1996 Fiscal Year Final Research Report Summary
Study on maturation and replication mechanisms of Pestivirus and hepatitis C virus
| Project/Area Number |
07456137
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Basic veterinary science/Basic zootechnical science
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| Research Institution | National Institute of Health |
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Principal Investigator |
MATSUURA Yoshiharu National Institute of Health, Department of Virology II,Section Chief, ウイルス第二部, 室長 (50157252)
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| 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)
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| Project Period (FY) |
1995 – 1996
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| Keywords | HCV, / Gene Expression, / Apoptosis |
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| 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
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[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)
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[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)
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[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).
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