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
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
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.