Functional dissection of the Autographa california nuclear polyhedrosis virus enhancer element hr5

The hr5 enhancer element of Autographa californica nuclear polyhedrosis virus stimulates transcription from baculovirus-delayed early promoters in the presence of the viral transactivator, IE1. Gel retardation analyses of interactions between a fragment of hr5 and extracts prepared from cells transfected with an IE1-encoding plasmid revealed the presence of three DNA-protein complexes. In order to better define the functional domains of the hr5 enhancer, we constructed a set of plasmids containing partial deletions in the enhancer element. These constructs were tested for both in vitro DNA binding activity and enhancer function in transient assays. The results indicated that the minimum sequence required for DNA-protein interactions was half of the conserved 24-bp palindrome that is contained within a 60-bp direct repeat (DR60). However, the minimum sequence required for enhancer function was a complete copy of DR60. Template challenge experiments indicated that IE1 bound with equal affinity with a complete or a half copy of DR60. The deletion analyses were confirmed by in vitro binding and transient expression assays with synthetic oligonucleotides.