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

Pseudovirion matrix and glycoprotein quantitation. Equal volumes of cell supernatants containing pseudovirions from three independent stocks were lysed in native lysis buffer (phosphate-buffered saline [PBS] with 0.025% NP-40) and then passed through a dot blot apparatus onto nitrocellulose (Whatman). Wells were washed 5ϫ with PBS before being blocked in PBS with 10% nonfat milk for 1 h. Dot blots were incubated with the mouse anti-VSV matrix monoclonal antibody (MAb) 23H12 (a gift from Douglas Lyles) and the human anti-EBOV GP MAb KZ52 (a gift from Erica Saphire and Dennis Burton) diluted in PBS with 10% nonfat milk and 0.15% Tween-20 overnight at 4°C. By using separate IRDyeconjugated secondary antibodies (Abs) (LI-COR) directed toward the primary Abs, we were able to detect both VSV matrix protein and EBOV within a single well for each sample. The signals were visualized and quantified using an Odyssey Imaging Station and Image Studio software (LI-COR), which has been shown to be more sensitive and quantitative than enhanced chemiluminescence (47) . Thermolysin sensitivity assay. VSV pseudovirions, normalized for GP expression, were incubated with 200 g/ml, 2-fold serial dilutions starting with 5 g/ml, or a single concentration of 1.25 g/ml of thermolysin (THL) at 37°C for 15 min. The reaction mixtures were immediately placed on ice and diluted 20-fold in growth medium containing 50 M phosphoramidon (Sigma), a THL inhibitor. THL-treated pseudovirions were evaluated for Vero cell transduction. GFP-positive cells were analyzed by flow cytometry.