Cell culture system for genotype1b hepatitis C virus

2011 Fiscal Year Final Research Report

• Project/Area Number: 21390235
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Gastroenterology
• Research Institution: National Institute of Infectious Diseases
• Principal Investigator: WAKITA Takaji 国立感染症研究所, ウイルス第二部, 部長 (40280789)
• Project Period (FY): 2009 – 2011
• Keywords: 肝臓学

Research Abstract

Hepatitis C virus(HCV) infection causes chronic liver diseases and is a worldwide health problem. Despite the increasing demand for the knowledge of viral replication and pathogenesis, detail analysis of viral life cycle has been hampered by the lack of efficient viral culture system. In this study, we aimed to establish genotype1b HCV culture system since genotype1b infection is most prevalent and resistant for interferon therapy. We isolate a new genotype1b HCV clone and constructed full-length cDNA of viral genome. By introduction of adaptive mutants, we could observe viral replication and infectious virus particle formation and secretion. We also constructed chimeric virus using genotype 1b and 2a. With this chimeric virus constructs, we analyzed the anti-viral sensitivity profiles. In conclusion, genotype1b HCV culture system is useful and important for anti-viral development and analysis of drug resistant profile of HCV.

Research Products

• [Journal Article] Replication and infectivity of a novel genotype 1b hepatitis C virus clone (2012)
  • Author(s): Date T, Morikawa K, Tanaka Y, Tanaka-Kaneko K, Sata T, Mizokami M, Wakita T
  • Journal Title: Microbiol Immunol
  • DOI: doi:10.1111/j.1348-0421.2012.00437.x
• [Journal Article] Detergent-induced activation of the hepatitis C virus genotype 1b RNA polymerase (2012)
  • Author(s): Weng L, Kohara M, Wakita T, Shimotohno K, Toyoda T
  • Journal Title: Gene Volume: 496(2) Pages: 79-87
• [Journal Article] Potent and selective inhibition of hepatitis C virus replication by novel phenanthridinone derivatives (2011)
  • Author(s): Salim MT, Aoyama H, Sugita K, Watashi K, Wakita T, Hamasaki T, Okamoto M, Urata Y, Hashimoto Y, Baba M
  • Journal Title: Biochem Biophys Res Commun Volume: 415(4) Pages: 714-9
• [Journal Article] Genetic Variation of the IL-28B Promoter Affecting Gene Expression (2011)
  • Author(s): Sugiyama M, Tanaka Y, Wakita T, Nakanishi M, Mizokami M
  • Journal Title: PLoS One Volume: 6(10) Pages: e26620
• [Journal Article] Hepatitis C virus reveals a novel early control in acute immune response (2011)
  • Author(s): Arnaud N, Dabo S, Akazawa D, Fukasawa M, Shinkai-Ouchi F, Hugon J, Wakita T, Meurs EF
  • Journal Title: PLoS Pathog Volume: 7(10) Pages: e1002289
• [Journal Article] Role of the endoplasmic reticulum-associated degradation(ERAD) pathway in degradation of hepatitis C virus envelope proteins and production of virus particles (2011)
  • Author(s): Saeed M, Suzuki R, Watanabe N, Masaki T, Tomonaga M, Muhammad A, Kato T, Matsuura Y, Watanabe H, Wakita T, Suzuki T
  • Journal Title: J Biol Chem Volume: 286(43) Pages: 37264-73
• [Journal Article] Development of recombinant hepatitis C virus with NS5A from strains of genotypes 1 and 2 (2011)
  • Author(s): Okamoto Y, Masaki T, Murayama A, Munakata T, Nomoto A, Nakamoto S, Yokosuka O, Watanabe H, Wakita T, Kato T
