Development of an anti-viral agent based on functional analysis of the human papillomavirus E2 protein

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The E2 protein of HPV is a DNA-binding protein that regulates the expression of the E6 and E7 genes and is believed to be extremely important in the analysis of the mechanism underlying viral carcinogenesis. Accordingly, we attempted to isolate the peptide that binds to the E2 protein and inhibit its transcription. First we isolated a phage clone binding to the E2 protein of HPV type 16. As a result of clonal analysis, we identified a peptide sequence containing tryptophan binding to the E2 protein. When tryptophan was replaced with alanine and the binding activity to the E2 protein was analyzed, the modified peptide was found to exhibit reduced binding activity. Furthermore, we produced a synthetic peptide by adding a nuclear localization signal to the isolated peptide sequence, and examined its effect on the transcriptional activity of the E2 by means of the luciferase assay ; it was found in this experiment that the transcriptional activity was decreased. These findings showed that the isolated peptide binding to the E2 protein inhibited the transcriptional activity. Then, we incorporated a nucleotide sequence obtained on the basis of this peptide sequence in the expression vector, forcibly expressed the peptide into the cells and determined its effect on the transcriptional activity of E2. However, to date, we have not discovered any sequence that efficiently inhibits the transcription activity. A possible reason for this is that such a peptide is not effectively expressed in the cells. Since the E2 protein is also involved in virus replication, we established an in viiro system for studying viral gene replication using the E2 protein. We added the above-described peptide using this system to examine whether or not gene replication would be inhibited. However, we found no inhibitory effect of the peptide on the virus replication. These results indicate that the peptide identified by us specifically inhibits viral transcription.