Fig. 3: VP35 interacts with the decapping complex through EDC4. | Nature Communications

Fig. 3: VP35 interacts with the decapping complex through EDC4.

From: A protein-proximity screen reveals Ebola virus co-opts the mRNA decapping complex through the scaffold protein EDC4

Fig. 3

a Pulldowns of HA-tagged mRNA decapping proteins using anti-HA beads in the presence of FLAG-tagged VP35 show VP35 coprecipitates only with EDC4 (lane 6). One of two biological replicates is shown. b Confirmation of VP35-EDC4 interaction by pulldown of FLAG-tagged VP35 using anti-FLAG beads. One of two biological replicates is shown. c Cells challenged with EBOV at MOI 1 were fixed at 20 hpi, labeled with VP35 and EDC4 antibodies, and imaged by super-resolution confocal microscopy (48 nm resolution in x-y plane). Optical slicing of z-stacks shows x-y axis (top left), x-z and y-z axes (lower and right panels). EDC4 puncta (green) were present throughout the cell and within VP35-positive inclusion bodies (red), with colocalized puncta in yellow. Z-planes spanned 500 nm. Focus planes of x-z and y-z axes are shown with dashed lines. Merged image includes CellMask (blue), EDC4 (green), and VP35 (red). Scale bar = 5 μm. Right Quantification of EDC4 puncta within 21 inclusion bodies of varying volumes. Linear regression yielded R² = 0.78, p = 9.51E-08. One technical replicate of one representative biological replicate is shown. d Cells were infected at MOI 0.5 to visualize both infected and uninfected cells. EDC4 puncta volumes were measured in VP35-positive infected (n = 99) and uninfected (n = 119) cells using Imaris software. Representative merged image and individual channels shown with quantification at right (unpaired t-test, p < 0.0001). e EDC4 levels in EBOV- or mock-infected cells were measured by immunoblot and quantitated relative to β-actin using image densitometry and area under the curve analysis. Values were scaled by 104 to account for low signal intensities. One representative biological replicate containing two technical replicates is shown. f Proximity ligation assay (PLA) with antibodies to EDC4 and VP35. Cells were challenged with EBOV or mock-infected and fixed at 20 hpi. Left shows PLA signal (magenta); right shows PLA merged with CellMask (blue). Scale bar = 25 μm. Quantification shown at right (full images in Supplementary Fig. 4b). One representative biological replicate with four technical replicates is shown. Data are presented as mean ± SD. Statistical significance was determined using an unpaired t-test (p = 0.0131). Source data are provided as a Source Data File.

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