TRANSCRIPTIONAL ENHANCER ACTIVITY OF HR5 REQUIRES DUAL-PALINDROME HALF SITES THAT MEDIATE BINDING OF A DIMERIC FORM OF THE BACULOVIRUS TRANSREGULATOR IE1

The hr5 enhancer element stimulates early viral transcription and may function as an origin of DNA replication for Autographa californica nu clear polyhedrosis virus (AcMNPV). The smallest functional unit of hr5 is a 28-bp repeat consisting of an imperfect palindrome (28-mer). To identify essential sequences and examine the molecular basis of hr5 ac tivity, the effects of site-directed mutations on transcriptional enha ncement by the 28-mer and binding of the AcMNPV transregulator IE1 wer e investigated. In transfection assays and infections with AcMNPV reco mbinants, activation of a basal viral promoter required sequences,with in both halves of the 28-mer. Basal promoter activation also required a critical spacing between these half sites. Mobility shift assays ind icated that hr5 probes containing a single 28-mer were bound by in vit ro-synthesized IE1. Competition assays using DNA fragments that contai ned mutated 28-mers demonstrated that both half sites were required fo r optimal binding of IE1. Similar assays using mutated 28-mer DNAs and nuclear extracts indicated that the relative affinity with which AcMN PV infection-specific proteins bound to the 28-mer was similar to that of in vitro-synthesized IE1. By using a combination of DNA binding an d antibody supershift assays, it was demonstrated that IE1 binds to th e 28-mer as a dimer. Collectively, these findings support a model in w hich symmetrical IE1 binding and simultaneous interaction with each ha lf site are required for IE1-mediated transcriptional enhancement by h r5. Thus, sequence-specific binding may be one of the mechanisms by wh ich IE1 directly or indirectly transregulates baculovirus gene express ion.