新規培養系を利用したB型肝炎ウイルス侵入機序・宿主因子の包括的解析

2019 Fiscal Year Annual Research Report

• Project/Area Number: 17H04085
• Research Institution: National Institute of Infectious Diseases
• Principal Investigator: 渡士 幸一 国立感染症研究所, 治療薬・ワクチン開発研究センター, 治療薬開発総括研究官 (40378948)
• Project Period (FY): 2017-04-01 – 2020-03-31
• Keywords: HBV

Outline of Annual Research Achievements

B型肝炎ウイルス(HBV)の細胞内侵入には侵入受容体NTCPが必要であるが、これがどのようにHBV侵入に関与するかはほとんど不明である。本研究ではNTCPと相互作用あるいは制御する因子を同定することを目的とした。これまで上皮成長因子受容体(EGFR)がNTCPと相互作用し、NTCPの侵入受容体に必要であることを報告してきた。そこでEGFRのどの機能がHBV感染に重要であるかを解析した。EGFRはリガンド刺激により、Ras/MAPK、Akt/PI3K、JAK/STATなどの下流情報伝達を活性化し、また一方でそれ自身がエンドサイトーシスすることが知られている。そこでこのいずれの機能がHBV感染をサポートするのに重要であるかを解析した。まずRas/MAPK、Akt/PI3K、JAK/STATの6種類の阻害剤を処理したが、これによっていずれもHBV感染に変化はなかった。一方エンドサイトーシスしないEGFR点変異体はHBV感染を媒介せず内在化もほとんど認められなかった。以上より、HBV侵入にはEGFRのエンドサイトーシスが必須であることが示唆された。またEGFRエンドサイトーシスに関わるAP2A1、EPS15をノックダウンすると、いずれも顕著なHBV感染の低下が認められた。またHBV preS1は細胞表面に吸着後、EGFRと共局在しながら経時的に初期エンドソーム、後期エンドソーム、リソソームへと局在変化していくことが観察された。後期エンドソームへの輸送に関わるSTAMのノックダウンによりHBV感染は低下し、またSTAMに阻害的に機能するLAPTM4Bは逆にHBV感染を上昇させた。このことはHBV感染に少なくとも後期エンドソームへの局在化が必要であることを示唆している。以上の結果より、HBV侵入にはEGFRが媒介するエンドソーム輸送経路が関与することを明らかにした。

