An unusual 30-kDa protein binding to the polyhedrin gene promoter of Autographa californica nuclear polyhedrosis virus

Abstract

Gel retardation assays using nuclear extracts from the Autographa californica nuclear polyhedrosis virus-infected Sf21 (Spodoptera frugiperda) insect cell line revealed a host factor (polyhedrin promoter-binding protein or PPBP) that binds to the polyhedrin gene promoter. A hexanucleotide sequence (AATAAA) within the promoter is important for binding in association with neighboring elements, some of which are contributed by the sequence TAAGTATT present at the transcription start point. PPBP was affinity purified and appears to be an unusual DNA-binding protein with respect to its stability (binding was obtained at NaCl concentrations and temperatures ranging from 0.2 to 2 M and 0 to 65 °C, respectively), high binding affinity (binding even in the absence of nonspecific DNA) with an apparent dissociation constant of ~3.7 × 10^-12 M, and high specificity (binding was unaffected in the presence of a 50,000 times excess of nonspecific DNA). From UV cross-linking and Southwestern analyses the molecular mass of PPBP was estimated to be ~30 kDa. PPBP is phosphorylated and may have a regulatory function because dephosphorylation abolished DNA binding activity. Differences in PPBP characteristics between nuclear extracts from Sf21 and from Bm5 (Bombyx mori) cell line suggested that additional factors may be involved in the interaction of PPBP with the polyhedrin promoter.