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2000 Fiscal Year Final Research Report Summary

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
KeywordsSV40 / AAV / CAPSID PROTEIN / VIRUS-LIKE PARTICLES / RECOMBINANT PROTEIN
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.

  • Research Products

    (18 results)

All Other

All Publications (18 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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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)

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      「研究成果報告書概要(和文)」より
  • [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)

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      「研究成果報告書概要(欧文)」より
  • [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
  • [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)

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      「研究成果報告書概要(欧文)」より
  • [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)

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      「研究成果報告書概要(欧文)」より
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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

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Published: 2002-03-26  

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