ASFV pA137R protein triggers inflammatory response by inducing NF-κB signaling pathway and facilitating NLRP3 inflammasome assembly

Abstract

African swine fever (ASF), caused by the African swine fever virus (ASFV), is characterized by high mortality in infected pigs. ASFV infection triggers severe inflammatory response in the host, which is a crucial contributor to the high lethality of this disease. However, the underlying mechanism by which ASFV infection induces inflammatory response is still poorly understood. In this study, we found that UV-inactivated ASFV induces interleukin-1β (IL-1β) production, suggesting that certain structural proteins incorporated in the virion possess the ability to trigger inflammatory response. Further investigations demonstrated that deletion of the ASFV A137R gene significantly inhibited the ASFV-induced upregulation of the mRNA transcription of various proinflammatory genes and phosphorylation of p65 and IκBα. Furthermore, the purified pA137R protein promoted the mRNA transcription of these proinflammatory genes and phosphorylation of p65 and IκBα. Additionally, pA137R protein interacted with the NACHT and LRR domains of NLRP3 through its N terminal 1–99 amino acid domain, thereby promoting the oligomerization of NLRP3 and ASC and subsequently facilitating NLRP3 inflammasome assembly. Collectively, our findings identify ASFV pA137R protein as a key proinflammatory determinant of ASFV, which not only advances our understanding of the molecular mechanisms underlying ASFV-induced inflammatory response but also provides new insights into ASFV pathogenesis.

Highlights

• •Deletion of A137R gene attenuates ASFV induced inflammatory responses.
• •ASFV pA137R triggers NF-κB activation and promotes NLRP3 inflammasome assembly.
• •The NTD domain of A137R interacts with the LRR-NACHT domain of NLRP3.

Introduction

African swine fever (ASF) is an acute, highly contagious disease caused by African swine fever virus (ASFV), characterized by extensive hemorrhaging in internal organs, high fever and dysfunction of the respiratory and nervous systems. Due to its high mortality rate and the lack of effective antiviral drugs and vaccines, ASFV exerts a profound impact on the pig industry around the world, particularly in China and other Asian countries (Tao et al., 2020; Mahanta et al., 2024; Li and Zheng, 2025). ASFV is a large icosahedral double-stranded DNA virus with a particle size of 200–300 nm. It has a genome with a length of 170–194 kb that contains over 150 open reading frames (Galindo and Alonso, 2017). ASFV primarily targets myeloid lineage cells, such as macrophages and monocytes. During infection, ASFV triggers severe inflammatory response and massive secretion of proinflammatory cytokines, a phenomenon termed “cytokine storm”, which plays an important role in severe tissue damage (Wang et al., 2020). Therefore, elucidating the mechanisms underlying ASFV-induced inflammatory response is critical for deepening our understanding of ASFV pathogenesis and providing a valuable insight for vaccine development.

Inflammatory responses play a crucial role in host antimicrobial processes, which consist of two core signaling pathways: signal 1 and signal 2. Signal 1 is mediated by Toll-like receptors (TLRs)-dependent NF-κB pathway activation, which upregulates the expression of multiple proinflammatory cytokines and several pattern recognition receptors (PRRs). Signal 2 is induced by different components of pathogens or multiple cellular perturbations, leading to the formation of multiprotein complexes termed the inflammasome. To date, various PRRs involved in inflammasome assembly have been identified, including members of the NLRPs family (NLRP1, NLRP3, NLRP6, etc.), NLRC4, AIM2 and IFI16. These PRRs recruit ASC and pro-caspase-1 to form inflammasomes, which trigger the maturation of IL-1β, IL-18 and gasdermin D (GSDMD), resulting in pyroptosis and the secretion of matured IL-1β and IL-18 (Barnett et al., 2023).

