ASFV infection induced inflammatory cytokines production

Previous studies have demonstrated that pro-inflammatory cytokines were increased in the serum after ASFV infection [34]. To assess whether ASFV infection induced an immune response in infected macrophages, porcine BMDMs were infected with ASFV (strain GS/CN/2018) at the indicated times. The results showed that IL-1β, IL-6, IL-18, IL-10, TNF-α, and IFN-γ were upregulated at both the mRNA and protein levels post-ASFV infection (Fig 1A and 1B). These pro-inflammatory cytokines increase over time during ASFV infection. Absolute quantitative PCR was employed to assess ASFV loads in BMDMs post ASFV infection, revealing an increase in ASFV genome copies over the infection course (Fig 1C). Consistently, as shown in Fig 1D, the secretion of matured IL-1β (17KDa) in supernatant was increased during ASFV infection.

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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.

https://doi.org/10.1371/journal.ppat.1013874.g001

These results indicated that ASFV infection induced the production of pro-inflammatory cytokines.

ASFV infection induced cell pyroptosis

IL-1β secretion was increased after ASFV infection, and its maturation correlated with the occurrence of cell pyroptosis. We further evaluated whether ASFV infection led to cellular pyroptosis. Cell viability results indicated that cell death was increased following ASFV (MOI = 1) infection in BMDMs (Fig 2A). Consistently, damage-associated molecular patterns (DAMPs) markers and lactate dehydrogenase (LDH) release, and Casp-1 activity, were also elevated in cell culture medium after ASFV infection (Fig 2B and 2C). Further, as shown in Fig 2D, we observed that compared to the control, positive control (LPS+Nigercin) and ASFV-GFP (ASFV genome containing GFP tag) treatment obviously triggered cell death (dead cells could be stained with propidium Iodide (PI)). Moreover, ASFV infection induced N-GSDMD, IL-1β, and Casp-1 maturation (Fig 2E). Electron microscopy results showed that ASFV infection induced cell swelling and cell membrane damage (Fig 2F). The fluorescence results also showed that ASFV infection induced the formation of ASC specks and the enrichment of mature N-GSDMD on the cell membrane (Fig 2G). ASC oligomerization was increased after ASFV infection (Fig 2H).

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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.

https://doi.org/10.1371/journal.ppat.1013874.g002

To verify the ability of ASFV to induce pyroptosis in other cell types., PBMCs were isolated and infected with ASFV. We found that ASFV infection induced the maturation of IL-1β and Casp-1 in PBMCs (Fig 2I). The production of pro-IL-1β mRNA is associated with NF-κB activation; thus, we further examined the phosphorylation of p65. The results revealed that phosphorylated IκBα and p65 were enhanced by ASFV infection (Fig 2I). PI staining results indicated the induction of cell death following ASFV infection (Fig 2J). Similarly, ASFV infection in PBMCs caused reduced cell viability and elevated Casp-1 activity (Fig 2K). Additionally, we observed a significant increase in pro-IL-1β mRNA in PBMCs following ASFV infection (Fig 2K). In addition, previous study showed that ASFV active Casp-3/Casp-4 to cleave GSDMA to regulate pyroptosis [35], therefore, we used Casp-3 and Casp-4 inhibitors to assess the function of ASFV-induced Casp-1-mediated pyroptosis. The results demonstrated that without Casp-3 and Casp-4, ASFV also induced pyroptosis (S1A-S1C Fig).

Taken together, these results suggested that ASFV infection activated Casp-1-mediated pyroptosis and IL-1β production in BMDMs.

ASFV infection induced cell pyroptosis via the NLRP3 inflammasome

AIM2 senses foreign pathogen DNA to activate inflammasome-mediated Casp-1 maturation, IL-1β production, and cell pyroptosis. Although ASFV is a DNA virus, it has been demonstrated that AIM2 is a pseudogene in pigs [36]. Therefore, we investigated whether ASFV-induced pyroptosis is associated with the NLRP3 inflammasome. ASFV-infected BMDMs were treated with VX765 (Casp-1 inhibitor) or MCC950 (NLRP3 inhibitor). As shown in Fig 3A and 3B, similar to LPS and Nigericin stimulation (LPS + Nig), ASFV-induced maturation and secretion of Casp-1, IL-1β, and GSDMD-N were significantly inhibited after treatment with VX765 or MCC950. Meanwhile, we observed that PI-stained dead cells were reduced after treatment with MCC950 or VX765 during ASFV infection (Fig 3C).

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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.

https://doi.org/10.1371/journal.ppat.1013874.g003

Furthermore, the short hairpin RNA (shRNA) of NLRP3 inflammasome components was used to assess cell pyroptosis and IL-1β production following ASFV infection. As shown in S2A Fig, the expression of ASC, NLRP3, and Casp-1 proteins was interfered by their specific shRNA. After specific shRNA transfection, we observed a significant decrease in IL-1β secretion during ASFV infection (Fig 3D–3F). Moreover, the levels of mature Casp-1, IL-1β, and N-GSDMD were decreased following ASFV infection after specific shRNA transfection. (Fig 3D–3F). PI staining results also showed that cell death was reduced after ASC, NLRP3, or Casp-1 shRNA transfection during ASFV infection (Fig 3G).

