Analysis of Early Genes in HPV maintenance replication: The Application to new anti-HPV drugs

• Project/Area Number: 17K16869
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Obstetrics and gynecology
• Research Institution: Keio University
• Principal Investigator: MURAKAMI Isao 慶應義塾大学, 医学部(信濃町), 助教 (70445237)
• Project Period (FY): 2017-04-01 – 2020-03-31
• Project Status: Completed (Fiscal Year 2019)
• Budget Amount: ¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000); Fiscal Year 2018: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000); Fiscal Year 2017: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
• Keywords: 低リスク型ヒトパピローマウイルス / 高リスク型ヒトパピローマウイルス / E1遺伝子 / 生活環 / E1 / E6 / E7 / p53 / K10 / MCM / HPV / 初期遺伝子 / 抗ウィルス薬

Outline of Final Research Achievements

Wild-type and mutated HPV11/16 genomes were maintained in NIKS using a modified growth protocol that allows genome maintenance and first commitment to differentiation to be studied.
Both wild-type HPV11/16 genomes were maintained during cell division, despite different growth rates and patterns of transcription. However, HPV16 genome levels rose post-confluence, while HPV11 genome copy number declined. Both HPV11/16 E1-deficient genomes were maintained pre-confluence, reinforcing the emerging view that maintenance-replication is E1-independent. Loss of functional E1 prevented HPV16 confluence-dependent genome amplification, indicative of a confluence-mediated switch in replication mode.

Academic Significance and Societal Importance of the Research Achievements

本研究の結果よりHPVは、宿主細胞の分化に伴いE1非依存性の複製からE1依存性の複製に移行していると考えられた。今までの研究によりHPVはE1依存性に維持複製されていると考えられていたが、今回の結果より基底細胞内での維持にはE1が不要であることが示唆された。
これらの結果はHPVにおける今までの概念を覆す結果と言える。今回確立した細胞株と実験手法を用いた初期遺伝子群の発現解析は、HPVのlife cycleの包括的な理解、さらにはHPV感染とそれに続いて起こるウイルス遺伝子発現の制御につながることが期待される。この実験系を用いてさらなる研究が可能であり、新たな知見が発見されることが期待される。

Research Products

• [Journal Article] Roles for E1-independent Replication and E6-mediated p53 Degradation During Low-Risk and High-Risk Human Papillomavirus Genome Maintenance (2019)
  • Author(s): Isao Murakami, Nagayasu Egawa, Heather Griffin, Wen Yin, Christian Kranjec, Tomomi Nakahara, Tohru Kiyono , John Doorbar
  • Journal Title: PLOS Pathogens Volume: 15 Issue: 5 Pages: e1007755-e1007755
  • DOI: 10.1371/journal.ppat.1007755
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] HPV検査の活用法 (2018)
  • Author(s): 村上功、岩田卓、森定徹、青木大輔
  • Journal Title: 臨床婦人科産科 Volume: 72 Pages: 150-157
• [Presentation] The HPV E4 is a candidate biomarker in cervical intraepithelial neoplasia grade 2 (2020)
  • Author(s): Murakami I, Iwata T, Egawa N, Saito M, Nakamura M, Morisada T, Tanaka K, Griffin H, Doorbar J, Aoki D
  • Organizer: The 33rd International Papillomavirus Conference and Clinical Workshop
  • Int'l Joint Research
• [Presentation] HPV E1 is dispensable for low and high-risk HPV maintenance and latent replication in keratinocytes but is necessary for genome amplification. (2018)
  • Author(s): Murakami I, Iwata T, Nakamura M, Morisada T, Tanaka K, Tanaka M, Aoki D
  • Organizer: 第70回日本産科婦人科学会学術講演会
• [Presentation] Nucleosome positioning on episomal human papillomavirus DNA in cultured cells. (2018)
  • Author(s): Murakami I, Iwata T, Egawa N, Nakamura M, Morisada T, Tanaka K, Doorbar J, Aoki D
  • Organizer: The 32nd International Papillomavirus Conference and Clinical Workshop
  • Int'l Joint Research
• [Presentation] E1 is dispensable for maintenance replication in keratinocytes, but necessary for sustenance of viral genome in confluent keratinocytes mediated by p53 degradation by E6. (2018)
  • Author(s): Egawa N, Murakami I, Griffin H, Doorbar J
  • Organizer: he 32nd International Papillomavirus Conference and Clinical Workshop
  • Int'l Joint Research
• [Presentation] HPV11 (low-risk) E6 directs p53 degradation in confluent keratinocytes and acts to sustain episomal copy number (2017)
  • Author(s): Murakami I, Nakamura M, Morisada T, Iwata T, Tanaka K, Tanaka M, Aoki D
  • Organizer: The 69th Annual Congress of the Japan Society of Obstetrics and Gynecology
  • Int'l Joint Research
• [Presentation] HPV11 (Low-risk) E6 directs p53 degradation in confluent keratinocytes, and like HPV16 (high-risk) E6, acts to sustain episomal copy number (2017)
  • Author(s): Murakami I, Egawa N, Griffin H, Doorbar J
  • Organizer: 2017 DNA Tumor Virus Meeting
  • Int'l Joint Research
• [Presentation] E1 is dispensable for low-risk and high-risk maintenance and latent replication in keratinocytes but is necessary for viral genome amplification (2017)
  • Author(s): Murakami I, Egawa N, Griffin H, Doorbar J
  • Organizer: 2017 DNA Tumor Virus Meeting
  • Int'l Joint Research
• [Presentation] Expression of difucosylated H-antigen in human uterine cervical carcinoma tissues detected by monoclonal antibody HMMC-1 (2017)
  • Author(s): Tanaka K, Murakami I, Iwata T, Aoki D, Iwamori M
  • Organizer: The 76th Annual Meeting of the Japanese Cancer Association