  • Journal Title: Biochem Biophys Res Commun Volume: 410(3) Pages: 404-9
• [Journal Article] Structural requirements of virion-associated cholesterol for infectivity, buoyant density and apolipoprotein association of hepatitis C virus (2011)
  • Author(s): Yamamoto M, Aizaki H, Fukasawa M, Teraoka T, Miyamura T, Wakita T, Suzuki T
  • Journal Title: J Gen Virol Volume: 92(Pt9) Pages: 2082-7
• [Journal Article] Production and characterization of HCV particles from serum-free culture (2011)
  • Author(s): Akazawa D, Morikawa K, Omi N, Takahashi H, Nakamura N, Mochizuki H, Date T, Ishii K, Suzuki T, Wakita T
  • Journal Title: Vaccine Volume: 29(29-30) Pages: 4821-8
• [Journal Article] In vivo adaptation of hepatitis C virus in chimpanzees for efficient virus production and evasion of apoptosis (2011)
  • Author(s): Saeed M, Shiina M, Date T, Akazawa D, Watanabe N, Murayama A, Suzuki T, Watanabe H, Hiraga N, Imamura M, Chayama K, Choi Y, Krawczynski K, Liang TJ, Wakita T, Kato T
  • Journal Title: Hepatology Volume: 54(2) Pages: 425-33
• [Journal Article] Hokuriku Liver Study Group. Malnutrition impairs interferon signaling through mTOR and FoxO pathways in patients with chronic hepatitis C (2011)
  • Author(s): Honda M, Takehana K, Sakai A, Tagata Y, Shirasaki T, Nishitani S, Muramatsu T, Yamashita T, Nakamoto Y, Mizukoshi E, Sakai Y, Yamashita T, Nakamura M, Shimakami T, Yi M, Lemon SM, Suzuki T, Wakita T, Kaneko S
  • Journal Title: Gastroenterology Volume: 141(1) Pages: 128-140
• [Journal Article] Development of mouse hepatocyte lines permissive for hepatitis C virus(HCV) (2011)
  • Author(s): Aly HH, Oshiumi H, Shime H, Matsumoto M, Wakita T, Shimotohno K, Seya T
  • Journal Title: PLoS One Volume: 6(6) Pages: e21284
• [Journal Article] Impaired antiviral activity of interferon alpha against hepatitis C virus 2a in Huh-7 cells with a defective Jak-Stat pathway (2010)
  • Author(s): Hazari S, Chandra PK, Poat B, Datta S, Garry RF, Foster TP, Kousoulas G, Wakita T, Dash S
  • Journal Title: Virol J Volume: 7(1) Pages: 36
• [Journal Article] 2', 5'-Oligoadenylate synthetase-like gene highly induced by hepatitis C virus infection in human liver is inhibitory to viral replication in vitro (2010)
  • Author(s): Ishibashi M, Wakita T, Esumi M
  • Journal Title: Biochem Biophys Res Commun Volume: 392(3) Pages: 397-402
• [Journal Article] Identification of hepatitis C virus genotype 2a replicon variants with reduced susceptibility to ribavirin (2010)
  • Author(s): Hmwe SS, Aizaki H, Date T, Murakami K, Ishii K, Miyamura T, Koike K, Wakita T, Suzuki T
  • Journal Title: Antiviral Res Volume: 85(3) Pages: 520-524
• [Journal Article] Requirement of cellular DDX3 for hepatitis C virus replication is unrelated to its interaction with the viral core protein (2010)
  • Author(s): Angus AG, Dalrymple D, Boulant S, McGivern DR, Clayton RF, Scott MJ, Adair R, Graham S, Owsianka AM, Targett-Adams P, Li K, Wakita T, McLauchlan J, Lemon SM, Patel AH
  • Journal Title: J Gen Virol Volume: 91(1) Pages: 122-32
• [Journal Article] Ethanol enhances hepatitis C virus replication through lipid metabolism and elevated NADH/NAD+ (2010)
  • Author(s): Seronello S, Ito C, Wakita T, Choi J
