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Study on maturation and replication mechanisms of Pestivirus and hepatitis C virus

Research Project

Project/Area Number 07456137
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field Basic veterinary science/Basic zootechnical science
Research InstitutionNational Institute of Health

Principal Investigator

MATSUURA Yoshiharu  National Institute of Health, Department of Virology II,Section Chief, ウイルス第二部, 室長 (50157252)

Co-Investigator(Kenkyū-buntansha) MANABE Noboru  Kyoto University, Faculty of Agriculture, Associate Professor, 農学部, 助教授 (80243070)
MIYAMURA Tatsuo  National Institute of Health, Department of Virology II,Director, ウイルス第二部, 部長 (90100099)
Project Period (FY) 1995 – 1996
Project Status Completed (Fiscal Year 1996)
Budget Amount *help
¥6,400,000 (Direct Cost: ¥6,400,000)
Fiscal Year 1996: ¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 1995: ¥3,500,000 (Direct Cost: ¥3,500,000)
KeywordsHCV, / Gene Expression, / Apoptosis / ペスティウイルス / ウイルスの成熟機構
Research Abstract

We constructed a full length cDNA clone of hepatitis C virus (HCV) from a blood sample of an HCV carrier and established a HepG2 cell line constitutively expressing HCV core protein. The cell line showed apoptotic changes in response to stimulation with anti-Fas monoclonal antibody. Cells treated with the antibody showed extensive cell rounding, shrinkage and cytoplasmic blebbing, and finally detached from plates. Fragmentation of the chromatin was observed in the nucleus and DNA ladders were detected. Apoptotic cell death was prevented by pretreatment with a specific inhibitor of the cysteine protease CPP32. While the specific inhibitor of interleukin-1b-converting enzyme did not show the preventive effect. The results suggest (i) that intracellular expression of HCV core protein makes cells prone to apoptotic death without upregulation of surface Fas expression, and (ii) that the CPP32 protease plays a part in the apoptosis effector pathway of HCV core expressing cells. HCV core prot … More ein may have a role in immune mediated liver cell injury.
To establish a system to produce minigene RNA of HCV in mammalian cells, we constructed a replication-deficient recombinant adenovirus expressing bacteriophage T7 RNA polymerase under the control of CAG promoter (AdexCAT7). After infection with AdexCAT7, various cell lines were transfected with plasmids carrying reporter luciferase gene under the control of T7 promoter. Expression of luciferase was highest in a cell line derived from human hepatocellular carcinoma. In this cell line, higher level of reporter gene expression was also observed by transfection of RNA synthesized in vitro. This hybrid-T7 adenovirus system to synthesize HCV minigenes in vivo may be useful to elucidate the molecular mechanism of HCV replication in human liver cells.
The core protein of HCV is expected to bind with viral sense RNA to form a nucleocapsid because of its high content of basic amino acid residues. It is very important to clarify the binding property to understand the replication mechanism of HCV.We synthesized various regions of viral and anti-viral RNA of HCV in vitro and transfected into HepG2 cells expressing HCV core protein. Cell lysates were immunoprecipitated with anti-core antibody. RNA was extracted from the immunoprecipitates of core protein and RNA,and polarity of the RNA was determined by dot blot or northern blot analysis. Specific binding of core protein was observed with a full length of positive sense HCV RNA,but not with negative sense. Deletion analysis of viral sense RNA revealed that 381nt (329-710nt from the 5'end) of the genomic RNA is responsible for specific binding with core protein. Furthermore, we prepared deletion mutants of core protein lacking each of basic amino acid clusters in core protein. A basic amino acid cluster of 14aa in core protein was shown to play an important role in binding with the viral RNA. Less

Report

(3 results)
  • 1996 Annual Research Report   Final Research Report Summary
  • 1995 Annual Research Report
  • Research Products

    (53 results)

All Other

All Publications (53 results)