Previous studies have shown that TLR1, TLR2, TLR3, TLR4 and TLR9 are critical for ASFV-induced NF-κB pathway activation, and NLRP3 is involved in ASFV-induced inflammasome assembly and proinflammatory cytokine secretion (Li et al., 2021). Additionally, several ASFV viral proteins, such as pE199L, pO61R (Song et al., 2020), B169L (Li et al., 2025) and pI177L (Wu et al., 2025), have been reported to induce NLRP3 inflammasome activation. However, the specific ASFV components recognized by TLRs are poorly characterized.

pA137R is a protein encoded by ASFV with a molecular mass of approximately 15 kDa. Expressed at the late stage of viral replication, this protein is incorporated into the virion as a structural component (Alcamí et al., 1993). Structurally, pA137R protein adopts a pistol-like conformation and has the ability to oligomerize, further assembling into an icosahedral architecture (Li et al., 2024). Notably, pA137R protein serves as one of the key virulence factors of ASFV (Gladue et al., 2021). First, it exhibits immune evasion by binding to TBK1 to trigger its autophagy-mediated lysosomal degradation, thereby inhibiting host antiviral immune responses (Sun et al., 2022). Second, ASFV infection induces the production of pA137R-specific antibodies in pigs, which promote viral infection via antibody-dependent enhancement (ADE), thereby exacerbating clinical symptoms in infected animals (Yang et al., 2024; Zhai et al., 2025).

In this study, we identified that ASFV pA137R acts as Pathogen-Associated Molecular Patterns (PAMPs) to activate the NF-κB pathway. Furthermore, ASFV pA137R protein interacts with NLRP3 to promote NLRP3 inflammasome assembly. These findings reveal a novel molecular mechanism by which ASFV triggers host inflammatory response, thereby advancing our understanding of ASFV pathogenesis.

Results

ASFV pA137R protein induces the production of proinflammatory cytokines

Our previous study showed that ASFV infection upregulates IL-1β mRNA transcription and triggers NLRP3-dependent secretion of proinflammatory cytokines (Li et al., 2021). Here, we first aimed to identify the specific ASFV components responsible for inducing host inflammatory response. Porcine alveolar macrophages (PAMs) were infected with ASFV, heat-inactivated or UV-inactivated ASFV. As shown in Fig. 1A and B, after inactivation, the virus completely lost its replication ability, and the secretion level of IL-1β was markedly reduced following both UV and heat inactivation of the virus, which illustrates that viral replication and protein expression are pivotal for inducing host inflammatory response. Notably, compared to heat-inactivated ASFV, UV-inactivated ASFV still induced a certain level of IL-1β production, suggesting that structural proteins involved in viral particles are sufficient to elicit IL-1β secretion.

Fig. 1.

ASFV pA137R protein induces inflammatory response. A PAMs were mock infected or infected with ASFV, UV- or heat-inactivated ASFV at an MOI of 1. 24 h later, the genomic copy numbers of ASFV were detected by qPCR. B PAMs were mock infected or infected with ASFV, UV- or heat-inactivated ASFV at an MOI of 1. 24 h later, the level of mature IL-1β in the supernatant was measured by ELISA. C Schematic representation of the construction of the ASFV A137R knockout strain. The A137R gene is replaced by a cassette containing a p72 promoter and GFP reporter gene. D The A137R gene was detected in the genomic DNA of ASFV-WT or ASFV-ΔA137R using A137R-specific primers by PCR assay. E Growth kinetics of ASFV-ΔA137R and ASFV-WT in PAMs. PAMs were infected with ASFV-ΔA137R or ASFV-WT (MOI = 0.01), and samples were taken from three independent experiments at the indicated time points and titrated. F PAMs were mock infected or infected with ASFV-WT or ASFV-ΔA137R at an MOI of 1. 24 h later, hemadsorption and green fluorescence were observed under microscope. Scale bars, 275 μm. G PAMs were infected with ASFV-WT or ASFV-ΔA137R. At 0, 12 or 24 hpi (hours post-infection), the level of mature IL-1β in the supernatant was detected.

H The genomic copy numbers of ASFV in (G) were detected by qPCR.

