Three positively charged binding sites on the eastern equine encephalitis virus E2 glycoprotein coordinate heparan...

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.