1997 Fiscal Year Final Research Report Summary
Analysis of persistent infection mechanism of RNA virus
| Project/Area Number |
08457101
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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 |
Virology
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| Research Institution | The Tokyo Metropolitan Institute of Medical Science |
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Principal Investigator |
KOHARA Michinori The Tokyo Metropolitan Institute of Medical Science, Department of Radiation Research, Head, 放射線医学研究部門, 研究員 (10250218)
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| Co-Investigator(Kenkyū-buntansha) |
KOHARA Kyoko The Tokyo Metropolitan Institute of Medical Science, Department of Radiation Res, 放射線医学研究部門, 研究員 (20225478)
WAKITA Takaji The Tokyo Metropolitan Institute of Medical Science, Department of Radiation Res, 放射線医学研究部門, 研究員 (40280789)
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| Project Period (FY) |
1996 – 1997
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| Keywords | Hepatitis C virus / HCV cDNA / Infectious clone / Immuno-electron microscopic study / HCV like-particle |
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| Research Abstract |
One of the major impediments to the structural analysis of the HCV genome and genetic analysis of viral replication has been lack of a reliable cell culture system permissive for HCV replication. Therefore, we established the HCV replication system by using entire HCV cDNA and T7 RNA polymerase recombinant adeno virus system.
Methods : the putative full-length HCV genome (1-9603) was transiently expressed by the adeno virus/T7 RNA polymerase hybrid expression system. We transfected to a HCV high sensitive cell line (IMY cell) with HCV entire cDNA clone and infected recombinant adeno virus. The HCV particle was characterized by immuno-electron microscopy. Results : The transfected HCV genome replicated in cells, as evidenced by appearance of progeny HCV RNA and detection of negative-strand viral RNA.Immuno-electron microscopic studies have shown that the HCV like-particle was detected in the transfected cells and culture medium. We show the replication process of HCV in the cell and clarified the structure of viral particle. Conclusion : The expression system using HCV entire cDNA clone and showed the importance of 3'X region for HCV replication in the cell. The elctron microscopy study revealed the dynamics of viral core particle in the cells. Moreover the core particle was proved to fold the hexagonal structure.
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[Publications] Tomiko Kashiwakuma, Akira Hasegawa, Tadahiro Kajita, Atumi Tanaka, Hiyoyuki Mori, Yohsuke Ohta, Eiji Tanaka, Kendo Kiyosawa, Takeshi Tanaka, Satoshi Tanaka, Nobu Hattori and Michinori Kohara: "Detection of hepatitis C virus specific core protein in serum of patients by a sensitive fluorescence enzyme immunoassay (FEIA). J.Immunological Methods" J.Immunological Methods. 190. 79-89 (1996)
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[Publications] G.Barba, F.Harper, T.Harada, M.Kohara, S.Goulinet, Y.Matsuura, G.Eder, Zs.Schaff, M.J.Chapman, T.Miyamura, and C.Brechot.: "Hepatitis C Virus Core Protein shows a Cytoplasmic Localization and Associates to Cellular Lipid Storage Droplets." Proc.Natl.Acad.Sci.USA. 94. 1200-1205 (1997)
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[Publications] Tokushige, K., Moradpour, D., Wakita, T., Geissler, M., Hayashi, N., Wand, JR.: "Comparison between cytomegalovirus promoter and elongation factor-1 alpha promoter-driven constructs in the establishment of cell lines expressing hepatitis C virus core protein" J.of Virological Methods. 64 (1). 73-80 (1997)
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