1997 Fiscal Year Annual Research Report

RNAウイルスの細胞内持続感染機構の解析(C型肝炎ウイルスの感染性cDNAクローンを用いて)

Research Project

Project/Area Number	08457101
Research Institution	The Tokyo Metropolitan Institute of Medical Science
Principal Investigator	小原道法 (財)東京都臨床医学総合研究所, 放射線医学研究部門, 研究員 (10250218)
Co-Investigator(Kenkyū-buntansha)	小原恭子 (財)東京都臨床医学総合研究所, 放射線医学研究部門, 研究員 (20225478) 脇田隆字 (財)東京都臨床医学総合研究所, 放射線医学研究部門, 研究員 (40280789)
Keywords	C型肝炎ウイルス / 全遺伝子のcDNA / 免疫電子顕微鏡法 / 遺伝子複製効率 / ウイルス様粒子
Research Abstract	研究目的.方法 C型肝炎ウイルス(HCV)の複製および病原性発現の過程を宿主因子との関係を含めて分子レベルで明らかにすることを目的としている。我々はHCVの感染複製における各遺伝子領域の役割を明らかにするために、HCV全遺伝子のcDNAをT7プロモーターの下流に導入した発現ベクターをHCV感受性細胞にトランスフエクションし、T7 RNAポリメラーゼ発現組換えアデノウイルスを感染させる事によりHCV蛋白質を発現させた。HCV蛋白質の発現は、各蛋白質に特異的な単クローン抗体により確認した。粒子形成の有無については免疫電子顕微鏡法により検討を行った。研究結果.考察 HCV遺伝子にコードされている全蛋白質が産製され、マイナス鎖RNAの存在が同定され、遺伝子RNAが複製している可能性が示唆された。HCV遺伝子の3'末端には98塩基の多くの分離株で塩基配列がよく保存された領域が存在する。この領域の有無と遺伝子複製効率について検討したところ、効率を制御している可能性が示された。さらに、培養上清中からもHCV-RNA及びコア蛋白質が検出されたので蔗糖密度勾配遠心により粒子の密度を検討した。分画にわけ、コア蛋白質量を定量したところ、密度1.05、1.10、1.25の3カ所にピークを示した。1.10、1.25の2カ所のピークはHCV遺伝子量のピークと一致した。また、免疫電子顕微鏡による検討から細胞質内に直径30-35nmの内部に核酸を有するコア粒子様構造物が、また小胞体内に50-60nmのウイルス様粒子の生成が認められた。培養上清中からも50-60nmの抗エンベロープ抗体と特異的に反応するウイルス様粒子の生成が認められた。培養上清中に存在するウイルス様粒子について現在感染性の有無について検討を続けている。

Research Products

(19 results)

All Other

All Publications (19 results)

[Publications] Barba,G.,: "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)
[Publications] Kamoshita,N.: "Genetic analysis of internal ribosomal entry site on hepatitis C virus RNA:implication for involvement of the highly-ordered structure and cell type specific trans-acting factors." Virology. 233. 9-18 (1997)
[Publications] Tanaka,T.: "Acute hepatitis caused by sexual or household transmission of GBV-C." J.Hepatology. 27. 1110-1112 (1997)
[Publications] Akiyama,H.: "Effects of cyclosporin A on hepatitis C virus infection in bone marrow transplantation patients." Bone Marrow Transplant. 20. 993-995 (1997)
[Publications] Wakita,T.: "Efficient conditional transgene expression in hepatitis C virus cDNA transgenic mice mediated by the Cre/loxP system." J.Biol.Chem.(in press).
[Publications] Kondo,S.: "Inhibition of telomerase increases the susceptibility of human malignant glioma cells to cisplatin-induced apoptosis." Oncogene. (in press). (1998)
[Publications] Kase,R.: "Immunohistochemical characterization of transgenic mice highly expressing human lysosomal α-galactosidase" Biochimica et Biophvsica Acta. (in press). (1998)
[Publications] Kondo,T.: "Identification and chacterization of nucleophosmin/B23/numatrin which binds the anti-oncogenic transcription factor IRF-1 and manifests oncogenic activity." Oncogene. 15. 1275-1281 (1997)
[Publications] Taniguchi,T.: "IRF-1:the transcription factor linking the interferon response and oncogenesis." Biochimica et Biophysica Acta. (in press). (1998)
[Publications] Tanaka,N.: "Type linterferons are essential mediators of apoptotic death in virally-infected cells." Genes to Cells. 3. 29-37 (1998)
[Publications] Nozawa,H.: "Functionally inactivating point mutation in the tumor suppressor IRF-1 gene was identified in human gastric cancer." Int.J.Cancer. (in press). (1998)
[Publications] Tanaka,T.: "Acute hepatitis caused by sexual or household transmission of GBV-C." J.Hepatology. 27. 1110-1112 (1997)
[Publications] Kondo,S.: "Antisense telomerase treatment;induction of two distinct pathways,apoptosis and differentiation." FASEB Journal. in press. (1998)
[Publications] Kondo,S.: "FADD gene therapy for malignant gliomas in vitro and in vivo." Human Gene Therapy. in press. (1998)
[Publications] Kondo,S.: "Retrovial Transfer of Cpp32 β Gene into Malignant Gliomas in vitro and in vivo." Cancer Research. 58. 962-967 (1998)
[Publications] Shimmoto,M.: "Generation and characterization of transgenic mice expressing a human mutant alpha-galactosidase with an R301Q substitution causing a variant form of Fabry disease" FEBS Letter. 417. 89-91 (1997)
[Publications] Ishii,S.: "α-Galactosidase transgenic mouse:Heterogeneous gene expression and posttranscriptional glycosilation in tissues." Glycoconjugate Jorunal. in press. (1998)
[Publications] 小原道法: "Modem Physician" C型肝炎とG型肝炎検査法の進歩, 5 (1997)
[Publications] 小原道法: "南江堂" C型肝癌発生とウイルス増殖, 4 (1997)

