C型肝炎ウイルス複製に関与する宿主因子の同定とウイルス複製機構の解析

• Project/Area Number: 19590799
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Gastroenterology
• Research Institution: National Institute of Infectious Diseases
• Principal Investigator: 相崎 英樹 National Institute of Infectious Diseases, ウイルス第二部, 主任研究官 (00333360)
• Co-Investigator(Kenkyū-buntansha): 鈴木 哲朗 国立感染研究所, ウイルス第二部, 室長 (00250184)
• Project Period (FY): 2007 – 2008
• Project Status: Completed (Fiscal Year 2008)
• Budget Amount: ¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000); Fiscal Year 2008: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000); Fiscal Year 2007: ¥2,730,000 (Direct Cost: ¥2,100,000、Indirect Cost: ¥630,000)
• Keywords: C型肝炎ウイルス / 複製複合体 / プロテオミックス / レプリコン / creatine kinase B / 界面性剤不溶性膜分画 / 脂質ラフト / サブゲノムレプリ / 界面活性剤不溶性膜分画 / 分子シャペロン / NS5B蛋白

Research Abstract

HCVは感染細胞内でウイルス蛋白と宿主因子からなる複製複合体を形成し効率良くウイルス複製を行っていると考えられている。本研究はプロテオミックス技術により、HCV複製複合体(RC)を構成する宿主因子を同定し、その機能を解析することを目的とした。
サブゲノムレプリコン(genotype 1b)を有するHuh7細胞からHCVRCを含む界面活性剤不溶性膜分画を抽出し、Huh7細胞と比較したところ、の結果、約1500個の蛋白が検出され、62個がレプリコン細胞で2倍以上蛋白量が増加しており、それらはHeat shock protein、Chaperone、Enzyme、Receptor signaling complex、Ubiquitin proteasome system proteinなどに分類された。
HCV複製複合体を構成する宿主因子として、細胞内のATP輸送、ATP/ADP濃度調節に働くcreatine kinase B(CKB)を同定した。遺伝子型1b及び2a由来のHCVレプリコン細胞において、CKB遺伝子のノックダウン、dominant negative CKBの強制発現あるいはCKB阻害剤cyclocreatine処理によりHCV RNA複製の有意な抑制が観察されCKBがHCV RNA複製調節に関与することが明らかとなった。さらに、セミインタクト細胞を用いたHCV replicaseアッセイ系において、CKB添加によりHCV複製が亢進されることを見出した。

Research Products

• [Journal Article] SYNCRIP (synaptotagmin-binding, cytoplasmic RNA-interacting protein) is a host factor involved in hepatitis C virus RNA replication (2009)
  • Author(s): Liu HM, Aizaki H, Choi KS, Machida K, Ou JJ, Lai MM.
  • Journal Title: Virology 386 Pages: 249-56
  • Peer Reviewed
• [Journal Article] Involvement of creatine kinase B in hepatitis C virus genome replication through interaction with the viral NS4A protein (2009)
  • Author(s): Hara H, Aizaki H, Matsuda M, Shinkai-Ouchi F, Inoue Y, Murakami K, Shoji I, Kawakami H, Matsuura Y, Lai MM, Miyamura T, Wakita T, Suzuki T.
  • Journal Title: J Virol. 84 Pages: 5137-47
  • Peer Reviewed
• [Journal Article] Proteasomal turnover of hepatitis C virus core protein is regulated by two distinct mechanisms : ubiquitin-dependent and ubiquitin-independent but PA2 8gamma-dependent (2009)
  • Author(s): Suzuki, R., Moriishi, K., Fukuda, K., Shirakura, M., Ishii, K., Wakita T., Miyamura, T., Matsuura, Y., Suzuki, T.
  • Journal Title: J. Virol. 83 Pages: 2389-2392
  • Peer Reviewed
• [Journal Article] Cellular vimentin content regulates the protein level of Hepatitis C virus core protein and the Hepatitis C virus production in cultured cells (2009)
  • Author(s): Nitahara-Kasahara Y, Fukasawa M, Shinkai-Ouchi F, Sato S, Suzuki T, Murakami K, Wakita T, Hanada K, Miyamura T, Nishijima M.