ASFV EP364R induced IL-1β production and cell pyroptosis

To elucidate the underlying mechanism of NLRP3 inflammasome-mediated cell pyroptosis during ASFV infection, we constructed a database of 185 ASFV-encoded proteins. We assayed their capacity to enhance IL-1β secretion in PK15 cells activated by NLRP3-inflammasome components. Through this screening process, ASFV EP364R was identified as a candidate to increase IL-1β secretion during the expression of NLRP3-inflammasome components ASC, NLRP3, pro-Casp-1, and pro-IL-1β (Fig 4A). Additionally, we also found that EP364R was a late-stage expression protein during ASFV infection and is localized in both the cytoplasm and nucleus of BMDMs (S3A Figs and 4B).

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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.

https://doi.org/10.1371/journal.ppat.1013874.g004

Furthermore, to evaluate the capacity of EP364R alone to induce IL-1β production, EP364R was transfected into BMDMs, and the induction of IL-1β secretion was significantly increased after EP364R expression (Fig 4C). The expression of EP364R reduced cell viability in a dose-dependent manner (Fig 4D). Consistently, LDH release and Casp-1 activity also were increased in a dose-dependent manner after EP364R transfection (Figs 4E and S3B).

Moreover, PI staining results indicated that EP364R-expressed cells undergo significant cell death (Fig 4F). Further, some cells exhibited pyroptotic features, such as rounded swelling, following LPS + Nig or EP364R stimulation (Fig 4G). Electron microscope results also showed that EP364R induced typical morphological features of pyropotosis, such as cell swelling and vesicle formation at the membrane edge (S3C Fig). To further confirm the capacityof EP364R to induce pyroptosis, cells were co-transfected with different doses of EP364R and NLRP3 inflammasome components. We found that EP364R enhanced the maturation of Casp-1, and IL-1β protein expression (Fig 4H). EP364R alone also induced GSDMD-N, Casp-1, and IL-1β expression (Fig 4I). Additionally, we observed that N-GSDMD and ASC oligomerization occurred when EP364R was present (Fig 4J). Consistently, GSDMD-N and ASC specks formed during EP364R expression (Fig 4K).

The first signal of NLRP3 inflammasome activation requires NF-κB activation; therefore, we further evaluated the effect of EP364R on p65 phosphorylation. The results showed that EP364R induced the phosphorylation of IκBα and p65 (S3D Fig). Furthermore, the mRNA levels of pro-inflammatory cytokines, including IL-1β, IL-6, and TNF-α, as well as IFN-β, were increased after EP364R expression (S3E Fig). In addition, EP364R also induced DAMPs release, such as Casp-1, CRT, and ATP (S3F Fig)

Taken together, these results indicated that EP364R induced pyroptosis and IL-1β production.

The EP364R protein induced inflammation in mice

To better determine the effect of the EP364R protein on inflammation, a Flag-EP364R and GFP stable overexpression porcine iPAM cells were constructed. Puromycin was employed to select the transfected cells until the 15th generation. The fluorescence, PCR, and WB were used to determine the constructed cells, and the results showed that the EP364R gene was successfully integrated into the cell genome, and the EP364R was stably expressed (Fig 5A and 5B).

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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.

https://doi.org/10.1371/journal.ppat.1013874.g005

Additionally, we observed that GSDMD-N, Casp-1, and IL-1β maturation occurred in EP364R-expressing cells (Fig 5B). Furthermore, the mRNA transcription and secretion of pro-inflammatory cytokines of IL-1β, IL-6, TNF-α, CXCL10, and IFN-β were increased in EP364R-expressed cells (Fig 5C and 5D).

Moreover, mice were infected with the purified EP364R lentivirus (Fig 5E), and we found that EP364R induced spleen enlargement (Fig 5F). Additionally, maturation of Casp-1 and IL-1β was observed in heart, liver, spleen, lung, and kidney (Fig 5G). IL-1β, TNF-α, IL-6, and IFN-β secretion were increased in the heart, liver, spleen, lung, kidney, and serum during EP364R expression (S4A and S4B Fig). Taken together, the results indicated that EP364R expression induced inflammation in mice.

ASFV EP364R deficiency impaired pyroptosis and IL-1β production

We next investigated the effects of EP364R on ASFV-induced pyroptosis and inflammation. EP364R shRNAs were determined to interfere with EP364R expression, as illustrated in Fig 6A and 6B. shEP364R-3 exhibited the strongest capacity to interfere with EP364R expression. EP364R and shEP364R-3 were co-transfected into iPAMs, and IL-1β secretion was measured. As shown in Fig 6C, siEP364R impaired ASFV-induced IL-1β secretion compared to siRNA control (siNC). Then, ASFV-infected BMDMs were subjected to EP364R shRNA treatment. The inhibition of EP364R expression significantly reduced ASFV-induced IL-1β secretion and Casp-1 expression (Fig 6D). This also motivates us to further investigate the effect of EP364R inhibition on ASFV-induced NF-κB activation; the results showed that the phosphorylation of p65 and IκBα induced by ASFV was inhibited when EP364R expression was disrupted (S5A Fig).

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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.

https://doi.org/10.1371/journal.ppat.1013874.g006

Furthermore, cell viability results showed that inhibition of EP364R expression reduced ASFV-induced cell viability loss (Fig 6E). Consistently, Casp-1 activity and LDH release also decreased after ASFV infection when EP364R expression was interfered (Fig 6F and 6G). Additionally, the PI staining results showed that interference with EP364R expression reduced ASFV-induced cell death (Fig 6H). We also found that ASC oligomerization was suppressed during ASFV infection when interference EP364R expression (Fig 6I). Consistently, the mRNA levels of IL-1β, IL-6, TNF-α, and CXCL10 were also inhibited during ASFV infection when EP364R expression was knocked down (Fig 6J).

