Identification of an enhancer-like element in the polyhedrin gene upstream region of Bombyx mori nucleopolyhedrovirus

Abstract

A series of deletions in the upstream region of the gene encoding polyhedrin (polh) of Bombyx mori nucleopolyhedrovirus (BmNPV) were generated in plasmid constructs and tested for transcription. In transient transfection assays in Bombyx mori-derived BmN cells with firefly luciferase as the reporter gene, a 293 bp fragment located 1·0 kb upstream with respect to the +1 ATG of polh showed 10-fold enhancement in expression from the minimal promoter. This increase in reporter activity was observed only when the fragment was positioned in cis with respect to the promoter and not in trans. The stimulation of reporter gene expression was independent of the orientation of the fragment and was due to increased transcription from the promoter. When placed upstream of another promoter, the viral very late gene p10 promoter, the enhancer brought about a 2-fold increase in expression. The region encompassing the enhancer was itself transcriptionally active, and transcripts corresponding to both of the encoded ORFs (N-terminal regions of ORF453 and ORF327, located in opposite orientations) were detected. Two AP1 sites (TGACTCG) in the 293 bp fragment did not appear to contribute to the enhancer function. Since repeat motifs, the hallmark of conventional enhancer sequences, were absent from this fragment, it is designated as an enhancer-like element. The influence of this region of the polh upstream sequence on expression from strong, very late viral promoters has not been reported previously.