Development of plaque assay to isolate cytopathic HCV clones

• Project/Area Number: 21590832
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Gastroenterology
• Research Institution: Tokyo Medical and Dental University
• Principal Investigator: OOOKA Shinya 東京医科歯科大学, 医学部・附属病院, 講師 (90361691)
• Co-Investigator(Kenkyū-buntansha): SAKAMOTO Naoya 東京医科歯科大学, 大学院・医歯学総合研究科, 寄付講座教員 (10334418) ; AZUMA Seishin 東京医科歯科大学, 医学部附属病院, 助教 (10376783) ; WATANABE Mamoru 東京医科歯科大学, 大学院・医歯学総合研究科, 教授 (10175127)
• Project Period (FY): 2009 – 2011
• Project Status: Completed (Fiscal Year 2011)
• Budget Amount: ¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000); Fiscal Year 2011: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2010: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2009: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
• Keywords: HCVレプリコン / HCV-JFH1培養系 / プラークアッセイ / 細胞障害効果 / ヒト肝臓キメラマウス / 小胞体ストレス / RNAポリメラーゼ

Research Abstract

HCV-JFH1 yields subclones that develop cytopathic plaques(Sekine-Osajima Y, et al., Virology 2008 ; 371 : 71). Here, we investigated viral amino acid substitutions in cytopathic mutant HCV-JFH1 clones and their characteristics in vitro and in vivo. The mutant viruses with individual C2441S, P2938S or R2985P signature substitutions, and with all three substitutions, showed significantly higher intracellular replication efficiencies and greater cytopathic effects than the parental JFH1 in vitro. The mutant HCV-inoculated mice showed significantly higher serum HCV RNA and higher level of expression of ER stress-related proteins in early period of infection. At 8 weeks post inoculation, these signature mutations had reverted to the wild type sequences. HCV-induced cytopathogenicity is associated with the level of intracellular viral replication and is determined by certain amino acid substitutions in HCV-NS5A and NS5B regions. The cytopathic HCV clones exhibit high replication competence in vivo but may be eliminated during the early stages of infection

Research Products

• [Journal Article] Inhibition of hepatitis C virus NS5B polymerase by S-trityl-L-cysteine derivatives (2012)
  • Author(s): Kaushik-Basu N, Sakamoto N
  • Journal Title: Eur J Med Chem Volume: 49 Pages: 191-199
  • DOI: 10.1016/j.ejmech.2012.01.010
  • Peer Reviewed
• [Journal Article] Age and total ribavirin dose are independent predictors of relapse after interferon therapy in chronic hepatitis C revealed by data mining analysis (2012)
  • Author(s): Kurosaki M, Sakamoto N, et al
  • Journal Title: Antivir Ther Volume: 17(1) Pages: 35-43
  • Peer Reviewed
• [Journal Article] Identification of novel N-(morpholine-4-carbonyloxy) amidine compounds as potent inhibitors against hepatitis C virus replication (2012)
  • Author(s): Kusano-Kitazume A, Sakamoto N, Okuno Y, Sekine-Osajima Y, Nakagawa M, Kakinuma S, Kiyonashi K, Nitta S, Murakawa M, Azuma S, Nishimura-Sakurai Y, Hagiwara M, Watanabe M
  • Journal Title: Antimicrob Agent Chemother Volume: 56(3) Issue: 3 Pages: 1315-23
  • DOI: 10.1128/aac.05764-11
  • Peer Reviewed / Open Access
• [Journal Article] Data mining model using simple and readily available factors could identify patients at high risk for hepatocellular carcinoma in chronic hepatitis C (2012)
  • Author(s): Kurosaki M, Sakamoto N, Nakagawa M, et al.
  • Journal Title: J Hepatol Volume: 56(3) Issue: 3 Pages: 602-608
  • DOI: 10.1016/j.jhep.2011.09.011
  • Peer Reviewed
• [Journal Article] The inhibitory effect on hepatitis C virus infection of a triterpenoid compound, with or without interferon-alpha (2011)
  • Author(s): Watanabe T, Sakamoto N, Nakagawa M, Kakinuma S, Itsui Y, Nishimura-Sakurai Y, Ueyama M, Funaoka Y, Kitazume A, Nitta S, Kiyohashi K, Murakawa M, Azuma S, Tsuchiya K, Oooka S, Watanabe M
  • Journal Title: Antimicrob Agents Chemother Volume: 55(6) Pages: 2537-2545
• [Journal Article] Association of gene expression involving innate immunity and genetic variation in IL28B with antiviral response (2011)
  • Author(s): Asahina Y, Sakamoto N, et al
  • Journal Title: Hepatology Volume: 55(1) Issue: 1 Pages: 20-29
  • DOI: 10.1002/hep.24623
  • Peer Reviewed
• [Journal Article] Analysis of the complete open reading frame of hepatitis C virus in genotype 2a infection reveals critical sites influencing the response to peginterferon and ribavirin therapy (2011)
  • Author(s): Kadokura M, Nakagawa M, Sakamoto N, Watanabe M, et al.
