE1 for DNA binding and suggested a defect in oligomerization. Consistent with loss of oligomerization, substitutions within a hydrophobic repeat (residues 543 to 568) at the IE1 C terminus abolished 28-mer binding and demonstrated that this helix-loop-helix-like domain is required for DNA interaction. These data confirm that IE1 contains separable domains for transactivation and oligomerization-dependent DNA binding. Furthermore, they support a model wherein hr-mediated transactivation by IE1 involves sequence-specific DNA binding that contributes to transcriptional stimulation by interaction with components of the basal transcription complex.

The Baculovirus Transcriptional Transactivator ie0 Produces Multiple Products by Internal Initiation of Translation

“…The N-terminal 135 amino acids (aa) have been shown to be an acidic activation domain that can be functionally replaced by the archetype acidic activation domain from the herpes simplex virus protein VP16 (Forsythe et al, 1998). The AcMNPV IE1 N-terminal domain has also been shown to be an acidic activation domain when placed in heterologous expression systems (Rodems et al, 1997;Slack and Blissard, 1997). In addition, the AcMNPV IE1 Cterminal domains have been shown to be involved in homodimer formation and DNA binding to enhancer regions (Olson et al, 2001).…”

Nineteen Baculovirus Open Reading Frames, Including LEF-12, Support Late Gene Expression

“…IE-1 transactivates early gene expression and may be involved in transactivating lef gene expression (15,16,34). However, it also recognizes and binds imperfect palindromes within hr regions and may therefore be directly involved in DNA replication (8,14,47). IE-2 has two known functions: transactivating early gene expression (4,5) and blocking cell cycle progression (44).…”

Identification and Characterization of a Putative Baculoviral Transcriptional Factor IE-1 from Choristoneura fumiferana Granulovirus

“…This finding confirms the data from nucleopolyhedral IE-1 (Olson et al, 2001;Wang et al, 2001). Available data on IE-1 proteins from NPVs propose that the C-terminal regions of these proteins play a critical role in DNA binding; the N-terminal regions of IE1, which are rich in acidic residues, have been hypothesized to be an acidic activation domain (Theilmann and Stewart, 1991;Rodems et al, 1997;Forsythe et al, 1998;Leisy and Rohrmann, 2000;Olson et al, 2001). Two stretches of amino acid sequences are well-conserved in the C-terminal regions of all granuloviral IE-1 proteins.…”

“…These mutants lose their oligomerization activity and hence suffer the loss of IE-1 hrdependant transactivation (Olson et al, 2001). The 28-mer palindrome is the minimal requirement for the enhancer activity of cis linked viral promoters (Guarino and Dong, 1994;Rodems et al, 1997;Kremer and Knebel-Morsdorf, 1998;Leisy and Rohrmann, 2000;Massari and Murre, 2000;Olson et al, 2001). Evidence demonstrates that it is essential for IE-1 dimers to interact through cooperative binding with both palindromic half sites of the 28-mer palindrome in order to stimulate the hr enhancer activity (Rodems et al, 1997;Kremer and Knebel-Morsdorf, 1998;Leisy and Rohrmann, 2000).…”

“…The 28-mer palindrome is the minimal requirement for the enhancer activity of cis linked viral promoters (Guarino and Dong, 1994;Rodems et al, 1997;Kremer and Knebel-Morsdorf, 1998;Leisy and Rohrmann, 2000;Massari and Murre, 2000;Olson et al, 2001). Evidence demonstrates that it is essential for IE-1 dimers to interact through cooperative binding with both palindromic half sites of the 28-mer palindrome in order to stimulate the hr enhancer activity (Rodems et al, 1997;Kremer and Knebel-Morsdorf, 1998;Leisy and Rohrmann, 2000). The model that is presented for the DNA binding by IE-1 proposes that the interaction of the IE-1 dimer across the 28-mer axis of symmetry makes simultaneous contact with both half sites (Olson et al, 2001).…”