Fig. 1: E2 mutations independently and uniquely alter EEEV-HS interactions in vitro.
A Relative infectivity of WT or mutant SINV/EEEV for CHO-K1, GAG-deficient CHO-pgsA-745, and HS-deficient CHO-pgsD-677 cells (n = 9 from 3 independent experiments). B Binding of chimeric WT and mutant viruses to CHO-K1 and CHO-pgsA-745 cells (n = 6 from 2 independent experiments, except for SINV/EEEV WT where n = 9 from 3 independent experiments). Viruses were allowed to incubate with cells on ice for 75 min, then cells were washed, and the bound virus was quantified by ddPCR as a ratio of genomes bound to cells versus the host Mmadhc gene. C Relative infectivity of WT or mutant SINV/EEEV on BHK-21 cells treated with heparinases (n = 6 from 2 independent experiments). D Relative indirect binding to heparin-agarose beads by WT and mutant SINV/EEEV. Viruses were incubated with collagen-agarose or heparin-agarose beads; unbound viruses were titered on BHK-21 cells (n = 6 from 2 independent experiments). E Apparent binding affinity showing the nonlinear fit of WT or mutant SINV/EEEV for heparin, with KD apparent and Hill slope (h) as a measure of cooperativity, where h > 1 shows increased cooperative binding and h < 1 shows decreased cooperative binding (n = 6 for mutants and n = 5 for WT from 2 independent experiments). F Genome-to-BHK PFU ratios for EEEV WT and mutant viruses (n = 3 independent experiments). G BHK-21 cells were infected with equal genomes of EEEV WT and mutant EEEVs, corresponding to a multiplicity of infection of 1 for WT (n = 6 from 2 independent experiments). Limit of detection indicated with dashed line. Means ± SD are shown. Significance determined by (A–C) two-way ANOVA with Tukey’s post-hoc tests, D Brown-Forsythe and Welch ANOVA with Dunnett’s post-hoc tests, F one-way ANOVA with Tukey’s post-hoc tests on log-transformed data, or G two-way repeated measures ANOVA with Tukey’s post-hoc tests. Exact p-values are indicated. Source data provided as a Source Data file.
