Differential gene expression mediated by late, very late and hybrid baculovirus promoters

A series of recombinant viruses was constructed to study the regulation of expression from the promoter (Pcap) for the major capsid-protein-encoding gene (vp39) of the baculovirus, Autographa californica nuclear polyhedrosis virus. Each virus of this series contains the cat reporter gene under the control of Pcap or portions thereof. Pcap regulation of cat was determined at the RNA and enzyme activity levels, and was compared to cat gene expression driven by Ppolh, the promoter of the polyhedrin gene (polh). Although optimum expression was achieved with a 464-bp region, which included all three Pcap transcription start points (tsp), a 99-bp segment of Pcap containing a single tsp was sufficient to direct late cat expression. Ppolh and Pcap-mediated regulation differed temporally; Pcap-cat constructs were expressed at 12 h post-infection (p.i.) and continued through 48 h p.i., whereas Ppolh-cat were not expressed at 12 h, but were initiated around 18 h p.i. and underwent a burst of expression between 24 and 48 h p.i. A recombinant virus carrying a hybrid and polh promoter (Pcappolh) was also constructed and studied; Pcappolh contained the two distal tsp of Pcap and a proximal tsp of Ppolh. Pcappolh exhibited both 'late' and 'very late' regulation; the distal tsp were regulated as late sites and the proximal tsp was regulated primarily as a very late site although it also showed a weak late response. Hybrid late/very late promoters should prove useful in optimizing foreign gene expression using baculovirus vectors.