Fig 1.
ASFV induced the production of proinflammatory cytokines.
(A and B) Bone-marrow-derived macrophages (BMDMs) were infected with ASFV at an MOI of 1 and harvested at 6, 12, and 24 h post-infection (hpi). The transcriptional levels (A) and secretion levels (B) of IL-1β, IL-6, IL-18, IL-10, TNF-α, and IFN-γ in the cells and cell culture supernatant were measured by qPCR and ELISA, respectively. (C) ASFV infected with BMDMs for indicated time point. The ASFV HAD50 in BMDMs was determined. (D) ASFV infected with BMDMs for indicated time point. ASFV p30 protein expression in BMDMs was detected by Western blot (WB). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001; statistical significance was determined by one-way ANOVA.
Fig 2.
ASFV infection induced pyroptosis.
(A–C) BMDMs were infected with ASFV (MOI = 0.1/1/10) for 24 h. Cell viability (A), Casp-1 activity (B), and LDH release (C) were measured using CCK-8 assay kit, Casp-1 activity assay kit, and LDH assay kit, respectively. (D) PAMs were infected with ASFV-GFP (MOI = 1) for 24 h, followed by propidium iodide (PI) staining to observe cell death. A positive control (LPS [60 ng/mL for 8 h] + nigericin [Nig, 2 μM for 2 h]) was included. (E) BMDMs were infected with ASFV (MOI = 1) for 24 h. Expression of pyroptosis marker proteins (Casp-1, IL-1β, and GSDMD-N) was analyzed by WB. (F) PAMs were infected with ASFV at an MOI of 1 for 24 h, fixed with 2% glutaraldehyde, and processed for transmission electron microscopy (TEM). Arrows indicate plasma membrane rupture. (G) iPAM cells were infected with ASFV at an MOI of 1. Formation of ASC specks and membrane localization of N-GSDMD were observed by immunofluorescence assay (IFA). (H) iPAM cells were infected with ASFV (MOI = 1) or positive control (LPS + Nig). ASC oligomerization was detected by WB using disuccinimidyl suberate (DSS) crosslinking. (I) PBMCs were infected with ASFV (MOI = 1) for 24 h. Expression of pyroptosis markers (Casp-1, IL-1β, N-GSDMD) and phosphorylation of IκBα and p65 in the NF-κB pathway were analyzed by WB. (J) PBMCs were infected with ASFV (MOI = 1) for 24 h, followed by PI staining to observe cell death. (K) PBMCs were infected with ASFV (MOI = 1) for 24 hours. Cell viability, Casp-1 activity, IL-1β transcription, and ASFV p72 mRNA level were assessed by CCK-8 assay, Casp-1 activity kit, qPCR, and qPCR, respectively. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001; statistical significance was determined by one-way ANOVA.
Fig 3.
ASFV induced pyroptosis via the NLRP3 inflammasome.
(A and B) PAMs were pretreated with the Casp-1 inhibitor VX765 (10 μM, 2 h) (A) or the NLRP3 inhibitor MCC950 (10 μM, 1 h) (B), followed by ASFV infection (MOI = 1) for 24 h. DMSO was used as a control. IL-1β secretion was measured by ELISA, and pyroptosis biomarkers (Casp-1, IL-1β, N-GSDMD) expression were determined by WB. (C) PAMs were pretreated with VX765 or MCC950 as in (A and B), infected with ASFV (MOI = 1) for 24 h, and subjected to PI staining. ASFV-induced cell death was observed. (D–F) PAMs were transfected with shASC (D), shCasp-1 (E), or shNLRP3 (F) for 72 h, followed by ASFV infection (MOI = 1) for 24 h. shNC was used as a negative control. ELISA used for IL-1β secretion detection, and pyroptosis biomarkers expression were determined by WB. (G) PAMs were transfected with shASC, shCasp-1, or shNLRP3 for 72 h, infected with ASFV (MOI = 1) for 24 h, and subjected to PI staining. LPS + Nig served as a positive control. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001; statistical significance was determined by one-way ANOVA.
Fig 4.
ASFV pEP364R induced IL-1β production and pyroptosis.