EP364R induced pyroptosis and IL-1β production via NLRP3 inflammasome

We further determined whether EP364R induced pyroptosis through NLRP3 signaling. EP364R-expressed cells were treated with MCC950 or VX765, as shown in Fig.7A and 7B, EP364R-induced IL-1β secretion, Casp-1, and GSDMD-N maturation were suppressed during NLRP3 and Casp-1 inhibitor treatment. PI staining results showed that cell death was reduced in EP364R-expressed cells with MCC950 or VX765 treatment compared to DMSO treatment (S6A Fig).

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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.

https://doi.org/10.1371/journal.ppat.1013874.g007

Furthermore, IL-1β secretion, Casp-1, and GSDMD-N maturation levels were significantly reduced after knockdown of Casp-1, ASC, or NLRP3 (Fig 7C-7E). Additionally, PI staining results showed that EP364R-induced cell death decreased following disruption of ASC, NLRP3, or Casp-1 expression (Fig 7F).

We next assessed the EP364R function in NLRP3 knockout cells and found that NLRP3 mRNA expression was decreased in NLRP3 knockout BMDMs after EP364R transfection (Fig 7G). Importantly, IL-1β secretion, Casp-1, and GSDMD-N maturation were significantly suppressed during EP364R expression in NLRP3 knockout cells (Fig 7H). PI staining results also indicated that EP364R-induced cell death was decreased after NLRP3 knockout (S6B Fig).

EP364R targets DDX3X to induce NLRP3 inflammasome activation

To analyze the mechanism of EP364R-induced cell pyroptosis, EP364R was expressed in porcine iPAMs and HEK293T and subjected to affinity purification mass spectrometry. The results showed that DDX3X interacted with EP364R (Fig 8A). The previous study reported that DDX3X was associated with NLRP3. Co-IP results showed that EP364R interacted with both porcine NLRP3 and porcine DDX3X (Fig 8B). Additionally, ASC interacted with Casp-1, NLRP3 with LPS + Nig or EP364R treatment was observed (Fig 8C), which suggested that EP364R activated NLRP3 inflammasome formation. We also found that porcine DDX3X was associated with porcine NLRP3 (Fig 8D). In addition, we also found that EP364R expressed by ASFV also interacts with DDX3X and NLRP3 (Fig 8E). Furthermore, EP364R co-localized with DDX3X was confirmed (Fig 8F). Importantly, DDX3X and ASC colocalized and formed specks after EP364R expression was observed (Fig 8G).

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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.

https://doi.org/10.1371/journal.ppat.1013874.g008

These results indicated that EP364R interacted with DDX3X to trigger NLRP3 inflammasome activation.

DDX3X deficiency impaired cell pyroptosis induced by ASFV and EP364R

To investigate the role of DDX3X protein in ASFV-induced cell pyroptosis, DDX3X and EP364R were knocked down using siRNA or shRNA (Fig 9A). PI staining results showed that disrupting DDX3X expression also decreased ASFV-induced cell death (Fig 9B) We found that ASFV-induced IL-1β secretion, Casp-1, and GSDMD-N maturation were decreased after DDX3X knockdown (Fig 9C). Furthermore, as shown in. S7A and S7B Fig, co-transfection of siDDX3X and shEP364R reduced ASFV-induced IL-1β secretion, Casp-1 and GSDMD-N maturation, and cell death.

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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.

https://doi.org/10.1371/journal.ppat.1013874.g009

To determine the function of DDX3X on EP364R-induced cell pyroptosis, DDX3X knockdown experiments were performed. As shown in Fig 9D, DDX3X expression inhibition reduced positive control (LPS+Nigericine)- and EP364R-induced IL-1β secretion compared to the negative control siRNA (siNC). Additionally, PI staining results also showed that DDX3X expression disruption impaired EP364R-induced cell death compared to siNC (Fig 9E). GSDMD-N and Casp-1 maturation also reduced after EP364R expression in DDX3X knockdown cells (Fig 9F). Similarly, DDX3X knockdown decreased EP364R-induced ASC oligomerization (Fig 9G). Importantly, EP364R-induced ASC specks were obviously suppressed after DDX3X knockdown (Fig 9H).

These results suggested that DDX3X was involved in EP364R-induced pyroptosis through NLRP3 inflammasome activation.

DDX3X acted as a bridge for EP364R-induced NLRP3-mediated pyroptosis

Next, the effect of DDX3X knockout on EP364R-induced cell pyroptosis was assessed. As shown in Fig 10A, DDX3X was successfully knocked out in HEK293T cells. IL-1β secretion was reduced in DDX3X knockout cells when EP364R expression (Fig 10B). PI staining results showed that EP364R-induced cell death was decreased in DDX3X knockout cells (Fig. 10C). Casp-1 and GSDMD-N maturation were reduced without DDX3X expression (Fig 10D). ASC oligomerization was reduced in DDX3X knockout cells with EP364R expression (Fig 10E). Furthermore, DDX3X expression was recovered in knockout cells, and EP364R-induced maturation of Casp-1 and GSDMD-N was restored (Fig 10F). In addition, IP staining results showed that the interaction of EP364R and NLRP3 was absent in DDX3X knockout cells (Fig 10G). Meanwhile, EP364R-induced ASC speck formation was abolished after DDX3X knockout (Fig 10H).

