複製活性を保持した精製複製複合体からＨＣＶ複製に関与する宿主因子の同定と解析

2015 Fiscal Year Annual Research Report

• Project/Area Number: 24591001
• Research Institution: National Institute of Infectious Diseases
• Principal Investigator: 相崎 英樹 国立感染症研究所, ウイルス第二部, 室長 (00333360)
• Project Period (FY): 2012-04-01 – 2016-03-31
• Keywords: C型肝炎ウイルス / 翻訳・複製調節

Outline of Annual Research Achievements

我々はC型肝炎ウイルス(HCV)の複製と翻訳が同じ膜小胞で起こっていることを見出したものの、その複製と翻訳の調節メカニズムについては不明である。そこで、膜小胞内でのHCV RNA複製に関与するだけでなく、粒子形成においても重要なNS5Aに注目し、NS5A結合膜蛋白の翻訳・複製における調節機構を解析した。
NS5A発現細胞からpull-down法によりNS5Aに結合する膜蛋白を精製し, プロテオーム解析等を行い、翻訳および複製過程に関与している蛋白としてembryonic lethal, abnormal vision, drosophila-like 1 (ELAVL1)を見出した。ELAVL1の細胞内発現を調べたところ、レプリコン細胞ではHCV複製複合体が存在すると考えられている界面活性剤不溶性分画(DRM)に移行していた。HCV翻訳・複製が低下したELAVL1ノックダウンレプリコン細胞に各種ELAVL1の欠損変異体を発現させた実験から、ELAVL1が HCV RNAと結合し、さらにNS3、NS5A蛋白と結合することで複製の場DRMへ移行するものと考えられた。また、膜分画のトリプシン処理、ヌクレアーゼ処理により、主に複製は二重膜小胞(DMV)内側、翻訳はDMVの外側で行われているものと考えられた。
以上の結果から、ELAVL1はゲノム複製により新規合成されたHCV RNAと結合して、ウイルスタンパク質翻訳を誘導した。さらに、ELAVL1は産生されたNSタンパク質と結合して、HCV RNAを複製複合体へリクルートするものと考えられた。ELAVL1はHCV RNAの翻訳・複製を調節する宿主因子である可能性が示唆された。

Research Products

• [Journal Article] Single-domain intrabodies against HCV Core inhibit viral propagation and Core-induced NF-κB Activation. (2016)
  • Author(s): Suzuki R, Saito K, Matsuda M, Sato M, Kanegae Y, Shi G, Watashi K, Aizaki H, Chiba J, Saito I, Wakita T, Suzuki T.
  • Journal Title: J Gen Virol. Volume: - Pages: in press
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Prolactin regulatory element binding protein is involved in hepatitis C virus replication compartment by interacting with NS4B. (2016)
  • Author(s): Kong L, Fujimoto A, Nakamura M, Aoyagi H, Matsuda M, Watashi K, Suzuki R, Arita M, Yamagoe S, Dohmae N, Suzuki T, Sakamaki Y, Ichinose S, Suzuki T, Wakita T, Aizaki H.
  • Journal Title: J Virol. Volume: 90 Pages: 3093-111
  • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] A Novel Tricyclic Polyketide, Vanitaracin A, Specifically Inhibits the Entry of Hepatitis B and D Viruses by Targeting Sodium Taurocholate Cotransporting Polypeptide. (2015)
  • Author(s): Kaneko M, Watashi K, Kamisuki S, Matsunaga H, Iwamoto M, Kawai F, Ohashi H, Tsukuda S, Shimura S, Suzuki R, Aizaki H, Sugiyama M, Park SY, Ito T, Ohtani N, Sugawara F, Tanaka Y, Mizokami M, Sureau C, Wakita T.
  • Journal Title: J Virol. Volume: 89 Pages: 11945-53
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Dysregulation of Retinoic Acid Receptor Diminishes Hepatocyte Permissiveness to Hepatitis B Virus Infection through Modulation of NTCP Expression. (2015)
  • Author(s): Tsukuda S, Watashi K, Iwamoto M, Suzuki R, Aizaki H, Okada M, Sugiyama M, Kojima S, Tanaka Y, Mizokami M, Li J, Tong S, Wakita T.
