Structure analysis of the major capsid proteins of human polyomaviruses 6 and 7 reveals an obstructed sialic acid binding site

Abstract

Human polyomavirus 6 (HPyV6) and HPyV7 are commonly found on human skin. We have determined the X-ray structures of their major capsid protein, VP1, at resolutions of 1.8 and 1.7 Å, respectively. In polyomaviruses, VP1 commonly determines antigenicity as well as cell-surface receptor specificity, and the protein is therefore linked to attachment, tropism, and ultimately, viral pathogenicity. The structures of HPyV6 and HPyV7 VP1 reveal uniquely elongated loops that cover the bulk of the outer virion surfaces, obstructing a groove that binds sialylated glycan receptors in many other polyomaviruses. In support of this structural observation, interactions of VP1 with α2,3- and α2,6-linked sialic acids could not be detected in solution by nuclear magnetic resonance spectroscopy. Single-cell binding studies indicate that sialylated glycans are likely not required for initial attachment to cultured human cells. Our findings establish distinct antigenic properties of HPyV6 and HPyV7 capsids and indicate that these two viruses engage nonsialylated receptors.

Importance: Eleven new human polyomaviruses, including the skin viruses HPyV6 and HPyV7, have been identified during the last decade. In contrast to better-studied polyomaviruses, the routes of infection, cell tropism, and entry pathways of many of these new viruses remain largely mysterious. Our high-resolution X-ray structures of major capsid proteins VP1 from HPyV6 and from HPyV7 reveal critical differences in surface morphology from those of all other known polyomavirus structures. A groove that engages specific sialic acid-containing glycan receptors in related polyomaviruses is obstructed, and VP1 of HPyV6 and HPyV7 does not interact with sialylated compounds in solution or on cultured human cells. A comprehensive comparison with other structurally characterized polyomavirus VP1 proteins enhances our understanding of molecular determinants that underlie receptor specificity, antigenicity, and, ultimately, pathogenicity within the polyomavirus family and highlight the need for structure-based analysis to better define phylogenetic relationships within the growing polyomavirus family and perhaps also for other viruses.

FIG 1

Architecture of HPyV6 and HPyV7 VP1 pentamers. (A and B) Overall folds of HPyV6 (A) and HPyV7 (B) VP1 pentamers shown in a ribbon representation, with VP1 monomers highlighted in magenta and gold, respectively. A dashed line represents the missing HPyV6 CD-loop residues, which are defined only by rather poor electron density due to structural flexibility. (C) Closeup view of the surface loop architectures of HPyV6 and HPyV7 VP1 monomers. The loops are colored as described for panels A and B, respectively. VP1 monomer structures were superposed using the secondary-structure-matching (SSM) superposition tool (45) in the program Coot (42).

FIG 2

Architecture of VP1 surface loops. Superposition and comparison of VP1 surface loop structures are shown for wukipolyomaviruses HPyV6 and KIPyV (PDB accession no. 3S7V) and orthopolyomavirus SV40 (PDB accession no. 3BWR). VP1 monomers were superposed using the secondary-structure-matching (SSM) superposition tool (45). (A) HI-, BC1-, and BC2-loops are highlighted in color in the overview. (B) Closeup view of the HI-loop receptor binding pocket. The sialic acid moiety (Neu5Ac) of the GM1 glycan in the SV40 VP1-GM1 pentasaccharide complex structure (PDB accession no. 3BWR) and the Y256 side chain of HPyV6 VP1 are shown in stick representation. The same view is taken for panels A and B. (C) Sequence alignment of the HI-loop region. Key residues interacting with the sialic acid moiety in the SV40-GM1 glycan and MCPyV VP1–α2,3-sialyllactosamine complex structures (PDB accession no. 4FMI) are highlighted in blue and cyan, respectively. Regions in which all VP1 structures align with root mean square deviation (RMSD) values of <1.0 Å (dark gray) and >1.5 Å (light gray) between Cα atoms are shaded.

FIG 3

Surface structures of VP1 pentamers. Closeup views of VP1 pentamer top-surface regions that are involved in sialic acid engagement in the case of MCPyV and SV40 are shown. Equivalent surface sections are shown in surface and cartoon representations for VP1 pentamers from HPyV6, HPyV7, and KIPyV (PDB accession no. 3S7V) and WUPyV (PDB accession no. 3S7X) (A) and from SV40 (PDB accession no. 3BWR) and MCPyV (PDB accession no. 4FMI) (B). HI-loop residues are highlighted on the surface representations according to the colors assigned to the respective viruses. Carbohydrates (Neu5Ac and Gal of α2,3-sialyllactosamine and GM1 pentasaccharide) in panel B are shown in stick representations (colored by atom type; carbons in orange, oxygen in red, and nitrogen in blue), and glycan-protein contacts (hydrogen bonding and salt bridges) are shown as dashed lines for MCPyV and SV40 VP1.

FIG 4

HPyV6 and HPyV7 VP1 do not engage sialic acids. (A to F) Cell binding analysis. (G) Saturation transfer difference (STD) NMR spectroscopy of HPyV6 and HPyV7 VP1 pentamers with α2,3- and α2,8-sialyllactose. (A to F) HeLa S3 (A to C) and 293TT (D to F) cells were subjected to mock treatment (PBS) or were pretreated with 0.2 U/ml Clostridium perfringens neuraminidase (NA), washed, and then incubated with Alexa Fluor 488-conjugated VP1 pentamers. VP1 pentamer binding was then analyzed by flow cytometry. Histograms represent the fluorescence intensity of Alexa Fluor 488 for 10,000 gated events in each case. Data for cells alone are colored gray and black for mock- and NA-treated cells, respectively. Three independent experiments were performed, and results of a typical experiment are presented. (B and E) JCPyV and murine polyomavirus (RA strain) VP1 pentamers are included as positive controls for neuraminidase-sensitive attachment (30, 51). (C and F) JCPyV L54F is a VP1 mutant with an abolished sialic acid binding site (37) and was used to test for sialic acid-independent cell binding. FITC, fluorescein isothiocyanate; max, maximum. (G) From top to bottom: ¹H reference spectrum of 50 μM HPyV7 VP1 with 1 mM α2,3- and α2,6-sialyllactose each; STD NMR difference spectrum recorded with the same sample; STD NMR difference spectrum of 50 μM HPyV6 VP1 with 1 mM (each) α2,3- and α2,8-sialyllactose. No significant saturation transfer to either capsid protein was observed. HDO peaks were truncated for clarity.

FIG 5

Electrostatic surface potentials of VP1 pentamer from the wuki- and orthopolyomavirus genera. Overall surface representations of HPyV6, HPyV7, and KIPyV (PDB accession no. 3S7V) and WUPyV (PDB accession no. 3S7X) (A) and SV40 VP1 (PDB accession no. 3BWR) and MCPyV (PDB accession no. 4FMI) (B) pentamers are colored according to electrostatic potential (calculated using APBS tool 2.1; 46), with blue and red corresponding to +7 kT and −7 kT, respectively. Views are equivalent in panels A and B and are shown from the top—the outer surface of the virion—along the 5-fold axis of the pentamer. Carbohydrates (GM1 pentasaccharide and Neu5Ac and Gal of α2,3-sialyllactosamine) in panel B are shown in yellow stick representations, and the glycan binding site is highlighted for clarity with a box.