Fig 2: KapB-mediated PB disassembly and ARE-mRNA reporter expression require the selective autophagy receptor NDP52.A: HUVECs were sequentially transduced, first with recombinant lentivirus expressing KapB or an empty vector control and selected with blasticidin (5 μg/mL), and second with recombinant lentivirus expressing shRNAs targeting NDP52 or a non-targeting control (NS) and selected with puromycin (1 μg/mL). Coverslips were fixed in 4% paraformaldehyde, permeabilized in 0.1% Triton X-100 and immunostained for Hedls (PBs; white), DAPI (nuclei, blue). Scale bar = 20 μm. Hedls puncta were quantified using CellProfiler and presented as number of Hedls puncta per cell, all cells counted are displayed. Results were plotted in GraphPad and a 2-way ANOVA was performed on the main column effects with a Tukey’s multiple comparison test, bar represents the mean; n = 3, * = P<0.05. B: HUVECs were transduced with recombinant lentivirus expressing an shRNA targeting NDP52 and selected with puromycin (1 μg/mL). Cells were treated with Torin (250 nM) or a DMSO control for 2 h prior to fixation in 4% paraformaldehyde and permeabilization in 0.1% Triton X-100. Samples were immunostained for Hedls (PBs; white), DAPI (nuclei, blue). Scale bar = 20 μm. Hedls puncta were quantified using CellProfiler and presented as number of Hedls puncta per cell, all cells counted are displayed. Results were plotted in GraphPad and a 2-way ANOVA was performed on the main column effects with a Tukey’s multiple comparison test, bar represents the mean; n = 3, **** = P<0.0001. C: HUVECs were transduced with recombinant lentiviruses expressing either an shRNA targeting NDP52 or a non-targeting control (NS) and selected with puromycin (1 μg/mL). Cells were treated with DMSO or Torin (250 nM) for 4 h prior to harvest in 2X Laemmli buffer. Samples were resolved by SDS-PAGE and immunoblot was performed for Dcp1a, Pat1b or DDX6. Samples were quantified by normalizing Dcp1a or Pat1b protein levels to the total protein in each lane using Image Lab (BioRad) and then to the NS DMSO control. Results were plotted in GraphPad and a 2-way ANOVA was performed, ±SEM; n = 3 (Dcp1a) n = 4 (Pat1b), * = P<0.05. D: HeLa Tet-Off cells were transduced with recombinant lentivirus expressing shRNAs targeting NDP52, OPTN, p62 or a NS control and selected with puromycin (1 μg/mL) then cells were co-transfected, treated with Dox and luciferase activity was recorded and analyzed as in Fig 4. Data were plotted in GraphPad as the mean fold change in the relative luciferase activity of each condition compared to vector NS or KapB NS; n = 4. An unpaired t-test was performed; * = P<0.05 ** = P<0.01. E: HUVECs were sequentially transduced first with recombinant lentivirus expressing shNDP52 targeting the 3’-UTR of NDP52 or a NS control and selected with blasticidin (5 μg/mL), and second, with recombinant lentivirus expressing KapB and either mCherry control (mCh) or RFP-NDP52. Coverslips were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and immunostained with Hedls (PBs, green). Scale bar = 20 μm. F: Samples from E were quantified; Hedls puncta were quantified using CellProfiler and presented as number of Hedls puncta per cell, all cells counted are displayed. Results were plotted in GraphPad and a 2-way ANOVA was performed on the main column effects with a Tukey’s multiple comparison test, bar represents the mean; n = 3, **** = P<0.0001.

Fig 3: Dcp1a protein levels are decreased by KapB expression and Torin treatment.A: HUVECs were treated with DMSO or Torin (250 nM) for 4 h prior to harvest. Samples were lysed in 2X Laemmli buffer and resolved by SDS-PAGE before immunoblotting for Xrn1, EDC4/Hedls, Dcp1a, and DDX6. Samples were quantified by normalizing the PB resident protein levels to the total protein in each lane and then the DMSO control using ImageLab (BioRad). Results were plotted in GraphPad and a one-way ANOVA was performed ±SEM; n = 3, ** = P<0.01. B: HUVECs were transduced with recombinant lentiviruses expressing either KapB or an empty vector control and selected with blasticidin (5 μg/mL). Cells were treated with DMSO or Bafilomycin A1 (BafA1, 10 nM) for 4 h prior to harvest in 2X Laemmli buffer. Samples were resolved by SDS-PAGE and immunoblot was performed for Dcp1a or p62 (autophagy marker). Samples were quantified by normalizing Dcp1a protein levels to the total protein in each lane using Image Lab (BioRad) and then to the vector DMSO control. Representative blot is from the same membrane, hashed line indicates skipped lanes. Results were plotted in GraphPad and a 2-way ANOVA was performed, ±SEM; n = 3, *** = P<0.001. C: HUVECs were transduced with recombinant lentiviruses expressing either KapB or an empty vector control and selected with blasticidin (5 μg/mL). Samples were harvested in 2X Laemmli buffer, resolved by SDS-PAGE and immunoblot was performed for Xrn1, Hedls/EDC4, or DDX6. Samples were quantified by normalizing PB protein levels to the total protein in each lane using Image Lab (BioRad) and then to the vector control. Results were plotted in GraphPad. D: HUVECs were sequentially transduced: first with recombinant lentiviruses expressing either shRNAs targeting Atg5 (shAtg5) or a non-targeting control (NS) and selected with puromycin (1 μg/mL), and second with either KapB or an empty vector control and selected with blasticidin (5 μg/mL). Samples were harvested in 2X Laemmli buffer and resolved by SDS-PAGE. Immunoblot was performed for Dcp1a, Atg5, and KapB. Samples were quantified by normalizing Dcp1a protein levels to the total protein in each lane using Image Lab (BioRad) and then to the vector NS control. Results were plotted in GraphPad and a 2-way ANOVA was performed, ±SEM; n = 3, * = P<0.05, **P = <0.01.

Fig 4: EBOV replication depends on multiple components of the decapping complex.a Depletion of DCP2 and EDC3 protein with siRNA. Corresponding bands on each blot (upper panels) were quantitated (lower panels). One of three biological replicates is shown. b Impact of depletion of DCP2 or EDC3 on EBOV infection measured through RNAFISH of viral mRNA. Samples were infected at an MOI of 0.8. Scale bar = 250 μm. c Quantification of b. Statistical analysis was performed using one-way ANOVA with multiple comparisons correction. Four technical replicates of one biological replicate are shown. Data are presented as mean ± SD. *P-value = 0.0111; **P-value = 0.0074; ****P-value < 0.0001. d, e DCP1A and DCP2 colocalize with VP35 during infection. Cells were challenged with EBOV at an MOI of 1, fixed at 20 hpi, stained with the indicated antibodies and imaged. Localization of d DCP1A and e DCP2 puncta in VP35 inclusion bodies was observed. Scale bars = 10 μm. Optical slicing of z-stacks were performed as described in Fig. 3 with the x-y axis at top left and x-z and y-z axes at lower and right panels and the focus of the x-z and y-z planes are indicated by dashed lines. One technical replicate of a representative biological replicate is shown. Source data are provided as a Source Data File.