  • Journal Title: J Biol Chem. Volume: 290 Pages: 5673-84
  • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Targeting cellular squalene synthase, an enzyme essential for cholesterol biosynthesis, is a potential antiviral strategy against hepatitis C virus. (2015)
  • Author(s): Saito K, Shirasago Y, Suzuki T, Aizaki H, Hanada K, Wakita T, Nishijima M, Fukasawa M.
  • Journal Title: J Virol. Volume: 89 Pages: 2220-32
  • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
• [Presentation] Prolactin regulatory element binding protein and Surfeit 4 are involved in hepatitis C virus replication compartment by interacting with NS4B. (2016)
  • Author(s): Kong L, Fujimoto A, Nakamura M, Aoyagi H, Matsuda M, Watashi K, Suzuki R, Arita M, Yamagoe S, Dohmae N, Suzuki T, Sakamaki Y, Ichinose S, Suzuki T, Wakita T, Aizaki H.
  • Organizer: Asian Pacific Association for the Study of the Liver
  • Place of Presentation: 東京
  • Year and Date: 2016-02-20 – 2016-02-24
  • Int'l Joint Research
• [Presentation] 肝炎ウイルス検査陽性者アンケート調査の受診勧奨効果 (2015)
  • Author(s): 中野卓二、橋本千樹、川部直人、村尾道人、嶋崎宏明、菅敏樹、中岡和徳、高川友花、大城昌史、高村知希、野村小百合、吉岡健太郎、中田佳穂里、青山政美、片岡博喜、相崎英樹、是永匡紹.
  • Organizer: 第41回日本肝臓学会西部会
  • Place of Presentation: 名古屋
  • Year and Date: 2015-12-03 – 2015-12-04
• [Presentation] Development of a novel vaccine platform based on Japanese encephalitis viruses. (2015)
  • Author(s): Suzuki R, Saga R, Fujimoto A, Watanabe N, Matsuda M, Hasegawa M, Watashi K, Aizaki H, Nakamura N, Konishi E, Kato T, Tajima S, Takasaki T, Takeyama H, Wakita T.
  • Organizer: 日本分子生物学会第38回年会
  • Place of Presentation: 神戸
  • Year and Date: 2015-12-01 – 2015-12-04
• [Presentation] Surfeit 4 is involved in HCV replication by interacting with NS4B. (2015)
  • Author(s): Lingbao K, Aoyagi H, Matsuda M, Fujimoto A, Watashi K, Suzuki R, Yamagoe S, Dohmae N, Sakamaki Y, Ichinose S, Suzuki T, Wakita T, Aizaki H.
  • Organizer: 日本ウイルス学会第63回学術集会
  • Place of Presentation: 福岡
  • Year and Date: 2015-11-22 – 2015-11-24
• [Presentation] Transfer hepatitis C virus between human liver cells and hepatic stellate cells. (2015)
  • Author(s): Aizaki H.
  • Organizer: The 2015 Italy-Japan Liver Workshop
  • Place of Presentation: Italy
  • Year and Date: 2015-10-26 – 2015-10-26
  • Int'l Joint Research
• [Presentation] A new fungi-derived vanitaracin A specifically inhibits the entry of hepatitis B and D viruses through interacting with NTCP. (2015)
  • Author(s): Kaneko M, Watashi K, Kamisuki S, Matsunaga H, Iwamoto M, Kawai H, Ohashi H, Tsukuda S, Shimura S, Suzuki R, Aizaki H, Sugiyama M, Sam-Yong P, Ohtani N, Sugawara F, Tanaka Y, Mizokami M, Wakita T.
  • Organizer: 日本ウイルス学会第63回学術集会
  • Place of Presentation: 福岡
  • Year and Date: 2015-10-22 – 2015-10-24
• [Presentation] Transfer Hepatitis C virus from human liver cells to hepatic stellate cells. (2015)
  • Author(s): Zaitsu T, Aoyagi H, Fujimoto A, Matsuda M, Watanabe N, Watashi K, Suzuki R, Fukuhara T, Matsuura Y, Sakamaki Y, Ichinose S, Wake K, Suzuki T, Iijima H, Matsuura T, Tamura K, Wakita T, Aizaki H.
