Figure 2

VP24 amino acids V170 and N171 are critical for interacting with NP. (a) A structural model of EBOV VP24 based on the crystal structure of Sudan and Reston virus VP24 (PDB 3VNE & 4D9O). Amino acids 169–176 are colored yellow on the surface diagram, and the inset highlights amino acids 169–173 with a ribbon and stick diagram. The arrow indicates a 90° rotation of the molecule around the Y-axis. (b) HEK 293 cells were co-transfected with pCAGGS-NP-FH and pCAGGS-Wild-type (WT) VP24 or a VP24 point mutant: pCAGGS-VP24 169–176A, pCAGGS-VP24 V170A, pCAGGS-VP24 N171A, or pCAGGS-VP24 V170A/N171A. Lysates were immunoprecipitated with mouse anti-FLAG or isotype control (Iso) antibodies, and immunoprecipitation (IP) and whole cell lysate (WCL) fractions were subjected to Western blot (WB) with mouse anti-FLAG, rabbit anti-VP24, or rabbit anti-β-tubulin antibodies. IP/Western blot data are representative of at least three independent experiments. Western blots were cropped for presentation; full-length blots are available in Supplementary Fig. 7. (c) Hela cells were co-transfected with pCAGGS-NP-FH and pCAGGS-Wild-type (WT) VP24 or a VP24 mutant: pCAGGS-VP24 169–176A, pCAGGS-VP24 V170A, pCAGGS-VP24 N171A, or pCAGGS-VP24 V170A/N171A. Following fixation and permeabilization, cells were stained with mouse anti-FLAG and rabbit anti-VP24 antibodies prior to staining with AlexaFluor 488 goat anti-mouse and AlexaFluor 546 goat anti-rabbit secondary antibodies. Coverslips were mounted with ProLong Gold Antifade Mountant containing DAPI to visualize the nuclei. Note that the extranuclear DAPI staining is an occasional artifact of transfection. White scale bars represent 10 uM.