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A sialic acid binding site in a human picornavirus.

Zocher G, Mistry N, Frank M, Hähnlein-Schick I, Ekström JO, Arnberg N, Stehle T - PLoS Pathog. (2014)

Bottom Line: Biochemical studies verified the relevance of the binding site and demonstrated a preference of CVA24v for α2,6-linked glycans.This preference can be rationalized by molecular dynamics simulations that show that α2,6-linked glycans can establish more contacts with the viral capsid.Our results form an excellent platform for the design of antiviral compounds to prevent AHC.

View Article: PubMed Central - PubMed

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

ABSTRACT
The picornaviruses coxsackievirus A24 variant (CVA24v) and enterovirus 70 (EV70) cause continued outbreaks and pandemics of acute hemorrhagic conjunctivitis (AHC), a highly contagious eye disease against which neither vaccines nor antiviral drugs are currently available. Moreover, these viruses can cause symptoms in the cornea, upper respiratory tract, and neurological impairments such as acute flaccid paralysis. EV70 and CVA24v are both known to use 5-N-acetylneuraminic acid (Neu5Ac) for cell attachment, thus providing a putative link between the glycan receptor specificity and cell tropism and disease. We report the structures of an intact human picornavirus in complex with a range of glycans terminating in Neu5Ac. We determined the structure of the CVA24v to 1.40 Å resolution, screened different glycans bearing Neu5Ac for CVA24v binding, and structurally characterized interactions with candidate glycan receptors. Biochemical studies verified the relevance of the binding site and demonstrated a preference of CVA24v for α2,6-linked glycans. This preference can be rationalized by molecular dynamics simulations that show that α2,6-linked glycans can establish more contacts with the viral capsid. Our results form an excellent platform for the design of antiviral compounds to prevent AHC.

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Glycan binding and attachment to CVA24v.(A) Overview of all glycans used in our incorporation experiment. The glycans 6SL and DSLNT bind well to CVA24v based on the electron density (green background). Very weak binding is observed for the LSTc, Sialyl-LewisX, 3SL and 3SLN (yellow background), and no binding could be detected for GM1, GM2, GD1a, GD1b, and GD3 (pink background). (B) The unbiased (Fo-Fc)-omit map (2.9σ, pink) revealed binding of the Neu5Ac entity (orange) of DSLNT and 6SL between two protomers with main interactions to the DE-loop and the HI-loop of clockwise rotated (cw) protomer. A galactose entity is shown (grey, not included into the deposited coordinates) which emphasize the direction of glycan binding towards the solvent. (C) Neu5Ac is recognized by hydrogen bonds to Y725, S727 and cwY830. The carbon atom C2 linking the adjacent glycan entity is marked.
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ppat-1004401-g002: Glycan binding and attachment to CVA24v.(A) Overview of all glycans used in our incorporation experiment. The glycans 6SL and DSLNT bind well to CVA24v based on the electron density (green background). Very weak binding is observed for the LSTc, Sialyl-LewisX, 3SL and 3SLN (yellow background), and no binding could be detected for GM1, GM2, GD1a, GD1b, and GD3 (pink background). (B) The unbiased (Fo-Fc)-omit map (2.9σ, pink) revealed binding of the Neu5Ac entity (orange) of DSLNT and 6SL between two protomers with main interactions to the DE-loop and the HI-loop of clockwise rotated (cw) protomer. A galactose entity is shown (grey, not included into the deposited coordinates) which emphasize the direction of glycan binding towards the solvent. (C) Neu5Ac is recognized by hydrogen bonds to Y725, S727 and cwY830. The carbon atom C2 linking the adjacent glycan entity is marked.

Mentions: Neu5Ac is required for infectivity of CVA24v; the roles of additional sugar moieties and the preferred Neu5Ac linkage have not been determined. In order to advance an understanding of the requirements for CVA24v binding to sialylated glycans, we derivatized CVA24v crystals with eleven physiologically relevant, commercially available sialyloligosaccharides (Figure 2A) that differ in glycan composition and linkage. We determined all structures to high resolution. In unbiased (2Fo-Fc)-omit maps, we observed a contoured electron density at a σ-level of 1.0 for the glycan only for α2,6-sialyllactose (6SL) and disialyllacto-n-tetraose (DSLNT) (Figure 2A), a hexasaccharide that carries an α2,3 and α2,6-linked Neu5Ac. In contrast, we observed very weak binding (corresponding to a σ–level of 0.7 in a (2Fo-Fc)-omit map) for α2,3-sialylated glycans, and no detectable binding for any β–branched or α2,8-α2,3-disialylated glycan. Thus, our analysis indicated that CVA24v preferentially engages glycans that contain α2,6-linked sialyloligosaccharide epitopes.


A sialic acid binding site in a human picornavirus.