  • Journal Title: Hepatol Int Volume: 5(3) Issue: 3 Pages: 789-799
  • DOI: 10.1007/s12072-011-9267-x
  • Peer Reviewed
• [Journal Article] Use of human hepatocyte-like cells derived from induced pluripotent stem cells as a model for hepatocytes in hepatitis C virus infection (2011)
  • Author(s): Yoshida T, Sakamoto N, et al
  • Journal Title: Biochem Biophys Res Commun Volume: 416(1-2) Issue: 1-2 Pages: 119-124
  • DOI: 10.1016/j.bbrc.2011.11.007
  • Peer Reviewed
• [Journal Article] A new method for induced fit docking (GENIUS) and its application to virtual screening of novel HCV NS3-4A protease inhibitors (2011)
  • Author(s): Takaya D, Sakamoto N, et al.
  • Journal Title: Bioorganic & Medicinal Chemistry Volume: 19(22) Issue: 22 Pages: 6892-6905
  • DOI: 10.1016/j.bmc.2011.09.023
  • Peer Reviewed
• [Journal Article] Serum interleukin-6 levels correlate with resistance to treatment of chronic hepatitis C infection with pegylated-interferon-alpha2b plus ribavirin (2011)
  • Author(s): Ueyama M, Sakamoto N, et al
  • Journal Title: Antivir Ther Volume: 16 Pages: 1081-1091
  • Peer Reviewed
• [Journal Article] Genome-wide association study identified ITPA/DDRGK1 variants reflecting thrombocytopenia in pegylated interferon and ribavirin therapy for chronic hepatitis C (2011)
  • Author(s): Tanaka Y, Sakamoto N, et al.
  • Journal Title: Human Molecular Genetics Volume: 20(17) Issue: 17 Pages: 3507-3516
  • DOI: 10.1093/hmg/ddr249
  • Peer Reviewed
• [Journal Article] Pretreatment Prediction of Anemia Progression by Peginterferon Plus Ribavirin Therapy in Chronic Hepatitis C : Decision-Tree Analysis (2011)
  • Author(s): Hiramatsu N, Sakamoto N, et al.
  • Journal Title: J Gastroenterol Volume: 46(9) Issue: 9 Pages: 1111-1119
  • DOI: 10.1007/s00535-011-0412-z
  • Peer Reviewed
• [Journal Article] Analysis of the complete open reading frame of genotype 2b hepatitis C virus in association with the response to peginterferon and ribavirin therapy (2011)
  • Author(s): Kadokura M, Nakagawa M, Sakamoto N, Watanabe M, et al.
  • Journal Title: PLoS One Volume: 6(9) Issue: 9 Pages: e24514-e24514
  • DOI: 10.1371/journal.pone.0024514
  • Peer Reviewed
• [Journal Article] Pretreatment prediction of response to peginterferon plus ribavirin therapy in genotype 1 chronic hepatitis C using data mining analysis (2011)
  • Author(s): Kurosaki M, Sakamoto N, et al.
  • Journal Title: J Gastroenterol Volume: 46(3) Issue: 3 Pages: 401-409
  • DOI: 10.1007/s00535-010-0322-5
  • NAID: 10029028096
  • Peer Reviewed
• [Journal Article] Adenovirus vector-mediated assay system for hepatitis C virus replication (2011)
  • Author(s): Yoshida T, Sakamoto N, et al
  • Journal Title: Nucleic Acids Res Volume: 39(10) Issue: 10 Pages: e64-e64
  • DOI: 10.1093/nar/gkr047
  • Peer Reviewed
• [Journal Article] Analysis of interferon signaling by infectious hepatitis C virus clones with substitutions of core amino acids 70 and 91 (2011)
  • Author(s): Funaoka Y, Sakamoto N, Suda G, Itsui Y, Nakagawa M, Kakinuma S, Watanabe T, Nitta S, Kitazume A, Kiyohashi K, Murakawa M, Azuma S, Tsuchiya K,Watanabe M
  • Journal Title: J Virol Volume: 85 Issue: 12 Pages: 5986-94
  • DOI: 10.1128/jvi.02583-10
  • Peer Reviewed
• [Journal Article] Inhibitory effect of a triterpenoid compound, with or without alpha interferon, on hepatitis C virus infection (2011)
  • Author(s): Watanabe T, Sakamoto N, Nakagawa M, Azuma S, Watanabe M, et al.
