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Structural and Functional Analysis of Murine Polyomavirus Capsid Proteins Establish the Determinants of Ligand Recognition and Pathogenicity.

Buch MH, Liaci AM, O'Hara SD, Garcea RL, Neu U, Stehle T - PLoS Pathog. (2015)

Bottom Line: By comparing electron density of the oligosaccharides within the binding pockets at various concentrations, we show that the [α-2,8]-linked sialic acid increases the strength of binding.Moreover, the amino acid exchanges have subtle effects on their affinity for the validated receptor GD1a.Our results indicate that both receptor specificity and affinity influence MuPyV pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany.

ABSTRACT
Murine polyomavirus (MuPyV) causes tumors of various origins in newborn mice and hamsters. Infection is initiated by attachment of the virus to ganglioside receptors at the cell surface. Single amino acid exchanges in the receptor-binding pocket of the major capsid protein VP1 are known to drastically alter tumorigenicity and spread in closely related MuPyV strains. The virus represents a rare example of differential receptor recognition directly influencing viral pathogenicity, although the factors underlying these differences remain unclear. We performed structural and functional analyses of three MuPyV strains with strikingly different pathogenicities: the low-tumorigenicity strain RA, the high-pathogenicity strain PTA, and the rapidly growing, lethal laboratory isolate strain LID. Using ganglioside deficient mouse embryo fibroblasts, we show that addition of specific gangliosides restores infectability for all strains, and we uncover a complex relationship between virus attachment and infection. We identify a new infectious ganglioside receptor that carries an additional linear [α-2,8]-linked sialic acid. Crystal structures of all three strains complexed with representative oligosaccharides from the three main pathways of ganglioside biosynthesis provide the molecular basis of receptor recognition. All strains bind to a range of sialylated glycans featuring the central [α-2,3]-linked sialic acid present in the established receptors GD1a and GT1b, but the presence of additional sialic acids modulates binding. An extra [α-2,8]-linked sialic acid engages a protein pocket that is conserved among the three strains, while another, [α-2,6]-linked branching sialic acid lies near the strain-defining amino acids but can be accommodated by all strains. By comparing electron density of the oligosaccharides within the binding pockets at various concentrations, we show that the [α-2,8]-linked sialic acid increases the strength of binding. Moreover, the amino acid exchanges have subtle effects on their affinity for the validated receptor GD1a. Our results indicate that both receptor specificity and affinity influence MuPyV pathogenesis.

No MeSH data available.


Related in: MedlinePlus

Binding of ligands to MuPyV VP1.The average simulated annealing Fobs-Fcalc electron density for the Neu5Aca-[α-2,3]-Gala in GT1a, GD1a, and DSLNT is plotted against ligand concentration. RA VP1 is colored blue, PTA VP1, is colored red, and LID VP1 is colored green. GT1a is displayed in a dotted line with triangles, GD1a in a dashed line with circles, and DSLNT in a solid line with squares. The error bars correspond to the standard deviation of the mean electron density observed in the five chains of VP1. A Electron density of GT1a, GD1a, and DSLNT in RA VP1. B Same as in A, but for PTA VP1. C Same as in A, but for LID VP1. D Comparison of GT1a-derived electron density in RA VP1, PTA VP1, and LID VP1. E Same as in D, but for GD1a. F Same as in D, but for DSLNT.
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ppat.1005104.g007: Binding of ligands to MuPyV VP1.The average simulated annealing Fobs-Fcalc electron density for the Neu5Aca-[α-2,3]-Gala in GT1a, GD1a, and DSLNT is plotted against ligand concentration. RA VP1 is colored blue, PTA VP1, is colored red, and LID VP1 is colored green. GT1a is displayed in a dotted line with triangles, GD1a in a dashed line with circles, and DSLNT in a solid line with squares. The error bars correspond to the standard deviation of the mean electron density observed in the five chains of VP1. A Electron density of GT1a, GD1a, and DSLNT in RA VP1. B Same as in A, but for PTA VP1. C Same as in A, but for LID VP1. D Comparison of GT1a-derived electron density in RA VP1, PTA VP1, and LID VP1. E Same as in D, but for GD1a. F Same as in D, but for DSLNT.

Mentions: The GT1a glycan exhibits the strongest binding in all three VP1 variants compared with DSLNT or GD1a (Fig 7A–7C), with no detectable difference between the strains (Fig 7D). This finding is in accord with our ganglioside add-back experiments in cell culture (Fig 2), which consistently showed higher levels of infection mediated by GT1a compared to GD1a. The stronger overall binding of GT1a can be attributed to the additional [α-2,8]-linked sialic acid present in GT1a (Neu5Acb), which contributes several interactions and an increased buried surface area. These contacts seem to outweigh the differences in van der Waals contacts with the side chains of E91 or V296, at least to the extent discernable in our assay.


