2005 Fiscal Year Final Research Report Summary
Analyzing the role of NS5A protein in regulation of interferon sensitivity using HCV replicon system.
| Project/Area Number |
16590583
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Gastroenterology
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| Research Institution | University of Yamanashi |
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Principal Investigator |
KITAHARA Fumiaki University of Yamanashi, Department of Research Interdisciplinary Graduate School of Medicine and Engineering, Research Associate, 大学院・医学工学総合研究部, 助手 (50303409)
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| Co-Investigator(Kenkyū-buntansha) |
ENOMOTO Nobuyuki University of Yamanashi, Department of Research Interdisciplinary Graduate School of Medicine and Engineering, Professor, 大学院・医学工学総合研究部, 教授 (20251530)
SAKAMOTO Minoru University of Yamanashi, Department of Research Interdisciplinary Graduate School of Medicine and Engineering, Research Associate, 大学院・医学工学総合研究部, 助手 (60324191)
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| Project Period (FY) |
2004 – 2005
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| Keywords | Hepatitis C Virus / NS5A protein / HCV replicon / interferon |
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| Research Abstract |
The number of amino acid substitutions in the interferon sensitivity-determining region (ISDR) in the nonstructural 5A (NS5A) gene of hepatitis C virus (HCV) is closely associated with the interferon response and viral load. Several HCV replicon-based studies have reported that ISDR sequences had an influence on viral replication in vitro. However, it is unclear how the variety of ISDR sequences affect HCV replication. Various clinically observed ISDR sequences were introduced into HCV replicons, and their contribution to viral replication was investigated using a colony formation assay and/or a transient replication assay. A mapping study of the ISDR was performed to identify the amino acid positions that critically affect replication. While no colonies were formed in colony formation assay using HCV replicons with few mutations (0, 1, and 3) in the ISDR, numerous colonies (>200) appeared using constructs with six mutations. Introduction of various distinct ISDR sequences with multipl
… More
e mutations resulted in replication enhancement in transient assays. A mapping study identified several specific sites in the ISDR that critically altered replication, including codon 2209 which was closely associated in patients with a strong response to IFN. ISDR sequences associated with a clinical interferon response and viral load modulated the replication of HCV replicons, suggesting the importance of the ISDR sequence in HCV infection.
Next, we studied expression profiles of ISGs in cells supporting subgenomic HCV replication (Huh7/Rep), and screened their activities to suppress HCV replication. Real-time PCR analyses showed that the expression levels of 23 ISGs were significantly lower in Huh7/Rep than naive Huh7 cells due to transcriptional suppression of the interferon-stimulated response element (ISRE). Furthermore, the expression level of ISGs was also decreased in the cured Huh7 cells in which replicon had been eliminated (cHuh7), indicating adaptation of the cells to support HCV replication by downregulating ISGs. On the other hand, expression of HCV replicon was significantly suppressed by overexpression of several ISGs including PKR, MxA, IRF-9, GBP-1, IFI-6-16, IFI-27, 25OAS and IRF-1. Knock down of GBP-1, IFI-6-16 and IFI-27 by short hairpin RNA resulted in increase of HCV replication. Less
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Research Products
(10 results)
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[Journal Article] Suppression of hepatitis C virus replication by cyclosporine A is mediated by blockade of cyclophilins.2005
Author(s)
Nakagawa M, Sakamoto N, Tanabe Y, Koyama T, Itsui Y, Takeda Y, Chen CH, Kakinuma S, Oooka S, Maekawa S, Enomoto N, Watanabe M.
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Journal Title
Gastroenterology 129(3)
Pages: 1031-1041
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Introduction of NS5A mutations enables subgenomic HCV replicon derived from chimpanzee-infectious HC-J4 isolate to replicate efficiently in Huh-7 cells.2004
Author(s)
Maekawa S, Enomoto N, Sakamoto N, Kurosaki M, Ueda E, Kohashi T, Watanabe H, Chen CH, Yamashiro T, Tanabe Y, Kanazawa N, Nakagawa M, Sato C, Watanabe M.
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Journal Title
J Viral Hepat. 11
Pages: 394-403
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Regulation of hepatitis C virus replication by interferon regulatory factor 1.2004
Author(s)
Kanazawa N, Kurosaki M, Sakamoto N, Enomoto N, Itsui Y, Yamashiro T, Tanabe Y, Maekawa S, Nakagawa M, Chen CH, Kakinuma S, Oshima S, Nakamura T, Kato T, Wakita T, Watanabe M.
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Journal Title
J Virol. 78
Pages: 9713-9720
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Site-specific mutation of the interferon sensitivity-determining region (ISDR) modulates hepatitis C virus replication
Author(s)
Kohashi T, Maekawa S, Sakamoto N, Watanabe H, Tanabe Y, Chen C, Nakagawa M, Kakinuma S, Kurosaki M, Itsui Y, Koyama T, Takeda Y, Sekine Y, Enomoto N, Watanabe M.
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Journal Title
J Viral Hepat. (In press)
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Expressional screening of interferon-stimulated genes for antiviral activity against hepatitis C virus replication
Author(s)
Itsui Y, Sakamoto N, Kurosaki M, Kanazawa N, Tanabe Y, Koyama T, Takeda Y, Nakagawa M, Kakinuma S, Sekine Y, Maekawa S, Enomoto N, Watanabe M.
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Journal Title
J Viral Hepat. (In press)
Description
「研究成果報告書概要(欧文)」より