  • Journal Title: J Biol Chem Volume: 285(2) Pages: 845-54
• [Journal Article] Infectivity of hepatitis C virus is influenced by association with apolipoprotein E isoforms (2010)
  • Author(s): Hishiki T, Shimizu Y, Tobita R, Sugiyama K, Ogawa K, Funami K, Ohsaki Y, Fujimoto T, Takaku H, Wakita T, Baumert TF, Miyanari Y, Shimotohno K
  • Journal Title: J Virol Volume: 84(22) Pages: 12048-57
• [Journal Article] Hepatitis C virus NS2 protein triggers endoplasmic reticulum stress and suppresses its own viral replication (2010)
  • Author(s): von dem Bussche A, Machida R, Li K, Loevinsohn G, Khander A, Wang J, Wakita T, Wands JR, Li J
  • Journal Title: J Hepatol Volume: 53(5) Pages: 797-804
• [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(2) Pages: 361-9
• [Journal Article] Production of infectious hepatitis C virus in primary cultures of human adult hepatocytes (2010)
  • Author(s): Podevin P, Carpentier A, Pene V, Aoudjehane L, Carriere M, Zaidi S, Hernandez C, Calle V, Meritet JF, Scatton O, Dreux M, Cosset FL, Wakita T, Bartenschlager R, Demignot S, Conti F, Rosenberg AR, Calmus Y
  • Journal Title: Gastroenterology Volume: 139(4) Pages: 1355-64
• [Journal Article] A disulfide-bonded dimer of the core protein of hepatitis C virus is important for virus-like particle production (2010)
  • Author(s): Kushima Y, Wakita T, Hijikata M
  • Journal Title: J Virol Volume: 84(18) Pages: 9118-27
• [Journal Article] Primary hepatocyte culture supports hepatitis C virus replication : a model for infection-associated hepatocarcinogenesis (2010)
  • Author(s): Banaudha K, Orenstein JM, Korolnek T, St Laurent GC 3rd, Wakita T, Kumar A
  • Journal Title: Hepatology Volume: 51(6) Pages: 1922-32
• [Journal Article] La protein required for internal ribosome entry site-directed translation is a potential therapeutic target for hepatitis C virus replication (2010)
  • Author(s): Shirasaki T, Honda M, Mizuno H, Shimakami T, Okada H, Sakai Y, Murakami S, Wakita T, Kaneko S
  • Journal Title: J Infect Dis Volume: 202(1) Pages: 75-85
• [Journal Article] Hepatitis C virus controls interferon production through PKR activation (2010)
  • Author(s): Arnaud N, Dabo S, Maillard P, Budkowska A, Kalliampakou KI, Mavromara P, Garcin D, Hugon J, Gatignol A, Akazawa D, Wakita T, Meurs EF
  • Journal Title: PLoS One Volume: 5(5) Pages: e10575
• [Journal Article] RNA polymerase activity and specific RNA structure are required for efficient HCV replication in cultured cells (2010)
  • Author(s): Murayama A, Weng L, Date T, Akazawa D, Tian X, Suzuki T, Kato T, Tanaka Y, Mizokami M, Wakita T, Toyoda T
  • Journal Title: PLoS Pathog Volume: 6(4) Pages: e1000885
• [Journal Article] Biological properties of purified recombinant HCV particles with an epitope-tagged envelope (2010)
  • Author(s): Takahashi H, Akazawa D, Kato T, Date T, Shirakura M, Nakamura N, Mochizuki H, Tanaka-Kaneko K, Sata T, Tanaka Y, Mizokami M, Suzuki T, Wakita T
  • Journal Title: Biochem Biophys Res Commun Volume: 395(4) Pages: 565-71
• [Journal Article] Production of infectious hepatitis C virus by using RNA polymerase I-mediated transcription (2010)
  • Author(s): Masaki T, Suzuki R, Saeed M, Mori K, Matsuda M, Aizaki H, Ishii K, Maki N, Miyamura T, Matsuura Y, Wakita T, Suzuki T