Research Progress Status

令和元年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

令和元年度が最終年度であるため、記入しない。

Research Products

• [Journal Article] Immunomodulatory Mechanism of Acyclic Nucleoside Phosphates in Treatment of Hepatitis B Virus Infection (2020)
  • Author(s): Murata K, Tsukuda S, Suizu F, Kimura A, Sugiyama M, Watashi K, Noguchi M, Mizokami M
  • Journal Title: Hepatology Volume: 71 Pages: 1533-1545
  • DOI: 10.1002/hep.30956
  • Peer Reviewed
• [Journal Article] Synthesis of nucleotide analogues, EFdA, EdA and EdAP, and the effect of EdAP on hepatitis B virus replication (2020)
  • Author(s): Kamata M, Takeuchi T, Hayashi E, Nishioka K, Oshima M, Iwamoto M, Nishiuchi K, Kamo S, Tomoshige S, Watashi K, Kamisuki S, Ohrui H, Sugawara F, Kuramochi K
  • Journal Title: Biosci Biotechnol Biochem Volume: 84 Pages: 217-227
  • DOI: 10.1080/09168451.2019.1673696
  • Peer Reviewed
• [Journal Article] Surfeit 4 Contributes to the Replication of Hepatitis C Virus Using Double-Membrane Vesicles (2020)
  • Author(s): Kong L, Aoyagi H, Yang Z, Ouyang T, Matsuda M, Fujimoto A, Watashi K, Suzuki R, Arita M, Yamagoe S, Dohmae N, Suzuki T, Suzuki T, Muramatsu M, Wakita T, Aizaki H
  • Journal Title: J Virol Volume: 94 Pages: e00858-19
  • DOI: 10.1128/JVI.00858-19
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Anti-viral effects of interferon-λ3 on hepatitis B virus infection in cell culture (2020)
  • Author(s): Yamada N, Murayama A, Shiina M, Aly HH, Iwamoto M, Tsukuda S, Watashi K, Tanaka T, Moriishi K, Nishitsuji H, Sugiyama M, Mizokami M, Shimotohno K, Muramatsu M, Murata K, Kato T
  • Journal Title: Hepatol Res Volume: 50 Pages: 283-291
  • DOI: 10.1111/hepr.13449
  • Peer Reviewed
• [Journal Article] Non-nucleoside hepatitis B virus polymerase inhibitors identified by an in vitro polymerase elongation assay (2020)
  • Author(s): Nakajima S, Watashi K, Fukano K, Tsukuda S, Wakae K, Aizaki H, Muramatsu M, Wakita T, Toyoda T
  • Journal Title: J Gastroenterol Volume: 55 Pages: 441-452
  • DOI: 10.1007/s00535-019-01643-0
  • Peer Reviewed
• [Journal Article] The machinery for endocytosis of epidermal growth factor receptor coordinates the transport of incoming hepatitis B virus to the endosomal network (2020)
  • Author(s): Iwamoto M, Saso W, Nishioka K, Ohashi H, Sugiyama R, Ryo A, Ohki M, Yun JH, Park SY, Ohshima T, Suzuki R, Aizaki H, Muramatsu M, Matano T, Iwami S, Sureau C, Wakita T, Watashi K
  • Journal Title: J Biol Chem Volume: 295 Pages: 800-807
  • DOI: 10.1074/jbc.AC119.010366
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Prolyl Isomerase Pin1 Regulates the Stability of Hepatitis B Virus Core Protein (2020)
  • Author(s): Nishi M, Miyakawa K, Matsunaga S, Khatun H, Yamaoka Y, Watashi K, Sugiyama M, Kimura H, Wakita T, Ryo A
  • Journal Title: Front Cell Dev Biol Volume: 8 Pages: 26
  • DOI: 10.3389/fcell.2020.00026
  • Peer Reviewed / Open Access
• [Journal Article] Engineering Cellular Biosensors with Customizable Antiviral Responses Targeting Hepatitis B Virus (2020)
  • Author(s): Matsunaga S, Jeremiah SS, Miyakawa K, Kurotaki D, Shizukuishi S, Watashi K, Nishitsuji H, Kimura H, Tamura T, Yamamoto N, Shimotohno K, Wakita T, Ryo A
  • Journal Title: iScience Volume: 23 Pages: 100867
  • DOI: 10.1016/j.isci.2020.100867
  • Peer Reviewed / Open Access
• [Journal Article] The Beginning of Ending Hepatitis C Virus: A Summary of the 26th International Symposium on Hepatitis C Virus and Related Viruses (2020)
  • Author(s): Shin EC, Han JW, Kang W, Kato T, Kim SJ, Zhong J, Kim S, Park SH, Sung PS, Watashi K, Park JY, Windisch MP, Oh JW, Wakita T, Han KH, Jang SK
