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Construction of Artificial Capsule by Using Novel Bio- Materials

Research Project

Project/Area Number 11470078
Research Category

Grant-in-Aid for Scientific Research (B).

Allocation TypeSingle-year Grants
Section一般
Research Field Virology
Research InstitutionTokyo Institute of Technology

Principal Investigator

HANDA Hiroshi  Frontier Collaborative Research Center Tokyo Institute of Technology Professor, フロンティア創造共同研究センター, 教授 (80107432)

Co-Investigator(Kenkyū-buntansha) WATANABE Hajime  Center for Integrative Bioscience, Okazaki National Research Institute, Associated Prof., 基礎生物学研究所, 助教授 (80212322)
ARISAKA Fumio  Graduate School of Bioscience and Biotechnology Tokyo Institute of Technology Associated Prof., 大学院・生命理工学研究科, 助教授 (80133768)
Project Period (FY) 1999 – 2000
Project Status Completed (Fiscal Year 2000)
Budget Amount *help
¥12,300,000 (Direct Cost: ¥12,300,000)
Fiscal Year 2000: ¥5,300,000 (Direct Cost: ¥5,300,000)
Fiscal Year 1999: ¥7,000,000 (Direct Cost: ¥7,000,000)
KeywordsSV40 / AAV / CAPSID PROTEIN / VIRUS-LIKE PARTICLES / RECOMBINANT PROTEIN / ウイルス粒子様粒子 / 組み換えプラスミド
Research Abstract

Adeno-associated virus (AAV) capsids are composed of three proteins, VP1, VP2 and VP3. Recombinant protein of VP2 and VP3 are prepared. The simian virus 40 capsid is composed of 72 pentamers of VP1 protein. Although the capsid is known to dissociate to pentamers in vitro following simultaneous treatment with reducing and chelating agents, the functional roles of disulfide linkage and calcium ion-mediated interactions are not clear. To elucidate the roles of these interactions, we introduced amino acid substitutions in VP1 at cysteine residues and at residues involved in calcium binding. We expressed the mutant proteins in a baculovirus system and analyzed both their assembly into virus-like particles (VLPs) in insect cells and the disassembly of those VLPs in vitro. We found that disulfide linkages at both Cys-9 and Cys-104 conferred resistance to proteinase K digestion on VLPs, although neither linkage was essential for the formation of VLPs in insect cells. In particular, reduction of the disulfide linkage at Cys-9 was found to be critical for VLP dissociation to VP1 pentamers in the absence of calcium ions, indicating that disulfide linkage at Cys-9 prevents VLP dissociation, probably by increasing the stability of calcium ion binding. We found that amino acid substitutions at carboxy-terminal calcium ion binding sites (Glu-329, Glu-330, and Asp-345) resulted in the frequent formation of unusual tubular particles as well as VLPs in insect cells, indicating that these residues affect the accuracy of capsid assembly. In addition, unexpectedly, amino acid substitutions at any of the calcium ion binding sites tested, especially at Glu-157, resulted in increased stability of VLPs in the absence of calcium ions in vitro. These results suggest that appropriate affinities of calcium ion binding are responsible for both assembly and disassembly of the capsid.

Report

(3 results)
  • 2000 Annual Research Report   Final Research Report Summary
  • 1999 Annual Research Report
  • Research Products

    (34 results)

All Other

All Publications (34 results)

