Mutational analysis of a baculovirus major late promoter

We have used a linker-scan mutation strategy to analyze Pcap99, the proximal promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) gene encoding the major capsid protein. A series of recombinant viruses expressing the cat reporter gene under the control of selected mutants of this promoter was constructed. Only mutations that altered bases within a region extending from 8 bp upstream to 6 bp downstream from a TAAG sequence had a significant effect on expression from the late gene promoter. A synthetic promoter consisting of only these 18 bp (Pcapmin) was sufficient to direct late expression. Aside from this small region surrounding the TAAG, no evidence for distinct late activating or repressing sequence elements was obtained. Experiments comparing and combining late and very late gene promoter sequences suggest that late expression is intrinsically determined by the presence and immediate context of a TAAG sequence and that very late expression [as previously shown in Ooi et al., J. Mol. Biol. 210 (1989) 721-736] results from additional modulation of TAAG-dependent expression by downstream promoter elements placed in an appropriate context. A compact combination promoter (95 bp), constructed by fusing Pcapmin to a linker-modified very late polyhedrin promoter, directs strong expression at late and very late times post-infection.