  • Journal Title: Antimicrob Agents Chemother Volume: 55(6) Issue: 6 Pages: 2537-2545
  • DOI: 10.1128/aac.01780-10
  • Peer Reviewed
• [Journal Article] Studies on virus kinetics using infectious fluorescence-tagged hepatitis C virus cell culture (2011)
  • Author(s): Yamamoto M, Sakamoto N, Nakamura T, Itsui Y, Nakagawa M, Nishimura-Sakurai Y, Kakinuma S, Azuma S, Kato T, Wakita T, Watanabe M
  • Journal Title: Hepatol Res Volume: 41 Issue: 3 Pages: 258-269
  • DOI: 10.1111/j.1872-034x.2010.00771.x
  • Peer Reviewed
• [Journal Article] Association between lipid accumulation and the cannabinoid system in Huh7 cells expressing HCV genes (2011)
  • Author(s): Toyoda M, Sakamoto N, et al
  • Journal Title: Int J Mol Med Volume: 27 Issue: 5 Pages: 619-624
  • DOI: 10.3892/ijmm.2011.622
  • Peer Reviewed
• [Journal Article] Association of IL28B variants with response to pegylated-interferon alpha plus ribavirin combination therapy reveals intersubgenotypic differences between genotypes 2a and 2b (2011)
  • Author(s): Sakamoto N, Nakagawa M, Azuma S, Watanabe M, et al.
  • Journal Title: J Med Virol Volume: 83 Issue: 5 Pages: 871-878
  • DOI: 10.1002/jmv.22038
  • Peer Reviewed
• [Journal Article] Cholestatic liver fibrosis and toxin-induced fibrosis are exacerbated in matrix metalloproteinase-2 deficient mice (2011)
  • Author(s): Onozuka I, Sakamoto N, Nakagawa M, Watanabe M, et al.
  • Journal Title: Biochem Biophys Res Commun Volume: 406 Issue: 1 Pages: 134-140
  • DOI: 10.1016/j.bbrc.2011.02.012
  • Peer Reviewed
• [Journal Article] Pre-treatment prediction of response to pegylated-interferon plus ribavirin for chronic hepatitis C using genetic polymorphism in IL28B and viral factors (2011)
  • Author(s): Kurosaki M, Sakamoto N, Nakagawa M, Watanabe M, et al.
  • Journal Title: J Hepatol Volume: 54 Issue: 3 Pages: 439-448
  • DOI: 10.1016/j.jhep.2010.07.037
  • Peer Reviewed
• [Journal Article] Sequences in the interferon sensitivity-determining region and core region of hepatitis C virus impact pretreatment prediction of response to PEG-interferon plus ribavirin : data mining analysis (2011)
  • Author(s): Kurosaki M, Sakamoto N, Nakagawa M, et al.
  • Journal Title: J Med Virol Volume: 83 Issue: 3 Pages: 445-452
  • DOI: 10.1002/jmv.22005
  • Peer Reviewed
• [Journal Article] Cell culture and in vivo analyses of cytopathic hepatitis C virus mutants (2010)
  • Author(s): Mishima K, Sakamoto N, Sekine-Osajima Y, Nakagawa M, Itsui Y, Azuma S, Kakinuma S, Kiyohashi K, Kitazume A, Tsuchiya K, Imamura M, Hiraga N, Chayama K, Wakita T, Watanabe M
  • Journal Title: Virology Volume: 405 Pages: 361-369
• [Journal Article] SR蛋白リン酸化酵素阻害剤によるHCV増殖抑制効果の解析 (2010)
  • Author(s): 唐鎌優子、坂本直哉、井津井康浩、中川美奈、藤田めぐみ、櫻井幸、柿沼晴、大岡真也、東正新、土屋輝一郎、小野木博、萩原正敏、渡辺守
  • Journal Title: 消化器内科 Volume: 51(6) Pages: 632-635
• [Journal Article] Inhibition of HCV replication by a specific inhibitor of serin-arginine-rich protein kinase. (2010)
  • Author(s): Yuko Karakama, Naoya Sakamoto, Shinva Oooka, et al.
