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

CLEC-expressing HEK293T cells were also used to evaluate the impact of N-linked glycans on our MLD deletion mutant, GP1⌬muc. In these studies, we compared the transduction of GP1⌬muc to that of 5G⌬muc. While our 5G⌬muc mutant was poorly expressed and resulted in low levels of transduction compared to the transduction of GP1⌬muc (see Fig. S2 in the supplemental material), the levels of 5G⌬muc transduction were about 50% of the levels observed with GP and sufficient to allow transduction studies to be performed (Fig. S6) . All five CLECs enhanced GP1⌬muc transduction, increasing entry by 4.5-to 22fold ( Fig. 4B to F) . In all cases, the loss of five of the N-linked glycans (5G⌬muc) on the glycan cap resulted in a decrease in transduction, with the reduction being more modest for transduction mediated by L DC-SIGN, ASGPRI, and L hMGL. As the better transduction of 5G⌬muc into cells expressing L DC-SIGN, ASG-PRI, and L hMGL cannot result from interactions with MLD glycans, this enhancement must be due to either the two remaining intact N-glycans on the GP1 core at N40 and N204 or the two N-glycans on GP2. Additionally, the differences in 5G⌬mucmediated transduction observed between L-SIGN and L DC-SIGN indicate that these CLECs have different ligand specificities, despite both binding to high-mannose oligosaccharides.