Sialic Acid-Containing Glycolipids Extend the Receptor Repertoire of Enterovirus-D68

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  1. Enterovirus-D68 strains carrying a VP1 mutation bind to sulfated glycosaminoglycans. (A) Phylogenetic tree of enterovirus-D68 (EV-D68) clades derived from the Nextstrain database (B) Heparin sulfate binding profile of various EV-D68 strains. Two independent arrays from two different batches of virus stocks were performed. Data displayed represent one independent experiment. (C) In a single-cycle viral replication assay, RD cells were pretreated with NaClO3 and infected with EV-D68 strains at multiplicity of infection (MOI) of 1. 10 μM of the replication inhibitor (S)-fluoxetine (SFX) targeting the viral protein 2C was used as positive control. Twenty-four hours postinfection (hpi), virus titers of cell lysates were determined by end point dilution. Data represent values from averaged technical replicates from three independent experiments ± the standard error of mean (SEM). Statistical analysis was performed with Student’s t test. (D) EV-D68 strains incubated with 1 mg/mL soluble heparin were used to infect RD cells at a multiplicity of infection (MOI) of 1. Cells were fixed 24 h hpi and stained for presence of double stranded RNA (green) and nuclei were visualized with Hoechst…

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  2. Enterovirus-D68 strains bind to SIA containing glycolipids. (A) Glycan structures containing α2,3-linked (black bars position 1–6), α2,6-linked N-linked glycans (white bars position 7–18), and ganglioside structures (cyan bars position A-M) containing α2,3- or α2,8-linked sialic acids used in the glycan array. (B) N-Glycan and glycolipid binding profile of various enterovirus-D68 strains. Two independent arrays from two different batches of virus stocks were performed. Data displayed represent one independent experiment.

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  3. Glycolipid depletion reduces Enterovirus-D68 replication. (A) In a single-cycle viral replication assay, RD cells were first pretreated with 5 μM GEN-Z123346 (Gen-Z) for 72 h (hours) or 100 mU/mL neuraminidase (NA) for 2 h. After pretreatment, RD cells were infected with Enterovirus-D68 (EV-D68) strains at multiplicity of infection (MOI) of 1. 10 μM of the replication inhibitor (S)-fluoxetine (SFX) targeting the viral protein 2C was used as positive control. Twenty-four hours postinfection, virus titers of lysates were determined by end point dilution. Data represent values from all technical replicates ± the standard error of mean (SEM). Statistical analysis was performed with one way ANOVA comparing the values to mock treatment. (B) EV-D68 strains were preincubated with the multivalent glycolipid mimics GM3 and GD3 and their corresponding backbone (BB, LAK) for 1 h at 33 °C. Afterward RD cells were infected with MOI 1 and 24 h postinfection virus titers of cell lysates were determined by end point dilution. Data represent values from averaged technical replicates from three independent experiments ± SEM. Statistical analysis was performed with One-Way ANOVA comparing the values…

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  4. Amino acids coordinating the sialic acid binding. Pocket containing the pocket factor (PDB: 4WM8) as well as the pocket accommodating 3′SLN (dark gray) and 6’SLN (white) (Overlay 5BNP and 6CVB) are shown. The VP1 protein is shown in cyan, VP3 in red and VP2 in green. (B) Close-up of the sialic acid binding pocket containing 3′SLN (dark gray) and 6’SLN (white) with the interaction residues. (C) Alignment of the amino acid residues in VP3 and VP1 that coordinate the sialic acid moiety.

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  5. Variation in pH and thermostability stability among Enterovirus-D68 strains. (A) Heparan sulfate (HS)-binding or (B) other enterovirus-D68 (EV-D68) strains were incubated at different pH and infectivity was assessed by end point dilution. Sigmoid Curve fit was used to calculate the 50% inhibitory pH value (IC50). Data displayed represents averages of technical replicates ± standard error of the mean (SEM) and are derived from three independent experiments. Acid stability of EV-D68 strains were compared to the lab-adapted Fermon strain and statistical significance was analyzed by One-way ANOVA with the Tukey post hoc test. (C) HS-binding EV-D68 strains or (D) other EV-D68 strains were incubated at different temperatures ranging from 20 to 60 °C for 15 min. Infectivity was assessed by end point dilution. Sigmoid Curve fit was used to calculate the IC50 temperature values. Data displayed represents averages of technical replicates ± SEM and are derived from three independent experiments. Thermostability of EV-D68 strains compared to the lab-adapted Fermon strain was analyzed by One-way ANOVA with the Tukey post hoc test. Asterisks indicate statistically significant differences P >…

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  6. Acid sensitivity of Enterovirus-D68 strains. (A) Hela cells were pretreated with 200 nM bafilomycin A1 (BAF-A1) for 1 h at 37 °C followed by infection with Enterovirus-D68 (EV-D68) strains at multiplicity of infection (MOI) 1. After 24 h, virus titers in supernatants were determined by end point dilution. Data displayed represents averages of technical replicates ± standard error of the mean (SEM) and are derived from three independent experiments. Statistical analysis was performed with Students t test comparing untreated to BAF-A1 treated infected cells. P > 0.05; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001 (B) Hela cells were pretreated with 200 nM BAF-A1 for 1h at 37 °C followed by infection with EV-D68 strains at MOI 1. After 24 h, cells were fixed and stained for the presence of double stranded RNA (green) and nuclei were visualized using Hoechst (blue). (C) Quantification of infection percentage comparing EV-D68 and human rhinovirus-B14 (RV-B14) infected Hela cells with untreated or BAF-A1 pretreatment. Data displayed represents averages of technical replicates ± SEM and are derived from three independent experiments. (D) Quantification of infection percentage…