Designing novel protease inhibitors based on the interference of mouse APOBEC3 with retroviral Gag-Pol autocatalysis

• Project/Area Number: 16H05199
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Virology
• Research Institution: Kindai University
• Principal Investigator: MIYAZAWA Masaaki 近畿大学, 医学部, 教授 (60167757)
• Co-Investigator(Kenkyū-buntansha): 博多 義之 近畿大学, 医学部, 講師 (30344500)
• Project Period (FY): 2016-04-01 – 2019-03-31
• Project Status: Completed (Fiscal Year 2018)
• Budget Amount: ¥14,430,000 (Direct Cost: ¥11,100,000、Indirect Cost: ¥3,330,000); Fiscal Year 2018: ¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000); Fiscal Year 2017: ¥5,200,000 (Direct Cost: ¥4,000,000、Indirect Cost: ¥1,200,000); Fiscal Year 2016: ¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000)
• Keywords: レトロウイルス / 宿主因子 / 複製阻害 / プロテアーゼ / APOBEC3 / 粒子成熟 / Gag-Pol前駆体 / 複製制限 / タンパク質相互作用 / プロセシング / HIV-1 / マウスレトロウイルス / ウイルス / タンパク質間相互作用 / ヒト免疫不全ウイルス / ウイルスプロテアーゼ / タンパク質プロセシング / 分子間相互作用

Outline of Final Research Achievements

Mouse APOBEC3 (mA3) inhibits murine leukemia virus (MuLV) replication through a deamination-independent mechanism in which the reverse transcription is considered the prime target process. However, other steps in virus replication can also be targeted by mA3. We have investigated the possible effect of mA3 on viral protease-mediated processes. We found that mA3 directly bound both mature viral protease and Pr180gag-pol precursor. Furthermore, the autoprocessing of Pr180gag-pol was impeded by mA3, resulting in reduced production of mature viral protease. Exon 5-containing and -lacking isoforms of mA3 equally exhibited this antiviral activity, and physiologically expressed levels of mA3 impeded Pr180gag-pol autocatalysis and Pr65gag processing. This blockade was independent of the deaminase activity and required the C-terminal half of mA3. These results suggest that Pr180gag-pol autoprocessing can be a critical step for mA3 to exert its deaminase-independent antiretroviral activity.

Academic Significance and Societal Importance of the Research Achievements

抗レトロウイルス薬の多剤併用により、先進国ではHIV-1感染者の生命予後が非感染者のそれに近付いているとされる。しかし、現在用いられている抗レトロウイルス薬では耐性株出現が不可避であり、若年感染者が短期間に治療を必要とするようになる現状からも、新たな作用機序を持つ抗HIV薬開発が喫緊の課題である。プロテアーゼ阻害薬は、活性部位への結合や二量体化の抑制を指標に開発されて来た。我々の研究成果を基礎に、Gag-Pol前駆体からのプロテアーゼ切り出しを阻害する薬物のシーズを宿主因子の分子構造から探ることにより、有効でかつ副作用の少ない新規抗レトロウイルス薬の開発に貢献できる可能性がある。

