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A structure-guided mutation in the major capsid protein retargets BK polyomavirus.

Neu U, Allen SA, Blaum BS, Liu Y, Frank M, Palma AS, Ströh LJ, Feizi T, Peters T, Atwood WJ, Stehle T - PLoS Pathog. (2013)

Bottom Line: We have characterized the receptor specificity, structure and infectivity of the human polyomavirus BKPyV, the causative agent of polyomavirus-associated nephropathy, and uncover a molecular switch for binding different carbohydrate receptors.The crystal structure of the BKPyV capsid protein VP1 in complex with GD3 reveals contacts with two sialic acid moieties in the receptor, providing a basis for the observed specificity.Mutation of this residue from lysine in BKPyV to serine in SV40 switches the receptor specificity of BKPyV from GD3 to GM1 both in vitro and in cell culture.

View Article: PubMed Central - PubMed

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

ABSTRACT
Viruses within a family often vary in their cellular tropism and pathogenicity. In many cases, these variations are due to viruses switching their specificity from one cell surface receptor to another. The structural requirements that underlie such receptor switching are not well understood especially for carbohydrate-binding viruses, as methods capable of structure-specificity studies are only relatively recently being developed for carbohydrates. We have characterized the receptor specificity, structure and infectivity of the human polyomavirus BKPyV, the causative agent of polyomavirus-associated nephropathy, and uncover a molecular switch for binding different carbohydrate receptors. We show that the b-series gangliosides GD3, GD2, GD1b and GT1b all can serve as receptors for BKPyV. The crystal structure of the BKPyV capsid protein VP1 in complex with GD3 reveals contacts with two sialic acid moieties in the receptor, providing a basis for the observed specificity. Comparison with the structure of simian virus 40 (SV40) VP1 bound to ganglioside GM1 identifies the amino acid at position 68 as a determinant of specificity. Mutation of this residue from lysine in BKPyV to serine in SV40 switches the receptor specificity of BKPyV from GD3 to GM1 both in vitro and in cell culture. Our findings highlight the plasticity of viral receptor binding sites and form a template to retarget viruses to different receptors and cell types.

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The K68S mutation targets BKPyV to the SV40 receptor GM1.STD difference spectra of (A) BKPyV K68S with GD3, (B and C, respectively) BKPyV K68S and SV40 with GM1. (D) SV40-GM1 off-resonance spectrum. A 50-fold excess of oligosaccharide was used for each spectrum. The off-resonance spectrum was scaled to 3%. Resonances labeled in the difference spectra with GM1 (B and C) receive considerable saturation transfer from BKPyV K68S and SV40. Regions with strong signal overlap are not labeled because they cannot be unambiguously assigned. Binding of BKPyV K68S to GD3, previously seen for WT BKPyV (Fig. 1C), is abolished by the mutation (A). Carbohydrate microarray analyses of recombinant VP1 of (E) BKPyV, (F) BKPyV K68S and (G) SV40 using 21 ganglioside-related saccharide probes, which included the b-series gangliosides as well as GM1 variants NeuNAc-GM1 and NeuNGc-GM1. The doses of probes arrayed per spot are indicated. Numerical scores of the binding signals are means of duplicate spots (with error bars). The complete list of probes and their sequences are in Supplemental Table S1.
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ppat-1003688-g005: The K68S mutation targets BKPyV to the SV40 receptor GM1.STD difference spectra of (A) BKPyV K68S with GD3, (B and C, respectively) BKPyV K68S and SV40 with GM1. (D) SV40-GM1 off-resonance spectrum. A 50-fold excess of oligosaccharide was used for each spectrum. The off-resonance spectrum was scaled to 3%. Resonances labeled in the difference spectra with GM1 (B and C) receive considerable saturation transfer from BKPyV K68S and SV40. Regions with strong signal overlap are not labeled because they cannot be unambiguously assigned. Binding of BKPyV K68S to GD3, previously seen for WT BKPyV (Fig. 1C), is abolished by the mutation (A). Carbohydrate microarray analyses of recombinant VP1 of (E) BKPyV, (F) BKPyV K68S and (G) SV40 using 21 ganglioside-related saccharide probes, which included the b-series gangliosides as well as GM1 variants NeuNAc-GM1 and NeuNGc-GM1. The doses of probes arrayed per spot are indicated. Numerical scores of the binding signals are means of duplicate spots (with error bars). The complete list of probes and their sequences are in Supplemental Table S1.

Mentions: To validate the conclusions derived from the structural comparisons, we introduced a K68S mutation into the BKPyV VP1 pentamer expression construct. Purified K68S pentamers were analyzed by STD NMR for binding to GD3 and GM1. Unlike the WT BKPyV-GD3 pair, almost no saturation transfer was observed for BKPyV K68S and GD3, indicating that the mutation virtually abolished binding to the disialic acid motif of GD3 (Fig. 5A). However, saturation transfer from BKPyV K68S VP1 to GM1 was as efficient as for the SV40 VP1-GM1 pair, which was included for comparison (Fig. 5B–D). This indicates that the K68S mutation switches the binding preference of BKPyV VP1 from GD3 to GM1. The STD NMR spectra of SV40 VP1 and BKPyV K68S VP1 with GM1 are almost indistinguishable, suggesting that GM1 engages in the same contacts with both proteins. Saturation transfer is primarily observed to protons of the NeuNAc 3R and Gal 4L rings. In addition, both the GalNAc and the NeuNAc methyl groups in GM1 received considerable saturation in the complexes, with the NeuNAc methyl group being more affected. Our observations are in good agreement with the crystal structure of the SV40 VP1-GM1 complex [16] and demonstrate that a single amino acid mutation suffices for BKPyV to adapt to the SV40 receptor.


