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Journal of Virology, January 2001, p. 61-72, Vol. 75, No. 1
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.
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
*
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.
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