  • Journal Title: Antimicrobial Agent and Chemotherapy Volume: 54 Pages: 3179-31869
  • Peer Reviewed
• [Journal Article] Mutations in the Interferon Sensitivity Determining Region and virological response to combination therapy with Pegylated-interferon alpha 2b plus ribavirin in patients with chronic hepatitis C-1b infection. (2010)
  • Author(s): Nakagawa M, Sakamoto N(equal contribution), et al.
  • Journal Title: J Gastroenterol (in press)
  • NAID: 10027216778
  • Peer Reviewed
• [Journal Article] Two flavonoids extracts from Glycyrrhizae radix inhibit in vitro hepatitis C virus replication (2009)
  • Author(s): Sekine-Osajima Y, Sakamoto N, Nakagawa M, Itsui Y, Tasaka M, Nishimura-Sakurai Y, Chen CH, Suda G, Mishima K, Onuki Y, Yamamoto M, Maekawa S, Enomoto N, Kanai T, Tsuchiya K, Watanabe M
  • Journal Title: Hepatol Res Volume: 39 Pages: 60-69
  • NAID: 10025324061
• [Journal Article] Comparison of HCV-associated gene expression and cell signaling pathways in cells with or without HCV replicon and in replicon-cured cells. (2009)
  • Author(s): Nishimura-Sakurai Y, Sakamoto N, et al
  • Journal Title: J Gastroentorol
  • NAID: 10027216127
  • Peer Reviewed
• [Journal Article] Antiviral effects of the interferon-induced protein GBP-1 and its interaction with the hepatitis C virus NS5B protein. (2009)
  • Author(s): Itsui Y, Sakamoto N(equal contribution)
  • Journal Title: Hepatology 50 Pages: 1727-1737
  • Peer Reviewed
• [Presentation] ITPA gene variant protects against treatment-induced anemia and improves viral clearance by pegylated interferon-alfa and ribavirin therapy in chronic hepatitis C patients (2011)
  • Author(s): Sakamoto N, Azuma S, Watanabe M, et al
  • Organizer: 62th. Annual Meeting of American Association for the Study of Liver Diseases
  • Place of Presentation: San Francisco
  • Year and Date: 2011-11-04
• [Presentation] MMP-2 and MMP-14 derived from donor cells enhance therapeutic efficacy of liver cell transplantation in mice (2011)
  • Author(s): Kakinuma S, Sakamoto N, Watanabe M, et al
  • Organizer: 62th. Annual Meeting of American Association for the Study of Liver Diseases
  • Place of Presentation: San Francisco
  • Year and Date: 2011-11-04
• [Presentation] A high-content screening assay using infectious fluorescence-tagged hepatitis C virus reveals candidates for small molecule inhibitors of viral entry (2011)
  • Author(s): Kusano-Kitazume A, Sakamoto N, Watanabe M, et al
  • Organizer: 62th. Annual Meeting of American Association for the Study of Liver Diseases
  • Place of Presentation: San Francisco
  • Year and Date: 2011-11-04
• [Presentation] Loww of Wnt5A promotes biliary differentiation of murine hepatic stem/progenitor cells (2011)
  • Author(s): Kiyohashi K, Sakamoto N, Watanabe M, et al
  • Organizer: 62th. Annual Meeting of American Association for the Study of Liver Diseases
  • Place of Presentation: San Francisco
  • Year and Date: 2011-11-04
• [Presentation] Antiviral effect of a novel interferon-inducible protein, IFI-27, against hepatitis C virus replication (2011)
  • Author(s): Itsuil Y, Sakamoto N, Watanabe M, et al
  • Organizer: 62th. Annual Meeting of American Association for the Study of Liver Diseases
  • Place of Presentation: San Francisco
  • Year and Date: 2011-11-04
• [Presentation] IL-6-mediated intersubgenotypic variation of interferon sensitivity in hepatitis C virus genotype 2a/2b chimeric clones (2011)
  • Author(s): Suda G, Sakamoto N, Azuma S, Watanabe M, et al
  • Organizer: 62th. Annual Meeting of American Association for the study of Liver Diseases
  • Place of Presentation: San Francisco
  • Year and Date: 2011-11-04
