LATE PROMOTER SELECTION IN THE BACULOVIRUS GP64 ENVELOPE FUSION PROTEIN GENE

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.