  • Journal Title: Viruses Volume: 12 Pages: 302
  • DOI: 10.3390/v12030302
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] N-Terminal PreS1 Sequence Regulates Efficient Infection of Cell-Culture-Generated Hepatitis B Virus (2020)
  • Author(s): Murayama A, Yamada N, Osaki Y, Shiina M, Aly HH, Iwamoto M, Tsukuda S, Watashi K, Matsuda M, Suzuki R, Tanaka T, Moriishi K, Suzuki T, Nishitsuji H, Sugiyama M, Mizokami M, Shimotohno K, Wakita T, Muramatsu M, Liang TJ, Kato T
  • Journal Title: Hepatology Volume: 73 Pages: 520-532
  • DOI: 10.1002/hep.31308
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] A global scientific strategy to cure hepatitis B (2019)
  • Author(s): Revill PA, Chisari FV, Block JM, Dandri M, Gehring AJ, Guo H, Hu J, Kramvis A, Lampertico P, Janssen HLA, Levrero M, Li W, Liang TJ, Lim SG, Lu F, Penicaud MC, Tavis JE, Thimme R, Arbuthnot P, Boonstra PA, Chang KM, Chen PJ, Glebe D, Guidotti LG, Fellay J, Ferrari C, Jansen L, Lau DTY, Lok AS, Maini MK, Mason W, et al.
  • Journal Title: Lancet Gastroenterol Hepatol Volume: 4 Pages: 545-558
  • DOI: 10.1016/S2468-1253(19)30119-0
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Retrotransposon elements among initial sites of hepatitis B virus integration into human genome in the HepG2-NTCP cell infection model (2019)
  • Author(s): Chauhan R, Shimizu Y, Watashi K, Wakita T, Fukasawa M, Michalak TI
  • Journal Title: Cancer Genet Volume: 235-236 Pages: 39-56
  • DOI: 10.1016/j.cancergen.2019.04.060
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Small Interfering RNA Screening for the Small GTPase Rab Proteins Identifies Rab5B as a Major Regulator of Hepatitis B Virus Production (2019)
  • Author(s): Inoue J, Ninomiya M, Umetsu T, Nakamura T, Kogure T, Kakazu E, Iwata T, Takai S, Sano A, Fukuda M, Watashi K, Isogawa M, Tanaka Y, Shimosegawa T, McNiven MA, Masamune A
  • Journal Title: J Virol Volume: 93 Pages: e00621-19
  • DOI: 10.1128/JVI.00621-19
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] An interferon-like small chemical compound CDM-3008 suppresses hepatitis B virus through induction of interferon-stimulated genes (2019)
  • Author(s): Furutani Y, Toguchi M, Shiozaki-Sato Y, Qin XY, Ebisui E, Higuchi S, Sudoh M, Suzuki H, Takahashi N, Watashi K, Wakita T, Kakeya H, Kojima S
  • Journal Title: PLoS One Volume: 14 Pages: e0216139
  • DOI: 10.1371/journal.pone.0216139
  • Peer Reviewed / Open Access
• [Journal Article] Bardoxolone methyl as a novel potent antiviral agent against hepatitis B and C viruses in human hepatocyte cell culture systems (2019)
  • Author(s): Nio Y, Sasai M, Akahori Y, Okamura H, Hasegawa H, Oshima M, Watashi K, Wakita T, Ryo A, Tanaka Y, Hijikata M
  • Journal Title: Antiviral Res Volume: 169 Pages: 104537
  • DOI: 10.1016/j.antiviral.2019.104537
  • Peer Reviewed
• [Journal Article] Pyrimidotriazine derivatives as selective inhibitors of HBV capsid assembly (2019)
  • Author(s): Toyama M, Sakakibara N, Takeda M, Okamoto M, Watashi K, Wakita T, Sugiyama M, Mizokami M, Ikeda M, Baba M
  • Journal Title: Virus Res Volume: 271 Pages: 197677
  • DOI: 10.1016/j.virusres.2019.197677
  • Peer Reviewed
• [Journal Article] Development of an in vitro cholestatic drug-induced liver injury evaluation system using HepG2-hNTCP-C4 cells in sandwich configuration (2019)
  • Author(s): Sakai Y, Okumura H, Iwao T, Watashi K, Ito K, Matsunaga T
  • Journal Title: Toxicol In Vitro Volume: 61 Pages: 104619
  • DOI: 10.1016/j.tiv.2019.104619
  • Peer Reviewed