Fig. 3: Passage of HS/protein receptor binding site mutants on cultured cells selects for mutations that impact binding of both receptors. | Nature Communications

Fig. 3: Passage of HS/protein receptor binding site mutants on cultured cells selects for mutations that impact binding of both receptors.

From: Three positively charged binding sites on the eastern equine encephalitis virus E2 glycoprotein coordinate heparan sulfate- and protein receptor-dependent infection

Fig. 3: Passage of HS/protein receptor binding site mutants on cultured cells selects for mutations that impact binding of both receptors.

A Infectivity of double SINV/EEEV mutants found in passaging experiment on CHO-K1, CHO-pgsA-745, or CHO-pgsD-677 cells. Relative infectivity compared to CHO-K1 was determined for all viruses (n = 9 from 3 independent experiments). B Binding of chimeric passaging mutant viruses to CHO-K1 and CHO-pgsA-745 cells(n = 6 from 2 independent experiments). (Data for single mutant parent viruses are the same as in Fig. 1B.) C Apparent binding affinity showing nonlinear fit of chimeric passaging mutants for heparin, with KD apparent and Hill slope (h) (n = 6 from 2 independent experiments). D, E Infectivity of passaging mutants in (D) K562 cells expressing EV, VLDLR, ApoER2 isoform 1, or ApoER2 isoform 2 (n = 9 from 3 independent experiments, except for the 71–77 and 156–157 mutants on K562-EV cells where n = 12 from 4 independent experiments) or E THP-1 EV or LDLR cells (n = 9 from 3 independent experiments) quantified by flow cytometry. (Data for single mutant parent viruses are the same as in Fig. 2A, B). F Binding of chimeric WT and mutant viruses to K562-EV or K562-VLDLR cells, performed as described in Fig. 2C (n = 6 from 2 independent experiments, except for 1561-57 where n = 9 from 3 independent experiments). Binding is expressed as a ratio to GAPDH and normalized to EV. (Data for single mutant parent viruses are the same as in Fig. 2C.) G Apparent binding affinity showing nonlinear fit of chimeric passaging mutants for VLDLR LA(1-2)-Fc, with KD apparent and h (n = 6 from 2 independent experiments). H Neutralization of chimeric passaging mutant viruses by VLDLR LA(1-2)-Fc (100–0.1 µg in tenfold dilutions and 0 µg control) in CHO-K1 and CHO-pgsA-745 cells (n = 4 from 2 independent experiments). Means ± SD are shown. Significance was determined by (A) one-way ANOVA with Bonferroni’s post-hoc tests and (B, D–F, and H) two-way ANOVA with Tukey’s post-hoc tests. Exact p-values are indicated. Source data provided as a Source Data file.

Back to article page