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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.

https://doi.org/10.1371/journal.ppat.1013874.g010

Taken together, these results suggested that DDX3X served as a bridge for EP364R-induced pyroptosis.

EP364R-induced cell pyroptosis without utilizing DDX3X helicase activity

Based on the results of the aggregation of DDX3X with NLRP3 and forming speckles were modulated by EP364R. Additionally, DDX3X possesses RNA helicase activity. We investigated whether the helicase activity of DDX3X was involved in EP364R-induced cell pyroptosis. We found that RK-33 (an inhibitor of helicase activity) did not affect EP364R-induced IL-1β secretion (S8A-S8B Fig). Furthermore, RK-33 failed to suppress EP364R-induced Casp-1 and GSDMD-N maturation (S8C Fig). The formation of EP364R-induced ASC oligomerization also remained unchanged after RK-33 treatment (S8D Fig). The fluorescence results showed that EP364R-induced ASC speckles formation remained unchanged with RK-33 treatment (S8E Fig). Moreover, EP364R-induced cell death caused by EP364R did not decrease with RK-33 treatment (S8F Fig). These results suggested that EP364R-triggered pyroptosis was independent of DDX3X helicase activity.

The mode of EP364R-DDX3X interaction

To identify the critical sites of EP364R-DDX3X interaction, Co-IP assays and protein molecular docking model were employed. Co-IP of the truncated DDX3X domain with EP364R was performed to identify the functional domains of DDX3X, the locations of the different domains of DDX3X: N-terminal (1–159aa), DEAD domain (160–410aa), Helicase (414–575aa), C-terminal (576–662aa), Helicase core (160–575aa) (S9A Fig). As shown in Fig 11A, EP364R interacted with all the DDX3X domain deletion mutants. We then found that the NACHT domain of porcine NLRP3 interacted with porcine DDX3X, which was similar to the results of the binding domains of human NLRP3 and human DDX3X [37] (Fig 11B). In addition, Co-IP results showed that EP364R interacted with the LRR and NACHT domains of NLRP3(Fig 11C).

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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.

https://doi.org/10.1371/journal.ppat.1013874.g011

To further define the binding interface, we conducted computational protein-protein docking using AlphaFold 3.0 for homology modeling. Neither EP364R nor porcine DDX3X has experimentally resolved crystal structures, so we generated AlphaFold-predicted models with confidence scores (S9B Fig). Due to low confidence scores in EP364R’s 305–366aa region, this segment was omitted from analysis. Docking simulations focused on EP364R’s nuclease domain (1–160aa), DNA-binding domain (161–304aa), and full-length structure in complex with DDX3X (S9B Fig). Key interaction patterns emerged from these simulations (S9C Fig):

Pose 1: Hydrogen bonds formed between EP364R residues Lys174, Met125, Val126, and Lys252 with DDX3X residues Asp165, Val561, Lys418, Asp426, and Tyr200, respectively.

Pose 2: Specific ionic interactions involved EP364R residues Lys259, Arg226, and Lys252 binding to DDX3X residues Asp433, Asp558, and Asp426, respectively.

Pose 3: Multimodal interactions were observed between EP364R residues Gln30, Lys154, Lys50, and Met156 and DDX3X residues Glu388, Asp521, Glu523, Glu449, Pro519, and Arg528.

The protein docking results showed that the binding sites of EP364R and DDX3X were located in different regions. We speculated that this was related to their spatial conformation. The previous study reported that DDX3X has disordered regions [37]. Therefore, we predicted the disordered regions of EP364R and porcine DDX3X and found that many sites where EP364R and DDX3X molecules dock were located within these disordered regions (Fig 11D).

These findings showed that both domains of EP364R mediated interactions with the spatial structure of DDX3X.

Natural product HAMNO targets EP364R to inhibit ASFV replication

Currently, there are no commercial vaccines or drugs available to prevent or treat ASF. We speculated that drugs may inhibit ASFV replication. Given the findings of the EP364R mode, we sought to screen natural products targeting EP364R. The EP364R mode was analyzed using MOE software. Inhibitor binding active site 1 including Phe3, Val5, Lys39, Gln40, Leu41, Phe42, Thr43, Leu47, Cys49, Ser53, Ile55, and Leu85, and site 2 including Glu256, Ile257, Leu258, Lys259, Asp260, His261, Ile277, Gln278, Ile279, and Val280, were found on the surface of EP364R (S10A Fig). Then, using Schrödinger software to perform compound docking on each compound in the chemical library (consisting of 23619 small molecules). Ultimately, 73 and 61 small molecules that bind to site 1 and site 2, respectively, were retained through affinity and structural diversity analysis, as well as visual binding mode analysis.

Further, cells treated with these retained small molecules separately were infected with ASFV-GFP. A natural product, HAMNO, was found to strike a balance between inhibiting ASFV replication and producing less cytotoxicity (S10B-S10D Fig). The binding location of HAMNO in the predicted structure of EP364R was analyzed to bind site 2 (S10E Fig). Partial views of the 3D and 2D conformations of HAMNO binding to EP364R were performed (S10F and S10G Fig), and the amino acids within 4Å surrounding the HAMNO were annotated. For example, HAMNO forms hydrogen bonds with E256, K259, and D260 in the structure of EP364R (S10G Fig). Meanwhile, the inhibition effect of HAMNO on ASFV replication was dose-dependent (S10H and S10I Fig). Additionally, cells treated with HAMNO at indicated times were infected with ASFV, and we found that it was more effective when cells were treated with HAMNO before ASFV infection (S10J and S10K Fig.).

