Structure and assembly of a T=1 virus-like particle in BK polyomavirus

Abstract

In polyomaviruses the pentameric capsomers are interlinked by the long C-terminal arm of the structural protein VP1. The T=7 icosahedral structure of these viruses is possible due to an intriguing adaptability of this linker arm to the different local environments in the capsid. To explore the assembly process, we have compared the structure of two virus-like particles (VLPs) formed, as we found, in a calcium-dependent manner by the VP1 protein of human polyomavirus BK. The structures were determined using electron cryomicroscopy (cryo-EM), and the three-dimensional reconstructions were interpreted by atomic modeling. In the small VP1 particle, 26.4 nm in diameter, the pentameric capsomers form an icosahedral T=1 surface lattice with meeting densities at the threefold axes that interlinked three capsomers. In the larger particle, 50.6 nm in diameter, the capsomers form a T=7 icosahedral shell with three unique contacts. A folding model of the BKV VP1 protein was obtained by alignment with the VP1 protein of simian virus 40 (SV40). The model fitted well into the cryo-EM density of the T=7 particle. However, residues 297 to 362 of the C-terminal arm had to be remodeled to accommodate the higher curvature of the T=1 particle. The loops, before and after the C-terminal short helix, were shown to provide the hinges that allowed curvature variation in the particle shell. The meeting densities seen at the threefold axes in the T=1 particle were consistent with the triple-helix interlinking contact at the local threefold axes in the T=7 structure.

FIG. 1.

Factors guiding disassembly of BKV VLPs. (A) Electron micrograph (negative stained) of recombinant BKV VLPs (0.5 mg/ml). To find dissociation conditions, the BKV VLPs (A) were equilibrated by dialysis against different concentrations of EDTA (10 to 100 mM), 2-ME (10 to 100 mM), and NaCl (0.15 to 1 M). All experiments were done overnight at room temperature, and the resulting appearance of the samples are shown in the electron micrographs B to E (negative stain). (B) Result from dialysis against 20 mM EDTA. (C) Result from dialysis against 30 mM 2-ME. (D) Result from dialysis against 20 mM EDTA and 30 mM 2-ME. (E) Result from dialysis against 20 mM EDTA, 30 mM 2-ME, and 0.6 M NaCl. Bars, 50 nm.

FIG. 2.

Effect of buffer composition on the shape of reassembled particles. When the VLPs had been dissociated, the sample (now containing free pentamers) was dialyzed again against buffers with different concentrations of a monovalent salt, NaCl (0.0 to 1.0 M), pH (7.0 to 7.8), and a divalent ion, Ca²⁺ (0.0 to 100.0 mM). Square: buffer conditions when less then 30% of the reassembled particles had a T=1 symmetry. Triangle: buffer conditions when more then 80% of the reassembled particles had a T=1 symmetry. All experiments were done at room temperature and overnight.

FIG. 3.

Cryo-electron micrograph of BKV VLPs that were reassembled after overnight equilibration against 5 mM Ca²⁺, 150 mM NaCl, and 10 mM Tris-HCl, pH 7.4. Two populations of BKV VLPs that differed in size can be seen (white arrow, small VLP; black arrow, large native-size VLP). Bar, 50 nm.

FIG. 4.

Cryo-EM three-dimensional reconstruction of the small BKV VLP from Fig. 3. The particle is viewed along the twofold axis. It has icosahedral symmetry, and the VP1 protein establishes an arrangement according to a T=1 lattice with protruding capsomer at each fivefold. Bar, 10 nm.

FIG. 5.

Fitting of the VP1 model to the density map of the larger T=7 particle structure. (A) The density map is viewed along the local sixfold axis, showing the VP1 model fitted to one fivefold pentamer and six local sixfold pentamers. Asterisk, the local threefold position. Bar, 10 nm. (B) Close-up view of the six unique monomers (α, α′, and α" at the local threefold; β, β′ around the icosahedral threefold; and γ at the twofold). The positions of the threefold and fivefold axes are marked as triangle and pentagons, respectively.

FIG. 6.

Fitting of the VP1 model to the density map of the T=1 structure. (A) The fivefold monomers from the larger T=7 particle were fitted into the density map of the T=1 particle. The core of the VP1 model fitted well, while the C-terminal arm was protruding out from the density map at the threefold. This was because the angle between capsomers in the T=1 structure was 38 degrees larger than the averaged angle between capsomers in the T=7 particle. (B) Parts of two pentamers are seen from the side. There were no structural changes made in the core of the VP1 protein, except for the CD-loop. The unchanged VP1 model is shown in yellow, whereas the modulations made to fit the T=1 structure is shown in red. The fivefold axis is marked with a pentagon.

FIG.7.

Interpentameric interactions in the T=1 and T=7 BKV VLP. (A) Left: VP1 model of the α (yellow), α′ (red), and α′ (green) monomer fitted to the density map of the T=7 particle at the local threefold. Right: close-up of the interpentameric contact “triple-helix bundle” at the local threefold. (B) Left: three T=1 VP1 models fitted to the density map of the T=1 particle, the particle is viewed along the threefold axis. Right: close-up of the interpentameric contact found at the threefold axis in the T=1 particle. (C) Close-up of the triple-helix bundle in the smaller T=1 particle, showing possible salt-bridges and hydrophobic interactions. Right, side view; left, top view. (D) The C-terminal arm from the T=1 particle (red) and the α monomer from the T=7 particle (yellow) are here superimposed to compare interpentameric interactions. The C-terminal arms interact with two monomers in the neighboring pentamer (blue). The C-terminal arms start with a helix at the threefold (in the T=1 particle) and at the local threefold in the T=7 particle (1). The helix is connected through a long loop (2) to the J-strand (4). The two possible calcium-binding amino acids in the C-terminal arm are also marked, Glu331 (3) and Asp346 (5). The threefold and fivefold are marked as triangle and pentagons, respectively. Bar, 10 nm.