  • Journal Title: Virology 383 Pages: 319-327
  • Peer Reviewed
• [Journal Article] A Critical Role of Virion-Associated Cholesterol and Sphingolipid in Hepatitis C Virus Infection (2008)
  • Author(s): Aizaki H, Morikawa K, Fukasawa M, Hara H, Inoue Y, Tani H, Saito K, Nishijima M, Hanada K, Matsuura Y, Lai MM, Miyamura T, Wakita T, Suzuki T.
  • Journal Title: J Virol. 82 Pages: 5715-24
  • Peer Reviewed
• [Journal Article] Dynamic behavior of hepatitis C virus quasispecies in a long-term culture of the three-dimensional radial-flow bioreactor system (2008)
  • Author(s): Murakami K, Inoue Y, Hmwe SS, Omata K, Hongo T, Ishii K, Yoshizaki S, Aizaki H, Matsuura T, Shoji I, Miyamura T. Suzuki T
  • Journal Title: J Virol Methods 148 Pages: 174-181
  • Peer Reviewed
• [Journal Article] Interaction of hepatitis C virus nonstructural protein 5A with core protein is critical for the production of infectious virus particles (2008)
  • Author(s): Masaki T, Suzuki R, Murakami K, Aizaki H, Ishii K, Murayama A, Date T, Matsuura Y, Miyamura T, Wakita T, Suzuki T.
  • Journal Title: J Virol. 82 Pages: 7964-76
  • Peer Reviewed
• [Journal Article] Characterzation of infectious hepatitis C virus from liver-derived cell lines (2008)
  • Author(s): Akazawa D, Date T, Morikawa K, Murayama A, Omi N, Takahashi H, Nakamura N, Ishii K, Suzuki T, Mizokami M, Mochizuki H, Wakita T.
  • Journal Title: Biochem. Biophys. Res. Commun. 377 Pages: 747-751
  • Peer Reviewed
• [Journal Article] Trans-encapsidation of hepatitis C virus subgenomic replicon RNA with viral structure proteins (2008)
  • Author(s): Ishii, K., Murakami, K., Hmwe, SS., Bin, Z., Li, J., Shirakura, M., Morikawa, K., Suzuki, R., Miyamura, T., Wakita, T., Suzuki, T.
  • Journal Title: Biochem. Biophys. Res. Commun. 371 Pages: 446-450
  • Peer Reviewed
• [Journal Article] Intramembrane processing by signal peptide peptidase regulates the membrane localization of hepatitis C virus core protein and viral propagation (2008)
  • Author(s): Okamoto K., Mori Y., Komoda Y., Okamoto T., Okochi M., Takeda M., Suzuki T., Moriishi K., Matsuura Y.
  • Journal Title: J. Virol. 82 Pages: 8349-8361
  • Peer Reviewed
• [Journal Article] A single amino acid mutation in hepatitis C virus NS5A disrupting FKBP8 interaction impairs viral replication (2008)
  • Author(s): Okamoto T., Omori H., Kaname Y., Abe T., Nishimura Y., Suzuki T., Miyamura T., Yoshimori T., Moriishi K., Matsuura Y.
  • Journal Title: J. Virol. 82 Pages: 3480-3489
  • Peer Reviewed
• [Journal Article] Identification and characterization of the human inhibitor of caspase-activated DNase gene promoter (2008)
  • Author(s): Omata, K., Suzuki, R., Masaki, T., Miyamura, T., Satoh, T., Suzuki, T.
  • Journal Title: Apoptosis 13 Pages: 929-937
  • Peer Reviewed
• [Journal Article] Virological characterization of HCV JFH-1 strain in lymphocytic cell lines (2008)
  • Author(s): Murakami, K., Kimura, T., Osaki, M., Ishii, K., Miyamura, T., Suzuki, T., Wakita, T., Shoii, I.