To investigate HAMNO has the function on ASFV or EP364R-induced pyroptosis and inflammation response, HAMNO was added to the cells accompany with ASFV or EP364R treatment. The results demonstrated that HAMNO decreases the IL-1β secretion and pyroptosis biomarkers (N-GSDMD, Casp-1, IL-1β) expression which induced by ASFV and EP364R (S10L and S10M Fig.). Moreover, cytokines of IL-1β, IL-18, and TNF-α transcription were suppressed during ASFV infection or EP364R transfection with HAMNO treatment (S10N Fig). Importantly, HAMNO treatment also inhibited the interaction of EP364R, NLRP3, and DDX3X (S10O Fig). Taken together, these findings suggested that HAMNO was a potential therapeutic agent that effectively inhibited ASFV replication via EP364R.

Materials and methods

Ethics statement

This study complies with local laws and institutional requirements. The animal study was approved by the Ethic Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (License No. LVRIAEC-2022–053).

Viruses and cells

The ASFV CN/GS/2018 strain was identified and preserved by the ASF Regional Laboratory of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The ASFV-GFP strain was preserved by the Lanzhou Veterinary Research Institute. The E. coli DH5α strain was purchased from Takara Bio Inc. Primary porcine alveolar macrophages (PAMs) were isolated from 30–40-day-old SPF piglets in our laboratory. HEK293T cells and iPAMs (3D4/21 cell line) were preserved in our laboratory. DDX3X gene knockout HEK293T cells were purchased from Beyotime. NLRP3 gene knockout mice were bred in our laboratory. All the cells are cultured with RPMI 1640 or DMEM (Gibco, USA) with 10% fetal bovine serum (FBS) (Aoqing Biotechnology Co.,LTD, Beijing, China).

Reagents, antibodies

Lipopolysaccharide (LPS), nigericin, MCC950, VX-765, DSS Crosslinker, and Protein A/G Magnetic Beads were purchased from MCE. RK-33 was purchased from TargetMol. Lipofectamine 2000, Opti-MEM, and Plasmocin treatment were purchased from Invitrogen. The one-step TB Green RT-PCR quantitative detection kit was purchased from Takara. Protease inhibitor PMSF and lactate dehydrogenase (LDH) activity detection kit were purchased from Solarbio. The NucleoBond ultra-rapid transfection-grade plasmid midi-prep kit Plus was purchased from MN (Macherey-Nagel). The Casp-1 activity detection kit and ATP detection kit were purchased from Beyotime. The CCK-8 kit and total RNA extraction reagent were purchased from Biosharp. Propidium iodide (PI) staining solution was purchased from BD Pharmingen. Dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich. The cytokines ELISA detection kit was purchased from Ximbio. The lentiviral infection enhancer polybrene was purchased from Hanbio Biotechnology. HA-Tag (26D11) mAb, Myc-Tag (19C2) mAb, and DDX3X antibody were purchased from Abmart. Monoclonal anti-FLAG M2 mouse antibody and anti-beta-actin mouse monoclonal antibody were purchased from Sigma-Aldrich. Horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (H + L) and HRP-labeled goat anti-mouse IgG (H + L) were purchased from ZSGB-BIO. IL-1β (D3U3E) Rabbit mAb, Cleaved-IL-1β (Asp116) (D3A3Z) Rabbit mAb, Casp-1 (D7F10) Rabbit mAb, Cleaved Casp-1 (Asp297) (D57A2) Rabbit mAb, Gasdermin D (E9S1X) Rabbit mAb, Cleaved Gasdermin D (Asp276) Crosslinker, and fluorescent secondary antibodies were purchased from CST. Anti-ASFV-p72 and Anti-ASFV-p30 antibidies were prepared and stored in our laboratory.

Plasmids and transfection

Eukaryotic expression plasmids Flag-EP364R, Myc-NLRP3, Myc-LRR, Myc-PYD, and Myc-NACHT were constructed and preserved in our laboratory. Eukaryotic expression plasmids HA-DDX3X, HA-ΔN, HA-Helicase core, HA-ΔC, HA-ΔDEAD, and HA-ΔHelicase were synthesized by Sangon Biotech. Lentiviral overexpression plasmids pHBLV-Vector and pHBLV-EP364R, as well as lentiviral packaging accessory plasmids psPAX2 and pMD2.G, were purchased from HANBIO. HEK293T cells were cultured in DMEM complete medium, while iPAMs cells and PAMs were cultured in 1640 complete medium at 37°C with 5% CO₂. When cell density reached 70%, transfection was performed. The plasmids to be transfected and Lipofectamine 2000 were separately mixed with Opti-MEM medium, and incubated at room temperature for 5 min, then plasmids and Lipofectamine 2000 were mixed and incubated at room temperature for another 15 min followed by added to the cells. The medium was replaced with complete medium at 5h post incubation at 37°C with 5% CO₂, and the cells were collected at 24 h post transfection.

