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

doi:10.1128/JVI.75.1.61-72.2001

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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 pentamersin vitro following simultaneous treatment with reducing and chelatingagents, the functional roles of disulfide linkage and calciumion-mediated interactions are not clear. To elucidate the rolesof these interactions, we introduced amino acid substitutionsin VP1 at cysteine residues and at residues involved in calciumbinding. We expressed the mutant proteins in a baculovirus systemand analyzed both their assembly into virus-like particles (VLPs)in insect cells and the disassembly of those VLPs in vitro. Wefound that disulfide linkages at both Cys-9 and Cys-104 conferredresistance to proteinase K digestion on VLPs, although neitherlinkage was essential for the formation of VLPs in insect cells.In particular, reduction of the disulfide linkage at Cys-9 wasfound to be critical for VLP dissociation to VP1 pentamers inthe absence of calcium ions, indicating that disulfide linkageat Cys-9 prevents VLP dissociation, probably by increasing thestability of calcium ion binding. We found that amino acid substitutionsat carboxy-terminal calcium ion binding sites (Glu-329, Glu-330,and Asp-345) resulted in the frequent formation of unusual tubularparticles as well as VLPs in insect cells, indicating that theseresidues affect the accuracy of capsid assembly. In addition,unexpectedly, amino acid substitutions at any of the calcium ionbinding sites tested, especially at Glu-157, resulted in increasedstability of VLPs in the absence of calcium ions in vitro. Theseresults suggest that appropriate affinities of calcium ion bindingare responsible for both assembly and disassembly of thecapsid.

^* 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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