Molecular analysis of a baculovirus regulatory gene

Abstract

To better understand the structure and function of a baculovirus regulatory gene, the nucleotide sequence of IE-N expressed by Autographa californica nuclear polyhedrosis virus was determined. The 2.0-kb PstI-EcoRV restriction fragment (97.5 to 98.9 mu) encodes the upstream regulatory sequences, open reading frame, and downstream sequences of the immediate early IE-N gene. Using a convenient restriction site, the 285-bp promoter of IE-N was divided into two functional regions as defined by transient expression assays of mutant sequences. The sequences of IE-N from -1 to -45 nt encoded a minimal promoter capable of directing low levels of transcription. The minimal promoter was fully responsive to positive regulation by IE-N. The upstream region from -46 to -285 nt contains two direct repeats which increased levels of IE-N gene expression. Computer-assisted translation of the IE-N sequence indicates that this fragment of DNA encodes a single long open reading frame with a predicted molecular weight of 47,000. The amino acid sequence of the predicted protein exhibits three motifs common to transcriptional regulators: a serine-threonine rich region, a proline-rich region, and a polyglutamine tract. IE-N autoregulates its own expression and stimulates both IE-1 and IE-0 in transient assays. The stimulation of IE-1 may account for the augmenting activity of IE-N in the IE-1-mediated trans-activation of the 39K promoter.