  • Journal Title: J Virol Volume: 84(11) Pages: 5824-35
• [Journal Article] HCV genotype 1b chimeric replicon with NS5B of JFH-1 exhibited resistance to cyclosporine A (2009)
  • Author(s): Abe K, Ikeda M, Ariumi Y, Dansako H, Wakita T, Kato N
  • Journal Title: Arch Virol Volume: 154(10) Pages: 1671-7
• [Journal Article] Evaluation of hepatitis C virus core antigen assays in detecting recombinant viral antigens of various genotypes (2009)
  • Author(s): Saeed M, Suzuki R, Kondo M, Aizaki H, Kato T, Mizuochi T, Wakita T, Watanabe H, Suzuki T
  • Journal Title: J Clin Microbiol Volume: 47(12) Pages: 4141-3
• [Journal Article] Identification of bisindolylmaleimides and indolocarbazoles as inhibitors of HCV replication by tube-capture-RT-PCR (2009)
  • Author(s): Murakami Y, Noguchi K, Yamagoe S, Suzuki T, Wakita T, Fukazawa H
  • Journal Title: Antiviral Res Volume: 83(2) Pages: 112-7
• [Journal Article] Double-stranded RNA-induced interferon-beta and inflammatory cytokine production modulated by hepatitis C virus serine proteases derived from patients with hepatic diseases (2009)
  • Author(s): Dansako H, Ikeda M, Ariumi Y, Wakita T, Kato N
  • Journal Title: Arch Virol Volume: 154(5) Pages: 801-10
• [Journal Article] Modification of hepatitis C virus 1b RNA polymerase to make a highly active JFH1-type polymerase by mutation of the thumb domain (2009)
  • Author(s): Weng L, Du J, Zhou J, Ding J, Wakita T, Kohara M, Toyoda T
  • Journal Title: Arch Virol Volume: 154(5) Pages: 765-73
• [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 Volume: 83(10) Pages: 5137-47
• [Journal Article] Hepatitis C virus nonstructural 4B protein modulates sterol regulatory element-binding protein signaling via the AKT pathway (2009)
  • Author(s): Park CY, Jun HJ, Wakita T, Cheong JH, Hwang SB
  • Journal Title: J Biol Chem Volume: 284(14) Pages: 9237-46
• [Presentation] Hepatitis C virus replication models and anti-viral development (2012)
  • Author(s): T Wakita
  • Organizer: The 1^<st> International Symposium on Latent TGF-beta Activation Reaction
  • Place of Presentation: RIKEN Kobe Inst, Ctr. For Delop Biol, Auditorium, Kobe
  • Year and Date: 2012-02-25
• [Presentation] Hepatitis C virus replication in vitro and persistent infection in vivo : mechanistic analysis and antiviral development (2012)
  • Author(s): T Wakita
  • Organizer: Challenges to overcome Emerging Infectious Diseases in South-eastern Asia
  • Place of Presentation: Siran Kaikan, Kyoto Uninversity. Kyoto
  • Year and Date: 2012-01-13
• [Presentation] Hepatitis C virus replication in vitro and persistent infection in vivo : mechanistic analysis and antiviral development (2011)
  • Author(s): T Wakita
  • Organizer: Singapore-Japan Forum on Emerging Concepts in Microbiology
  • Place of Presentation: National University of Singapore, Singapore
  • Year and Date: 20111115-16
• [Presentation] HCV JFH-1キメラ株を用いたNS5a阻害剤の株特異的抗ウイルス活性の評価 (2011)
  • Author(s): 加藤孝宣、政木隆博、脇田隆字
  • Organizer: 第15回日本肝臓学会大会
  • Place of Presentation: 福岡国際会議場
  • Year and Date: 20111020-21
• [Presentation] HCV JFH-1株のチンパンジー感染実験で得られた適応変異株の機能解析 (2011)
  • Author(s): 鈴木哲朗、脇田隆字
  • Organizer: 第15回日本肝臓学会大会
  • Place of Presentation: 福岡国際会議場
  • Year and Date: 20111020-21