  • [Publications] Suzuki R.: "Nuclear localization of the truncated hepatitis C virus core protein deleted in its hydrophobic C-terminus." J.Gen.Virol.76. 53-61 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Harada T.: "Characterization of an established human hepatoma cell line constitutively expressing non-structural proteins of hepatitis C virus by transfection of viral cDNA." J.Gen.Virol.76. 1215-1221 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Kakimi K.: "Helper T cell epitopes in hepatitis C virus core region recognized by BALB/c and C57BL/6 mice." J.Gen.Virol.76. 1205-1214 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Suzuki T.: "In vivo and in vitro trans-cleavage activity of hepatitis C virus serine proteinase expressed by recombinant baculoviruses." J.Gen.Virol.76. 3021-3029 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Shoji I.: "Proteolytic activity of NS3 serine proteinase of hepatitis C virus efficiently expressed in Escherichia coli." Hepatology. 22. 1648-1655 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Koike K.: "Expression of hepatitis C virus envelope proteins in transgenic mice." J.Gen.Virol.76. 3031-3038 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Makimura M.: "Induction of antibodies against structural proteins of hepatitis C virus in mice using recombinant adenovirus." Vaccine. 14. 28-34 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Suzuki T.: "Hepatitis C virus infection and hepatocellular carcinoma." Liver. 16. 221-224 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Suzuki N.: "Poly (Tri or Bi)-cistronic Phytoreoviral segments translatable in both plant and insect cells." J.Virol.70. 8155-8159 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Aizaki H.: "Mother to child transmission of a hepatitis C virus variant with an insertional mutation in its hypervariable region." J.Hepatol.25. 608-613 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Koike K.: "Sialadenitis resembling Sjogren′s syndrome in hepatitis C virus transgenic mice." Proc.Natl.Acad.Sci.USA.94. 233-236 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Barba G.: "Hepatitis C virus core protein shows a cytoplasmic localization and associates to cellular lipid storage droplets." Proc.Natl.Acad.Sci.USA.(印刷中).

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Ruggieri A.: "Sensitization to Fas-mediated Apoptosis by Hepatitis C Virus Core Protein." Virology. (印刷中).

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Moriya K.: "Hepatitis C virus core protein induces hepatic steatosis in transgenic mice." J.Gen Virol.(印刷中).

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Yap C-C: "A hybrid baculovirus-T7 RNA polymerase system for recovery of an infectious virus from cDNA." Virology. (印刷中).

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Missale G.: "Effects of inteferon treatment on the anti-viral T cell response in hepatitis C virus genotype 1b and genotype 2-infected patients." Hepatology. (印刷中).

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] 勝二郁夫: "増刊号"分子肝炎ウイルス病学"" 日本臨床, 30-35 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] 松浦善治: "増刊号"分子肝炎ウイルス病学"" 日本臨床, 51-56 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] 相崎英樹: "増刊号"分子肝炎ウイルス病学"" 日本臨床, 80-84 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] 槙村美保: "増刊号"分子肝炎ウイルス病学"" 日本臨床, 91-94 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] 松浦善治: "増刊号"分子肝炎ウイルス病学"" 日本臨床, 112-116 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] 松浦善治: "Medical Practice" 文光堂, 1625-1627 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] 松浦善治: "ウイルス" 日本ウイルス学会, 105-115 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Matsuura Y.: "Vaccine 97.(Chanock R.M.,Ginsberg H.S.,Brown F.,and Lemer R.A.ed.)" Cold Spring Harbor Laboratory Press,New York.(印刷中),

