Structural and functional analysis of natural capsid variants suggests sialic acid-independent entry of BK polyomavirus

Abstract

BK polyomavirus (BKPyV) is an opportunistic pathogen that uses the b-series gangliosides GD1b and GT1b as entry receptors. Here, we characterize the impact of naturally occurring VP1 mutations on ganglioside binding, VP1 protein structure, and virus tropism. Infectious entry of single mutants E73Q and E73A and the triple mutant A72V-E73Q-E82Q (VQQ) remains sialic acid dependent, and all three variants acquire binding to a-series gangliosides, including GD1a. However, the E73A and VQQ variants lose the ability to infect ganglioside-complemented cells, and this correlates with a clear shift of the BC2 loop in the crystal structures of E73A and VQQ. On the other hand, the K69N mutation in the K69N-E82Q variant leads to a steric clash that precludes sialic acid binding. Nevertheless, this mutant retains significant infectivity in 293TT cells, which is not dependent on heparan sulfate proteoglycans, implying that an unknown sialic acid-independent entry receptor for BKPyV exists.

Keywords: CP: Microbiology; CP: Molecular biology; capsid; glycan; polyomavirus; receptor; structure; tropism.

Graphical abstract

Figure 1

Patient-derived variants of BKPyV have distinct infectious profiles (A and B) Infectivity assays in 293TT and RS cells. Cells were inoculated with different variants or WT PSVs. Infection was characterized by quantification of GFP⁺ cells. Bar corresponds to the median. Each condition was done in quadruplicate. Significant differences were tested through one-way ANOVA followed by Dunnett’s test with “WT” condition used as the control group. ^∗∗∗∗p < 0.0001, ^∗∗∗p < 0.001, ^∗∗p < 0.01, ^∗p < 0.05. Representative images of cells infected by each PSV in both cell lines are associated with each panel. Scale bar: 100 μm. (C) Comparison of MALDI-TOF-MS profiles of permethylated glycosphingolipids from HEK-293-TT and RS cells. GSLs are present as d18:1/C16:0 (Cer^∗) and d18:1/C24:0 (Cer^∗∗) isomers. yellow circles, galactose; blue circles, glucose; yellow squares, N-acetylgalactosamine; purple diamonds, N-acetylneuraminic acid. (D and E) Percentage of remaining infection by PSVs after neuraminidase treatment in 293TT and RS cells. Bar corresponds to the median. Each condition was done in quadruplicate. Representative images of cells with and without neuraminidase infected with different PSVs are associated with each panel. Scale bar: 100 μm.

Figure 2

BKPyV variants have distinct glycan binding profiles (A) Results table showing the ganglioside-focused glycan microarray data including the list of glycan probes, their sequences and lipid moieties, their fluorescence binding scores, and relative binding intensities (“heatmap”) elicited with the His-tagged BKPyV VP1s. Lipid moieties: Cer, ceramide; DH, 1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine (DHPE). Fluorescence binding signals are shown as means of duplicate spots at 5 fmol per spot; “–” indicates fluorescence intensity less than 1. Errors represent half of the difference between the two values. Color patterns for relative binding intensity: blue, <10%; yellow, 10%–30%; orange, 30%–70%; red, 70%–100%. 100%, the maximum binding score observed across the five experiments. (B) Histograms representing fluorescent intensities of Alexa 647 variant or WT VLPs during cell-binding assays on ganglioside-supplemented LNCaP cells. LNCaP cells alone are shown in a filled gray histogram for negative control, and other conditions with VLPs are represented by colored histograms. (C) Median fluorescence intensity comparison for binding of each VLP to LNCaP cells supplemented with different gangliosides. Bar corresponds to the median. Each condition was done in quadruplicate. Significant differences were tested through one-way ANOVA followed by Dunnett’s test with “no gg” condition used as the control group. ^∗∗∗∗p < 0.0001, ^∗∗∗p < 0.001, ^∗∗p < 0.01, ^∗p < 0.05. (D and E) Infectivity assays on ganglioside-supplemented LNCaP or GM95 cells with WT, E73Q, E73A, and VQQ PSV (D) and WT, VQQ, and N-Q PSV (E). Infection was characterized through quantification of GFP⁺ cells. Bar corresponds to the median. Each condition was done in quadruplicate.