Gene screen assays

Plasmids encoding NLRP3, pro-Caspase-1, pro-IL-1β and ASC were co-transfected with either ASFV gene-encoding plasmids or an empty vector (EV) into iPAM or PK-15 cells. After 24 h, the supernatants were collected and lyophilized. The resulting powder was reconstituted in PBS, and the concentration of porcine IL-1β was measured using a commercial ELISA kit according to the manufacturer’s instructions. The results were compared with those obtained from supernatants of cells transfected with the EV.

Co-IP and western blotting analysis

HEK293T cells were transfected with target plasmids, lysed, and centrifuged. Input samples were prepared by boiling lysates with loading buffer. For Co-IP, lysates were incubated with target antibodies or control IgG overnight at 4°C, followed by Protein A/G Magnetic Beads for another 8 h. Beads were washed, boiled with loading buffer, and analyzed by Western Blotting (WB)

RNA isolation and qPCR

Trizol lysis buffer was added to ASFV CN/GS/2018-infected BMDMs and lyse at room temperature for 10 min. Add chloroform, vortex vigorously for 15 s, let stand for 15 min, and centrifuge at 12000 r/min for 20 min at 4°C. Carefully transfer the upper aqueous phase to a new EP tube, add pre-cooled isopropanol, mix, and let stand at room temperature for 30 min, then centrifuge at 12000 r/min for 15 min at 4°C and discard the supernatant, wash with pre-cooled 75% ethanol, and centrifuge at 12000 r/min for 15 min at 4°C. Discard the supernatant, air-dry the ethanol, dissolve the RNA in DEPC water and store at −80°C or proceed directly to RT-qPCR using the one-step amplification kit. For absolute quantification, use cDNA as the template for RT-qPCR. One-step RT-qPCR amplification kit was employed to amplify the targeting genes. For absolute quantification, the total 20 µL volume of reaction system include: 2 µL template, 0.5 µL probe, 1 µL for each primer (10 µM), 10 µL mix buffer, and 5.5 µL ddH₂O. The reaction program was set as: 94°C for 30 s; 40 cycles of 94°C for 5 s, 60°C for 30 s; 95°C for 15 s, 60°C for 1 min, 95°C for 1 s. For relative quantification, reaction system include: 200 µL template, 0.4 µL for each primer (10 µM), 0.4 µL enzyme, 5 µL 2X Mix, 2.8 µL ddH₂O. The reaction program set as: 95°C for 30 s; 40 cycles of 95°C for 5 s, 60°C for 30 s.

The sequences of primers and probes used in this study are listed in Table 1.

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Table 1. Primers for qPCR.

https://doi.org/10.1371/journal.ppat.1013874.t001

siRNA or shRNA interference

siRNAs or shRNA targeting DDX3X, EP364R, ASC, NLRP3, or Casp-1 were designed (sequences in Table 1), transfected into cells, and knockdown efficiency was validated. Briefly, for DDX3X-targeting siRNA, a total of 5 nmol siRNA was dissolved in DEPC water to prepare a 50 μM stock solution. Then, 1 μl of the siRNA was transfected into cells for 48 hours, followed by treatment with LPS+Nigericin or transfection with EP364R to activate NLRP3. For shRNA targeting EP364R, ASC, NLRP3, or Casp-1, cells were transfected for 48 hours and then infected with ASFV (MOI = 1). Samples were collected 24 hours post-infection for ELISA or Western blot analysis.

Lentiviral preparation and Infection

HEK293T cells were seeded at 5 × 10⁶ cells/T75 flask and transfected with mix including 10 µg packaging plasmid psPAX2, 10 µg pMD2.G, and 10 µg target plasmid pHBLV-EP364R or pHBLV-Vector. The medium was replaced at 6 h post-transfection. Viral supernatant was collected at 48 h and 72 h post-transfection, filtered through a 0.45 µm filter, and centrifuged at 72,000 g for 120 min at 4°C. Then, HEK293T cells were seeded in a 96-well plate with 1 × 10⁴ cells/well. Lentivirus was serially 10-fold diluted, and added to the cells for overnight. After replacing the medium, GFP expression was observed, and the virus titer (TU/mL) was calculated. The viral pellet was resuspended in 500 µL fresh medium and stored at −80°C. For mice infection with lentivirus, 24 BALB/c mice (4-week-old) were randomly divided into Mock, PBS, LV-Vector, and LV-EP364R groups (n = 6). Mice were injected via the tail vein with 5 × 10⁷TU lentivirus twice at 48 h intervals. At 96 h post injection, mice were sacrificed. Serum was collected for ELISA detection, tissues were collected for RNA and protein extraction, and histopathological analysis.

Enzyme-Linked Immunosorbent Assay (ELISA)

Blank control (Mock), treatment groups, and positive controls (LPS+Nigericin) were set up. Samples were freeze-thawed twice at −80°C, after vacuum freeze drying, resuspended in PBS, and centrifuged at 5000 r/min for 10 min. ELISA was employed to measure IL-1β, IL-6, IL-18, IL-10, IFN-γ, CRT, and TNF-α levels in the supernatant samples based on the instruction of the Ximbio ELISA kit instructions.