Zocher G, Mistry N, Frank M, Hähnlein-Schick I, Ekström JO, Arnberg N, Stehle T - PLoS Pathog. (2014)

Glycan binding and attachment to CVA24v.(A) Overview of all glycans used in our incorporation experiment. The glycans 6SL and DSLNT bind well to CVA24v based on the electron density (green background). Very weak binding is observed for the LSTc, Sialyl-LewisX, 3SL and 3SLN (yellow background), and no binding could be detected for GM1, GM2, GD1a, GD1b, and GD3 (pink background). (B) The unbiased (Fo-Fc)-omit map (2.9σ, pink) revealed binding of the Neu5Ac entity (orange) of DSLNT and 6SL between two protomers with main interactions to the DE-loop and the HI-loop of clockwise rotated (cw) protomer. A galactose entity is shown (grey, not included into the deposited coordinates) which emphasize the direction of glycan binding towards the solvent. (C) Neu5Ac is recognized by hydrogen bonds to Y725, S727 and cwY830. The carbon atom C2 linking the adjacent glycan entity is marked.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4199766&req=5

ppat-1004401-g002: Glycan binding and attachment to CVA24v.(A) Overview of all glycans used in our incorporation experiment. The glycans 6SL and DSLNT bind well to CVA24v based on the electron density (green background). Very weak binding is observed for the LSTc, Sialyl-LewisX, 3SL and 3SLN (yellow background), and no binding could be detected for GM1, GM2, GD1a, GD1b, and GD3 (pink background). (B) The unbiased (Fo-Fc)-omit map (2.9σ, pink) revealed binding of the Neu5Ac entity (orange) of DSLNT and 6SL between two protomers with main interactions to the DE-loop and the HI-loop of clockwise rotated (cw) protomer. A galactose entity is shown (grey, not included into the deposited coordinates) which emphasize the direction of glycan binding towards the solvent. (C) Neu5Ac is recognized by hydrogen bonds to Y725, S727 and cwY830. The carbon atom C2 linking the adjacent glycan entity is marked.
Mentions: Neu5Ac is required for infectivity of CVA24v; the roles of additional sugar moieties and the preferred Neu5Ac linkage have not been determined. In order to advance an understanding of the requirements for CVA24v binding to sialylated glycans, we derivatized CVA24v crystals with eleven physiologically relevant, commercially available sialyloligosaccharides (Figure 2A) that differ in glycan composition and linkage. We determined all structures to high resolution. In unbiased (2Fo-Fc)-omit maps, we observed a contoured electron density at a σ-level of 1.0 for the glycan only for α2,6-sialyllactose (6SL) and disialyllacto-n-tetraose (DSLNT) (Figure 2A), a hexasaccharide that carries an α2,3 and α2,6-linked Neu5Ac. In contrast, we observed very weak binding (corresponding to a σ–level of 0.7 in a (2Fo-Fc)-omit map) for α2,3-sialylated glycans, and no detectable binding for any β–branched or α2,8-α2,3-disialylated glycan. Thus, our analysis indicated that CVA24v preferentially engages glycans that contain α2,6-linked sialyloligosaccharide epitopes.

Bottom Line: Biochemical studies verified the relevance of the binding site and demonstrated a preference of CVA24v for α2,6-linked glycans.This preference can be rationalized by molecular dynamics simulations that show that α2,6-linked glycans can establish more contacts with the viral capsid.Our results form an excellent platform for the design of antiviral compounds to prevent AHC.

View Article: PubMed Central - PubMed

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

ABSTRACT
The picornaviruses coxsackievirus A24 variant (CVA24v) and enterovirus 70 (EV70) cause continued outbreaks and pandemics of acute hemorrhagic conjunctivitis (AHC), a highly contagious eye disease against which neither vaccines nor antiviral drugs are currently available. Moreover, these viruses can cause symptoms in the cornea, upper respiratory tract, and neurological impairments such as acute flaccid paralysis. EV70 and CVA24v are both known to use 5-N-acetylneuraminic acid (Neu5Ac) for cell attachment, thus providing a putative link between the glycan receptor specificity and cell tropism and disease. We report the structures of an intact human picornavirus in complex with a range of glycans terminating in Neu5Ac. We determined the structure of the CVA24v to 1.40 Å resolution, screened different glycans bearing Neu5Ac for CVA24v binding, and structurally characterized interactions with candidate glycan receptors. Biochemical studies verified the relevance of the binding site and demonstrated a preference of CVA24v for α2,6-linked glycans. This preference can be rationalized by molecular dynamics simulations that show that α2,6-linked glycans can establish more contacts with the viral capsid. Our results form an excellent platform for the design of antiviral compounds to prevent AHC.

Show MeSH
Related in: MedlinePlus