• [Presentation] HCV感染に伴う宿主細胞の脂質代謝の変化と代謝産物のメタボロミクス解析 (2011)
  • Author(s): 相崎英樹、鈴木哲朗、脇田隆字
  • Organizer: 第47回日本肝臓学会総会
  • Place of Presentation: ホテルグランパシフィック
  • Year and Date: 20110602-03
• [Presentation] HCV RNA replication and drug development (2011)
  • Author(s): T Wakita
  • Organizer: The 8th APASL Single Topic Conference Beijing
  • Place of Presentation: China
  • Year and Date: 2011-10-07
• [Presentation] Hepatitis C Virus Infection and Replication, annual meeting of Prof (2011)
  • Author(s): T Wakita
  • Organizer: Juei-Low Sung's Research Foundation
  • Place of Presentation: Taipei, Taiwan
  • Year and Date: 2011-08-06
• [Presentation] HCV replication in vitro, The 7th Single Topic Conference : Hepatitis C Virus (2010)
  • Author(s): T Wakita
  • Organizer: Asia Pacific Association of the Study of the Liver(APASL)
  • Place of Presentation: Makuhari Messe, Chiba, Japan
  • Year and Date: 20101217-18
• [Presentation] ESCRT小胞輸送系のHCV産生への関与 (2010)
  • Author(s): 有海康雄、黒木美沙緒、土方誠、Qi Yue、池田正徳、脇田隆字、下遠野邦忠、加藤宜之
  • Organizer: 日本ウイルス学会第58回学術集会
  • Place of Presentation: あわぎんホール
  • Year and Date: 20101107-09
• [Presentation] HCVの増殖適応変異とその意義 (2010)
  • Author(s): 加藤孝宣、岡本有加、村山麻子、政木隆博、脇田隆字
  • Organizer: 日本ウイルス学会第58回学術集会
  • Place of Presentation: あわぎんホール
  • Year and Date: 20101107-09
• [Presentation] HCV replication and persistent infection, Cold Spring Harbor Asia Conference on Emerging Infectious Diseases : Emerging Viruses and the Control of Viruses (2010)
  • Author(s): T Wakita
  • Organizer: Cold Spring Harbor Asia Conference
  • Place of Presentation: Suzhou Dushu Lake Conference Center, Suzhou, China
  • Year and Date: 20101018-22
• [Presentation] HCVNS5A蛋白のリン酸化に関与する新規セリン/スレオニンキナーゼの探索 (2010)
  • Author(s): 政木隆博、松永智子、高橋宏隆、加藤孝宣、宮村達男、遠藤弥重太、澤崎達也、脇田隆字、鈴木哲朗
  • Organizer: 第46回日本肝臓学会総会
  • Place of Presentation: ホテルメトロポリタン山形
  • Year and Date: 20100527-28
• [Presentation] HCV replication and virus particle formation in cell culture (2010)
  • Author(s): T Wakita
  • Organizer: International Symposium for Information Exchange in Bio Industry : Inflamation and Cancer : International Symposium on Current Insight into Prevalent Diseases
  • Place of Presentation: POSTECH Biotech Center, Pohang, Korea
  • Year and Date: 2010-02-22
• [Presentation] HCV cell culture system and antiviral development. International Symposium on Hepatocellular Carcinoma (2010)
  • Author(s): T Wakita
  • Organizer: Kagoshima University Faculty of Medicine
  • Place of Presentation: Kagoshima, Japan
  • Year and Date: 2010-02-19
• [Presentation] HCV粒子形成に関与する脂肪滴周辺膜蛋白の同定 (2009)
  • Author(s): 相崎英樹、後藤耕司、山本真民、佐藤慈子、高橋信弘、深澤征義、花田賢太郎、松浦善治、宮村達男、脇田隆字、鈴木哲朗
  • Organizer: 日本ウイルス学会第57回学術集会
  • Place of Presentation: 都市センターホテル
  • Year and Date: 20091025-27
• [Presentation] C型肝炎ウイルスの感染粒子形成機構 (2009)
  • Author(s): 鈴木哲朗、脇田隆字
  • Organizer: 第13回日本肝臓学会大会
  • Place of Presentation: 国立京都国際会館
  • Year and Date: 20091014-16
• [Presentation] 肝炎ウイルス基礎研究 (2009)
  • Author(s): 脇田隆字
  • Organizer: 第45回日本肝臓学会総会
  • Place of Presentation: 神戸ポートピアホテル
  • Year and Date: 20090604-05
• [Presentation] HCV cell culture system (2009)
  • Author(s): T Wakita
  • Organizer: Asian Pacific Digestive Week(APDW2009)
  • Place of Presentation: Taipei, Taiwan
  • Year and Date: 2009-09-29