Oligomerization Detection

Cells were crosslinked with DSS, pelleted, and analyzed by WB to detect oligomerized proteins. Briefly, after treatment, cells were collected by centrifugation at 2,000 rpm for 5 min at 4°C. The cell pellet was resuspended in 300 μL of PBS, 100 μL of this suspension was centrifuged and the pellet was lysed in cell lysis buffer for 4 hours. After lysis, the sample was centrifuged at 12,000 rpm for 10 min at 4°C. The supernatant was collected, mixed with protein loading buffer, and boiled at 100°C for 10 min to prepare the input sample. The remaining 200 μL of cell suspension was centrifuged, and the pellet was resuspended and treated with 2 mM disuccinimidyl suberate (DSS), followed by incubation in a metal bath at 37°C for 30 min. After crosslinking, protein loading buffer was added directly to the sample without further processing to prepare the pellet sample. Both input and pellet samples were subjected to WB analysis.

Immunofluorescence Assay (IFA)

Adherent cells were cultured in confocal dishes until reaching 50% confluence, followed by transfection with plasmid or viral infection for 24 hours. Cells were then fixed with 4% paraformaldehyde at room temperature for 15 min. After removal of the fixative, permeabilization was performed using 0.1% Triton X-100 for 20 min at room temperature. The solution was discarded, and cells were blocked with 10% FBS at 37°C for 1 h. After blocking, the cells were incubated with primary antibody overnight at 4°C. The antibody was then removed, and the cells were washed four times with PBST, 5 min each. Subsequently, cells were incubated with a fluorescence-conjugated secondary antibody for 45 min at room temperature, protected from light. After removal of the secondary antibody, the cells were washed four times with PBST in the dark, 5 min per wash. Then, DAPI staining was performed for 10 min at room temperature under light protection. After discarding the DAPI solution, the cells were washed twice with PBST in the dark, 3 min each. Finally, PBST was added to cover the cells, and samples were imaged using a laser scanning confocal microscope.

Transmission electron microscope (TEM) assay

Cells were seeded into 6-well plates and infected with ASFV at an MOI of 1, and the cells were harvested and fixed with 2% glutaraldehyde in PBS for 1 hour at 24 hpi. The samples were dehydrated, embedded, and stained according to standard procedures. The samples were observed and analyzed using a transmission electron microscope.

Isolation and Culture of BMDMs from NLRP3^-/- Mice

Wild-type and NLRP3^-/- mice were sacrificed by cervical dislocation, and incubated in 75% ethanol for 5 min, Then, the hind limbs were isolated, and muscle was removed in sterile PBS. Cut the ends of the femur and tibia, and flush out the bone marrow with induction medium using a syringe, and repeatedly pipette to disperse cell clusters. Cells were filtered using a 70 µm cell strainer and centrifuged at 1500 r/min for 10 min, discard the supernatant and the cells in resuspend in BMDM induction medium. The collected cells were seed for 3 days (do not change medium during this process). The medium was half replaced at 3 days and entirely replaced at 5 days, respectively, post seed. The cells can be used for experiments on day 7.

LDH Release Assay

Cells were collected (minimum of 10⁴ cells) and lysed by adding extraction buffer (1 mL per 5 × 10⁶ cells), followed by ultrasonic disruption on ice (200 W power, 3 s sonication pulses with 10 s intervals, repeated 30 times). The lysate was centrifuged at 12,000 rpm and 4 °C for 10 min, and the supernatant was collected and kept on ice for subsequent use. The supernatant was mixed with the LDH detection working solution, prepared according to the instructions of the Solaibao (Beyotime)-LDH-Assay-Kit, and incubated at room temperature (approximately 25 °C) for 30 min in the dark (samples may be wrapped in aluminum foil and gently shaken on a horizontal or rocking shaker). Absorbance was measured at 490 nm.

Caspase-1 activity assay

Cells were collected and washed once with PBS. Lysis buffer was added (100 µL per 2 × 10⁶ cells), and the pellet was resuspended and incubated on ice for 15 min. The lysate was centrifuged at 12,000 rpm and 4 °C for 20 min, and the supernatant was transferred to a pre-chilled tube. The reaction system and standards were prepared according to the instructions of the Beyotime-Caspase-1-Activity-Assay-Kit. The mixture was incubated at 37 °C for 60–120 min. Once a noticeable color change was observed, absorbance was measured at 405 nm. Further data analysis and statistics were performed.

ATP level assay

Cells were collected, and 200 µL of lysis buffer per well (equivalent to 1/10 of the original 2 mL culture medium volume) was added to lyse the cells. The lysate was centrifuged at 12,000 rpm and 4 °C for 5 min, and the supernatant was collected for subsequent measurement. Reagents were thawed on ice before use. The detection reagent and standards were prepared according to the instructions of the Beyotime-ATP-Assay-Kit. Luminescence was measured using a luminometer, and relative light unit (RLU) values were recorded for further data analysis and statistics.

CCK-8 cell viability assay

Cells were seeded in a 96-well plate at a density of 5,000 cells per well in 100 µL of medium and cultured under appropriate conditions. According to experimental requirements, plasmid transfection or viral infection was performed. After 24 h, 10 µL of CCK-8 solution was added to each well. A well containing culture medium without cells was included as a blank control, and LPS plus Nigericin (LPS + Nig)-treated cells were used as a positive control. The plate was incubated at 37°C in a 5% CO₂ atmosphere for 1 h. Absorbance was measured at 450 nm, and data analysis was performed according to the instructions provided by the Biosharp CCK-8 assay kit.

