A sialic acid binding site in a human picornavirus

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.

Figure 1. CVA24v in complex with its glycan receptor.

The capsid structure of CVA24v with the capsid proteins VP1 (light blue), VP2 (green), VP3 (red) is shown in a surface representation. VP4 is located inside the capsid not visible in this figure. The Neu5Ac entity (black) is located at a positively charged, solvent exposed region of VP1. The atoms of one pentameric section (left) are colored according to the electrostatic potential using a color scale from red to blue. The adjacent pentameric section (right) was colored according to the distance from the center of the capsid, ranging from blue (122 Å) to red (162 Å).

Figure 2. 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.

Figure 3. CVA24v binding inhibition assay and MD simulations.

(A) ³⁵S-labeled CVA24v virions showed substantially reduced binding to human corneal epithelial cells when the virions were pre-incubated with α2,6-linked glycans 6SL and DSLNT, while the effect on α2,3-linked glycans 3SL and 3SLN was less pronounced. (B) Histogram of calculated interaction scores confirmed the preference of α2,6-linked glycans and is in line with the structural and biochemical observations.