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. Author manuscript; available in PMC: 2020 Apr 8.
Published in final edited form as: Cell Rep. 2019 Jul 9;28(2):460–471.e5. doi: 10.1016/j.celrep.2019.06.022

Figure 4. CHIKV residue E1–80 impacts virion fusion dynamics, pH sensitivity and cholesterol dependence.

Figure 4.

(A) CHIKV binding assay. BHK-21 cells were incubated with an equivalent of 100 particles per cell of purified ZsGreen E1-V80, A, and L variants (based on viral genomes) at 4 °C and in the presence of 20 mM ammonium chloride for 5 or 60 min. Then, cells were washed extensively with PBS, and RNA genomes were quantified by RT-qPCR. Data represent three independent experiments, each with internal duplicates, *p<0.05. (B) CHIKV fusion-from-without. Purified ZsGreen viruses were bound to BHK-21 cells at a MOI of 10 for 1 h, at 4 °C. After binding, cells were treated at the indicated pHs for 2 min at 37 °C. pH was neutralized, and the amount of ZsGreen expressing cells was determined 16 hpi. Data represent two independent experiments, each with internal triplicates, * p<0.05, ** p<0.01, *** p < 0.001. (C) Effect of lysosomotropic drugs on CHIKV mutant variants. BHK-21 cells were pre-treated for 3 h with increasing concentrations of ammonium chloride (left panel) or Bafilomycin A1 (right panel), and then infected with purified ZsGreen viruses at a MOI of 1. Data represent two independent experiments, each with internal triplicates, * p<0.05, ** p<0.01, *** p < 0.001. (D) Ammonium chloride by-pass assay. BHK-21 cells were pre-incubated for 1 h, at 4 °C. Purified ZsGreen E1-V80, V80A, and V80L variants (MOI= 0.1) were added to the cells and incubated for 1 h, at 4 °C. Then, cells were incubated at 37 °C, and 20 mM ammonium chloride was added at the indicated time points. Data represent two independent experiments, each with internal duplicates, ** p<0.01, *** p < 0.001, **** p < 0.0001. (E) Cholesterol depletion. BHK-21 cells were treated with increased concentrations of Methyl-β-cyclodextrin (MβCD) for 1 h at 37 °C. Infections with ZsGreen-viruses were performed at a MOI of 1 for 1 h, and subsequently complete media with 20 mM ammonium chloride was added to impair viral spreading. Data represent three independent experiments, each with internal triplicates, ***p < 0.001. (F) Cholesterol repletion. Cholesterol depleted BHK-21 cells were treated with media containing 200 μg/ml soluble cholesterol or media alone. Infections with ZsGreen viruses were performed at a MOI of 1 for 1 h, and subsequently complete media with 20 mM ammonium chloride was added. Data represent two independent experiments, each with internal triplicates, ** p<0.01, *** p<0.001, **** p < 0.0001. All data represents the mean and SEM. P values were determined by one or two-way ANOVA with Bonferroni post-hoc test. See also Figure S4.