Early transcription of the ie-1 transregulator gene of Autographa californica nuclear polyhedrosis virus is regulated by DNA sequences within its 5' noncoding leader region

Abstract

The ie-1 gene of Autographa californica nuclear polyhedrosis virus (AcMNPV) encodes a transregulatory protein (IE1) which accelerates the expression of early and late virus genes. Transcription of ie-1 occurs immediately upon infection from a conserved CAGT motif and continues into the late phases. To examine the mechanisms by which ie-1 expression is regulated, cis-acting control elements within the ie-1 promoter were identified by constructing hybrid early promoters and by using site-directed mutagenesis. The ie-1 upstream activating region, extending from nucleotide -546 to the TATA element at -34, stimulated ie-1 basal promoter activity more than 1,000-fold when transfected into uninfected Spodoptera frugiperda SF21 cells. However, when introduced into the genome of AcMNPV recombinants, the ie-1 upstream activating region had only a minimal twofold effect early in infection. Instead, maximum steady-state levels of early ie-1 RNAs required sequences within the 5' noncoding leader region extending from +11 to +24 relative to the RNA start site (+1). The +11 to +24 noncoding region did not influence the stability of ie-1 transcripts. When assayed by in vitro transcription, deletion of the +11 to +24 region reduced the levels of ie-1 runoff RNAs. Thus, this downstream activating sequence controlled the rate of early ie-1 transcription. A larger overlapping region from +11 to +36 affected steady-state levels of ie-1 RNAs late (24 h) in infection. Deletion of sequences that included the conserved CAGT start site abolished early ie-1 transcription. Thus, ie-1 is the first example of an early baculovirus gene in which essential cis-acting regulatory elements reside within the 5' noncoding region and include sequences comprising the RNA start site.