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Screening of small molecules was performed at the New England Regional Centers of Excellence for Biodefense and Emerging Infectious Diseases at Harvard Medical School. Infection was assayed using VSV pseudotyped viruses encoding GFP or luciferase. Experiments with native ebolavirus were performed under BSL-4 conditions at the United States Army Medical Research Institute for Infectious Diseases. Cells were infected with EboV Zaire-Mayinga GFP and growth was measured by mean fluorescence. EboV GP_Δ™ is a derivative of EboV GP in which the transmembrane domain has been replaced by a GCN4-derived trimerization domain followed by a His6 tag for purification. Late endosomes/lysosomes (LE/LY) were isolated by differential centrifugation and further purified by Percoll density gradient centrifugation. LE/LY were disrupted by incubation with methionine methyl ester and coated onto high binding ELISA plates. Following attachment, unbound LE/LY membranes were removed and plates were blocked. Bound membranes were incubated with the indicated amounts of native or thermolysin-cleaved EboV GP_Δ™ protein. Unbound EboV GP_Δ™ protein was removed, membranes were washed and bound EboV GP_Δ™ protein was recovered in SDS loading buffer and analyzed by immunoblot using GP1 antiserum. Where applicable, membranes were pre-incubated with 3.0, 3.47, 3.18 or vehicle prior to the addition of EboV GP_Δ™. To analyze EboV GP_Δ™ binding to NPC1, LE/LY membranes were dissolved in 10mM CHAPSO and NPC1 was recovered by immunoprecipitation and the immune complexes were analyzed by immunoblot probed with EboV GP1 antiserum.

A standard lipid mixing reaction contained 45 μL of unlabeled t-SNARE liposomes and 5 μL of v-SNARE liposomes labeled with NBD and rhodamine, and was conducted in a 96-well microplate at 37 °C.^{18 (link)} The NBD-fluorescence (excitation: 460 nm; emission: 538 nm) was measured every 2 min in a BioTek Synergy HT microplate reader. For content mixing assays,^{11a (link),19 (link)} unlabeled t-SNARE liposomes were directed to fuse with sulforhodamine B-loaded v-SNARE liposomes. The sulforhodamine B fluorescence (excitation: 565 nm; emission: 585 nm) was measured every 2 min. At the end of the reaction, 10 μL of 10% CHAPSO was added to each sample.
To assess the regulatory activities of SNARE-binding molecules, v-and t-SNARE liposomes were mixed with the regulators and immediately loaded into a preheated microplate (37 °C) to initiate fusion. The fusion reactions were performed as we previously described,^{11a (link),15a (link)} except that no low-temperature preincubation was included in any of the fusion reactions in this study. Fusion data were presented as the percentage of maximum fluorescence change. The maximum fusion rate within the first 10 min of liposome fusion reaction was used to represent the initial rate of a fusion reaction. Full accounting of statistical significance was included for each figure based on at least three independent experiments.
To introduce macromolecular crowding agents, Ficoll 70 (GE Healthcare), bovine serum albumin (BSA, Fisher), and Dextran 70 (Fluka) were separately dissolved in the reconstitution buffer. To remove impurities, BSA was dialyzed overnight in a dialysis bag against the reconstitution buffer with Bio-Beads (Bio-Rad) added. The final concentration of each crowding agent in the reactions was 100 mg/mL. SM proteins promote fusion with such potency that it is critical to start all fusion reactions immediately after mixing (less than 1 min). Otherwise, SM protein-containing liposomes would fuse during the preparation period, yielding inaccurate initial fluorescence readings.