  • [Publications] M.Hoque,K.Ishizu,Handa, et al.: "Nuclear transport of the major capsid protein is essential for the capsid formation of adeno-associated virus."J.Virol.. 73. 7912-7915 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] M.Hoque,K.Ishizu,Handa, et al.: "Chimeric virus-like particle formation of adeno-associated virus."Biochem.Biophysic.Res.Commun.. 266. 371-376 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] M.Takayama,H.Handa, et al.: "Transfer of SV40 temperature-sensitive early gene into human epidermal keratinocytes by the recombinant adenovirus vector"In Vitro Cell.Dev.Biol.. 36. 110-116 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] N.Shimizu,H.Watanabe,H.Handa, et al.: "High-performance affinity beads for identifying drug receptors"Nature Biotechnology. 18. 877-881 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] S.Guo,Y.Yamaguchi,T.Wada,H.Handa, et al.: "A regulator of transcriptional elongation controls vertebrate neuronal development"Nature. 408. 366-369 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] K-I,Ishizu,H.Watanabe,H.Handa, et al.: "Roles of Disulfide Linkage and Calcium Ion-Mediated Interactions in Assembly and Disassembly by Virus-Like Particles Composed of Siman Virus 40 VP1 Capsid Protein"Journal of Virology. 75. 61-72 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] K.Nagata,H.Handa (Eds.): "Real-Time Analysis of Biomolecular Interactions Application of BIACORE"Springer. 256 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] 半田宏,石井俊輔,山本雅之,藤井義明 共編: "ゲノムからの情報発現-転写因子とその機能"シュプリンガー・フェアラーク東京. 238 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] S.Aizawa, M.Nakano, O.Iwase, M.Yaguchi, M.Hiramoto, H.Hoshi, R.Nabeshima, D.Shima, H.Handa and K.Toyama.: "Bone marrow stroma from refractory anemia of myelodysplastic syndrome is defective in its ability to support normal CD34-positive cell proliferation and differentiation in vitro."Leuk.Res.. 23. 239-246 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Y.Yamaguchi, T.Takagi, T.Wada, K.Yano, A.Furuya, S.Sugimoto, J.Hasegawa and H.Handa.: "NELF, a multiple complex containing RD, cooperates with DSIF to repress RNA plymerase II elongation."Cell. 97. 41-51 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Y.Yamaguchi, T.Wada, D.Watanabe, T.Takagi, J.Hasegawa and H.Handa: "Structure and function of the human transcription elongation factor DSIF."J.Biol.Chem.. 274. 8085-8092 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] S.Satoh, M.Hijikata, H.Handa and S.Shimotono.: "Caspase-mediated cleavage of eukaryotic translation initiation factor subunit 2α."Biochem.J.. 342. 65-70 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] M.Hashimoto, K.Chayama, M.Kobayashi, A.Tsubota, Y.Arase, S.Saitou, Y.Suzuki, K.Ikeda, M.Matsuda, H.Koike, M.Kobayashi, H.Handa, and H.Kumada: "Fluctuations of hepatitis C virus load are not related to amino acid substitutions in hyper variable region I and interferon sensitivity determining region."J.Med.Virol.. 58. 247-255 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] M.Hoque, K.Ishizu, A.Matsumoto, S-I.Han, F.Arisaka, M.Takayam, K.Suzuki, T.Kanda, K.Kato, H.Watanabe and H.Handa: "Nuclear transport of the major capsid protein is essential for the capsid formation of adeno-associated virus."J.Viorl.. 73. 7912-7915 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] M.Hoque, N.Shimizu, K-I.Ishizu, H.Yajima, F.Arisaka, K.Suzuki, H.Watanabe, and H.Handa: "Chimeric virus-like particle formation of adeno-associated virus."Biochem.Biophysic.Res.Commun.. 266. 371-376 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] M.Takayama, E.Kim, M.Kidokoro, K.Shimamura, K.Shiroki, H.Yajima, S.Ito, H, Handa and S.Inokuchi.: "Transfer of SV40 temperature-sensitive early gene into human epidermal keratinocytes by the recombinant adenovirus vector."In Vitro Cell.Dev.Biol.. 36. 110-116 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] N.Shimizu, K.Sugimoto, J.Tang, M.Hiramoto, S.Aizawa, A.Oomori, H.Watanabe, H.Tanaka, H.Kawaguchi and H.Handa.: "High performance affinity beads for identification of drug receptors."Nature Biotechnol.. 18. 877-881 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] K-I.Ishizu, H.Watanabe, S-I.Han, S-N.Kanesashi, M.Hoque, H.Yajima, K.Kataoka and H.Handa.: "The roles of disulfide linkage and calcium ion-mediated interactions in assembly and disassembly by virus-like particles composed of SV40 VP1 capsid protein."J.Virol.. 75. 61-72 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] M.Takayama,H.Handa, et al.: "Transfer of SV40 temperature-sensitive early gene into human epidermal keratinocytes by the recombinant adenovirus vector"In Vitro Cell.Dev.Biol.. 36. 110-116 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] K.Araki-Sasaki,H.Handa, et al.: "Sabstance P-Induced cadherin expression and its signal transduction in a cloned human corneal epithelial cell line"J.Cell.Physiol.. 182. 371-376 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] T.Wada,Y.Yamaguchi,H.Handa, et al.: "FACT relieves DSIF/NELF-mediated inhibition of transcription elongation and reveals functional differences between P-TEFb and TFIIH"Mol.Cell. 5. 1067-1072 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] N.Shimizu,H.Watanabe,H.Handa, et al.: "High-performance affinity beads for indentifying drug receptors"Nature Biotechnology. 18. 877-881 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] S.Guo,Y.Yamaguchi,T.Wada,H.Handa, et al.: "A regulator of transcriptional elongation controls vertebrate neuronal development"Nature. 408. 366-369 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] K-I,Ishizu,H.Watanabe,H.Handa, et al.: "Roles of Disulfide Linkage and Calcium Ion-Mediated Interactions in Assembly and Disassembly by Virus-Like Particles Composed of Siman Virus 40 VP1 Capsid Protein"Journal of Virology. 75. 61-72 (2001)

