Direct Intracellular Visualization of Ebola Virus-Receptor Interaction by In Situ Proximity Ligation

Proximity ligation is sensitive to perturbations in the GP_CL-NPC1 domain C interface. (A) VSV mNG-P particles bearing EBOV GP or EBOV GP^{T83M/K114E/K115E} were internalized into U2OS^NPC1-eBFP2 cells for 60 min followed by cell fixation, permeabilization, and PLA. Cells were analyzed by fluorescence microscopy; data points represent the percentage of VSV⁺/NPC1⁺/PLA⁺ compartments per individual cell; bars depict the pooled averages and standard deviations (± SD) for all cells from two independent experiments (n ≥ 40). An unpaired two-tailed t test was used to compare VSV⁺/NPC1⁺/PLA⁺ compartments of cells infected by VSV bearing either EBOV GP or EBOV GP^{T83M/K114E/K115E} (****, P < 0.0001). Group means calculated from the percentage of VSV⁺/NPC1⁺/PLA⁺ vesicles were compared by Cohen’s d effect size (d > 1.3). (B) U2OS^NPC1-eBFP2 cells were preincubated with the inhibitor 3.47 (1 μM) for 60 min at 37°C followed by VSV mNG-P EBOV GP uptake for 60 min and PLA. Data points were acquired and analyzed as described in panel A. (C) After preincubation of U2OS^NPC1-eBFP2 and wild-type U2OS cells with increasing 3.47 concentrations, cells were infected with VSV mNG-P EBOV GP for 16 h. Infection was measured by automated counting of mNG⁺ cells and normalized to infection obtained in the absence of 3.47. Averages ± SD for six technical replicates pooled from two independent experiments are displayed. Data were subjected to nonlinear regression analysis to derive 3.47 concentration at half-maximal inhibition of infection (IC₅₀ ± 95% confidence intervals for nonlinear curve fit).

Detection of GP_CL-NPC1 binding by PLA requires endosomal cleavage of GP. (A) VSV mNG-P particles bearing EBOV GP were incubated with either U2OS^NPC1-eBFP2 cells pretreated with E-64d (100 mM for 6 h at 37°C) (left) or U2OS CatB/L-KO cells ectopically overexpressing Flag-tagged NPC1 (right). Cells were fixed following virus incubation (1 h at 37°C), permeabilized, and subjected to PLA. Data points represent the percentage of VSV⁺/NPC1⁺/PLA⁺ compartments per individual cell; bars depict the average ± SD for all data points pooled from two independent experiments (n ≥ 30). Points with reduced transparency represent values outside the 10th to 90th percentiles. An unpaired two-tailed t test was used to compare VSV⁺/NPC1⁺/PLA⁺ compartments of wild-type cells with either inhibitor-treated (left) or KO (right) cells infected by EBOV GP-decorated VSV (****, P < 0.0001). Group means calculated from the percentage of VSV⁺/NPC1⁺/PLA⁺ vesicles were compared by Cohen’s d effect size. (B) Cells described in panel A were exposed to VSV mNG-P EBOV GP uptake for 1 h at 37°C. After fixation, viral particles, NPC1, and GP_CL were visualized by fluorescence microscopy. Representative images from two independent experiments are shown. (C) VSV mNG-P particles bearing EBOV GP were treated in vitro with thermolysin (THL) or cathepsin L (CatL), respectively. Viral particles were taken up into U2OS^NPC1-eBFP2 cells followed by fixing of the cells and subjecting them to PLA. Data points represent the percentage of VSV⁺/NPC1⁺/PLA⁺ compartments per individual cell; bars depict the average ± SD for all data points pooled from two independent experiments (n ≥ 25). To compare VSV⁺/NPC1⁺/PLA⁺ compartments of cells which endocytosed VSV studded with either uncleaved or THL/CatL-cleaved GP, an unpaired two-tailed t test was used (****, P < 0.0001). Cohen’s d effect size was used to compare the group means calculated from the percentages of VSV⁺/NPC1⁺/PLA⁺ vesicles (d > 1.3). (D) U2OS^NPC1-eBFP2 cells were (not) preincubated with E-64d (as described in panel A), and VSV mNG-P particles bearing EBOV GP were treated in vitro with THL. Following virus internalization into U2OS^NPC1-eBFP2, cells were fixed and subjected to PLA. Data points represent the percentage of VSV⁺/NPC1⁺/PLA⁺ compartments per individual cell; bars depict the average ± SD for all data points pooled from two independent experiments (n ≥ 25). Points with reduced transparency represent values outside the 10th to 90th percentiles. To compare VSV⁺/NPC1⁺/PLA⁺ compartments of E-64d-treated cells with those of untreated cells which endocytosed VSV studded with either uncleaved or THL-cleaved GP, an unpaired two-tailed t test was used (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001). Cohen’s d effect size was used to compare the group means calculated from the percentages of VSV⁺/NPC1⁺/PLA⁺ vesicles.

In situ proximity ligation decouples GP_CL-NPC1 interaction from post-binding entry steps. (A) VSV mNG-P particles bearing EBOV GP or EBOV GP^L529A/I544A were internalized into U2OS^NPC1-eBFP2 cells for 60 min followed by PLA. Data points represent the percentage of triple-positive compartments per individual cell; bars depict the average ± SD for all data points pooled from two independent experiments (n ≥ 37). Data analyses included an unpaired two-tailed t test to compare VSV⁺/NPC1⁺/PLA⁺ vesicles of cells which internalized VSV decorated with wild-type or mutant GP (ns, P > 0.05). Cohen’s d effect size was used to compare the group means calculated from the percentages of VSV⁺/NPC1⁺/PLA⁺ compartments. (B) ADI-15946 was incubated with VSV EBOV GP particles and exposed to U2OS^NPC1-eBFP2 (right). After virus uptake for 1 h at 37°C, cells were fixed, and viral particles, NPC1, and bound antibodies were visualized by fluorescence microscopy. Representative images from two independent experiments are shown. Virions were preincubated with increasing amounts of KZ52, mAb100, ADI-15946, or ADI-16061 and then exposed to U2OS^NPC1-eBFP2 cells for 16 h at 37°C (left). The number of infected cells was determined by automated counting of mNG⁺ cells and normalized to infection obtained in the absence of antibodies. Averages ± SD for six technical replicates pooled from two independent experiments are shown. (C) VSV mNG-P EBOV GP virions were complexed with KZ52, mAb100, ADI-15946, or ADI-16061 (50 μg/ml and 100 μg/ml) for 1 h at room temperature. Following internalization into U2OS^NPC1-eBFP2, cells were fixed and subjected to in situ PLA. Data points represent the percentage of VSV⁺/NPC1⁺/PLA⁺ compartments per individual cell; bars show the average ± SD for all data points pooled from two independent experiments (n ≥ 22). VSV⁺/NPC1⁺/PLA⁺ vesicles were analyzed by unpaired two-tailed t test (ns, P > 0.05; *, P < 0.05; **, P < 0.01; ****, P < 0.0001) comparing cells which were exposed to virion-antibody complexes to cells exposed to untreated virus. Group means calculated from the percentage of VSV⁺/NPC1⁺/PLA⁺ vesicles were also compared by Cohen’s d effect size.