  • Journal Title: J. Gen. Virol. 89 Pages: 1587-1592
  • Peer Reviewed
• [Journal Article] ヒト肝癌細胞の三次元培養化に伴う遺伝子発現変動の網羅的解析 (2008)
  • Author(s): 葛岡健太郎, 岩田耕一郎, 吉崎佐矢香, 相崎英樹, 鈴木哲朗, 長尾垣
  • Journal Title: 東京医大誌 66 Pages: 212-223
  • NAID: 10024169293
  • Peer Reviewed
• [Journal Article] (2008)
  • Author(s): 鈴木哲朗, 政木隆博, 相崎英樹
  • Journal Title: ウイルス(日本ウイルス学会)
• [Journal Article] (2008)
  • Author(s): Aizaki H, Suzuki T.
  • Journal Title: Structure-based Study of Viral Replication(World Scientific)
• [Journal Article] A Critical Role of Virion-Associated Cholesterol and Sphingolipid in Hepatitis C Virus Infection (2008)
  • Author(s): Aizaki H, Morikawa K, Fukasawa M, Hara H, Inoue Y, Tani H, Saito K, Nishijima M, Hanada K, Matsuura Y, Lai MM, Miyamura T, Wakita T, Suzuki T.
  • Journal Title: J Virol. (In press)
  • Peer Reviewed
• [Journal Article] Dynamic behavior of hepatitis C virus quasispecies in a long-term culture of the three-dimensional radial-flow bioreactor system. (2008)
  • Author(s): Murakami K, Inoue Y, Hmwe SS, Omata K, Hongo T, Ishii K, Yoshizaki S, Aizaki H, Matsuura T, Shoji I, Miyamura T, Suzuki T
  • Journal Title: J Virol Methods 148 Pages: 174-181
  • Peer Reviewed
• [Journal Article] A Single Amino Acid Mutation in Hepatitis C Virus NS5A Disrupting FKBP8 Interaction Impairs Viral Replication. (2008)
  • Author(s): Okamoto T, Omori H, Kaname Y, Abe T, Nishimura Y, Suzuki T, Miyamura T, Yoshimori T, Moriishi K, Matsuura Y.
  • Journal Title: J. Virol. 82 Pages: 3480-3489
  • Peer Reviewed
• [Journal Article] Trans-encapsidation of hepatitis C virus subgenomic replicon RNA with viral structure proteins. (2008)
  • Author(s): Ishii K, Murakami K, Hmwe SS, Zhang B, Li J, Shirakura M, Morikawa K, Suzuki R, Miyamura T, Wakita T, Suzuki T.
  • Journal Title: Biochem Biophys Res Commun. (In press)
  • Peer Reviewed
• [Journal Article] Transcriptomic comparison of human hepatoma Huh-7 cell clones with different hepatitis C virus replication efficiencies. (2007)
  • Author(s): Inoue Y, Murakami K, Hmwe SS, Aizaki H, Suzuki T.
  • Journal Title: Jpn J Infect Dis. 60 Pages: 173-178
  • Peer Reviewed
• [Journal Article] Hepatitis C viral life cycle. (2007)
  • Author(s): Suzuki T, Ishii K, Aizaki H, Wakita T
  • Journal Title: Adv Drug Deliv Rev 59 Pages: 1200-1212
• [Journal Article] Molecular biology of hepatitis C virus. (2007)
  • Author(s): Suzuki T., Aizaki H, Murakami K, Shoji I, Wakita T
  • Journal Title: J Gastroenterol 42 Pages: 411-423
  • NAID: 40016676275
• [Journal Article] Human Butyrate-Induced Transcript 1 Interacts with Hepatitis C Virus NS5A and Regulates Viral Replication. (2007)
  • Author(s): Taguwa S, Okamoto T, Abe T, Mori Y, Suzuki T, Moriishi K, Matsuura Y.
  • Journal Title: J. Virol. 82 Pages: 2631-2641
  • Peer Reviewed
• [Journal Article] E6AP ubiquitin ligase mediates ubiquitylation and degradation of hepatitis C virus core protein. (2007)
  • Author(s): Shirakura M, Murakami K, Ichimura T, Suzuki R, Shimoji T, Fukuda K, Abe K, Sato S, Fukasawa M, Yamakawa Y, Nishijima M, Moriishi K, Matsuura Y, Wakita T, Suzuki T, Howley PM, Miyamura T, Shoji I.