• [Presentation] Prediction model of ribavirin-induced anemia incorporating ITPA genotype could identify chronic hepatitis C patients at high risk of relapse among virological responders to pegylated-interferon and ribavirin (2011)
  • Author(s): Kurosaki M, Sakamoto N, et al
  • Organizer: 62th. Annual Meeting of American Association for the Study of Liver Diseases
  • Place of Presentation: San Francisco
  • Year and Date: 2011-11-04
• [Presentation] IL28B and ITPA gene variants correlate with treatment efficacy in pegylated-interferon plus ribavirin therapy for chronic hepatitis C (2011)
  • Author(s): Matsuura K, Sakamoto N, et al
  • Organizer: 62th. Annual Meeting of American Association for the Study of Liver Diseases
  • Place of Presentation: San Francisco
  • Year and Date: 2011-11-04
• [Presentation] Mutations in the interferon sensitivity determining region of HCV, age and total ribavirin dose is an independent predictor of relapse among early virological responders to peg-interferon plus ribavirin therapy (2011)
  • Author(s): Kurosaki M, Sakamoto N, et al
  • Organizer: 62th. Annual Meeting of American Association for the study of Liver Diseases
  • Place of Presentation: San Francisco
  • Year and Date: 2011-11-04
• [Presentation] Development of an RNA polymerase I-driven adenoviral vector and its application in an HCV replication assay (2011)
  • Author(s): Yoshida T, Sakamoto N, et al
  • Organizer: 18th. International Meeting on Hepatitis C Virus & Related Viruses
  • Place of Presentation: Seattle
  • Year and Date: 2011-09-08
• [Presentation] Analysis of interferon signaling by infectious hepatitis C virus clones with substitutions of core amino acids 70 and 91 (2011)
  • Author(s): Sakamoto N, Watanabe M, et al
  • Organizer: 18th. International Meeting on Hepatitis C Virus & Related Viruses
  • Place of Presentation: Seattle
  • Year and Date: 2011-09-08
• [Presentation] HCV-NS4B targets STING and abrogates RIG-I-mediated type-I interferon- dependent innate immune response (2011)
  • Author(s): Nitta S, Sakamoto N, Watanabe M, et al
  • Organizer: 18th. International Meeting on Hepatitis C Virus & Related Viruses
  • Place of Presentation: Seattle
  • Year and Date: 2011-09-08
• [Presentation] Development of RNA pol-driven adenovirus vector expressing hepatitis C virus replicon (2011)
  • Author(s): Yamagishi Y, Sakamoto N, et al
  • Organizer: Experimental Biology 2011
  • Place of Presentation: Washington
  • Year and Date: 2011-04-13
• [Presentation] 細胞障害性HCV-JFH1における遺伝子変異の機能解析とin vivo感染動態の検討 (2010)
  • Author(s): 三島果子、坂本直哉、筬島裕子、中川美奈、柿沼晴、陳正新、脇田隆字、今村道雄、茶山一彰、渡辺守
  • Organizer: (一般口演)第46回日本肝臓学会総会
  • Place of Presentation: 山形
  • Year and Date: 2010-05-27
• [Presentation] Establishment and genetic analyses of cytopathogenic HCV-JFH1 mutants by plaque-forming assay (2009)
  • Author(s): Kako Mishima, Naoya Sakamoto, Yuko Sekine-Osajima, Mina Nakagawa, Megumi Tasaka, Yuki Nishimura-Sakurai, Yasuhiro Itsui, Takaji Wakita, and Mamoru Watanabe
  • Organizer: 16th. International Meeting on Hepatitis C Virus & Related Viruses
  • Place of Presentation: Nice, France
  • Year and Date: 2009-10-03
• [Presentation] 細胞傷害性HCV-JFH1subcloneの単離と機能解析.(ワークショップ3) (2009)
  • Author(s): 三島果子坂本直哉中川美奈井津井康浩筬島裕子田坂めぐみ櫻井幸須田剛生山本満千植山真由美渡辺貴子船岡祐介渡辺守
  • Organizer: 第45回日本肝臓学会総会
  • Place of Presentation: 神戸
  • Year and Date: 2009-06-04
• [Presentation] 細胞障害性HCV-JFH1subcloneの単離と機能解析
  • Author(s): 三島果子、坂本直哉、筬島裕子、中川美奈、藤田めぐみ、櫻井幸、須田剛生、唐鎌優子、山本満千、渡辺貴子、植山真由美、船岡祐介、幾世橋佳、新田沙由梨、北詰晶子、井津井康浩、柿沼晴、陳正新、脇田隆字、渡辺守
  • Organizer: 京都(ポスター)
• [Patent(Industrial Property Rights)] C型肝炎ウイルスの増殖を抑制する医薬組成物 (2011)
  • Inventor(s): 坂本直哉、渡辺守、北詰晶子、萩原正敏、奥野友紀子
  • Industrial Property Rights Holder: 東京医科歯科大学
  • Industrial Property Number: 2011-194082
  • Filing Date: 2011-09-06