AN UNUSUAL 30-KDA PROTEIN-BINDING TO THE POLYHEDRON GENE PROMOTER OF AUTOGRAPHA-CALIFORNICA NUCLEAR POLYHEDROSIS-VIRUS

Gel retardation assays-using nuclear extracts from the Autographa cali fornica nuclear polyhedrosis virus-infected Sf21 (Spodoptera frugiperd a) insect cell line revealed a host factor (polyhedrin promoter-bindin g protein or PPBP) that binds to the polyhedrin gene promoter. A hexan ucleotide sequence (AATAAA) within the promoter is important for bindi ng in association with neighboring elements, some of which are contrib uted 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 co ncentrations and temperatures ranging from 0.2 to 2 m and 0 to 65-degr ees-C, respectively), high binding affinity (binding even in the absen ce of nonspecific DNA) with an apparent dissociation constant of appro ximately 3.7 x 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 wa s estimated to be approximately 30 kDa. PPBP is phosphorylated and may have a regulatory function because dephosphorylation abolished DNA bi nding activity. Differences in PPBP characteristics between nuclear ex tracts from Sf21 and from Bm5 (Bombyx mori) cell line suggested that a dditional factors may be involved in the interaction of PPBP with the polyhedrin promoter.