A structure-guided mutation in the major capsid protein retargets BK polyomavirus.

Neu U, Allen SA, Blaum BS, Liu Y, Frank M, Palma AS, Ströh LJ, Feizi T, Peters T, Atwood WJ, Stehle T - PLoS Pathog. (2013)

The K68S mutation targets BKPyV to the SV40 receptor GM1.STD difference spectra of (A) BKPyV K68S with GD3, (B and C, respectively) BKPyV K68S and SV40 with GM1. (D) SV40-GM1 off-resonance spectrum. A 50-fold excess of oligosaccharide was used for each spectrum. The off-resonance spectrum was scaled to 3%. Resonances labeled in the difference spectra with GM1 (B and C) receive considerable saturation transfer from BKPyV K68S and SV40. Regions with strong signal overlap are not labeled because they cannot be unambiguously assigned. Binding of BKPyV K68S to GD3, previously seen for WT BKPyV (Fig. 1C), is abolished by the mutation (A). Carbohydrate microarray analyses of recombinant VP1 of (E) BKPyV, (F) BKPyV K68S and (G) SV40 using 21 ganglioside-related saccharide probes, which included the b-series gangliosides as well as GM1 variants NeuNAc-GM1 and NeuNGc-GM1. The doses of probes arrayed per spot are indicated. Numerical scores of the binding signals are means of duplicate spots (with error bars). The complete list of probes and their sequences are in Supplemental Table S1.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3795024&req=5

ppat-1003688-g005: The K68S mutation targets BKPyV to the SV40 receptor GM1.STD difference spectra of (A) BKPyV K68S with GD3, (B and C, respectively) BKPyV K68S and SV40 with GM1. (D) SV40-GM1 off-resonance spectrum. A 50-fold excess of oligosaccharide was used for each spectrum. The off-resonance spectrum was scaled to 3%. Resonances labeled in the difference spectra with GM1 (B and C) receive considerable saturation transfer from BKPyV K68S and SV40. Regions with strong signal overlap are not labeled because they cannot be unambiguously assigned. Binding of BKPyV K68S to GD3, previously seen for WT BKPyV (Fig. 1C), is abolished by the mutation (A). Carbohydrate microarray analyses of recombinant VP1 of (E) BKPyV, (F) BKPyV K68S and (G) SV40 using 21 ganglioside-related saccharide probes, which included the b-series gangliosides as well as GM1 variants NeuNAc-GM1 and NeuNGc-GM1. The doses of probes arrayed per spot are indicated. Numerical scores of the binding signals are means of duplicate spots (with error bars). The complete list of probes and their sequences are in Supplemental Table S1.
Mentions: To validate the conclusions derived from the structural comparisons, we introduced a K68S mutation into the BKPyV VP1 pentamer expression construct. Purified K68S pentamers were analyzed by STD NMR for binding to GD3 and GM1. Unlike the WT BKPyV-GD3 pair, almost no saturation transfer was observed for BKPyV K68S and GD3, indicating that the mutation virtually abolished binding to the disialic acid motif of GD3 (Fig. 5A). However, saturation transfer from BKPyV K68S VP1 to GM1 was as efficient as for the SV40 VP1-GM1 pair, which was included for comparison (Fig. 5B–D). This indicates that the K68S mutation switches the binding preference of BKPyV VP1 from GD3 to GM1. The STD NMR spectra of SV40 VP1 and BKPyV K68S VP1 with GM1 are almost indistinguishable, suggesting that GM1 engages in the same contacts with both proteins. Saturation transfer is primarily observed to protons of the NeuNAc 3R and Gal 4L rings. In addition, both the GalNAc and the NeuNAc methyl groups in GM1 received considerable saturation in the complexes, with the NeuNAc methyl group being more affected. Our observations are in good agreement with the crystal structure of the SV40 VP1-GM1 complex [16] and demonstrate that a single amino acid mutation suffices for BKPyV to adapt to the SV40 receptor.

Bottom Line: We have characterized the receptor specificity, structure and infectivity of the human polyomavirus BKPyV, the causative agent of polyomavirus-associated nephropathy, and uncover a molecular switch for binding different carbohydrate receptors.The crystal structure of the BKPyV capsid protein VP1 in complex with GD3 reveals contacts with two sialic acid moieties in the receptor, providing a basis for the observed specificity.Mutation of this residue from lysine in BKPyV to serine in SV40 switches the receptor specificity of BKPyV from GD3 to GM1 both in vitro and in cell culture.

View Article: PubMed Central - PubMed

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

ABSTRACT
Viruses within a family often vary in their cellular tropism and pathogenicity. In many cases, these variations are due to viruses switching their specificity from one cell surface receptor to another. The structural requirements that underlie such receptor switching are not well understood especially for carbohydrate-binding viruses, as methods capable of structure-specificity studies are only relatively recently being developed for carbohydrates. We have characterized the receptor specificity, structure and infectivity of the human polyomavirus BKPyV, the causative agent of polyomavirus-associated nephropathy, and uncover a molecular switch for binding different carbohydrate receptors. We show that the b-series gangliosides GD3, GD2, GD1b and GT1b all can serve as receptors for BKPyV. The crystal structure of the BKPyV capsid protein VP1 in complex with GD3 reveals contacts with two sialic acid moieties in the receptor, providing a basis for the observed specificity. Comparison with the structure of simian virus 40 (SV40) VP1 bound to ganglioside GM1 identifies the amino acid at position 68 as a determinant of specificity. Mutation of this residue from lysine in BKPyV to serine in SV40 switches the receptor specificity of BKPyV from GD3 to GM1 both in vitro and in cell culture. Our findings highlight the plasticity of viral receptor binding sites and form a template to retarget viruses to different receptors and cell types.

Show MeSH
Related in: MedlinePlus