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

Abstract

Naturally circulating strains of eastern equine encephalitis virus (EEEV) bind heparan sulfate (HS) receptors and this interaction has been linked to its neurovirulence. Previous studies associated EEEV-HS interactions with three positively charged amino acid clusters on the E2 glycoprotein. One of these sites has recently been reported to be critical for binding EEEV to very-low-density lipoprotein receptor (VLDLR), an EEEV receptor protein. The proteins apolipoprotein E receptor 2 (ApoER2) isoforms 1 and 2, and LDLR have also been shown to function as EEEV receptors. Herein, we investigate the individual contribution of each HS interaction site to EEEV HS- and protein receptor-dependent infection in vitro and EEEV replication in animals. We show that each site contributes to both EEEV-HS and EEEV-protein receptor interactions, providing evidence that altering these interactions can affect disease in mice and eliminate mosquito infectivity. Thus, multiple HS-binding sites exist in EEEV E2, and these sites overlap functionally with protein receptor interaction sites, with each type of interaction contributing to tissue infectivity and disease phenotypes.

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). Viruses were titrated on all three cell types, and percent infectivity compared to CHO-K1 was determined for each cell type. 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 minutes, 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 Genome-to-BHK PFU ratios for EEEV WT and mutant viruses (n = 3 independent experiments). F 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). All error bars show standard deviation (SD). 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, (E) one-way ANOVA with Tukey’s post-hoc tests on log-transformed data, or (F) two-way repeated measures ANOVA with Tukey’s post-hoc tests, *(p<0.05), ** (p<0.01) ***(p<0.001), ****(p<0.0001).

Fig. 2.. E2 mutations that disrupt E2 HS-binding sites also interfere with EEEV-protein receptor interactions.

A-B Infection of chimeric eGFP reporter WT and mutant SINV/EEEV viruses quantified by flow cytometry. A K562 cells expressing empty vector (EV), VLDLR, ApoER2 isoform1, or ApoER2 isoform2; infection expressed as fold-increase (n = 9 from 3 independent experiments, except for EV where n = 15 from 5 independent experiments). B THP-1 cells expressing EV or LDLR; infection expressed as percent infectivity (n = 9 from 3 independent experiments). C Binding of chimeric WT and mutant viruses to K562-EV or K562-VLDLR cells (n = 12 from 4 independent experiments for WT, n = 9 from 3 independent experiments for 84–119 and 156–157, and n = 6 from 2 independent experiments for 71–77). Viruses were allowed to incubate with cells on ice for 75 minutes, then cells were washed, and the bound virus was quantified by qPCR, then expressed as a ratio to GAPDH and normalized to EV. D Neutralization of chimeric eGFP reporter WT and mutant viruses by VLDLR LA(1–2)-Fc or LDLRAD3 LA1-Fc (100–0.1 μg/mL in 10-fold dilutions and 0 μg control) in Vero cells (n = 4 from 2 independent experiments). E Neutralization of chimeric eGFP reporter WT and mutant viruses by VLDLR LA(1–2)-Fc in CHO-K1 and CHO-pgsA-756 cells (n = 4 from 2 independent experiments, except for WT where n = 9 from 3 independent experiments). F Neutralization of chimeric eGFP reporter WT by heparin or BSA control (2,000–2 μg/mL in 10-fold dilutions and control) in cells overexpressing protein receptors (n = 6 from 2 independent experiments) quantified by flow cytometry. Significance was determined by two-way ANOVA with Tukey’s post-hoc tests. Error bars show SD, except for (B) where error bars show standard error of the mean (SEM); *(p<0.05), **(p<0.01), ***(p<0.001), ****(p<0.0001).

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). Viruses were titrated on all three cell types, and percent infectivity compared to CHO-K1 was determined for each cell type. B Binding of chimeric passaging mutant viruses to CHO-K1 and CHO-pgsA-745 cells, performed as described in Fig. 1B (n = 6 from 2 independent experiments) with bound virus expressed as the ratio to Mmadhc. (Data for single mutant parent viruses are the same as in Fig. 1B.) C-D Infectivity of passaging mutants in (C) 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 = 15 from 5 independent experiments) or (D) 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.) E 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.) F Neutralization of chimeric passaging mutant viruses by VLDLR LA(1–2)-Fc (100–0.1μg in 10-fold dilutions and 0 μg control) in CHO-K1 and CHO-pgsA-745 cells (n = 4 from 2 independent experiments). Significance was determined by (A) one-way ANOVA with Bonferroni’s post-hoc tests and (B-F) two-way ANOVA with Tukey’s post-hoc tests. Error bars show SD, except for (E) where error bars show SEM; *(p<0.05), **(p<0.01), ***(p<0.001), ****(p<0.0001).

Fig. 4.. Mutation of HS/protein receptor binding sites attenuates EEEV disease.

A-C Groups of five CD-1 mice were infected with equal genomes of WT and mutant EEEV viruses equivalent to 10³ WT PFU in fp. (n = 15 mice from 3 independent experiments). A Survival. B Weight change. C Clinical signs. D-E Groups of five CD-1 mice were infected ic. with equal genomes of WT and mutant SINV/EEEV virus equivalent to 10⁴ WT PFU (n = 15 mice from 3 independent experiments for SINV/EEEV WT and SINV/EEEV 84–119, and n= 10 mice from 2 independent experiments for SINV/EEEV 71–77 and SINV/EEEV 156–157). D Survival. E Weight change. F Clinical signs. All error bars show SEM. Significance determined by log-rank test; *(p<0.05), ** (p<0.01) ***(p<0.001), ****(p<0.0001).

Fig. 5.. Mutation of HS/protein receptor binding sites diminishes protection conferred by the receptor decoy inhibitor.

Survival data for mice that were treated intraperitoneally with 100 μg of VLDLR LA(1–2)-Fc (dashed line) or LDLRAD3 LA1-Fc (solid line) as a control. Six hours after treatment, mice were infected fp. with equal genomes of WT and mutant EEEV viruses equivalent to 10³ WT PFU (n = 6 mice from 2 independent experiments for LDLRAD3 LA1-Fc and n = 8 mice from 2 independent experiments for VLDLR LA(1–2)-Fc). A EEEV WT (FL93–939). B EEEV 71–77. C EEEV 84–119. D EEEV 156–157. Significance of survival for mice treated with control versus VLDLR LA(1–2)-Fc was determined by log-rank test; *(p<0.05), **(p<0.01), ***(p<0.001).

Fig. 6.. Location of protein receptor- and HS-binding residues on a ribbon model of the EEEV E2 trimer.

A ribbon model of the structure of EEEV E2 proteins as they appear in the E1/E2 trimeric spike. Side chains are displayed for residues involved in HS-binding (blue), residues identified in structural analysis as being involved in binding to one VLDLR molecule^– (red), and residues that are involved in both HS-binding and are direct contacts for VLDLR-binding (purple). Side chains for residues mutated during in vitro passage are lime green (K562-VLDLR) or cyan (BHK-21). A Top view. B Side view. Figures were made using UCSF ChimeraX^,.