  • Organizer: 日本ウイルス学会第63回学術集会
  • Place of Presentation: 福岡
  • Year and Date: 2015-10-22 – 2015-10-24
• [Presentation] Multimerization of hepatitis B virus core protein regulated by microtubules. (2015)
  • Author(s): Iwamoto M, Watashi K, Sugiyama M, Suzuki R, Aizaki H, Tanaka Y, Mizokami M, Ohtani N, Koiwai O, Wakita T.
  • Organizer: 日本ウイルス学会第63回学術集会
  • Place of Presentation: 福岡
  • Year and Date: 2015-10-22 – 2015-10-24
• [Presentation] Identification of a flavonoid derivative that directly acts on hepatitis B virus surface protein and inhibits viral entry. (2015)
  • Author(s): Tsukuda S, Watashi K, Hamada Y, Isogawa M, Iwamoto M, Suzuki R, Aizaki H, Kojima S, Sugiyama M, Saito A, Tanaka Y, Mizokami M, Wakita T.
  • Organizer: 日本ウイルス学会第63回学術集会
  • Place of Presentation: 福岡
  • Year and Date: 2015-10-22 – 2015-10-24
• [Presentation] Bivalent vaccine platform based on Japanese encephalitis virus (JEV) elicit neutralizing antibodies against JEV and Hepatitis C virus. (2015)
  • Author(s): Saga R, Fujimoto A, Watanabe N, Matsuda M, Hasegawa M, Watashi K, Aizaki H, Nakamura N, Konishi E, Kato T, Tajima S, Takasaki T, Takeyama H, Wakita T, Suzuki R.
  • Organizer: 日本ウイルス学会第63回学術集会
  • Place of Presentation: 福岡
  • Year and Date: 2015-10-22 – 2015-10-24
• [Presentation] Flutamide-mediated Disruption of Lipid Droplets Impairs the Host Permissiveness to Hepatitis C virus Production. (2015)
  • Author(s): Ohashi H, Watashi K, Nakajima S, Sulyi K, Suzuki R, Aizaki H, Fukasawa M, Kamisuki S, Sugawara F, Ohtani N, Wakita T.
  • Organizer: 日本ウイルス学会第63回学術集会
  • Place of Presentation: 福岡
  • Year and Date: 2015-10-22 – 2015-10-24
• [Presentation] Japanese encephalitis virus (JEV)-subviral particles harboring HCV neutralization epitopes elicit neutralizing antibodies against JEV and HCV. (2015)
  • Author(s): Saga R, Fujimoto A, Watanabe N, Matsuda M, Hasegawa M, Watashi K, Aizaki H, Nakamura N, Konishi E, Kato T, Takeyama H, Wakita T, Suzuki R.
  • Organizer: International Symposium on Hepatitis C Virus and Related Viruses.
  • Place of Presentation: Strasbourg, France
  • Year and Date: 2015-10-09 – 2015-10-13
  • Int'l Joint Research
• [Presentation] Prolactin regulatory element binding protein promotes the formation of HCV membranes replication compartment by interacting with NS4B. (2015)
  • Author(s): Kong L, Fujimoto A, Aoyagi H, Matsuda M, Watashi K, Suzuki R, Yamagoe S, Dohmae N, Suzuki T, Suzuki T, Wakita T, Aizaki H.
  • Organizer: International Symposium on Hepatitis C Virus and Related Viruses.
  • Place of Presentation: Strasbourg, France
  • Year and Date: 2015-10-09 – 2015-10-13
  • Int'l Joint Research
• [Presentation] Identification of NTCP-targeting small molecules that inhibit HBV entry by cell-based and in vitro technologies. (2015)
  • Author(s): Watashi K, Tsukuda S, Shimura S, Kaneko M, Sluder A, Peel M, Kamisuki S, Matsunaga S, Morishita R, Futamura Y, Kondoh Y, Suzuki R, Aizaki H, Sugiyama M, Borroto-Esoda K, Ryo A, Sugawara F, Osada H, Tanaka Y, Mizokami M, Wakita T.
