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: NDP52 immunoprecipitates with the PB resident protein Pat1b.A: HEK293T cells were transfected with plasmids expressing pcDNA-FLAG-NDP52 with either pLJM1-GFP (control), pLJM1-GFP-Dcp1a, or pLJM1-GFP-Pat1b for 48 h. Cells were harvested in lysis buffer and incubated with a FLAG mouse antibody (CST) overnight at 4°C. Immunoprecipitation was performed and samples were eluted from the magnetic beads through boiling in 4X Laemmli buffer (BioRad) with 10% v/v beta-mercaptoethanol. Samples were resolved by SDS-PAGE. A representative blot of three independent experiments is shown. B: HUVECs were infected with rKSHV.219 via spinoculation and infection was allowed to proceed for 96 h. Cells were harvested in 2X Laemmli buffer. Samples were resolved by SDS-PAGE and immunoblot was performed for Dcp1a or Pat1b. Samples were quantified by normalizing Dcp1a or Pat1b protein levels to the total protein in each lane using Image Lab (BioRad) and then to Mock. Results were plotted in GraphPad and an unpaired t-test was performed, ±SEM; n = 3, ** = P<0.01. C: Model of PB disassembly induced by KSHV/KapB. KapB increases the phosphorylation of Beclin at Serine 90 to enhance autophagic flux during KSHV latency while Torin induces autophagic flux via the inhibition of mTORC1/2 and dephosphorylation of the ULK complex. Enhanced activation of autophagic flux by either mechanism results in PB disassembly that is dependent on the selective autophagy receptor, NDP52. In our model, we predict that NDP52 recruits a PB component(s) to the nascent autophagosome. Our top candidate for this recruitment is the PB scaffolding protein, Pat1b, as NDP52 co-immunoprecipitated with Pat1b and Pat1b protein level decreases after KSHV infection and Torin treatment. Steady-state levels of Dcp1a also decreased in response to KapB expression, KSHV infection, or Torin treatment, suggesting that Dcp1a decreases may be mediated by its partial interaction with Pat1b despite observations that Dcp1a did not co-immunoprecipitate with NDP52. Autophagic degradation of Pat1b and Dcp1a, both key PB scaffolding proteins, leads to PB disassembly. Figure created with Biorender.

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.