Identification of a novel transcription factor, ACF, in cultured avian fibroblast cells that interacts with a Marek's disease virus late gene promoter.

Interactions between factors in duck and chick embryo fibroblast (DEF and CEF, respectively) nuclear extracts and the Marek's disease virus (MDV) gp57-65 gene promoter were investigated. Results of in vitro transcription and gel mobility-shift assays indicated that multiple cellular factors interact with 5'-flanking sequences of the MDV gp57-65 gene. One sequence-specific DNA binding activity (termed ACF for avian cell factor(s)) was identified by interaction of DEF and CEF nuclear extract proteins with a particular site (nucleotides -193 to -177) in the MDV gp57-65 gene promoter. Binding of ACF to its apparent recognition sequence, contained within the 17-bp oligonucleotide 5'-CTAGTTTACTTGTTTGT-3' (ACF-12), was highly sequence-specific. Radiolabeled ACF-12 oligonucleotide bound significant ACF protein in the presence of a 400-fold molar excess of unlabeled nonspecific competitor DNA. A similar amount of specific competitor completely abolished ACF binding to probe DNA. Deletion of the ACF binding site from MDV gp57-65 gene promoters linked to a chloramphenicol acetyltransferase (CAT) reporter gene reduced expression of CAT activity by twofold relative to that seen with a gp57-65 promoter-CAT construct containing an intact ACF binding site. Transfection inhibition assays using double-stranded ACF binding site competitors reduced steady-state levels of gp57-65 mRNA in MDV infected cells by over twofold relative to those in control infected cells. Introduction of a similar amount of nonspecific double-stranded oligonucleotide had no adverse effect on gp57-65 mRNA levels. These data suggest that ACF is important for efficient expression of gp57-65.