Structure-function analysis of the human JC polyomavirus establishes the LSTc pentasaccharide as a functional receptor motif

Abstract

The human JC polyomavirus (JCV) causes a fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML), in immunocompromised individuals. Current treatment options for PML are inadequate. Sialylated oligosaccharides and the serotonin receptor are known to be necessary for JCV entry, but the molecular interactions underlying JCV attachment remain unknown. Using glycan array screening and viral infectivity assays, we identify a linear sialylated pentasaccharide with the sequence NeuNAc-α2,6-Gal-β1,4-GlcNAc-β1,3-Gal-β1,4-Glc (LSTc) present on host glycoproteins and glycolipids as a specific JCV recognition motif. The crystal structure of the JCV capsid protein VP1 was solved alone and in complex with LSTc. It reveals extensive interactions with the terminal sialic acid of the LSTc motif and specific recognition of an extended conformation of LSTc. Mutations in the JCV oligosaccharide-binding sites abolish cell attachment, viral spread, and infectivity, further validating the importance of this interaction. Our findings provide a powerful platform for the development of antiviral compounds.

Figure 1. LSTc is a functional, specific receptor motif for JCV

(A) Glycan microarray analysis of JCV VP1 showing highly selective binding to LSTc. Numerical scores for the binding intensity are shown as means of fluorescence intensities of duplicate spots at 2 (in blue) and 5 (in red) fmol/spots. Error bars represent half of the difference between the two values. (B) Structures of selected glycans present on the glycan microarray. The oligosaccharide sequence of the LSTc probe is shown, as well as those of similar compounds that were not bound. (C) LSTc inhibits JCV Infection. JC virus was pre-incubated with LSTb or LSTc, and complexes were added to SVG-A cells for infection. Infected cells were quantified based on nuclear VP1 staining. The data represent the average number of infected cells per visual field for 8 fields of view from an experiment performed in triplicate. Error bars indicate SD of triplicate samples. *, P<0.05.

Figure 2. Structure of JCV VP1 in complex with LSTc

(A) Structure of the JCV VP1 pentamer in complex with LSTc. The protein is shown in cartoon representation, with one VP1 monomer highlighted in pink and the other monomers depicted in gray. The LSTc oligosaccharide is drawn as a stick model, and colored according to atom type (nitrogens in blue, oxygens in red, and carbons in orange). (B) Close-up view of the LSTc binding site. JCV VP1 and LSTc are drawn as in (A). A composite annealed omit difference density map of LSTc is shown contoured at 3.0 σ for 2.0 Å around LSTc.

Figure 3. Interactions between JCV VP1 and LSTc

(A) Interactions between JCV VP1 and the terminal NeuNAc of LSTc. JCV VP1 is shown as a cartoon, with side chains interacting with LSTc in stick representation. Waters are represented with spheres. Residues forming direct hydrogen bonds to NeuNAc are colored teal and residues forming van der Waals contacts or water-mediated hydrogen bonds are colored pink. Direct hydrogen bonds between JCV VP1 and NeuNAc are shown as black dashed lines, water-mediated hydrogen bonds or bonds between protein atoms are colored gray. Intramolecular hydrogen bonds within the oligosaccharide are orange. (B) Interactions between JCV VP1 and other parts of LSTc. (C) The cartoon represents structural features of oligosaccharides that are required for JCV binding. These were extracted from our glycan microarray data. Crossed-out sugar residues would produce steric clashes. (D) Structural basis for JCV VP1 specificity for LSTc. JCV is shown in surface representation, with residues interacting with LSTc colored according to their change in surface accessibility upon LSTc binding (gray < 1 Ų change, light teal 1–10 Ų change, dark teal > 10 Ų change). The branching substitutions at LSTc that abolish binding are indicated as black hexagons, indicating where they would clash with protein or LSTc atoms. (E) Structural changes in JCV VP1 upon LSTc binding. The structures of unliganded (gray) and liganded (pink) JCV VP1 were superposed using the β-sandwich core residues. Hydrogen bonds only present in unliganded VP1 are indicated with green dashes, those only present in the complex are colored black.

Figure 4. Growth, Infectivity and Binding of JCV VP1 Mutants

(A) Point mutations introduced into JCV VP1. LSTc is shown in stick representation. (B) Growth of JCV VP1 wild-type and mutant viruses. SVG-A cells were transfected with linearized DNA from JCV VP1 wild-type and mutant constructs. Transfected cells were fixed and stained at day 4 post-transfection, then at 3-day intervals for 22 days by indirect immunofluorescence. Transfected or infected cells were quantified based on nuclear VP1 staining. Each data point represents the average number of infected cells per visual field for 10 fields of view for 3 independent experiments. Error bars indicate SD. (C) Infectivity of supernatants from JCV VP1 wild-type and mutant viruses. SVG-A cells were inoculated with supernatants harvested from infected cells at day 22 from (B). Cells were fixed and stained by indirect immunofluorescence at 72 h post-infection and quantified based on nuclear VP1 staining. The results are presented as the average number of infected cells per visual field for 10 visual fields from 3 individual samples performed in triplicate. Error bars indicate SD. (D) Growth of JCV VP1 wild-type and N123A. N123A was analyzed for viral growth as in (B). Each data point represents the average of number of infected cells per visual field for 10 fields of view for 3 independent experiments. Error bars indicate SD. *, P<0.05. (E) Infectivity of supernatants from JCV VP1 wild-type and N123A. N123A was analyzed for infectivity as in (C). Error bars indicate SD. *, P<0.05. (F) Cell-binding analysis of JCV wild-type and mutant pentamers. SVG-A cells were incubated with His-tagged wild-type or mutant pentamers and a Penta His Alexa Fluor 488 antibody. Cells were fixed and pentamer binding was analyzed by flow cytometry. Histograms represent the fluorescence intensity of Alexa 488 for antibody alone (filled) and pentamer samples (open) for 10,000 gated events. Data are grouped into two histograms based on mutants that propagate (bottom) or do not propagate (top) in SVG-A cells.

Figure 5. Comparison of oligosaccharide binding sites of JCV and SV40 VP1

(A) JCV VP1 in complex with LSTc. (B) SV40 VP1 in complex with GM1. The Glc in GM1 does not contact the protein and was omitted for clarity. The proteins are shown in surface representation, with the BC- and HI-loops also indicated in cartoon representation. Residues contributing to ligand binding or specificity are shown in stick representation. They are colored gray when they are in the same conformation in the two proteins. Residues that are not conserved and assume different conformations are colored pink for JCV and blue for SV40. A blue sphere indicates the Cα position of G131 in SV40. The carbohydrate ligands are shown as orange sticks. Key hydrogen bonds are shown as black dashes.