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1999 Fiscal Year Final Research Report Summary

Functional interaction between gene expression and DNA replication of HPV lifecyle

Research Project

Project/Area Number 10470076
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field Virology
Research InstitutionKYOTO UNIVERSITY

Principal Investigator

SAKAI Hiroyuki  Institute for Virus Research, Kyoto University Department of, ウイルス研究所, 助教授 (80281731)

Co-Investigator(Kenkyū-buntansha) ISHIMOTO Akinori  Institute for Virus Research, Kyoto University Department of Viral Oncology, Professor, ウイルス研究所, 教授 (50073127)
Project Period (FY) 1998 – 1999
Keywordspapillomavirus / tumor virus / cervical cancer / viral replication
Research Abstract

Gene expression and genome amplification of human papillomavirus (HPV) are tightly regulated by the status of host cell differentiation. In basal layer cells, weak expression of viral early genes and low copy number of viral genome are detectable. In the course of keratinocyte differentiation, the levels of viral gene expression and DNA amplification are upregulated, and the viral particle production can be detected only in fully differentiated cells. It is necessary, therefore, to analyze the mechanisms of HPV gene function and replication in the system that reflects the keratinocyte differentiation program.
The organotypic culture of keratinocytes is developed to reconstitute the skin structure, in which HPV DNA can express viral genes and replicate in differentiation specific manner. We adapted an organotypic culture system developed by Dr. Kuroki et al. for HPV analysis. HPV 18 genomic DNA was introduced into human primary keratinocytes by lipofection, and then the cells were used f … More or organotypic culture. In the basal layer cells, HPV DNA was difficult to detect by in situ hybridization, although it was detectable by Southern blot analysis. The DNA amplification could be found in the differentiated cells, in which transglutaminase, one of the differentiation markers, was expressing. mRNA expression level was also enhanced through cell differentiation. The result indicated that the organotypic culture system was suitable for the analysis of differentiation specific HPV function.
The gene function of HPV was analyzed in the organotypic culture system. Two major viral oncoproteins, E6 and E7, were expressed in keratinocytes and the effects on cell proliferation and differentiation were analyzed. In normal condition, cell division was restricted only in the basal layer, but E6/E7 expressing cells could proliferated even in partially differentiated layer. However, differentiation program was maintained intact even with E6/E7 expression. We are now expanding the analysis for other viral genes and low-risk type E6/E7 genes.
We observed a linkage between gene expression and DNA replication in the organotypic culture. Both functions are regulated mainly by LCR of HPV genome, in which viral E2 protein is involved. We obtained evidence that the E2 protein functioned as a mediator between both activities. We are now investigating the involvement of E2 protein in differentiation specific HPV replication mechanism. Less

  • Research Products

    (10 results)

All Other

All Publications (10 results)

  • [Publications] Nishimura, A. And Sakai, H. et. al.: "Mechanisms of human papillomavirus E2 mediated reprssion viral oncogene expression and cervical cancer cell growth inhibition"J. Virol.. 74. 3752-3760 (2000)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Yochinouchi M. and Sakai, H. et. al.: "Analysis by multiplex PCR of the physical status of human papillomavirus type 16 DNA in cervical cancers"J. Clin. Microbiol.. 37. 3514-3517 (1999)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Ono, T. and Sakai, H. et. al.: "Functional association between the nef gene product and gag-pol region of HIV-1"FEBS Letters. 466. 233-238 (2000)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Tayar, L. and Sakai, H. et. al.: "nduction of B-cell lymphoma in BALB/c nude mice with an ecotropic, B-tropic helper virus present in the murine AIDS virus stock"J. Virol.. 73. 1640-1644 (1999)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Zhang, X. and Sakai, H. et al: "phosphorothioate hammerhead ribozymes targeting a conserbed sepuence in the V3 loop region inhibit HIV-1 entry"Antisense Nucleic Acid Drug Dev.. 8. 441-450 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Nishimura, A., Ono, T., Ishimoto, A., Dowhanick, J.J., Frizzell, M.A., Howley, P.M., and H Sakai: "Mechanisms of human papillomavirus E2 mediated repression of viral oncogene expression and cervical cancer cell growth inhibition."J. Virol.. 74. 3752-3760 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Yoshinouchi, M., Hongo, A., Nakamura, K., Kodama, J., Itoh, S., Sakai, H., and Kudo, T.: "Analysis by multiplex PCR of the physical status of human papillomavirus type 16 DNA in cervical cancers."J. Clin. Microbiol.. 37. 3514-3517 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Tayar, L., Higo, K., Kubo, Y., Wang, Y., Lu, L.M., Zhang, F., Iwatani, Y., Wang, L., Ono, T., Maeda,: "Induction of B-cell lymphoma in BALB/c nude mice with an ecotropic, B-tropic helper virus present in the murine AIDS virus stock."J. Virol.. 73. 1640-1644 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Ono, T., Iwatani, Y., Nishimura, A., Ishimoto, A, and Sakai, H.: "Functional association between the nef gene product and gag-pol region of HIV-1."FEBS Letters. 466. 233-238 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Zhang, X., Iwatani, Y., Shimayama, T., Yamada, R., Koito, A., Xu, Y., Sakai, H., Uchiyama, T., and H: "Phosphorothioate hammerhead ribozymes targeting a conserved sequence in the V3 loop region inhibit HIV-1 entry."Antisense Nucleic Acid Drug Dev.. 8. 441-450 (1998)

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 2001-10-23  

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