DNA-DEPENDENT TRANSREGULATION BY IE1 OF AUTOGRAPHA-CALIFORNICA NUCLEAR POLYHEDROSIS-VIRUS - IE1 DOMAINS REQUIRED FOR TRANSACTIVATION AND DNA-BINDING

IE1 is the principal early transregulator of Autographa californica mu lticapsid nuclear polyhedrosis virus (AcMNPV), The 582-residue protein stimulates viral transcription and binds as a dimer to 28-bp palindro mic repeats (28-mers) comprising the AcMNPV homologous region (hr) tra nscription enhancers. To define IE1 domains responsible for hr-depende nt transactivation, we first constructed a series of IE1 fusions to th e DNA binding domain of the yeast GAL4 transactivator. In transfection assays, GAL4-IE1 fusions stimulated transcription from a TATA-contain ing AcMNPV promoter only upon cis linkage to GAL4 DNA binding sites. I E1 N-terminal residues 8 to 118 were sufficient for GAL4-binding-site- dependent transactivation. To identify IE1 residues required for hr in teraction, we tested a series of IE1 mutations for 28-mer binding by u sing electrophoretic mobility shift assays. Deletion of IE1 residues o ther than the N-terminal transactivation domain eliminated 28-mer bind ing. Of 14 insertion mutations, only IE1(I425) and IE1(I553) failed to bind the 28-mer either as homodimers or as heterodimers with function al IE1. In contrast to insertion IE1(I425), IE1(I553) also failed to c ompete with wild-type IE1 for DNA binding and suggested a defect in ol igomerization, Consistent with loss of oligomerization, substitutions within a hydrophobic repeat (residues 543 to 568) at the IE1 C terminu s 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-m ediated transactivation by IE1 involves sequence-specific DNA binding that contributes to transcriptional stimulation by interaction with co mponents of the basal transcription complex.