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Journal of Virology, January 2001, p. 61-72, Vol. 75, No. 1
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.1.61-72.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Roles of Disulfide Linkage and Calcium Ion-Mediated Interactions in Assembly and Disassembly of Virus-Like Particles Composed of Simian Virus 40 VP1 Capsid Protein

Ken-Ichiro Ishizu,1 Hajime Watanabe,2 Song-Iee Han,1 Shin-Nosuke Kanesashi,1 Mainul Hoque,1 Hiroaki Yajima,3 Kohsuke Kataoka,4 and Hiroshi Handa4,*

Frontier Collaborative Research Center4 and Faculty of Bioscience and Biotechnology,1 Tokyo Institute of Technology, Midori-ku, Yokohama 226-8501, Center for Integrative Bioscience, Okazaki National Research Institutes, Myodaiji, Okazaki 444-8585,2 and Kirin Brewery Co., Ltd., Kanazawa-ku, Yokohama 236,3 Japan

Received 17 July 2000/Accepted 25 September 2000

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.

* Corresponding author. Mailing address: Frontier Collaborative Research Center, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8503, Japan. Phone: 81-45-924-5872. Fax: 81-45-924-5145. E-mail: hhanda{at}bio.titech.ac.jp.

Journal of Virology, January 2001, p. 61-72, Vol. 75, No. 1
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.1.61-72.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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