The early baculovirus he65 promoter: On the mechanism of transcriptional activation by IE1.

We have initiated studies on the mechanism of early transcriptional activation of the early he65 promoter during infection with Autographa californica multicapsid nuclear polyhedrosis virus. This analysis is based on a comparison of the sequences required for he65 promoter activation with those sequences that support specific protein binding. The he65 promoter is located immediately downstream of the homologous region (hr) 4a. The sequences of hr4a are characterized by two imperfect palindromes of 24 bp. The results of transient expression assays indicate promoter activation in the presence of both the proximal palindrome and the known viral trans-regulator IE1. The results of mobility shift assays and DNaseI footprinting analyses reveal differences in specific protein binding at and close to the proximal palindrome depending on whether the nuclear protein extracts are prepared from uninfected or infected cells. The analysis of the protein binding complex at the proximal inverted repeat with extracts from infected cells suggests the involvement of both IE1 and IE0 as oligomers. The minimal protein binding sequences include the left half-site of the 24 bp repeat with 9 additional bp of the flanking sequences. The right half-site of the repeat also directs binding although with lower affinity as confirmed by phenanthroline-copper footprinting assays. Both half-sites of the repeat are thus essential for he65 promoter activation, suggesting that IE1 acts via cooperative binding. We conclude that the proximal inverted repeat is able to interact with both IE1 and IE0 although IE1 is sufficient for activation at least in transient expression assays. Copyright 1998 Academic Press.