  • Journal Title: J. Virol. 81 Pages: 1174-1185
  • Peer Reviewed
• [Journal Article] Involvement of the PA28gamma-dependent pathway in insulin resistance induced by hepatitis C virus core protein. (2007)
  • Author(s): Miyamoto H, Moriishi K, Moriya K, Murata S, Tanaka K, Suzuki T, Miyamura T, Koike K, Matsuura Y.
  • Journal Title: J. Virol. 81 Pages: 1727-1735
  • Peer Reviewed
• [Journal Article] Critical role of PA28gamma in hepatitis C virus-associated steatogenesis and hepatocarcinogenesis. (2007)
  • Author(s): Moriishi K, Mochizuki R, Moriya K, Miyamoto H, Mori Y, Abe T, Murata S, Tanaka K, Miyamura T, Suzuki T, Koike K, Matsuura Y.
  • Journal Title: Proc.Natl.Acad.Sci.USA. 104 Pages: 1661-1666
  • Peer Reviewed
• [Journal Article] GBV-B as a pleiotropic virus:distribution of GBV-B in extrahepatic tissues in vivo. (2007)
  • Author(s): Ishii K, lijima S, Kimura N, Lee Y, J, Ageyama N, Yagi S, Yamaguchi K, Maki N, Mori K, Yoshizaki S, Machida S, Suzuki T, Iwata N, Sata T, Terao K, Miyamura T, Akari H.
  • Journal Title: Microbes Infect. 9 Pages: 515-521
  • Peer Reviewed
• [Journal Article] Rapid genome sequencing of RNA viruses. (2007)
  • Author(s): Mizutani T, Endoh D, Shirato K, Shimizu H, Fukushi S, Saijo M, Sakai K, Kwang L, Ito M, Nerome R, Takasaki T, Ishii K, Suzuki T, Kurane I, Morikawa S, Nishimura H.
  • Journal Title: Emerg.Infect.Dis. 13 Pages: 322-324
  • NAID: 120005761147
  • Peer Reviewed
• [Journal Article] The NS3 helicase and NS5B-to-3'X regions are important for efficient hepatitis C virus strain JFH-1 replication in Huh7 cells. (2007)
  • Author(s): Murayama A, Date T, Morikawa K, Akazawa D, Miyamoto M, Kaga M, Ishii K, Suzuki T, Kato T, Mizokami M, Wakita T.
  • Journal Title: J.Virol. 81 Pages: 8030-8040
  • Peer Reviewed
• [Presentation] Interaction of hepatitis C virus nonstructural protein 5A with core protein is critical for the production of infectious virus particles (2008)
  • Author(s): Suzuki T.
  • Organizer: The 50th Anniversary Meeting of the International Association for the Study of the Liver
  • Place of Presentation: San Francisco, USA
• [Presentation] Involvement of creatine kinase B in hepatitis C virus genome replication through interaction with the viral NS4A protein (2008)
  • Author(s): Hara H, Aizaki H, Matsuda M, Murakami K, Shoji I, Miyamura T, Wakita T, Suzuki T.
  • Organizer: 15th International Symposium on Hepatitis C Virus and Related Viruses
  • Place of Presentation: SanAntonio, USA
• [Presentation] The C-terminal serine of NS5A is a determinant of NS5A-core protein interaction and HCV production (2008)
  • Author(s): Masaki R, Suzuki R, Murakami K, Aizaki H, Ishii K Miyamura T, Wakita T, Suzuki T.
  • Organizer: 15th International Symposium on Hepatitis C Virus and Related Viruses.
  • Place of Presentation: SanAntonio, USA
• [Presentation] Critical roles of virion-associated cholesterol and sphingolipids in the viral infectivity. (2007)
  • Author(s): Aizaki H, Suzuki T, et. al.
  • Organizer: 14^<th> International Meeting on Hepatitis C Virus and Related Viruses
  • Place of Presentation: Glasgow,UK
• [Presentation] Analysis of NS5A region for hepatitis C virus particle production. (2007)
  • Author(s): Masaki T, Suzuki T, et. al.
