Figure 1.
Structure of MCPyV VP1 in complex with sialylated oligosaccharides.
(A, B) The protein is shown in cartoon representation, with one VP1 monomer highlighted in cyan and the other monomers depicted in gray. The oligosaccharides are drawn as stick models. Nitrogen and oxygen atoms are colored blue and red, respectively, and carbon atoms are colored orange for 3SLN, light orange for DSL and yellow for GD1a. (C) Interactions between MCPyV VP1 and the Neu5Ac-α2,3-Gal motif of DSL. The protein surface is colored grey, the protein backbone is depicted in cartoon representation and colored grey, and side chains interacting with the ligand are shown in stick representation and colored by element. Hydrogen bonds and salt bridges are shown as dashed lines, and water molecules mediating hydrogen bonds are indicated as dark blue spheres. (D) Schematic representation of the oligosaccharide structures used in this study. The binding epitope is highlighted in color.
Table 1.
Data collection and refinement statistics.
Figure 2.
MCPyV VP1 epitope mapping of DSL.
1H-NMR-spectra showing the binding epitope of DSL binding to MCPyV VP1 in solution. Top: DSL reference spectrum, bottom: STD spectrum in the presence of MCPyV VP1. Assignments are color-coded according to the DSL cartoon. Signals in the STD spectrum belong to the Neu5Ac-α2,3-Gal motif in DSL only. One methyl group and residual HDO peaks are truncated. Spectra were recorded at 283 K.
Figure 3.
Sialic acid binding site of MCPyV VP1 is crucial for infectivity.
(A) Sialic acid binding site mutantions impair hemagglutination. Hemagglutination assays were performed by mixing a suspension of sheep red blood cells with varying doses of purified wild-type (WT) or VP1 mutant MCPyV capsids. (B) Pseudovirus transduction of sialic acid binding site mutants. Human A549 cells were inoculated with WT or mutant VP1 pseudovirions carrying an encapsidated GLuc reporter plasmid. Three days after inoculation, the culture supernatants were tested for GLuc activity (measured in relative light units, RLUs). (C) Cell attachment of sialic acid binding site mutants. A549 cell suspensions were mixed with 150 ng of WT or mutant VP1 capsids for 2 hours at 4°C. The cells were then washed and subjected to Western blotting using a polyclonal serum specific for MCPyV VP1. In the lanes labeled “Input,” 50 ng of VP1 was loaded into each lane. In the lanes labeled “Bound,” 2/3rds of the washed cell pellet was loaded into each lane. (D) Cell attachment of sialic acid binding site mutants depends on GAGs. A549 cell suspensions (5×104/well) were treated with a mixture of heparinase I (875 mU), heparinase III (87.5 mU), and chondroitinase ABC (35 mU) or mock treated in 50 µl of digestion buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 4 mM CaCl2 and 0.1% BSA) for one hour at 37°C. Then, 200 ng of WT or mutant VP1 capsids diluted in 200 µl of Opti-MEM were added and the mixture was incubated for 2 hours at 4°C. The cells were then washed and subjected to Western blotting using a polyclonal serum specific for MCPyV VP1.
Figure 4.
Plasticity in sialic acid recognition by polyomaviruses.
(A) Overlay of ligand binding sites of MCPyV VP1 (pdb 4FMH), SV40 VP1 (pdb 3BWR) and mPyV VP1 (pdb 1VPS). The proteins are shown in cartoon representation and colored grey, while the sialic acid residues are depicted in stick representation and colored according to complex, with the MCPyV-DSL complex colored cyan, the SV40-GM1 complex colored blue and the mPyV complex colored green. The SV40 and mPyV ligands are semi-transparent. (B–D) Specific interactions of MCPyV VP1 (B), SV40 VP1 (C) and mPyV VP1 (D) with sialic acid. The protein surface is shown in grey. The protein backbones are depicted in cartoon representation, residues in contact with sialic acid are shown in stick representation and colored according to complex as in (A). Residues in equivalent positions are underlined and those contributed from a second VP1 monomer are indicated with an asterisk. Sialic acid is shown in stick representation and colored orange. Hydrogen bonds and salt bridges are shown as dashed lines. (E) Structure-based sequence alignment of the receptor-binding surface loops of MCPyV, SV40 and mPyV VP1. Residues interacting with sialic acid are highlighted as in (B–D).
Table 2.
Variable interactions with sialic acid among polyomavirus VP1 proteins.