Research Products

• [Int'l Joint Research] National Institutes of Health(米国)
• [Journal Article] A leucine zipper-based peptide hybrid delivers functional Nanog protein inside the cell nucleus. (2019)
  • Author(s): Yoshiyuki Hakata, Hiroyuki Michiue, Takashi Ohtsuki, Masaaki Miyazawa and Mizuki Kitamatsu
  • Journal Title: Bioorganic & Medicinal Chemistry Letters Volume: 29 Issue: 7 Pages: 878-881
  • DOI: 10.1016/j.bmcl.2019.02.004
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] フレンドウイルス感染マウスにおける白血病発症機構 (2019)
  • Author(s): 塚本 徹雄、河原佐智代、宮澤正顯
  • Journal Title: Bio Clinica Volume: 印刷中
• [Journal Article] B7-H3 negatively modulates CTL-mediated cancer immunity (2018)
  • Author(s): Yonesaka K, Haratani K, Takamura S, Sakai H, Kato R, Takegawa N, Takahama T, Tanaka K, Hayashi H, Takeda M, Kato S, Maenishi O, Sakai K, Chiba Y, Okabe T, Kudo K, Hasegawa Y, Kaneda H, Yamato M, Hirotani K, Miyazawa M, Nishio K, Nakagawa K.
  • Journal Title: Clin Cancer Res. Volume: - Issue: 11 Pages: 2653-2664
  • DOI: 10.1158/1078-0432.ccr-17-2852
  • Peer Reviewed
• [Journal Article] SIV targeting of CXCR3+CD4+ T cells in secondary lymphoid organs associates with robust CXCL10 expression in monocyte/macrophage subsets. (2017)
  • Author(s): Fujino, M., H. Sato, T. Okamura, A. Uda, S. Takeda, N. Ahmed, S. Shichino, T. Shiino, Y. Saito, S. Watanabe, C. Sugimoto, M. Kuroda, M. Ato, Y. Nagai, S. Izumo, K. Matsushima, M. Miyazawa, A. Ansari, F. Villinger, and K. Mori
  • Journal Title: Journal of Virology Volume: 印刷中 Issue: 13
  • DOI: 10.1128/jvi.00439-17
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Repression of miRNA function mediates inflammation-associated colon tumorigenesis. (2017)
  • Author(s): Yoshikawa T., J. F. Wu, M. Otsuka, T. Kishikawa, N. Suzuki, A. Takata, M. Ohno, R. Ishibashi, M. Yamagami, R. Nakagawa, N. Kato, M. Miyazawa, J. Han, and K. Koike
  • Journal Title: Gastroenterology Volume: 152 Issue: 3 Pages: 631-643
  • DOI: 10.1053/j.gastro.2016.10.043
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Functional polymorphisms and molecular evolution of mouse APOBEC3 (2016)
  • Author(s): 宮澤 正顯，博多 義之，武田 英里，李 君，河原 佐智代
  • Journal Title: 生化学 Volume: 88 Issue: 5 Pages: 582-592
  • DOI: 10.14952/SEIKAGAKU.2016.880582
  • ISSN: 0037-1017
  • Year and Date: 2016-10-25
  • Peer Reviewed
• [Journal Article] Specific niches for lung-resident memory CD8+ T cells at the site of tissue regeneration enable CD69-independent maintenance. (2016)
  • Author(s): Takamura, S., H. Yagi, Y. Hakata, C. Motozono, S. R. McMaster, T. Masumoto, M. Fujisawa, T. Chikaishi, J. Komeda, J. Itoh, M. Umemura, A. Kyusai, M. Tomura, T. Nakayama, D. L. Woodland, J. E. Kohlmeier, and M. Miyazawa
  • Journal Title: The Journal of Experimental Medicine Volume: 213 Issue: 13 Pages: 3057-3073
  • DOI: 10.1084/jem.20160938
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Presentation] Mouse APOBEC3 interferes with proteolytic autoprocessing of Gag-Pol polyprotein and inhibits Gag processing. (2018)
  • Author(s): Yoshiyuki Hakata, Jun Li, Takahiro Fujino, Yuki Tanaka, Rie Shimizu, and Masaaki Miyazawa
  • Organizer: The 30th International Workshop on Retroviral Pathogenesis
  • Int'l Joint Research
• [Presentation] The requirement of B cell-intrinsic TLR7 for the production of retrovirus-neutralizing antibodies is overcome by T helper cells in an epitope-specific manner. (2018)
  • Author(s): Sachiyo Tsuji-Kawahara and Masaaki Miyazawa
  • Organizer: The 30th International Workshop on Retroviral Pathogenesis
  • Int'l Joint Research
• [Presentation] Fate determination with a selected epitope facilitates follicular helper T cell differentiation and protection against acute retroviral infection. (2018)
  • Author(s): Shigeki Kato, Chihiro Motozono, Sachiyo Kawahara and Masaaki Miyazawa
