THE EARLY BACULOVIRUS HE65 PROMOTER - ON THE MECHANISM OF TRANSCRIPTIONAL ACTIVATION BY IE1

We have initiated studies on the mechanism of early transcriptional ac tivation of the early he65 promoter during infection with Autographa c alifornica multicapsid nuclear polyhedrosis virus. This analysis is ba sed on a comparison of the sequences required for he65 promoter activa tion with those sequences that support specific protein binding The he 65 promoter is located immediately downstream of the homologous region (hd 4a. The sequences of hr4a are characterized by two imperfect pali ndromes of 24 bp. The results of transient expression assays indicate promoter activation in the presence of both the proximal palindrome an d the known viral cans-regulator IE1. The results of mobility shift as says and DNaseI footprinting analyses reveal differences in specific p rotein binding at and close to the proximal palindrome depending on wh ether the nuclear protein extracts are prepared from uninfected or inf ected cells. The analysis of the protein binding complex at the proxim al inverted repeat with extracts from infected cells suggests the invo lvement of both IE1 and IE0 as oligomers. The minimal protein binding sequences include the left half-site of the 24 bp repeat with 9 additi onal bp of the flanking sequences. The right half-site of the repeat a lso directs binding although with lower affinity as confirmed by phena nthroline-copper footprinting assays. Both half-sites of the repeat ar e thus essential for he65 promoter activation, suggesting that IE1 act s via cooperative binding. We conclude that the proximal inverted repe at is able to interact with both IE1 and IE0 although IE1 is sufficien t for activation at least in transient expression assays. (C) 1998 Aca demic Press.