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

Abstract

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.Simian virus 40 (SV40), a member of the Papovaviridae, is a small, nonenveloped tumorigenic virus. Its genome consists of double-stranded circular DNA of 5,243 bp, which encodes three structural proteins (VP1, VP2, and VP3) and two nonstructural proteins (large T antigen and small T antigen). The structural proteins are expressed in late infection, and viral capsids are formed in the nucleus of infected susceptible host cells (32). The SV40 capsid is about 45 to 50 nm in diameter, and its major component is VP1. The capsid is formed by the arrangement of 72 VP1 pentamers in a Tϭ7d icosahedral lattice. The three-dimensional structure of the SV40 virion has been elucidated by X-ray crystallography, which showed that the capsid is formed from three nonequivalent types of interactions between pentameric capsomeres (␣-␣Ј, ␤-␤Ј, and ␥-␥) (20, 30).SV40 capsids, as well as those of the closely related murine polyomavirus, dissociate to VP1 pentamers following treatment with dithiothreitol (DTT) and EGTA in vitro (2-4, 7), indicating that disulfide linkage and calcium ion-mediated interactions between pentamers are important for capsid formation in these viruses. Consistent with these observations, crystallographic analysis of SV40 virions has identified disulfide linkage between Cys-104 and Cys-104 in ␤-␤...

The VP2/VP3 Minor Capsid Protein of Simian Virus 40 Promotes the in Vitro Assembly of the Major Capsid Protein VP1 into Particles

“…2A, upper panel, pH 5.0, open arrowheads) (9). The diameter of these tubes was ϳ40 -45 nm, and they were apparently infinitely long although disrupted during preparation.…”

“…Expression and Purification of Capsid Proteins-The construction of the SV40 VP1-expressing baculovirus and expression of the VP1 protein in Spodoptera frugiperda (Sf9) cells have been described (9,25,26). Briefly, VP1 protein assembled into particles (VP1-VLPs) in Sf9 cells was purified in two successive fractionations by cesium chloride density gradient centrifugation.…”

“…When indicated, the samples were preincubated with 49 and 490 ng or 4.9 g of mouse monoclonal anti-FLAG M2 antibody (Sigma). The samples were loaded onto a 0.8% agarose gel, run in 50 mM Tris acetate (pH 8.1) for 2 h at 4°C, and transferred onto a polyvinylidene fluoride membrane in 50 mM NaOH by a standard capillary transfer method (9). VP1, HF-VP2, and the anti-FLAG antibody were detected by immunoblotting with anti-VP1, anti-FLAG, and antimouse IgG antibodies, respectively.…”

“…Particles-We have established a system to evaluate conditions for the assembly of highly purified recombinant VP1 pentamers into particles by dialyzing a VP1-containing solution against various buffers, with subsequent EM observation (9,25,26). As we showed previously, VP1 pentamers do not assemble into particles under physiological salt and pH conditions (150 mM NaCl, pH 7.0) (Fig.…”

“…The properties of VP1-VLPs are very similar to those of wild type virions in that they dissociate into pentamers following treatment with a calcium-chelating agent (EGTA) under reducing conditions in vitro (22)(23)(24). Previously, we prepared highly purified SV40 VP1 pentamers (9,25,26), which assembled into morphologically heterogeneous particles under various nonphysiological conditions (26). Such particles include T ϭ 7d (triangulation number 7 dextro) spherical particles, small T ϭ 1 (triangulation number 1) particle composed of 12 pentamers, and long tubular structures of 40 -45 nm in diameter (26).…”