Comprehensive Functional Analysis of N-Linked Glycans on Ebola Virus GP1

Lennemann, Nicholas J.; Rhein, Bethany A.; Ndungo, Esther; Chandran, Kartik; Qiu, Xiangguo; Maury, Wendy

Removal of glycans shielding the RBD does not allow for NPC1 binding. Enzymatic removal of the glycan cap and MLD allows interaction of GP with the endosomal receptor NPC1 (23, 24) . Consequently, we postulated that exposing the RBD by disrupting NGS within the GP1 core and MLD might enhance the binding of GP to NPC1. In an NPC1 binding assay, a soluble form of the second luminal domain (C loop) of NPC1, which directly interacts with residues in the RBD of EBOV GP (23) , was used to bind VSV pseudovirions. Treatment of GP with THL, which is commonly used to mimic GP cleavage by CatB/-L in removing the MLD and glycan cap (20, 21) , resulted in binding of C loop over a wide range of concentrations (Fig. 3C) . However, exposure of the RBD through deglycosylation (7Gm8G) did not lead to binding of the NPC1 C loop above the background level, indicating that loss of the N-linked glycans on GP1 does not abrogate the need for endosomal proteolysis that is required for receptor interactions. This result suggests that, while N-glycans on GP may interfere with the ability of the RBD to interact with this endosomal receptor, glycan interference is not the sole explanation for the inability of GP to bind to NPC1.