Direct Visualization of Ebola Virus Fusion Triggering in the Endocytic Pathway

Spence, Jennifer S.; Krause, Tyler B.; Mittler, Eva; Jangra, Rohit K.; Chandran, Kartik

Antibodies and inhibitors. ZMapp was a gift from John Dye (USAM-RIID). For production of KZ52, various heavy-and light-chain domains of KZ52 were chemically synthesized as codon-optimized sequences for expression in human cells by Epoch Life Sciences and cloned into pMAZ-IgH (containing the expression cassette of human ␥1 chain constant domains) and pMAZ-IgL (containing the expression cassette of human light-chain constant domains) vectors (63) , respectively, using a Gibson assembly kit (New England Biolabs). Sanger sequencing confirmed the identity of the cloned vectors. Antibody production and purification were carried out as described earlier (64), with modifications. Briefly, suspension-adapted HEK293-Freestyle cells-grown in suspension cultures in Freestyle F17 expression media (Thermo Scientific)-were cotransfected with pMAZ-IgH-KZ52 and pMAZ-IgL-KZ52 (1:1 ratio) vectors using polyethyleneimine. At 6 to 7 days posttransfection, the supernatant was collected and incubated overnight at 4°C with protein A agarose resin (GenScript Corporation) in the presence of cOmplete Mini EDTA-free protease inhibitor cocktail (Roche Life Sciences). After the resin was washed with PBS (pH 8.0), bound IgG was eluted with 100 mM glycine (pH 3.0) into 1 M Tris-HCl (pH 8.0). The eluate was concentrated and buffer exchanged with PBS by use of an Amicon centrifugal filter concentrator (Millipore) with a molecular weight cutoff of 30,000 Da. Purified antibody was stored at Ϫ20°C. Protein concentration and yield were determined by UV absorbance, and purity was determined by SDS-PAGE. For labeling coverslip-bound virus, KZ52 was conjugated with Zenon Alexa Fluor 555 (Life Technologies) according to the manufacturer's instructions.