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

AUTOR(ES)

Rodems, S M

RESUMO

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.

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