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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 modes of GT1a to the different MuPyV strains.A Superposition of the GT1a-binding mode of RA (GT1a in sky blue), PTA (dark blue), and LID (pale blue). The Neu5Acb-[α-2,8]-Neu5Aca-[α-2,3]-Gala motif is shown in solid sticks, together with the adjacent GalNAc moiety. 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 Close view of the van-der-Waals contacts introduced by the E91 side chain present in PTA and LID (C), but not in RA (B). Hydrogen bonds are shown in grey, van-der-Waals contacts in cyan. D & E Close view of the hydrophobic contact mediated by V296 in RA and PTA (D), but not by A296 in LID (E). The 4.0 Å hydrophobic contact is not present in the LID strain, whose pocket is opened to the right. Hydrogen bonds are shown in grey, hydrophobic contacts are shown in gold.
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ppat.1005104.g005: Binding modes of GT1a to the different MuPyV strains.A Superposition of the GT1a-binding mode of RA (GT1a in sky blue), PTA (dark blue), and LID (pale blue). The Neu5Acb-[α-2,8]-Neu5Aca-[α-2,3]-Gala motif is shown in solid sticks, together with the adjacent GalNAc moiety. 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 Close view of the van-der-Waals contacts introduced by the E91 side chain present in PTA and LID (C), but not in RA (B). Hydrogen bonds are shown in grey, van-der-Waals contacts in cyan. D & E Close view of the hydrophobic contact mediated by V296 in RA and PTA (D), but not by A296 in LID (E). The 4.0 Å hydrophobic contact is not present in the LID strain, whose pocket is opened to the right. Hydrogen bonds are shown in grey, hydrophobic contacts are shown in gold.

Mentions: Because the differences in tumorigenicity and host spread among strains have been mapped to the glycan binding pocket of VP1, and because GT1a appears to be particularly efficient in facilitating productive infection, we set out to determine how the three strains engage GT1a. By solving the crystal structures of RA and LID VP1 complexed with GT1a using the identical strategy used for the PTA-GT1a complex, we found that the overall binding mode of GT1a is very similar across the three strains (Fig 5A), with a conserved binding mode of the [α-2,8]-linked Neu5Acb. Although the replacement of glutamate with glycine at position 91 leads to a contact area decrease of 33 Å2 in RA, the orientation of GT1a in this strain is not altered (compare Fig 5B and 5C). Likewise, the substitution of valine with alanine at position 296 in LID removes a hydrophobic contact but does not affect the conformation of GT1a (Fig 5E; S2 Fig).


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 modes of GT1a to the different MuPyV strains.A Superposition of the GT1a-binding mode of RA (GT1a in sky blue), PTA (dark blue), and LID (pale blue). The Neu5Acb-[α-2,8]-Neu5Aca-[α-2,3]-Gala motif is shown in solid sticks, together with the adjacent GalNAc moiety. 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 Close view of the van-der-Waals contacts introduced by the E91 side chain present in PTA and LID (C), but not in RA (B). Hydrogen bonds are shown in grey, van-der-Waals contacts in cyan. D & E Close view of the hydrophobic contact mediated by V296 in RA and PTA (D), but not by A296 in LID (E). The 4.0 Å hydrophobic contact is not present in the LID strain, whose pocket is opened to the right. Hydrogen bonds are shown in grey, hydrophobic contacts are shown in gold.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4608799&req=5

ppat.1005104.g005: Binding modes of GT1a to the different MuPyV strains.A Superposition of the GT1a-binding mode of RA (GT1a in sky blue), PTA (dark blue), and LID (pale blue). The Neu5Acb-[α-2,8]-Neu5Aca-[α-2,3]-Gala motif is shown in solid sticks, together with the adjacent GalNAc moiety. 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 Close view of the van-der-Waals contacts introduced by the E91 side chain present in PTA and LID (C), but not in RA (B). Hydrogen bonds are shown in grey, van-der-Waals contacts in cyan. D & E Close view of the hydrophobic contact mediated by V296 in RA and PTA (D), but not by A296 in LID (E). The 4.0 Å hydrophobic contact is not present in the LID strain, whose pocket is opened to the right. Hydrogen bonds are shown in grey, hydrophobic contacts are shown in gold.
Mentions: Because the differences in tumorigenicity and host spread among strains have been mapped to the glycan binding pocket of VP1, and because GT1a appears to be particularly efficient in facilitating productive infection, we set out to determine how the three strains engage GT1a. By solving the crystal structures of RA and LID VP1 complexed with GT1a using the identical strategy used for the PTA-GT1a complex, we found that the overall binding mode of GT1a is very similar across the three strains (Fig 5A), with a conserved binding mode of the [α-2,8]-linked Neu5Acb. Although the replacement of glutamate with glycine at position 91 leads to a contact area decrease of 33 Å2 in RA, the orientation of GT1a in this strain is not altered (compare Fig 5B and 5C). Likewise, the substitution of valine with alanine at position 296 in LID removes a hydrophobic contact but does not affect the conformation of GT1a (Fig 5E; S2 Fig).

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