(A) PK-15 cells, with activated NLRP3 inflammasome components, were transfected with 2 μg of each of the 185 plasmids encoding ASFV proteins. IL-1β secretion was screened by ELISA to identify proteins inducing high IL-1β levels. (B) iPAMs cells were transfected with 2 μg of EP364R plasmid. At 24 h post-transfection (h.p.t), cells were fixed and the subcellular localization of EP364R was observed by confocal microscopy. Scale bars, 20 μm. (C) BMDMs cells were transfected with 2 μg of EP364R plasmid. IL-1β secretion in the supernatant was measured by ELISA at 24 h. (D) iPAMs were transfected with increasing concentrations (0.25, 0.5, 1.0 μg) of EP364R plasmid, with 1 μg of empty vector (EV) plasmid as a control. At 24 h, 10 μl of CCK-8 reagent was added, followed by incubation at 37°C for 1 hour. Absorbance was measured at 450 nm. (E) iPAMs were transfected with 2 μg of EP364R plasmid. Lactate dehydrogenase (LDH) release was detected using an LDH assay kit at 0, 12, and 24 h. (F) iPAMs were transfected with 2 μg of EP364R plasmid, alongside blank control, positive control [LPS (60 ng/mL for 8 h) + Nigericin (Nig, 2 μM for 2 h)], liposome (4 μl) control, and negative control (EV, 2 μg). At 24 h, cells were stained with propidium iodide (PI) and cell death was observed by fluorescence microscopy. (G) Cell morphology was observed by microscopy 24 h after transfection of iPAMs with 2 μg of EP364R plasmid. (H) iPAMs were co-transfected with plasmids for NLRP3 (3 μg), pro-IL-1β (3 μg), ASC (1 μg), pro-Casp-1 (1 μg), and increasing amounts of EP364R (0, 2, 4, 6 μg). Cells were harvested and lysed 24 h p.t., and IL-1β secretion was analyzed by WB. (I) iPAMs were transfected with 2 μg of EP364R plasmid, with LPS + Nig treatment as a positive control. Cells were lysed 24 h p.t., and the expression of Casp-1, IL-1β, and GSDMD-N was analyzed by WB. (J) iPAMs were transfected with 2 μg of EP364R plasmid, with LPS + Nig as a positive control. At 24 h p.t., cell lysates were centrifuged. The supernatant was prepared as the ‘input’ sample. The cell pellet was resuspended in 100 μl PBS, cross-linked with 2 mM DSS at 37°C for 30 min, and then directly mixed with protein loading buffer to prepare the ‘pellet’ sample. ASC and GSDMD-N oligomerization were detected by WB. (K) iPAMs were transfected with 2 μg of EP364R plasmid. At 24 h p.t., cells were fixed, and ASC speck formation and GSDMD-N localization were observed by confocal microscopy. Scale bars, 20 μm. A P value less than 0.05 was considered statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 5.
pEP364R triggered inflammation in mice.
(A) The pHBLV-EP364R lentiviral overexpression plasmid (10 μg) was co-transfected with lentiviral packaging helper plasmids psPAX2 (10 μg) and pMD2.G (10 μg) into HEK293T cells. Supernatants containing lentivirus were collected at 48 and 72 h p.t., and purified, and used to infect iPAMs. Stable puromycin-resistant cells expressing green fluorescent protein (GFP) were selected. Stable EP364R expression was confirmed by WB and PCR. (B) Casp-1, IL-1β, and GSDMD-N in EP364R-overexpressed iPAMs (LV-EP364R) were detected by WB, iPAMs expressing only GFP and resistance genes served as negative controls (LV-Vector), and Mock cells were normal iPAMs. (C-D) Transcriptional and secretion levels of cytokines IL-1β, IL-6, TNF-α, CXCL10, and IFN-β were measured by qPCR and ELISA in EP364R-overexpressed iPAMs. (E) Successful EP364R overexpressed lentivirus was purified and determined by WB at 72 h p.t. (F) Mice were intravenously injected twice with 5 × 107 TU of purified EP364R overexpression lentivirus, with a 48-h interval. Mice were randomly selected from each group and euthanized, and pathological changes in the heart, liver, spleen, lung, and kidney were examined. (G) Mice from the LV-EP364R and LV-Vector groups were randomly selected and euthanized. Tissues (heart, liver, spleen, lung, kidney; 1 g each) were harvested, homogenized, and proteins were extracted using tissue lysis buffer. Expression of IL-1β was detected by WB. And p value less than 0.05 was considered statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 6.
ASFV pEP364R deficiency impaired pyroptosis and IL-1β production.
