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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

Differences in receptor binding patterns across glycans.A Superposition of the binding modes of GD1a to RA (light green), PTA (dark green), and LID (pale green). The sequence of GD1a is shown in the inset. The Neu5Aca-[α-2,3]-Gala motif is shown in solid sticks, together with the adjacent GalNAc moiety. In all figures, GT1a bound to PTA is overlaid as a grey ghost for comparison, with Neu5Acb omitted for clarity. Deviations exceeding the atomic error of the structure and alignment rmsd values are only found in the stem region of the sugar, starting at Galb. All superpositionings were carried out in PyMOL [61] using ‘align’ for the protein chains only. Surface, E91 and V296 are from PTA/GT1a. All ‘align’ rmsd values are below 0.16 Å. B & C Comparison of the DSLNT binding modes to RA (red), PTA (orange), and LID (violet). In PTA-DSLNT, [α-2,6]-branching causes a 15–20° psi angle shift of the GlcNAc moiety compared to GD1a and GT1a, resulting in a 1 Å sideward twist movement of the stem. In RA-DSLNT, combination of this shift with a 15° shift in the phi angle results in a downward movement of GlcNAc and its branching Neu5Acc compared to PTA-DSLNT. In LID, the shift is already observable for Gala and results in the loss of ordered density for GlcNAc. All angles were analyzed with CARP. The sequence of DSLNT is shown in the inset of panel C.
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ppat.1005104.g006: Differences in receptor binding patterns across glycans.A Superposition of the binding modes of GD1a to RA (light green), PTA (dark green), and LID (pale green). The sequence of GD1a is shown in the inset. The Neu5Aca-[α-2,3]-Gala motif is shown in solid sticks, together with the adjacent GalNAc moiety. In all figures, GT1a bound to PTA is overlaid as a grey ghost for comparison, with Neu5Acb omitted for clarity. Deviations exceeding the atomic error of the structure and alignment rmsd values are only found in the stem region of the sugar, starting at Galb. All superpositionings were carried out in PyMOL [61] using ‘align’ for the protein chains only. Surface, E91 and V296 are from PTA/GT1a. All ‘align’ rmsd values are below 0.16 Å. B & C Comparison of the DSLNT binding modes to RA (red), PTA (orange), and LID (violet). In PTA-DSLNT, [α-2,6]-branching causes a 15–20° psi angle shift of the GlcNAc moiety compared to GD1a and GT1a, resulting in a 1 Å sideward twist movement of the stem. In RA-DSLNT, combination of this shift with a 15° shift in the phi angle results in a downward movement of GlcNAc and its branching Neu5Acc compared to PTA-DSLNT. In LID, the shift is already observable for Gala and results in the loss of ordered density for GlcNAc. All angles were analyzed with CARP. The sequence of DSLNT is shown in the inset of panel C.

Mentions: The previously identified MuPyV receptor GD1a is similar to a truncated GT1a structure, containing only a Neu5Aca-[α-2,3]-Gala motif instead of Neu5Acb-[α-2,8]-Neu5Aca-[α-2,3]-Gal. The disaccharide engages all three strains in a very similar manner (Fig 6A). Neither the longer E91 side chain (in PTA and LID) nor the shorter A296 side chain (in LID) result in an altered conformation of the ligand.


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)

Differences in receptor binding patterns across glycans.A Superposition of the binding modes of GD1a to RA (light green), PTA (dark green), and LID (pale green). The sequence of GD1a is shown in the inset. The Neu5Aca-[α-2,3]-Gala motif is shown in solid sticks, together with the adjacent GalNAc moiety. In all figures, GT1a bound to PTA is overlaid as a grey ghost for comparison, with Neu5Acb omitted for clarity. Deviations exceeding the atomic error of the structure and alignment rmsd values are only found in the stem region of the sugar, starting at Galb. All superpositionings were carried out in PyMOL [61] using ‘align’ for the protein chains only. Surface, E91 and V296 are from PTA/GT1a. All ‘align’ rmsd values are below 0.16 Å. B & C Comparison of the DSLNT binding modes to RA (red), PTA (orange), and LID (violet). In PTA-DSLNT, [α-2,6]-branching causes a 15–20° psi angle shift of the GlcNAc moiety compared to GD1a and GT1a, resulting in a 1 Å sideward twist movement of the stem. In RA-DSLNT, combination of this shift with a 15° shift in the phi angle results in a downward movement of GlcNAc and its branching Neu5Acc compared to PTA-DSLNT. In LID, the shift is already observable for Gala and results in the loss of ordered density for GlcNAc. All angles were analyzed with CARP. The sequence of DSLNT is shown in the inset of panel C.
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Related In: Results  -  Collection

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ppat.1005104.g006: Differences in receptor binding patterns across glycans.A Superposition of the binding modes of GD1a to RA (light green), PTA (dark green), and LID (pale green). The sequence of GD1a is shown in the inset. The Neu5Aca-[α-2,3]-Gala motif is shown in solid sticks, together with the adjacent GalNAc moiety. In all figures, GT1a bound to PTA is overlaid as a grey ghost for comparison, with Neu5Acb omitted for clarity. Deviations exceeding the atomic error of the structure and alignment rmsd values are only found in the stem region of the sugar, starting at Galb. All superpositionings were carried out in PyMOL [61] using ‘align’ for the protein chains only. Surface, E91 and V296 are from PTA/GT1a. All ‘align’ rmsd values are below 0.16 Å. B & C Comparison of the DSLNT binding modes to RA (red), PTA (orange), and LID (violet). In PTA-DSLNT, [α-2,6]-branching causes a 15–20° psi angle shift of the GlcNAc moiety compared to GD1a and GT1a, resulting in a 1 Å sideward twist movement of the stem. In RA-DSLNT, combination of this shift with a 15° shift in the phi angle results in a downward movement of GlcNAc and its branching Neu5Acc compared to PTA-DSLNT. In LID, the shift is already observable for Gala and results in the loss of ordered density for GlcNAc. All angles were analyzed with CARP. The sequence of DSLNT is shown in the inset of panel C.
Mentions: The previously identified MuPyV receptor GD1a is similar to a truncated GT1a structure, containing only a Neu5Aca-[α-2,3]-Gala motif instead of Neu5Acb-[α-2,8]-Neu5Aca-[α-2,3]-Gal. The disaccharide engages all three strains in a very similar manner (Fig 6A). Neither the longer E91 side chain (in PTA and LID) nor the shorter A296 side chain (in LID) result in an altered conformation of the ligand.

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