FIG 13.

Pteropus alecto tetherin restricts the release of Ebola and Marburg virus-like particles (VLPs). P. alecto tetherin isoforms A (PA), B (PB), and C (PC), co-transfected with an (A) Ebola or (B) Marburg virus construct. Mammalian HEK293T cells were co-transfected with 200 ng of Ebola or Marburg virus plasmid expression construct encoding a VP40-eGFP protein which generates VLPs, and 0 to 200 ng of the tetherin plasmid expression vector. VLPs were harvested at 48 h and concentrated by ultracentrifugation through a sucrose gradient. VLP and cell lysates were subjected to SDS-PAGE and Western blot analysis. VLPs were detected with a mouse anti-GFP 4B10 primary antibody and a goat anti-mouse Alexa Fluor 680 fluorophore-conjugated fluorescent secondary antibody. Representative Western blots are shown. The extent of VLP restriction was quantitated by densitometric analysis of Western blots comparing the relative ratios of VLPs present in the viral lysates and cell lysates from n = 5 independent assays. Error bars represent the standard deviation. A nonparametric Wilcoxon rank-sum test was performed to calculate the statistical significance of the restriction of Ebola and Marburg VLPs in the 200 ng tetherin isoform A, B, and C treatment groups; P < 0.01 in all cases. SN, cell culture supernatant; CL, cell culture lysate.