(A) iPAMs were transfected with 2 μg of small hairpin RNA (shEP364R-1, -2, or -3) plasmids. At 72 h p.t., cells were transfected with 2 μg of EP364R plasmid for 24 h. small hairpin RNA Nonspecific shRNA (shNC) as a negative control. RNA was extracted for qPCR, and cells were lysed for WB analysis to screen for the most effective shEP364R. (B) iPAMs were transfected with 2 μg of shEP364R-3 plasmid. At 72 h p.t., cells were infected with ASFV (MOI = 1). EP364R transcription levels were validated by qPCR 24 h p.i (hours post infection). (C) iPAMs were transfected with 50 nM of the selected siEP364R. At 72 hours post-transfection, cells were infected with ASFV at an MOI of 1. The culture supernatants were collected 24 hours post-infection, processed through repeated freeze-thaw cycles and lyophilization, and then analyzed by ELISA to quantify secreted IL-1β. (D) iPAMs were transfected with 2 μg of shEP364R-3 plasmid. At 72 h p.t., cells were infected with ASFV (MOI = 1) for 24 h. IL-1β secretion was measured by ELISA, and expression of Casp-1, IL-1β, and GSDMD-N was analyzed by WB. (E) iPAMs were transfected with 2 μg of shEP364R-3 plasmid. At 72 h p.t., cells were infected with ASFV at MOI = 1 for 24 h. CCK-8 reagent (10 μl) was added, followed by incubation at 37°C for 1 h, and absorbance was measured at 450 nm. LPS and Nigericin stimulation group as a positive control. (F) iPAMs were transfected with 2 μg of shEP364R-3 or shNC plasmid. At 72 h p.t., cells were infected with ASFV at MOI = 1 for 24 h. Intracellular Casp-1 activity was measured using a Caspase-1 activity assay kit. (G) iPAMs were transfected with 2 μg of shEP364R-3 plasmid. At 72 h p.t., cells were infected with ASFV at MOI = 1 for 24 h. LDH release was detected using an LDH assay kit. (H) iPAMs were transfected with 2 μg of shEP364R-3 plasmid. At 72 h p.t., cells were infected with ASFV at MOI = 1 for 24 h. Cells were stained with PI (10 μl) and cell death was observed by fluorescence microscopy. (I) iPAMs were transfected with 2 μg of shEP364R-3 plasmid. At 72 h p.t., cells were infected with ASFV at MOI = 1 for 24 h. Cell lysates were centrifuged; the supernatant was prepared as the ‘input’ sample. The pellet was resuspended in PBS, cross-linked with DSS, incubated at 37°C for 30 min, and mixed with loading buffer to prepare the ‘pellet’ sample. ASC oligomerization was detected by WB. (J) iPAMs were transfected with 2 μg of shEP364R-3 plasmid. At 72 h p.t., cells were infected with ASFV at MOI = 1 for 24 h. RNA was extracted, and transcriptional levels of IL-1β, IL-6, TNF-α, CXCL10, and EP364R were measured by qPCR. A P value less than 0.05 was considered statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 7.
EP364R induced pyroptosis and IL-1β production via the NLRP3 inflammasome.
(A) iPAMs were pretreated with the NLRP3 inhibitor MCC950 (10 μM) for 1 h, then transfected with 3 μg of EP364R plasmid. IL-1β secretion was measured by ELISA and expression of Casp-1, IL-1β, and GSDMD-N was analyzed by WB 24 h p.t. (B) iPAMs were pretreated with the Casp-1 inhibitor VX765 (10 μM) for 2 h, then transfected with 3 μg of EP364R plasmid. IL-1β secretion was measured by ELISA and expression of Casp-1, IL-1β, and GSDMD-N was analyzed by WB 24 h p.t. (C-E) iPAMs were transfected with 3 μg of shNLRP3 (C), shASC (D), or shCasp-1 (E) plasmids. At 72 h p.t., cells were transfected with 3 μg of EP364R plasmid. IL-1β secretion was measured by ELISA and expression of Casp-1, IL-1β, and GSDMD-N was analyzed by WB 24 h later. (F) iPAMs were transfected with 3 μg of shNLRP3, shASC, or shCasp-1 plasmids. At 72 h p.t., cells were transfected with 3 μg of EP364R plasmid. At 24 h p.t., cells were stained with PI (10 μl) and cell death was observed by microscopy. (G) Bone marrow cells were isolated from NLRP3-/- knockout mice and differentiated for 7 days in medium containing L929 cell-conditioned supernatant. Cells were then transfected with 3 μg of EP364R plasmid. Transcriptional levels of EP364R and NLRP3 were measured by qPCR 24 h p.t., showing no significant difference in EP364R transcription but confirmed absence of NLRP3. (H) Bone marrow-derived macrophages (BMDMs) from NLRP3-/- mice (prepared as in G) were transfected with 3 μg of EP364R plasmid. IL-1β secretion was measured by ELISA and expression of Casp-1, IL-1β, and GSDMD-N was analyzed by WB 24 h p.t. A P value less than 0.05 was considered statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 8.
