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

Since complete deglycosylation of the core domains of GP1 did not negatively impact the expression of GP or the transduction of pseudotyped virions, we systematically combined N-linked glycan mutations in the MLD with our 7G mutant. Surprisingly, we were able to disrupt all NGS within GP1 without affecting expression (7Gm8G) ( Fig. 2A ; see also Fig. S2E in the supplemental material). Comparison of the 7Gm8G mutant and peptide N-glycosidase F (PNGase F)-treated GP with immunoblot analysis confirmed that the 7Gm8G mutant lacked all N-linked glycans (Fig. S1C ). Pseudovirion transduction of Vero cells was significantly enhanced in all GP mutants that lacked the N40 glycan ( Fig. 2B; Fig. S2D ), and is shown in light gray, RBD is shown in red, and MLD structure that has not been solved is represented as a gray sphere. PBD ID 3CSY. (B) Linear model of EBOV GP. The disulfide bond between GP1 and GP2 is indicated, as well as the locations of N-linked glycans (marked with "Ys") in the GP1 and -2 domains, and the known protease cleavage sites are noted. SP, signal peptide; RBD, receptor-binding domain; MLD, mucin-like domain; IFL, internal fusion loop; HR1 and -2, heptad repeats 1 and 2; TM, transmembrane domain. (C) Alignment of predicted N-linked glycan sites within the GP1 core of the five Ebola virus species. N-X-S/T sequons are highlighted with a black background.