  • Organizer: The 30th International Workshop on Retroviral Pathogenesis
  • Int'l Joint Research
• [Presentation] Expression of galectin-9 in normal bone marrow and Friend virus-induced erythroleukemia cells. (2018)
  • Author(s): Tetsuo Tsukamoto and Masaaki Miyazawa
  • Organizer: The 30th International Workshop on Retroviral Pathogenesis
  • Int'l Joint Research
• [Presentation] エピトープ特異性によるCD4陽性エフェクターT細胞機能の制御と腫瘍免疫 (2018)
  • Author(s): 宮澤 正顯
  • Organizer: 第69回日本皮膚科学会中部支部学術大会
  • Invited
• [Presentation] Mouse APOBEC3 disrupts proteolytic autoprocessing of retroviral Gag-Pol precursors through direct binding to the protease domain (2017)
  • Author(s): Masaaki Miyazawa, Yoshiyuki Hakata, Jun Li, Takahiro Fujino, and Yuki Tanaka
  • Organizer: The 29th International Workshop on Retroviral Pathogenesis
  • Int'l Joint Research
• [Presentation] A Checkpoint Molecule B7-H3 as a Novel Immune Therapy Target for Non-Small Cell Lung Cancer (NSCLC) (2017)
  • Author(s): K. Yonesaka, K. Kudoh, S. Takamura, H. Sakai, R. Kato, K. Haratani, T. Takahama, K. Tanaka, H. Hayashi, H. Kaneda, M. Takeda, O. Maenishi, M. Yamato, M. Miyazawa, K. Nishio, K. Nakagawa
  • Organizer: 18th World Conference on Lung Cancer
  • Int'l Joint Research
• [Presentation] T細胞受容体親和性による感染防御性CD4陽性T細胞分化の制御 (2017)
  • Author(s): 宮澤 正顯
  • Organizer: 第26回日本組織適合性学会大会
  • Invited
• [Presentation] T-helper cell fate determination and Toll-like receptor 7 overriding by priming with a selected retroviral epitope. (2016)
  • Author(s): Miyazawa, M., Motozono, C., Tsuji-Kawahara, S., and Kato, S.
  • Organizer: The 28th International Workshop on Retroviral Pathogenesis
  • Place of Presentation: Bourbon Orleans Hotel, New Orleans, Louisiana, U.S.A.
  • Year and Date: 2016-12-05
  • Int'l Joint Research
• [Presentation] Vaccine-elicited preferential induction of germinal center follicular helper T cells is associated with protection against retroviral infection. (2016)
  • Author(s): Motozono, C., Tsuji-Kawahara, S., Takamura, S., Yagi, H., Miyazawa, M.
  • Organizer: The 44th Annual Meeting of the Japanese Society for Immunology
  • Place of Presentation: Sapporo Convention Center, Sapporo
  • Year and Date: 2016-11-18
• [Presentation] Autophagy-independent maintenance of resident memory CD8+ T cells in the lung. (2016)
  • Author(s): Takamura S. and Miyazawa, M.
  • Organizer: The 44th Annual Meeting of the Japanese Society for Immunology
  • Place of Presentation: Sapporo Convention Center, Sapporo
  • Year and Date: 2016-11-18
• [Presentation] B cell-intrinsic TLR7 signaling is required to prevent the generation of recombinant viruses follwoing retrovirus infection. (2016)
  • Author(s): Tsuji-Kawahara, S., Kawasaki, Y., Motozono, C., Takamura, S., and Miyazawa, M.
  • Organizer: The 44th Annual Meeting of the Japanese Society for Immunology
  • Place of Presentation: Sapporo Convention Center, Sapporo
  • Year and Date: 2016-11-18
• [Presentation] Functional evolution of retrovirus restriction enzyme APOBEC3: Genetic polymorphisms and isoforms. (2016)
  • Author(s): Miyazawa, M.
  • Organizer: The 2nd International Symposium on Molecular Basis of Virus-Host Interactions
  • Place of Presentation: ACU and Sapporo Convention Center, Sapporo
  • Year and Date: 2016-10-22
  • Int'l Joint Research / Invited
• [Presentation] CD4陽性T細胞認識エピトープが決める感染防御免疫応答の行方 (2016)
  • Author(s): 宮澤正顯
  • Organizer: 2016年度眼科COI学術講演会
  • Place of Presentation: ヒルトン大阪, 大阪市
  • Invited
• [Book] 解明病理学第3版 (2017)
  • Author(s): 青笹克之、加藤光保、菅野 祐幸編 宮澤 正顯 章分担
  • Total Pages: 818
  • Publisher: 医歯薬出版
  • ISBN: 9784263731789
• [Remarks] 近畿大学医学部免疫学教室
  • URL: https://www.med.kindai.ac.jp/immuno/
• [Remarks] 近畿大学医学部免疫学教室
  • URL: http://www.med.kindai.ac.jp/immuno/
• [Funded Workshop] The 30th International Workshop on Retroviral Pathogenesis (2018)