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

Citation
Ki. Ishizu et al., 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, J VIROLOGY, 75(1), 2001, pp. 61-72
Citations number
33
Categorie Soggetti
Microbiology
Journal title
JOURNAL OF VIROLOGY
ISSN journal
0022538X → ACNP
Volume
75
Issue
1
Year of publication
2001
Pages
61 - 72
Database
ISI
SICI code
0022-538X(200101)75:1<61:RODLAC>2.0.ZU;2-V
Abstract
The simian virus 40 capsid is composed of 72 pentamers of VP1 protein. Alth ough the capsid is known to dissociate to pentamers in vitro following simu ltaneous 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 subs titutions in VP1 at cysteine residues and at residues involved in calcium b inding. We expressed the mutant proteins in a baculovirus system and analyz ed both their assembly into virus-like particles (VLPs) in insect cells and the disassembly of those VLPs in vitro. We found that disulfide linkages a t 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 i nsect 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 di ssociation, probably by increasing the stability of calcium ion binding. We found that amino acid substitutions at carboxy-terminal calcium ion bindin g 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 th at these residues affect the accuracy of capsid assembly. In addition, unex pectedly, amino acid substitutions at any of the calcium ion binding sites tested, especially at Glu-157, resulted in increased stability of VLPs in t he absence of calcium ions in vitro. These results suggest that appropriate affinities of calcium ion binding are responsible for both assembly and di sassembly of the capsid.