  • Organizer: International Meeting, Molecular Biology of Hepatitis B Viruses.
  • Place of Presentation: Dolce Bad Nauheim, Germany
  • Year and Date: 2015-10-04 – 2015-10-08
  • Int'l Joint Research
• [Presentation] Cilnidipine inhibits the early phase of HBV infection in a NTCP independent mechanism. (2015)
  • Author(s): Tsukuda S, Watashi K, Iwamoto M, Suzuki R, Aizaki H, Tanaka Y, Wakita T.
  • Organizer: International Meeting, Molecular Biology of Hepatitis B Viruses.
  • Place of Presentation: Dolce Bad Nauheim, Germany
  • Year and Date: 2015-10-04 – 2015-10-08
  • Int'l Joint Research
• [Presentation] Identification of a new tricyclic polyketide, vanitaracin A, which inhibits the entry of HBV, HDV, and nucleoside analog-resistant HBV through targeting NTCP. (2015)
  • Author(s): Kaneko M, Watashi K, Kamisuki S, Mtsunaga H, Iwamoto M, Kawai F, Ohashi H, Tsukuda S, Shimura S, Suzuki R, Aizaki H, Sugiyama M, Sam-Yong P, Ohtani N, Sugawara F, Tanaka Y, Mizokami M, Sureau C, Wakita T.
  • Organizer: International Meeting, Molecular Biology of Hepatitis B Viruses.
  • Place of Presentation: Dolce Bad Nauheim, Germany
  • Year and Date: 2015-10-04 – 2015-10-08
  • Int'l Joint Research
• [Presentation] B型肝炎ウイルスに対する直接標的薬proanthocyanidinおよび宿主標的薬vanitalacinの感染阻害効果および作用機序の解析. (2015)
  • Author(s): 渡士幸一、九十田千子、金子学、松永大輝、紙透伸治、齊藤安貴子、鈴木亮介、相崎英樹、杉山真也、溝上雅史、菅原二三男、田中靖人、脇田隆字.
  • Organizer: 第25回抗ウイルス療法学会総会、
  • Place of Presentation: 東京
  • Year and Date: 2015-05-22 – 2015-05-24
• [Presentation] Neoechinulin Bのliver X receptor転写阻害効果の同定およびそれによるC型肝炎ウイルス複製制御機構の解析. (2015)
  • Author(s): 渡士幸一、中嶋翔、紙透伸治、Jesus Izaguirre-Carbonell、Andrew Tae-Jun Kwon、鈴木治和、九十田千子、岡田麻衣子、鈴木亮介、相崎英樹、菅原二三男、脇田隆字.
  • Organizer: 第25回抗ウイルス療法学会総会
  • Place of Presentation: 東京
  • Year and Date: 2015-05-22 – 2015-05-24
• [Presentation] 新規真菌二次代謝産物 SF177 の B 型肝炎ウイルス感染阻害活性および NTCP トランスポーター阻害活性の同定. (2015)
  • Author(s): 金子学, 渡士幸一，紙透伸治, 岩本将士，河合文啓, 鈴木亮介，相崎英樹，朴三用, 菅原二三男, 脇田隆字.
  • Organizer: 第25回抗ウイルス療法学会総会
  • Place of Presentation: 東京
  • Year and Date: 2015-05-22 – 2015-05-24
• [Presentation] HCV複製複合体を含む膜小胞の形成メカニズムの解析. (2015)
  • Author(s): 相崎英樹、脇田隆字.
  • Organizer: 第51回日本肝臓学会総会
  • Place of Presentation: 熊本
  • Year and Date: 2015-05-21 – 2015-05-22
• [Book] C型肝炎治療における新時代の幕開け、C型肝炎ウイルスの複製・増殖のメカニズム (2016)
  • Author(s): 相崎英樹、脇田隆字
  • Total Pages: 4
  • Publisher: 医薬ジャーナル社
• [Book] 肝炎ウイルス検査のすすめ；身近にひそむ危険を防ぐ (2016)
  • Author(s): 相崎英樹、脇田隆字
  • Total Pages: 2
  • Publisher: 国民生活センター