Eight base pairs encompassing the transcriptional start point are the major determinant for baculovirus polyhedrin...

The effects of mutations within the 92-bp region immediately upstream from the translational initiation ATG of the polyhedrin gene of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) were determined by plasmid transient expression assays in the presence of wild-type (wt) AcMNPV DNA. Clustered point mutations were generated by substitution of 10-bp stretches of the polyhedrin promoter/leader region with a 10-bp HindIII linker. Three of these linker scan (LS) mutations in the region from nucleotides (nt) -62 to -84 (relative to the original polyhedrin ATG at +1, +2, +3) had no effect or a mild stimulatory effect on reporter gene expression. One mutation immediately upstream (nt -52 to -60) from the transcription start point (at nt -50) reduced expression four-fold. Three overlapping mutations affecting 8 bp from nt -44 to -51 (encompassing the transcriptional start point) reduced expression by 1000-fold. A 1000-fold reduction was also observed for a total deletion of nt -1 and -92. Five LS mutations between nt -1 and -43 each reduced expression two- to five-fold, whereas combining an LS mutation and a 9-bp deletion mutation in the leader reduced expression approx. nine-fold. Reversing the orientation of the reporter gene along with the wt 92-bp upstream polyhedrin promoter/leader sequences resulted in slightly higher expression levels than those observed for the normal orientation indicating that all the essential cis-acting promoter elements, with the possible exception of long-range enhancer sequences, are located downstream from nt -92. Sequences of the AcMNPVhr5 enhancer (homologous region No. 5 of AcMNPV) had only a minor effect on expression from the polyhedrin promoter in transient assays. The results show that 8 bp encompassing the transcriptional start point, a sequence which is conserved in all late AcMNPV promoters, is essential for polyhedrin gene expression. Additional nucleotides within the leader region are necessary for optimal expression.