TIM-1-mediated transduction requires PtdSer binding. (A) Transduction of TIM-1+ H3 cells with EBOV pseudovirions in the presence of increasing concentrations of PtdSer or PtdChl liposomes. (B) Transduction of EBOV and LASV pseudovirions into Vero cells in the presence of increasing EGTA concentrations. EGTA in PBS plus 10% FBS was incubated on Vero cells for 1 h before pseudovirions were added for 4 h. Medium was replaced, and transduction was assessed after 24 h. (C) Transduction of EBOV pseudovirions into empty-vector- or TIM-4-transfected HEK 293T cells relative to TIM-1 transduction. (D) AnxV binds to EBOV pseudovirions. Increasing concentrations of HA-tagged AnxV were incubated with ELISA plates prebound with EBOV pseudovirions (filled) or untreated (open). (E) HEK 293T supernatants containing HA-tagged soluble Axl, mSAP, TIM-1, TIM-4, or mutants of TIM-1, N114D or D115N, were incubated with ELISA plates prebound with EBOV pseudovirions or not treated. Relative protein amounts present in supernatants are shown below in a representative Western blot using anti-HA antisera. (F) Binding of TIM-1 and N114D from supernatants to ELISA plates prebound with PtdSer (filled) or PtdChl (open) liposomes (50 μM). (G) Binding of HA-tagged TIM-1 in the presence of IgG2a, MAb ARD5, MAb A6G2, PtdSer, or PtdChl liposomes or 2 mM EGTA to ELISA plates prebound with EBOV pseudovirions. Approximate background absorbance is shown with a dashed line. Data are shown as means ± SEM for at least three replicates. Significance was calculated using one-sample t-test comparison to 100 for panels A to C or a two-sample t test for panel G (**, P < 0.001; *, P < 0.01).