| Project/Area Number |
09470134
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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 |
Gastroenterology
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| Research Institution | Mie University |
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Principal Investigator |
WATANABE Shozo Mie University, Health Administration Center, Professor, 保健管理センター, 教授 (20134934)
|
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| Co-Investigator(Kenkyū-buntansha) |
IKOMA Jiro Mie University, School of Medicine, Instructor, 医学部・附属病院, 助手 (80283521)
KAITO Masahiro Mie University, School of Medicine, Instructor, 医学部, 講師 (70214244)
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| Project Period (FY) |
1997 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥13,100,000 (Direct Cost: ¥13,100,000)
Fiscal Year 1999: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1998: ¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 1997: ¥9,000,000 (Direct Cost: ¥9,000,000)
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| Keywords | hepatitis C virus (HCV) / HCV RNA / HCV core particle / HCV virion / IMY cell / immunogold electron microscopy / C型肝炎ウイルス / 回転焼付け法 / RTD-PCR / 金コロイド免疫電顕法 / HCVコア蛋白質 |
|---|
| Research Abstract |
Hybrid cells derived from human hepatocytes and HepG2 cells (IMY cells) were transfected with entire HCV cDNA including a novel 98-nucleotide sequence donwstream from the poly(U) stretch at the 3' end of the genome, and then the cells were infected with recombinant vaccinia virus or adenovirus expressing T7 RNA polymerase. Replication of the transfected HCV genome in the cells was evidenced by detecting the cellular expression of all HCV proteins and the presence of both positive-and negative-strand HCV RNAs. In the cells, clusters of 30-35 nm electron-dense viral core-like particles at the cytoplasmic amorphous matrix area and 50-60 nm virus-like particles within dilated cisternae of the endoplasmic reticulum were observed by conventional passing through electron microscopy. Some of the latter particles showed the budding feature of a virion into the cisternae of the endoplasmic reticulum. Indirect immunogold electron microscopy using antibodies to HCV core protein and HCV envelope pr
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otein revealed that 30-35 nm particles were HCV core particles and 50-60 nm particles were HCV virions. As to the culture media of these cells, negatively stained 55 - 65 nm spherical virus-like particles with surface spike-like projections were visualized in 1.12 - 1.15 g/ml sucrose density gradient fractions which contained the highest titers of HCV RNA. Indirect immunogold electron microscopy using anti-HCV envelope antibody characterized that these particles were HCV virions, as observed specific gold labeling to antibody haloes on the surface of HCV particles, and the virus morphology was similar to that in the HCV RNA-positive human plasma previously reported by us.
These findings show convincingly that HCV RNA replication and virus particle production are processed in the culture IMY cells introduced the full-length HCV cDNA by using transfection/infection protocol, and suggest that core proteins of HCV are synthesized and assembled into the core particles in the cytoplasm, and then bud into the cisternae of the endoplasmic reticulum to form the enveloped HCV virions. Less
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