The upstream promoter region of the Autographa californica multicapsid
nuclear polyhedrosis virus (AcMNPV) gp64 gene contains five copies of
TAAG, the conserved sequence found at the transcriptional initiation
sites of almost all baculovirus late genes. In AcMNPV-infected Sf9 cel
ls, late transcription initiation is detected from only two upstream T
AAG sites and not from three downstream TAAG sites. To examine several
models for preferential TAAG site utilization, we constructed a serie
s of recombinant AcMNPV baculoviruses that contain promoter region seq
uences from the gp64 gene fused to a chloramphenicol acetyl transferas
e reporter gene. Promoter-reporter constructs were inserted in the pol
yhedrin locus. To test a scanning model in which TAAG sites are sequen
tially selected according to their location in the region, we generate
d recombinant viruses in which the highly transcribed sites were inact
ivated by point mutations. Transcription from the mutant promoter cons
tructs was compared qualitatively and quantitatively to transcription
from the wild-type gp64 promoter. Inactivation of the upstream TAAG si
tes did not result in increased transcription from the downstream TAAG
sites, suggesting that immediate context, rather than position, deter
mines promoter utilization. To lest this hypothesis, we made a series
of minimal promoter constructs containing decreasing quantities of the
sequences immediately flanking one of the active gp64 TAAG sites. Rep
orter constructs containing a gp64 TAAG site and greater than or equal
to 12 bp of flanking sequence on both sides were transcribed at near
wild-type levels. Constructs with less flanking sequence (9 or 6 bp of
flanking sequence) were accurately transcribed, but at substantially
lower levels, and transcription was not detected from constructs conta
ining only 3 bp of flanking sequence. These results suggest that nucle
otides immediately flanking the TAAG site (4-6 bp) are necessary for b
asal promoter activity while additional flanking sequences (greater th
an or equal to 12 bp) are required for late promoter activation and re
gulation. To further examine late promoter selection, we constructed r
ecombinant AcMNPV baculoviruses that contain heterologous late promote
rs from the gp64 gene of the related virus Orgyia pseudotsugata MNPV (
OpMNPV). TAAG sites that serve as functional late promoters in OpMNPV
were found to mediate transcription initiation at only basal levels in
the context of the AcMNPV genome, suggesting that late promoter activ
ation may be virus specific within the family Baculoviridae. (C) 1997
Academic Press.