    • Related Report
      2000 Annual Research Report
  • [Publications] K.Nagata,H.Handa (Eds.): "Real-Time Analysis of Biomolecular Interactions Application of BIACORE"Springer. 256 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] 半田宏,石井俊輔,山本雅之,藤井義明 共編: "ゲノムからの情報発現-転写因子とその機能"シュプリンガー・フェアラーク東京. 238 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Y.Yamaguchi et.al.: "NELF,a multisubunit complex containing RD,cooperates with DSIF to repress RNA polymerase II elongation"Cell. 97. 41-51 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] B.R.Cairns et.al.: "Two functionally distinct forms of RSC nucleosome-remodeling complex,containing essential AT hook,BAH,and bromodomains"Mol.Cell. 4・5. 715-723 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] A.M.Dudley,C.Ragelle,&F.Winston: "The Spt components of SAGA facilitate TBP binding to a promoter at a post-activator-brinding step in vivo"Genes Dev.. 13・22. 2940-2945 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] P.B.Kamaruitsky,B.Michel & S.Buratowski: "TFIIO-specitic yeast TAF40 is essential for the majority of RNA polymerase II-mediated transcription in vivo"Genes Dev. 13. 2484-2489 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] C.R.Rodriguez et.al.: "Kin 28,the TFIIH-associated carboxy-terminal domain Kinase,facilitate the recruitment of mRNA processing in machinery to RNA polymene II"Mol.Cell.Biol. 20. 104-112 (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] M.Hogue,H.HANDA et.al.: "Nudear Transport of the Major Capsid Protein is Essential for Adeno-Associated Virus Capsid Formation"Journal of Virology. 73. 7912-7915 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] 半田 宏 共著: "環境ホルモン汚染対策〜測定・評価から企業対応まで〜"株)エヌ・ティー・エス. 426 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] 村松正實 山本 雅 編: "実験医学別冊新訂 新遺伝子工学ハンドブック改訂第3版"羊工社. 360 (1999)

    • Related Report
      1999 Annual Research Report

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Published: 1999-04-01   Modified: 2016-04-21  

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