EP364R induced NLRP3 inflammasome activation by targeting DDX3X.
(A) iPAMs and HEK293T cells were transfected with 5 μg of EP364R plasmid. Cells were lysed 24 h p.t., and immunoprecipitation (IP) samples were prepared. After protein electrophoresis and Coomassie Blue staining, target bands were excised and sent for mass spectrometry analysis. (B) iPAMs were transfected with 5 μg of EP364R plasmid. Cells were lysed 24 h p.t. A 100 μl aliquot of the supernatant was saved as ‘input’. The remaining supernatant was incubated with anti-Flag mouse monoclonal antibody or control IgM at 4°C for 8 h, followed by incubation with pre-washed beads for another 8 h at 4°C. Beads were washed with PBS and resuspended to prepare IP or control samples. Interaction between EP364R and DDX3X or NLRP3 was detected by WB. (C) iPAMs were transfected with 5 μg of EP364R plasmid. Cells were lysed 24 h p.t. A 100 μl aliquot was saved as ‘input’. The remaining lysate was incubated with anti-ASC rabbit antibody or control IgG at 4°C for 8 h, followed by incubation with beads for another 8 h at 4°C. Beads were washed and resuspended to prepare IP and control samples. NLRP3 inflammasome assembly was detected by WB. (D) In iPAMs lysates from LPS + Nig-induced inflammatory cells, a 100 μl aliquot was saved as ‘input’. The remaining supernatant was incubated with anti-NLRP3 rabbit antibody, anti-DDX3X mouse antibody, or control IgG at 4°C for 8 h, followed by incubation with beads for another 8 h at 4°C. Beads were washed and resuspended. Interaction between porcine DDX3X and NLRP3 was detected by WB. (E) PAMs were infected with ASFV, then cells were lysed 48 h post infection. A 100 μl aliquot of the supernatant was saved as ‘input’. The remaining supernatant was incubated with EP364R polyclonal antibody or control IgM at 4°C for 8 h, followed by incubation with pre-washed beads for another 8 h at 4°C. Beads were washed with PBS and resuspended to prepare IP or control samples. Interaction between EP364R and DDX3X or NLRP3 was detected by WB. (F) HEK293T cells were transfected with 2 μg of Flag-EP364R plasmid. At 24 h p.t., cells were fixed, and co-localization of Flag-EP364R and DDX3X was assessed by immunofluorescence assay (IFA). (F) HEK-293T cells were transfected with 2 μg of Flag-EP364R plasmid. At 24 h p.t., cells were fixed, and co-localization of DDX3X with ASC specks was assessed by IFA.
Fig 9.
DDX3X deficiency impaired EP364R-induced pyroptosis.
(A) HEK293T cells were transfected with small interfering RNA (siDDX3X-1, -2, or -3). At 48 h p.t., cells were transfected with 2 μg of EP364R plasmid for 24 h. RNA was extracted for qPCR and cells were lysed for WB analysis to screen for the most effective siDDX3X. (B) Porcine alveolar macrophages (PAMs) were transfected with siDDX3X-3. At 48 h p.t., cells were infected with ASFV at MOI = 1 for 24 h. IL-1β secretion was measured by ELISA, and expression of IL-1β and GSDMD-N was detected by WB. (C) PAMs were transfected with siDDX3X-3. At 48 h p.t., cells were infected with ASFV at MOI = 1 for 24 h. Cells were stained with PI (10 μl) and cell death was observed by fluorescence microscopy. (D) HEK293T cells were transfected with siDDX3X-3. At 48 h p.t., cells were transfected with 2 μg of EP364R plasmid. IL-1β secretion was measured by ELISA 24 h later. (E) HEK293T cells were transfected with siDDX3X-3. At 48 h p.t., cells were transfected with 2 μg of EP364R plasmid. At 24 h p.t., cells were stained with PI (10 μl) and cell death was observed by fluorescence microscopy. (F) HEK293T cells were transfected with siDDX3X-3. At 48 h p.t., cells were transfected with 5 μg of EP364R plasmid. Expression of IL-1β and GSDMD-N was analyzed by WB 24 h later. (G) HEK-293T cells were transfected with siDDX3X-3. At 48 h p.t., cells were transfected with 5 μg of EP364R plasmid. At 24 h p.t., cell lysates were centrifuged; the supernatant was the ‘input’. The pellet was resuspended in PBS, cross-linked with DSS at 37°C for 30 min, and mixed with loading buffer to prepare the ‘pellet’ sample. ASC oligomerization was detected by WB. (H) HEK293T cells were transfected with siDDX3X-3. At 48 h p.t., cells were transfected with 2 μg of EP364R plasmid. ASC speck formation was assessed by IFA 24 h later. And p value less than 0.05 was considered statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 10.