  • Organizer: 14^<th> International Meeting on Hepatitis C Virus and Related Viruses
  • Place of Presentation: Glasgow,UK
• [Presentation] Cellular vimentin content affects the protein level of hepatitis C virus core protein and the activity of hepatitis C virus production in cultured cells. (2007)
  • Author(s): Fukasawa M, Suzuki T, et. al.
  • Organizer: 14^<th> International Meeting on Hepatitis C Virus and Related Viruses
  • Place of Presentation: Glasgow,UK
• [Presentation] An adaptive mutation in E2 glycoprotein allows an efficient production of HCV particles with the epitope-tagged envelope. (2007)
  • Author(s): Takahashi H, Suzuki T, et. al.
  • Organizer: 14^<th> International Meeting on Hepatitis C Virus and Related Viruses
  • Place of Presentation: Glasgow,UK
• [Presentation] The cholesterol and sphingolipids of hepatitis C virus particles play critical roles in the viral infectivity (2007)
  • Author(s): Aizaki H, Fukasawa M, et. al.
  • Organizer: The 58^<th> annual meeting of the American association for the study of liver diseases
  • Place of Presentation: Boston
• [Presentation] Analysis of NS5A region important for hepatitis C virus particle production. (2007)
  • Author(s): Masaki T, Suzuki T, et. al.
  • Organizer: The 58^<th> annual meeting of the American association for the study of liver diseases
  • Place of Presentation: Boston
• [Presentation] Production of infectious hepatitis C virus by using RNA polymerase I-mediated transcription. (2007)
  • Author(s): Masaki T, Suzuki T, et. al.
  • Organizer: 第66回日本癌学会学術総会
  • Place of Presentation: 横浜
• [Presentation] 脂質のC型肝炎ウイルス感染、粒子形成における役割 (2007)
  • Author(s): 相崎 英樹、原弘 道、森川 賢一、宮村 達男、脇田 隆字、鈴木 哲朗
  • Organizer: 第43回日本肝臓学会総会
  • Place of Presentation: 東京
• [Presentation] 遺伝子型2a/2b間でのキメラウイルスの作製および性状解析 (2007)
  • Author(s): 伊達 朋子、鈴木 哲朗, 他
  • Organizer: 第55回日本ウイルス学会学術集会
  • Place of Presentation: 札幌
• [Presentation] C型肝炎ウイルスコア蛋白質のシグナルペプチドペプチダーゼによるプロセシングの生物学的意義. (2007)
  • Author(s): 森 嘉生、鈴木 哲朗, 他
  • Organizer: 第55回日本ウイルス学会学術集会
  • Place of Presentation: 札幌
• [Presentation] HCV JFH-1株の複製および感染性ウイルス粒子形成に重要な領域の解析 (2007)
  • Author(s): 村山 麻子、鈴木 哲朗, 他.
  • Organizer: 第55回日本ウイルス学会学術集会
  • Place of Presentation: 札幌
• [Presentation] HCVコア蛋白と結合する新規宿主因子hnRNP H1/H2の同定と相互作用解析 (2007)
  • Author(s): 阿部 克俊、鈴木 哲朗, 他
  • Organizer: 第55回日本ウイルス学会学術集会
  • Place of Presentation: 札幌
• [Presentation] 新規阻害剤探索のためのHCV感染増殖アッセイ系の構築とその評価 (2007)
  • Author(s): 上西 理恵、鈴木 哲朗, 他
  • Organizer: 第55回日本ウイルス学会学術集会
  • Place of Presentation: 札幌
• [Presentation] 細胞培養系により産生されたHCVウイルスの免疫原性に関する検討 (2007)
  • Author(s): 尾見 法昭、鈴木 哲朗, 他
  • Organizer: 第55回日本ウイルス学会学術集会
  • Place of Presentation: 札幌
• [Presentation] C型肝炎ウイルス脂質成分の感染における役割 (2007)
  • Author(s): 相崎 英樹、鈴木 哲朗, 他
  • Organizer: 第55回日本ウイルス学会学術集会
  • Place of Presentation: 札幌
• [Book] C型肝炎に関する最近の情報 (2008)
  • Author(s): 相崎 英樹、鈴木 哲朗、多田 有希、岡部 信彦、脇田 隆字
  • Publisher: 結核予防会機関誌「複十字」