Identification of proteins binding to the F441 locus of polyomavirus B enhancer that are required for its activity in embryonic carcinoma cells

Abstract

A point mutation at nucleotide 5233 of the polyomavirus (A2 strain) enables it to overcome growth restriction in undifferentiated embryonal carcinoma cells. We analysed the binding of nuclear proteins from F9 cells to a 38 bp region that spans this site of mutation and encompasses two copies of the bovine papillomavirus core sequence, CCACCC, and characterized this domain by mutational analysis. Our results showed that the F441 mutation creates a sequence motif which binds TEF-1 or a TEF-1-like protein from F9 cells more strongly than its wild-type counterpart and increases its activity by about 10-fold. Another protein identified as CP1 binds with increased affinity in the presence of the F441 mutation to the CAT box-like sequences which is contiguous with the downstream CCACCC box. Point mutations within these two motifs that abolished binding in vitro also impaired the activity of the F441 locus in vivo. As neither the wild-type sequence without the F441 mutation, nor the F441 template without the CAT box has appreciable activity in vivo, interaction between these two elements is required for function. At a higher level of organization, this interaction is probably extended to factors bound to other domains in the A and B enhancer.