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Suzuki R., Matsuura Y., Suzuki T., Ando A., Chiba J., Harada S., Saito I., Miyamura T.: "Nuclear localization of the truncated hepatitis C virus core protein deleted in its hydrophobic C-terminus." J.Gen. Virol.76. 53-61 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Harada T., Kim D.W., Sagawa K., Suzuki T., Takahashi K., Saito I., Matsuura Y., Miyamura T.: "Characterization of an established human hepatoma cell line constitutively expressing non-structural proteins of hepatitis C virus by transfection of viral cDNA." J.Gen. Virol.76. 1215-1221 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Kakimi K., Kuribayashi K., Iwashiro M., Masuda T., Kakai M., Ling W., Kubo Y., Kobayashi H., Higo K., Seki M., Honda Y., Yamada E., Matsuura Y., Miyamura T., Okumura M., Ishimoto A.: "Helper T cell epitopes in hepatitis C virus core region recognized by BALB/c and C57BL/6 mice." J.Gen. Virol.76. 1205-1214 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Suzuki T., Sato M., Chieda S., Shoji I., Harada T., Yamakawa Y., Watabe S., Matsuura Y.Miyamura T.: "In Vivo and In Vitro Trans-Cleavage Activity of Hepatitis C Virus Serine Proteinase Expressed by Recombinant Baculoviruses." J.Gen. Virol.76. 3021-3029 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Shoji I., Suzuki T., Chieda S., Sato M., Harada T., Chiba T., Matsuura Y., Miyamura T.: "Proteolytic activity of NS3 serine proteinase of hepatitis C virus efficiently expressed in Escherichia coli." Hepatology. 22. 1648-1655 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Koike, K., Moriya, K., Ishibashi, K., Matsuura, Y., Suzuki, T., Iino, S., Kurokawa, K., Miyamura T.: "Expression of hepatitis C virus envelope proteins in transgenic mice" J.Gen. Virol.76. 3031-3038 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Makimura M., Akino N., Miyake S., Takamori K., Matsuura Y., Miyamura T., Saito I.: "Induction of antibodies against structural proteins of hepatitis C virus in mice using recombinant adenovirus." Vaccine. 14. 28-34 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Suzuki T., Matsuura Y., Harada T., Makimura M., Suzuki R., Ando A., Chiba J., Saito I., Miyamura T.: "Hepatitis C virus infection and hepatocellular carcinoma" Liver. 16. 221-224 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Suzuki N., Sugawara M., Nuss D.L., Matsuura Y.: "Poly (Tri of Bi) -cistronic Phytoreoviral segments translatable in both plant and insect cells" J.Virol.70. 8155-8159 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Aizaki H., Saito A., Kusakawa I., Ashiwara H., Nagamori S., Toda G., Matsuura Y., Miyamura T.: "Mother to child transmission of a hepatitis C virus variant with an insertional mutation in its hypervariable region." J.Hepatol. 25. 608-613 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Koike K., Moriya K., Ishibashi K., Yotsuyanagi H., Shintani Y., Hujie H., Matsuura Y., Kurosawa K., Miyamura T.: "Sialadenitis resembling Sjogren's syndrome in hepatitis C virus transgenic mice." Proc. Natl. Acad. Sci. USA., 94. 233-236 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Barba G., Harper F., Harada T., Kohara M., Goulinet S., Matsuura Y., Eder G., Schaff Zs., Chapmann M.J., Miyamura T., Brechot C.: "Hepatitis C virus core protein shows a cytoplasmic localization and associates to cellular lipid storage droplets." Proc. Natl. Acad. Sci. USA. (in press).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Ruggieri A., Harada T., Matsuura Y., Miyamura T.: "Sensitization to Fas-mediated apoptosis by hepatitis C virus core protein" Virology. (in press).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Moriya K., Yotsuyanagi H., Ishibashi K., Shintani Y., Fujie H., Matsuura Y., Miyamura T., Koike K.: "Hepatitis C virus core protein induces hepatic steatosis in transgenic mice." J.Gen. Virol.(in press).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Yap C-C., Ishii K., Aoki Y., Aizaki H., Tani H., Shimizu H., Ueno Y., Miyamura T., Matsuura Y.: "A hybrid baculovirus-T7 RNA polymerase system for recovery of an infectious virus from cDNA." Virology. (in press).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Missale G., Matsuura Y., Miyamura T., Houghton M., Ferrari C.: "Effects of interferon treatment on the anti-viral T cell response in hepatitis C virus genotype 1b and genotype 2-infected patients." Hepatology. (in press).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Suzuki T.: "Hepatitis C virus infection and hepatocellular carcinoma." Liver. 16. 221-224 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Aizaki H.: "Mother to child transmission of a hepatitis C virus variant with an insertional mutation in its hypervariable region." J.Hepatol.25. 608-613 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Koike K.: "Sialadenitis resembling Sjogren's syndrome in hepatitis C virus transgenic mice." Proc.Natl.Acad.Sci.USA.(印刷中). (1997)

    • Related Report
      1996 Annual Research Report
  • [Publications] Barba G.: "Hepatitis C virus core protein shows a cytoplasmic localization and associates to cellular lipid storage droplets." Proc.Natl.Acad.Sci.USA.(印刷中). (1997)

    • Related Report
      1996 Annual Research Report
  • [Publications] Ruggieri A.: "Sensitization to Fas-mediated Apoptosis by Hepatitis C Virus Core Protein." Virology. (印刷中). (1997)

    • Related Report
      1996 Annual Research Report
  • [Publications] Manabe N.: "Signal transmission of granulosa cell apoptosis in the atretic antral follicles in the pig ovaries." J.Reprod.Dev.(印刷中). (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Matsuura Y.: "Vaccine 97" Chanock R.M.,Ginsberg H.S.,Brown F.,and Lerner R.A.(印刷中),

    • Related Report
      1996 Annual Research Report
  • [Publications] Shoji, I.: "Proteolytic activity of NS3 serine proteinase of hepatitis C virus efficiently expressed in Escherichia coli." Hepatology. 22. 1648-1655 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Suzuki, T.: "In vitro and in vivo trans-cleavage activity of hepatitis C virus serine protease expressed by recombinant baculoviruses." J. Gen. Virol.76. 3021-3029 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Makimura, M.: "Induction of antibodies against HCV structural proteins in mice using recombinant adenovirus." Vaccine. (in press).

    • Related Report
      1995 Annual Research Report
  • [Publications] Koike, K.: "Expression of hepatitis C virus envelope proteins in transgenic mice." J. Gen. Virol.76. 3031-3038 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Harada, T.: "Characterization of an established human hepatoma cell line constitutively expressing nonstructural proteins of hepatitis C virus by transfection of viral cDNA." J. Gen. Virol.76. 1215-1221 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Suzuki, R.: "Nuclear localization of the truncated hepatitis C virus core protein deleted in its hydrophobic C-terminus." J. Gen. Virol.76. 53-61 (1995)

    • Related Report
      1995 Annual Research Report

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Published: 1995-04-01   Modified: 2016-04-21  

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