1997 Fiscal Year Annual Research Report

RNAウイルスの細胞内持続感染機構の解析(C型肝炎ウイルスの感染性cDNAクローンを用いて)

Principal Investigator

小原 道法 (財)東京都臨床医学総合研究所, 放射線医学研究部門, 研究員 (10250218)

Research Products

[Publications] Barba,G.,: "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)

[Publications] Kamoshita,N.: "Genetic analysis of internal ribosomal entry site on hepatitis C virus RNA:implication for involvement of the highly-ordered structure and cell type specific trans-acting factors." Virology. 233. 9-18 (1997)

[Publications] Tanaka,T.: "Acute hepatitis caused by sexual or household transmission of GBV-C." J.Hepatology. 27. 1110-1112 (1997)

[Publications] Akiyama,H.: "Effects of cyclosporin A on hepatitis C virus infection in bone marrow transplantation patients." Bone Marrow Transplant. 20. 993-995 (1997)

[Publications] Wakita,T.: "Efficient conditional transgene expression in hepatitis C virus cDNA transgenic mice mediated by the Cre/loxP system." J.Biol.Chem.(in press).

[Publications] Kondo,S.: "Inhibition of telomerase increases the susceptibility of human malignant glioma cells to cisplatin-induced apoptosis." Oncogene. (in press). (1998)

[Publications] Kase,R.: "Immunohistochemical characterization of transgenic mice highly expressing human lysosomal α-galactosidase" Biochimica et Biophvsica Acta. (in press). (1998)

[Publications] Kondo,T.: "Identification and chacterization of nucleophosmin/B23/numatrin which binds the anti-oncogenic transcription factor IRF-1 and manifests oncogenic activity." Oncogene. 15. 1275-1281 (1997)

[Publications] Taniguchi,T.: "IRF-1:the transcription factor linking the interferon response and oncogenesis." Biochimica et Biophysica Acta. (in press). (1998)

[Publications] Tanaka,N.: "Type linterferons are essential mediators of apoptotic death in virally-infected cells." Genes to Cells. 3. 29-37 (1998)

[Publications] Nozawa,H.: "Functionally inactivating point mutation in the tumor suppressor IRF-1 gene was identified in human gastric cancer." Int.J.Cancer. (in press). (1998)

[Publications] Tanaka,T.: "Acute hepatitis caused by sexual or household transmission of GBV-C." J.Hepatology. 27. 1110-1112 (1997)

[Publications] Kondo,S.: "Antisense telomerase treatment;induction of two distinct pathways,apoptosis and differentiation." FASEB Journal. in press. (1998)

[Publications] Kondo,S.: "FADD gene therapy for malignant gliomas in vitro and in vivo." Human Gene Therapy. in press. (1998)

[Publications] Kondo,S.: "Retrovial Transfer of Cpp32 β Gene into Malignant Gliomas in vitro and in vivo." Cancer Research. 58. 962-967 (1998)

[Publications] Shimmoto,M.: "Generation and characterization of transgenic mice expressing a human mutant alpha-galactosidase with an R301Q substitution causing a variant form of Fabry disease" FEBS Letter. 417. 89-91 (1997)

[Publications] Ishii,S.: "α-Galactosidase transgenic mouse:Heterogeneous gene expression and posttranscriptional glycosilation in tissues." Glycoconjugate Jorunal. in press. (1998)

[Publications] 小原道法: "Modem Physician" C型肝炎とG型肝炎検査法の進歩, 5 (1997)

[Publications] 小原道法: "南江堂" C型肝癌発生とウイルス増殖, 4 (1997)

小原道法 (財)東京都臨床医学総合研究所, 放射線医学研究部門, 研究員 (10250218)