Elliot J. Androphy

The specific DNA recognition sequence of the bovine papillomavirus E2 protein is an E2-dependent enhancer.

The upstream regulatory region (URR) of the bovine papillomavirus (BPV) genome contains an enhancer that is activated by a BPV E2 gene product. We have previously found that a bacterially derived E2 fusion protein specifically interacted with several fragments of URR DNA, suggesting that E2 may activate transcription by directly binding to the enhancer. Each of the bound fragments contains at least one copy of a conserved motif (ACCN6GGT). To determine if this motif is required and sufficient for specific E2 binding, we have now constructed a bacterial expression vector that encodes a full‐length E2 peptide and developed a refinement of the McKay DNA immunoprecipitation assay that allows the determination, to the nucleotide level, of the minimum sequence required for specific binding. The results show that the E2 recognition sequence is a single copy of this motif and that the variant ACCGN4CGGT is bound with greater affinity than the minimum ACCN6GGT motif. An oligonucleotide encoding the motif was able to inhibit E2‐dependent trans‐activation in a transient transfection assay, indicating that the virally encoded E2 also interacts with this sequence in mammalian cells. When present in two or more copies, but not in a single copy, the E2 binding element had intrinsic enhancer activity but only in cells expressing E2. The results indicate that the conserved motif alone is sufficient for E2‐mediated enhancement and that the binding of E2 to the motif is probably required for efficient enhancement. Since a single motif did not have a significant enhancer activity, it is likely that bound E2 molecules act cooperatively in activating transcription.

Papillomavirus-associated inductions of cellular proteins in mouse C127 cells: Correlation with the presence of open reading frame E2

Bovine papillomavirus type 1 (BPV-1) readily transforms mouse C127 cells, conferring the ability to grow in soft agar and to form tumors in athymic (nu/nu) mice. Electrophoresis of total cellular proteins from these BPV-transformed lines on ultra-high resolution, giant two-dimensional gels displays the presence of novel, papillomavirus-related protein phenotypes. Analysis of the established BPV-1-transformed C127 cell lines, ID13 and ID14, reveals a set of six proteins which are either absent or synthesized at extremely low levels in the parental cell line. One of these proteins is also present in v-ras-transformed C127 cells, but none of the others are found in cells transformed by a variety of viral oncogenes, including the polyomavirus middle T, v-mos, or v-fes. The genome of BPV-1 contains two separate open reading frames (ORFs), E5 and E6, which can act independently to transform C127 cells. In addition, trans-activator and repressor proteins encoded respectively by the full-length and carboxy-terminal E2 ORF regulate the level of expression of other BPV-1 genes. We examined 34 cell lines transformed by intact and subgenomic recombinant DNAs of BPV-1. Cells harboring BPV-1 DNAs engineered to eliminate the expression of ORFs E4, E5, E6, or E7 display five of the PV-associated proteins, but these proteins are not seen in lines lacking the full E2 ORF. Moreover, G418-selected nontransformed cells expressing E2 cDNA from an SV40 promoter exhibit these proteins at high levels. Surprisingly, these proteins are also present in cells containing BPV-1 DNAs with amino-terminal E2 deletions, suggesting that these PV-associated proteins represent novel cellular responses to a factor encoded within the E2-C gene region.