The host factor polyhedrin promoter binding protein (PPBP) is involved in transcription from the baculovirus polyhedrin gene promoter.
Hypertranscription and temporal expression from the Autographa californica nuclear polyhedrosis (AcNPV) baculovirus polyhedrin promoter involves an alpha-amanitin-resistant RNA polymerase and requires a trans-acting viral factor(s). We previously reported that a 30-kDa host factor, polyhedrin promoter binding protein (PPBP), binds with unusual affinity, specificity, and stability to the transcriptionally important motif AATAAATAAGTATT within the polyhedrin (polh) initiator promoter and also displays coding strand-specific single-stranded DNA (ssDNA)-binding activity (S. Burma, B. Mukherjee, A. Jain, S. Habib, and S. E. Hasnain, J. Biol. Chem. 269:2750-2757, 1994; B. Mukherjee, S. Burma, and S. E. Hasnain, J. Biol. Chem. 270:4405-4411, 1995). We now present evidence which indicates that an additional factor(s) is involved in stabilizing PPBP-duplex promoter and PPBP-ssDNA interactions. TBP (TATA box binding protein) present in Spodoptera frugiperda (Sf9) cells is characteristically distinct from PPBP and does not interact directly with the polh promoter. Replacement of PPBP cognate sequences within the polh promoter with random nucleotides abolished PPBP binding in vitro and also failed to express the luciferase reporter gene in vivo. Phosphocellulose fractions of total nuclear extract from virus-infected cells which support in vitro transcription from the polh promoter contain PPBP activity. When PPBP was sequestered by the presence of oligonucleotides containing PPBP cognate sequence motifs, in vitro transcription of a C-free reporter cassette was affected but was restored by the exogenous addition of nuclear extract containing PPBP. When PPBP was mopped out in vivo by a plasmid carrying PPBP cognate sequence present in trans, polh promoter-driven expression of the luciferase reporter was abolished, demonstrating that binding of PPBP to the polh promoter is essential for transcription.
Eukaryotic Gene Expression Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.
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Ghosh
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Jain
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Mukherjee
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Habib
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Hasnain
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002458:Cell Fractionation; D002460:Cell Line; D002482:Cellulose / Q000031:analogs & derivatives; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D017930:Genes, Reporter; D019476:Insect Proteins; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009687:Nuclear Proteins / Q000378:metabolism*; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D000077183:Occlusion Body Matrix Proteins; D011401:Promoter Regions, Genetic; D018411:Spodoptera; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics*; D015678:Viral Structural Proteins
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D019476:Insect Proteins; D009687:Nuclear Proteins; D000077183:Occlusion Body Matrix Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; C085605:polyhedrin promoter-binding protein, insect; C009829:polyhedrin protein, Nucleopolyhedrovirus; D002482:Cellulose; C008881:phosphocellulose; D008156:Luciferases