DDX3X acted as a bridge for EP364R-induced NLRP3-mediated pyroptosis.
(A) WB analysis of lysates from DDX3X knockout cells. (B) DDX3X knockout cells were transfected with 2 μg of EP364R plasmid. IL-1β secretion was measured by ELISA 24 h p.t. (C) DDX3X knockout cells were transfected with 5 μg of EP364R plasmid. At 24 h p.t., cells were stained with PI (10 μl) and cell death was observed by fluorescence microscopy. (D) DDX3X knockout cells were transfected with 5 μg of EP364R plasmid. Expression of IL-1β and GSDMD-N was detected by WB 24 h p.t. (E) DDX3X knockout cells were transfected with 5 μg of EP364R plasmid. At 24 h p.t., cell lysates were centrifuged; the supernatant was the ‘input’. The pellet was resuspended in PBS, cross-linked with DSS at 37°C for 30 min, and mixed with loading buffer to prepare the ‘pellet’ sample. ASC and NLRP3 oligomerization were detected by WB. (F) DDX3X knockout cells were co-transfected with 5 μg each of EP364R and DDX3X plasmids. Expression of IL-1β and GSDMD-N was detected by WB 24 h p.t. (G) DDX3X knockout cells were co-transfected with 5 μg each of EP364R and NLRP3 plasmids. At 24 h p.t., cells were lysed. A 100 μl aliquot was saved as ‘input’. The remaining lysate was incubated with anti-Flag mouse antibody or control IgM at 4°C for 8 h, followed by incubation with beads for another 8 h at 4°C. Beads were washed and resuspended. Interaction between NLRP3 and EP364R was detected by WB. (H) DDX3X knockout cells were transfected with 2 μg of EP364R plasmid. ASC speck formation was observed by IFA 24 h p.t. And p value less than 0.05 was considered statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 11.
Interaction mode between EP364R and DDX3X.
(A) HEK293T cells were co-transfected with 5 μg of EP364R plasmid and 5 μg of plasmids encoding different HA-tagged DDX3X domains (HA-DDX3X, HA-ΔN, HA-Helicase core, HA-ΔC, HA-ΔDEAD, HA-ΔHelicase). Cells were lysed 24 h p.t. A 100 μl lysed samples were detected as ‘input’. The remaining lysate was incubated with anti-Flag mouse antibody or control IgM at 4°C for 8 h, followed by incubation with beads for another 8 h at 4°C. Beads were washed and subjected to WB. (B) HEK293T cells were co-transfected with 5 μg of HA-DDX3X plasmid and 5 μg of plasmids encoding different myc-tagged NLRP3 domains (myc-LRR, myc-PYD, myc-NACHT). Cells were lysed 24 h p.t. A 100 μl lysed samples were detected as ‘input’. The remaining lysate was incubated with anti-Flag antibody (presumably targeting the tag on DDX3X or co-precipitating partner) or control IgM at 4°C for 8 h, followed by incubation with beads. Beads were washed and resuspended. Interaction between NLRP3 domains and DDX3X was detected by WB. (C) HEK293T cells were co-transfected with 5 μg of EP364R plasmid and 5 μg of plasmids encoding different myc-tagged NLRP3 domains (myc-LRR, myc-PYD, myc-NACHT). Cells were lysed 24 h p.t. A 100 μl lysed samples were detected as ‘input’. The remaining lysate was incubated with anti-Flag mouse antibody or control IgM at 4°C for 8 h, followed by incubation with beads. Beads were washed and resuspended. Interaction between NLRP3 domains and EP364R was detected by WB. (D) Intrinsically disordered regions in porcine DDX3X and porcine NLRP3 proteins were predicted using the PONDR software.
Fig 12.
Mechanistic schematic of EP364R induced pyroptosis.
Upon infection of domestic swine tissues, the ASFV-encoded protein EP364R binds to a specific spatial structure of DDX3X, which triggers the oligomerization of the NLRP3 inflammasome. This activation leads to the cleavage and activation of Casp-1, which in turn executes pyroptotic cell death and facilitates the release of pro‑inflammatory cytokines and damage‑associated molecular patterns (DAMPs). This cascade is likely a key driver of the cytokine storm‑mediate tissues damage during infection. HAMNO, a small molecule targeting EP364R, inhibits ASFV replication through disruption of the EP364R-DDX3X interaction.
Table 1.
Primers for qPCR.