Structural and Functional Analysis of Murine Polyomavirus Capsid Proteins Establish the Determinants of Ligand Recognition and Pathogenicity.

Buch MH, Liaci AM, O'Hara SD, Garcea RL, Neu U, Stehle T - PLoS Pathog. (2015)

Binding of ligands to MuPyV VP1.The average simulated annealing Fobs-Fcalc electron density for the Neu5Aca-[α-2,3]-Gala in GT1a, GD1a, and DSLNT is plotted against ligand concentration. RA VP1 is colored blue, PTA VP1, is colored red, and LID VP1 is colored green. GT1a is displayed in a dotted line with triangles, GD1a in a dashed line with circles, and DSLNT in a solid line with squares. The error bars correspond to the standard deviation of the mean electron density observed in the five chains of VP1. A Electron density of GT1a, GD1a, and DSLNT in RA VP1. B Same as in A, but for PTA VP1. C Same as in A, but for LID VP1. D Comparison of GT1a-derived electron density in RA VP1, PTA VP1, and LID VP1. E Same as in D, but for GD1a. F Same as in D, but for DSLNT.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4608799&req=5

ppat.1005104.g007: Binding of ligands to MuPyV VP1.The average simulated annealing Fobs-Fcalc electron density for the Neu5Aca-[α-2,3]-Gala in GT1a, GD1a, and DSLNT is plotted against ligand concentration. RA VP1 is colored blue, PTA VP1, is colored red, and LID VP1 is colored green. GT1a is displayed in a dotted line with triangles, GD1a in a dashed line with circles, and DSLNT in a solid line with squares. The error bars correspond to the standard deviation of the mean electron density observed in the five chains of VP1. A Electron density of GT1a, GD1a, and DSLNT in RA VP1. B Same as in A, but for PTA VP1. C Same as in A, but for LID VP1. D Comparison of GT1a-derived electron density in RA VP1, PTA VP1, and LID VP1. E Same as in D, but for GD1a. F Same as in D, but for DSLNT.
Mentions: The GT1a glycan exhibits the strongest binding in all three VP1 variants compared with DSLNT or GD1a (Fig 7A–7C), with no detectable difference between the strains (Fig 7D). This finding is in accord with our ganglioside add-back experiments in cell culture (Fig 2), which consistently showed higher levels of infection mediated by GT1a compared to GD1a. The stronger overall binding of GT1a can be attributed to the additional [α-2,8]-linked sialic acid present in GT1a (Neu5Acb), which contributes several interactions and an increased buried surface area. These contacts seem to outweigh the differences in van der Waals contacts with the side chains of E91 or V296, at least to the extent discernable in our assay.

Bottom Line: By comparing electron density of the oligosaccharides within the binding pockets at various concentrations, we show that the [α-2,8]-linked sialic acid increases the strength of binding.Moreover, the amino acid exchanges have subtle effects on their affinity for the validated receptor GD1a.Our results indicate that both receptor specificity and affinity influence MuPyV pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany.

ABSTRACT
Murine polyomavirus (MuPyV) causes tumors of various origins in newborn mice and hamsters. Infection is initiated by attachment of the virus to ganglioside receptors at the cell surface. Single amino acid exchanges in the receptor-binding pocket of the major capsid protein VP1 are known to drastically alter tumorigenicity and spread in closely related MuPyV strains. The virus represents a rare example of differential receptor recognition directly influencing viral pathogenicity, although the factors underlying these differences remain unclear. We performed structural and functional analyses of three MuPyV strains with strikingly different pathogenicities: the low-tumorigenicity strain RA, the high-pathogenicity strain PTA, and the rapidly growing, lethal laboratory isolate strain LID. Using ganglioside deficient mouse embryo fibroblasts, we show that addition of specific gangliosides restores infectability for all strains, and we uncover a complex relationship between virus attachment and infection. We identify a new infectious ganglioside receptor that carries an additional linear [α-2,8]-linked sialic acid. Crystal structures of all three strains complexed with representative oligosaccharides from the three main pathways of ganglioside biosynthesis provide the molecular basis of receptor recognition. All strains bind to a range of sialylated glycans featuring the central [α-2,3]-linked sialic acid present in the established receptors GD1a and GT1b, but the presence of additional sialic acids modulates binding. An extra [α-2,8]-linked sialic acid engages a protein pocket that is conserved among the three strains, while another, [α-2,6]-linked branching sialic acid lies near the strain-defining amino acids but can be accommodated by all strains. By comparing electron density of the oligosaccharides within the binding pockets at various concentrations, we show that the [α-2,8]-linked sialic acid increases the strength of binding. Moreover, the amino acid exchanges have subtle effects on their affinity for the validated receptor GD1a. Our results indicate that both receptor specificity and affinity influence MuPyV pathogenesis.

No MeSH data available.


Related in: MedlinePlus