The E2 open reading frame of bovine papilloma virus 1 (BPV‐1) has been shown to encode both positive and negative acting transcriptional regulatory factors. The DNA binding properties of these factors were analysed to investigate the mechanism by which they might regulate viral gene expression. Polypeptides corresponding to the full‐length E2 product and a shorter protein thought to represent the repressor function were synthesized in vitro by translation of T7 polymerase generated transcripts. Using rabbit antisera generated against synthetic peptides from the E2 open reading frame, it was possible to immunoprecipitate each of these products and show that each was capable of binding the same specific sequence located at several sites in the BPV‐1 genome. This DNA binding property was mapped to a conserved carboxy‐terminal domain of 101 amino acids by analysis of truncated polypeptides synthesized from the E2 open reading frame.

Carcinogenic progression of a human papillomavirus (HPV)-infected cell is often associated with integration of the viral genome in a manner which results in the loss of expression of the viral regulatory protein E2. One function of E2 is the regulation of expression of the viral oncogenes, E6 and E7. Introduction of the bovine papillomavirus type 1 (BPV-1) E2 transactivator (E2-TA) in HeLa cells, an HPV type 18 (HPV-18)-positive cervical carcinoma cell line results in growth arrest. In this study, we have found that the HPV-16 and HPV-18 E2 proteins share with BPV-1 E2-TA the ability to suppress HeLa cell growth. This property was not observed for the BPV-1 E2 transcriptional repressor (E2-TR). Analysis of various mutant E2 proteins for growth suppression revealed a requirement for the intact transactivation and DNA binding domains. A HeLa cell line (HeLa-tsE2) which expressed a conditional mutant E2 protein that was functional only at the permissive temperature (32؇C) was established, permitting an analysis of the molecular and cellular consequences of E2 expression. Our data indicate that one mechanism by which E2 suppresses cellular growth is through repression of E6 and E7 expression, thereby enabling the cellular targets of E6 and E7 to resume regulation of the cell cycle.

Replication of bovine papillomavirus‐1 (BPV‐1) DNA requires two viral gene products, the E1 protein and the full‐length E2 protein. The 48 kDa E2 protein is a site‐specific DNA‐binding protein that binds to several sites which lie adjacent to the BPV‐1 origin of replication. The 85 amino acid C‐terminal domain contains the specific DNA binding and dimerization properties of the protein. The approximately 200 amino acid N‐terminal domain is crucial for transcriptional activation. Both of these domains are highly conserved among different papillomaviruses. An internal hinge region separates the two functional domains. The region varies in amino acid sequence and length among the E2 proteins of different papillomaviruses. A series of mutations were constructed within the E2 open reading frame which delete various regions of the conserved DNA binding and transactivation domains as well as the internal hinge region. Two mutated E2 proteins that lack portions of the conserved DNA‐binding domain but which support DNA replication were identified using transient replication assays. These mutated E2 proteins were unable to function as transcriptional activators. Conversely, two E2 proteins containing large deletions of the hinge region were able to activate transcription, but were defective for replication. Thus, the replication and transactivation functions of the E2 protein are separable.

The papilloma virus E2 transcriptional trans-activator is representative of a class of transcriptional modulators that activate transcription through direct binding to cis-acting DNA sequences. In this study we measured the capacity for this mammalian virus factor to function in Saccharomyces cerevisiae.

The N-terminal domain of the bovine papillomavirus type 1 E2 protein is important for viral DNA replication, for transcriptional transactivation, and for interaction with the E1 protein. To determine which residues of this 200-amino-acid domain are important for these activities, single conservative amino acid substitutions have been generated in 17 residues that are invariant among all papillomavirus E2 proteins. The resulting mutated E2 proteins were tested for the ability to support viral DNA replication, activate transcription, and cooperatively bind to the origin of replication with the E1 protein. We identified five mutated proteins that were completely defective for transcriptional activation and either were defective or could support viral DNA replication at only low levels. However, several of these proteins could still interact efficiently with the E1 protein. In addition, we identified several mutated proteins that were unable to efficiently cooperatively bind to the origin with the E1 protein. Although a number of the mutated proteins demonstrated wild-type activity in all of the functions tested, only 3 out of 17 mutated viral genomes were able to induce foci in a C127 focus formation assay when the mutations were generated in the background of the entire bovine papillomavirus type 1 genome. This finding suggests that the E2 protein may have additional activities that are important for the viral life cycle.