The glycan shield of alphaherpesvirus glycoprotein B modulates host co-receptor binding

Abstract Alphaherpesviruses can infect a wide range of cell types and are notorious for their neurotropism. They enter cells through the fusion activity of the essential viral glycoprotein gB. Interactions of gB with co-receptors Myelin Associated Glycoprotein (MAG), paired immunoglobulin-like type 2 receptor alpha (PILRα) and Non-muscle Myosin Heavy Chain IIA (NMHC-IIA) influence cell tropism and infectivity. Both MAG and PILRα are known to bind to sialic acids, highlighting the potential role for gB glycosylation in mediating the co-receptor interactions. Here, using glycoproteomics, cryo electron microscopy and surface plasmon resonance, we study the specificity of the three co-receptor interactions with gB from herpes simplex virus type 1 (HSV-1), type 2 (HSV-2), and varicella zoster virus (VZV). We show that all gB variants are heterogeneously N-glycosylated at six or seven predicted sites with moderate degrees of sialylation, complemented with extensive O-linked glycosylation at the disordered N-terminus and a flexible loop within domain II. All three gB variants bind to MAG with similar affinity, mediated by sialic acids on gB O-glycans. In contrast, PILRα binds only to HSV-1 and HSV-2, but not VZV gB. PILRα binding is also mediated by sialic acids on gB O-glycans, of which we identify multiple candidate sites in HSV-1 and HSV-2, and none in VZV gB. NMHC-IIA binds to HSV-1 gB and HSV-2 gB with high affinity through a glycan-independent interaction, and not to VZV gB. These data reveal divergent co-receptor specificity and interaction strength and provide an improved biophysical basis to understand human neurotropic herpesvirus infection.