Protein-protein docking and targeting EP364R inhibitors virtual screening

No crystal structures are currently available for the EP364R protein (369 aa) or porcine DDX3X (662 aa). In this study, we predicted their structures using AlphaFold 3.0. Structural assessment of EP364R revealed that the region spanning residues 305–366 exhibited low prediction confidence and was therefore excluded. The remaining structured regions of EP364R—comprising a nuclease domain (residues 1–160) and a DNA-binding domain (residues 161–304)—were used for molecular docking. The DDX3X model exhibited high overall confidence and was docked as a full-length structure.

Based on binding affinity estimates from molecular docking, 100 binding modes were generated for the EP364R/DDX3X complex. The calculated binding affinities between DDX3X and the nuclease domain of EP364R ranged from –71.4790 kcal/mol to –23.6872 kcal/mol, with a median value of –44.3487 kcal/mol. Affinities between DDX3X and the DNA-binding domain of EP364R ranged from –68.0725 kcal/mol to –19.8363 kcal/mol, with a median of –40.4307 kcal/mol.

Using these median values as thresholds, we selected 50 binding poses for further analysis: those with affinities lower than –44.3487 kcal/mol for the nuclease domain, lower than –40.4307 kcal/mol for the DNA-binding domain, and lower than –52.7687 kcal/mol for full-length EP364R. Protein–protein interaction interfaces within the complex were analyzed using MOE (2022). The results revealed the following specific residue interactions:

In pose 1, Gln30, Lys154, Lys50, and Met156 of EP364R form interactions with Glu388, Asp521, Glu523, Glu449, Pro519, and Arg528 of DDX3X.

In pose 2, Lys259, Arg226, and Lys252 of EP364R interact with Asp433, Asp558, and Asp426 of DDX3X.

In pose 3, Lys174, Met125, Val126, and Lys252 of EP364R form hydrogen bonds with Asp165, Val561, Lys418, Asp426, and Tyr200 of DDX3X.

Potential small molecule-binding sites on the EP364R protein were predicted using MOE, which identified two promising pockets—Site1 located in the nuclease domain and Site2 in the DNA-binding domain—suitable for small molecule binding. These sites were therefore selected for subsequent virtual screening. Site1 consists of the following residues: PHE3, VAL5, LYS39, GLN40, LEU41, PHE42, THR43, LEU47, CYS49, SER53, ILE55, LEU85. Site2 is composed of: GLU256, ILE257, LEU258, LYS259, ASP260, HIS261, ILE277, GLN278, ILE279, VAL280.

Molecular docking of each compound from the T001 library against the predicted structure of EP364R was performed using Schrödinger. Affinity scores were calculated for each compound. Among them, 6,001 compounds were docked into Site1, with binding affinities ranging from –8.9309 kcal/mol to –4.1953 kcal/mol. For Site2, 5,546 compounds were successfully docked, exhibiting affinity scores between –8.5520 kcal/mol and –3.8417 kcal/mol.

Due to variations in affinity distributions, compounds with binding affinities lower than –6 kcal/mol were selected, yielding a total of 310 compounds: 157 binding to Site1 and 153 to Site2. To assess structural diversity, the 157 compounds bound to Site1 and the 153 bounds to Site2 were subjected to structure-based clustering in MOE using the Jarvis-Patrick algorithm with a fingerprint similarity threshold of 70%. This grouped the Site1 set into 108 clusters and the Site2 set into 104 clusters.

The binding poses of the 108 clusters from Site1 and the 104 clusters from Site2 were visually inspected to eliminate unreasonable conformations. After curation, 73 compounds from Site1 and 61 from Site2 were retained for further analysis. These selected molecules were then applied to ASFV-treated cells for functional screening. Finally, the binding mode of HAMNO to EP364R was analyzed via 2D and 3D interaction visualization.

Virus assays

PAMs or BMDMs were seed into 6-well plates at 6 × 10⁵ cells/well. After 4 h of culture at 37°C, add 50 µL of virus to each well. Continue culturing in a 37°C, 5% CO₂ incubator for 96 h, then collect all cells and medium, and store at −80°C until use. ASFVs were determined using qPCR assay. DNA extraction was performed using the method recommended by the manufacturer (DP315-F, TIANGEN BIOTECH Co.,LTD, Beijing, China). Absolute quantification RT-PCR primers for ASFV detection: 5’- GATACCACAAGATCAGCCGT-3’ (Forward), 5’-CTGCTCATGGTATCAATCTTATCGA-3’ (reverse), the TaqMan probes 5’FAM-CCACGGGAGGAATACCAACCCAGTG-TAMRA-3’. DNA amplification was performed for 39 cycles of 6 s at 95 °C and 11 s at 58 °C. The HAD assay was used to evaluate the infectivity of ASFV. Briefly, PAMs (5 × 10⁴) were seeded into 96-well plates, and infected with ASFV at an MOI of 0.01. At 48 hpi, porcine red blood cells (5 × 10⁵) were added to each well, and the “rosettes” of red blood cells was observed using an optical microscope.

Biosafety statement

All experiments involving ASFV infection were performed in a biosafety level 3 (P3) facility at Lanzhou Veterinary Research Institute (LVRI) of the Chinese Academy of Agricultural Sciences (CAAS). This study was approved by the Ministry of Agriculture and Rural Affairs and were conducted in accordance with the recommendations in the Guidelines for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of the PRC.

Statistical analysis

Data were analyzed using GraphPad software (statistical significance using the student’s t test and ANOVA methods). Significance levels: * (p < 0.05), ** (p < 0.01), *** (p < 0.001), **** (p < 0.0001).