E2蛋白質をターゲットとしたHPV感染症に対する抗ウイルス薬剤開発への基礎的検討(1 子宮頸癌の発生と進展 : その制御を目指して)

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Epidemiologic data suggests that the chronic HPV infection is one of main risk factors for the development of cervical cancer, which is the third most common cause of cancer death in women all over the world. In the viral life cycle, the HPV protein E2 can function as both a transcriptional activator and repressor, probably by interaction with cellular transcriptional factors. Integration of human papillomavirus 16 DNA is considered to be a major genetic change in cervical carcinogenesis, and disruption of E2 protein in this process results in the overexpression of E6 and E7 genes. Thus, it is important to evaluate the physical status of HPV 16 E2 gene in cervical dysplasia and cervical cancer. We showed that the E2 gene was in-tact by in situ PCR in cervical dysplasia specimen. The conventional PCR method revealed that the E2 gene was retained in approximately 60-70% in cervical dysplasia and even carcinoma in situ. These data suggested that HPV 16 E2 gene was retained in the most of precancerous lesions of the cervix. We demonstrated the low level of HPV 16 E2 protein stimulatory for trans-activation by the luciferase as-say. Though it is important to elucidate the transcriptional activity of the E2 gene after initial stage of infection, it is difficult to perform experimentally because no propagation system was available for HPV 16. We therefore focused on the cottontail rabbit papillomavirus (CRPV) because CRPV is available and CRPV can infect into cultured cells. We found that low levels of the CRPV E2 protein were stimulatory for trans-activation, whereas high levels were inhibitory. In vitro infection assay by CRPV, we demonstrated that E2 protein was working as a transactivator after infection, suggesting that low levels of E2 protein transactivated E6 and E7 genes in the initial stage of infection. Thus, the E2 protein is a good target for developing the antiviral drugs. The infection assay may be useful for the identification of compounds which can be used for the inhibition of papillomavirus gene expression. According to this scenario, we aimed at the isolation of peptides that bind to HPV 16 E2 protein and inhibit the transcriptional activity. We screened the phage display library to isolate clones that bind to the HPV 16 E2 protein. These peptides were composed of tryptophan rich amino acids. Those binding specificity to the E2 protein was confirmed by enzyme-linked immunoassay. We have tested the inhibitory effect with synthetic peptides conjugated with FITC by the luciferase assay using the HPV16 E2 expression vector and the reporter vector that contained cognate site of E2 binding sites. At first, we showed these synthetic peptides were expressed in the nuclei of the cultured cells. Pep-2 showed transcriptional repression in a dose dependent manner. The alanine scanning peptide showed no inhibitory transcriptional effect by the luciferaseassay, suggesting that tryptophan rich sequences were associated with protein-protein interaction. In summary, we have isolated peptides that bind to HPV16 E2 protein. Isolated peptides showed the inhibitory effect on the transcriptional activity by the luciferase assay. We showed that tryptophan rich sequences were important for not only the binding to the E2 protein but also the transcriptional activity. The amino acid sequence composed of peptides could be candidates for the basic structure of anti-viral agents in future.
社団法人日本産科婦人科学会の論文
2002-08-01

E2蛋白質をターゲットとしたHPV感染症に対する抗ウイルス薬剤開発への基礎的検討(1 子宮頸癌の発生と進展 : その制御を目指して)

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