Figure 3

Mutations of amino acids 72 and 73 lead to structural changes in the BC loop conformation (A) Superposition of variant E73Q (PDB: 8AGO), E73A (PDB: 8AGH), and VQQ (PDB: 8AH0) VP1 pentamer structures. One monomer for each structure is highlighted in orange for E73Q, green for E73A, and blue for VQQ. (B) Superposition of variant VP1 pentamers with BKPyV WT VP1 pentamer in association with GD3 oligosaccharides (PDB: 4MJ0). WT VP1 monomer is highlighted in pink, and GD3 molecules are represented by yellow sticks. (C) Zoom on the BC loops of highlighted VP1 monomers. Amino acids 72, 73, and 82 are represented as sticks, and color-coding is as follows: pink, WT VP1; orange, E73Q; blue, VQQ; green, E73A. (D–F) Amino acids 70 to 76 shown as sticks in electron density for variant E73Q (D, orange), E73A (E, green), and VQQ (F, blue). The figure was created using PyMOL.

Figure 4

Structure of N-Q VP1 pentamer (A) Structure of N-Q VP1 pentamer (PDB: 8AH1). VP1 monomer is highlighted in purple. (B) Superposition of N-Q VP1 pentamer onto WT VP1 pentamers associated with GD3 oligosaccharides (PDB: 4MJ0). WT VP1 monomer is highlighted in pink, and GD3 molecules are represented by yellow sticks. (C) Zoom on the BC loops of highlighted VP1 monomers. Amino acids 69 and 82 are represented as sticks. Pink, WT VP1; blue, N-Q VP1. (D) Focus on amino acids in position 69 and their interaction with Neu5Ac. Hydrogen bonds are represented by black dashed lines between K69 and hydroxyl groups of Neu5Ac. The figure was created using PyMOL.

Figure 5

Infectious entry of N-Q and VQQ variant PSV does not involve GAGs (A) Infectivity assays in 293TT cells in the presence of 100 μg/mL of heparin or chondroitin sulfate A/C with BKPyV WT (2 x 10⁶ pEGFP copies/well), VQQ (2 x 10⁷ pEGFP copies/well), and N-Q (2 x 10⁶ pEGFP copies/well) PSVs. HPV16 (2 x 10⁶ pEGFP copies/well) and AAV2 (5 x 10⁵ GE/well) PSVs serve as positive control. Error bars correspond to standard deviations (n = 4). ^∗p < 0.05; #p < 0.001; ns, not significant by ANOVA and Dunnet’s post-hoc test comparing treatment conditions against infection in medium alone (Mock). (B) Infectivity assays in 293TT cells treated with heparinase I/III or chondroitinase ABC with BKPyV WT (2 x 10⁶ pEGFP copies/well), VQQ (2 x 10⁷ pEGFP copies/well), and N-Q (2 x 10⁶ pEGFP copies/well) PSVs. HPV16 (2 x 10⁶ pEGFP copies/well) and AAV2 (5 x 10⁵ GE/well) PSVs serve as positive control. Error bars correspond to standard deviations (n = 4). ^∗p < 0.05; #p < 0.001; ns, not significant by ANOVA and Dunnet’s post-hoc test comparing treatment conditions against infection in medium alone (Mock).

Figure 6

Possible entry mechanisms of N-Q and VQQ variants This figure illustrates one plausible model for the infectious entry of WT and variant BKPyV analyzed in this study. We hypothesize that WT BKPyV could use a two-step mechanism for infection involving (1) binding to b-series gangliosides and potentially to ɑ2-3 sialic acid-bearing glycans, followed by (2) interaction with an unknown co-receptor to potentiate entry into cells. Both the VQQ and N-Q variants had significantly reduced infectivity compared with WT, involving two different mechanisms. Variant VQQ showed broader ganglioside binding, but this was not sufficient for infectious entry in ganglioside-supplemented cells, clearly indicating the use of a co-receptor for the variant entry into cells. However, the VQQ variant retained some infectivity, suggesting that the interaction with this putative co-receptor was reduced, but not entirely abrogated, by the BC2 loop shift observed in the VQQ VP1 structure. The N-Q variant lost binding to sialylated glycans, but it retained some infectivity, suggesting that in certain conditions, BKPyV may interact directly with the unknown receptor for entry. The figure was created with BioRender.com.