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

To determine the role of GP1 N-glycans in EBOV GP-dependent entry, a library of over 40 individual and combinatorial mutations were made to disrupt the N-linked glycan sites (NGS; N-X-S/T sequons) within the GP1 subunit. We generated NGS mutations in GP1 of both GP and the MLD-deleted GP (GP1⌬muc) (for details of all mutations, see Table S1 in the supplemental material). NGS mutants were expressed in HEK293T cells and pseudotyped onto VSV⌬G-eGFP (expressing enhanced green fluorescent protein). The relative expression levels of wild-type (WT) or NGS mutant GP were determined by assessing the GP-to-VSV matrix ratios present in cell supernatants by dot blot analysis. In addition, the transduction efficiencies of the pseudovirions were assessed in Vero cells.