To identify which viral structural protein mediates NLRP3 inflammasome activation, we screened a panel of individual ASFV proteins and found that pA137R protein significantly activated the NLRP3 inflammasome (data not shown). To further confirm the effect of pA137R protein on inducing inflammatory response, we generated an A137R gene-deleted mutant virus (ASFV-ΔA137R) based on the virulent ASFV HLJ/18 strain by homologous recombination, in which the A137R gene was replaced with a p72 promoter-EGFP cassette (Fig. 1C). The mutant virus was purified through 10 rounds of limiting dilution and verified by PCR (Fig. 1D) and sequencing. Consistent with our design, the A137R-specific fragment could not be amplified from ASFV-ΔA137R genome and no other unintended genetic modifications were detected. We next evaluated the growth kinetics of ASFV-WT and ASFV-ΔA137R in PAMs. The results showed no significant difference in viral replication between ASFV-WT and ASFV-ΔA137R, indicating that pA137R protein does not affect viral replication (Fig. 1E). Additionally, ASFV-ΔA137R infection resulted in efficient GFP expression driven by the p72 promoter, and the hemadsorption characteristics of ASFV remained intact (Fig. 1F). To directly assess the impact of pA137R protein on inflammatory cytokine production, PAMs were infected with ASFV-WT or ASFV-ΔA137R. As shown in Fig. 1G and H, deletion of the A137R gene significantly decreased ASFV-induced IL-1β secretion, while viral replication remained unaffected. Taken together, these results demonstrate that ASFV pA137R protein is a key viral component that triggers host inflammatory response.

ASFV pA137R protein activates NF-κB pathway

Inflammatory pathway is divided into signal 1 (the NF-κB pathway) and signal 2 (inflammasome pathway). To clarify the role of ASFV pA137R protein in inflammatory signaling, we first investigated its effects on signal 1 (the NF-κB pathway). For this purpose, PAMs were infected with ASFV-WT or ASFV-ΔA137R and the mRNA levels of multiple NF-κB-regulated genes were measured at different time points post-infection. As shown in Fig. 2A–E, compared to ASFV-WT, ASFV-ΔA137R infection resulted in significantly attenuated transcription of several NF-κB target genes, including Il-1b, Il-6, Tnf-α and Nlrp3. Furthermore, we found that ASFV-ΔA137R infection induced lower phosphorylation levels of p65 and IκBα compared to ASFV-WT infection (Fig. 2F). Conclusively, these results indicate that ASFV pA137R protein is critical for the upregulation of proinflammatory genes expression upon ASFV infection.

Fig. 2.

Deletion of ASFV A137R gene impaired ASFV-induced NF-κB pathway activation in infected PAMs. A–D PAMs were infected with ASFV-WT or ASFV-ΔA137R at an MOI of 1. At 0, 6, 12, 24 hpi, total mRNA of PAMs was extracted and the mRNA levels of Il-1b (A), Il-6 (B), Tnf-α (C) and Nlrp3 (D) were measured by qPCR. E The genomic copy numbers of ASFV in (A–D) were detected by qPCR. F PAMs were mock infected, infected with ASFV-WT or ASFV-ΔA137R at an MOI of 1 for 24 h or stimulated with LPS for 12 h (20 μg/mL). Then, cells were lysed and phosphorylation of IκBα and p65 were detected by western blotting. Grayscale values were quantified using ImageJ.

Based on the above findings, we hypothesized that ASFV pA137R protein was one of the key viral components responsible for ASFV-triggered NF-κB pathway activation. To verify this hypothesis, we first examined the effect of intracellularly expressed pA137R protein on the NF-κB signaling pathway. The results showed that intracellularly expressed pA137R protein did not affect TLR4-induced NF-κB promoter activation (Fig. 3A). A previous study demonstrated that ASFV pA137R protein localizes on the outer capsid of intracellular ASFV virions (Yang et al., 2024). Therefore, we speculated that capsid-localized pA137R protein may activate the NF-κB signaling pathway via PRRs on the plasma membrane of host cells. To test this speculation, PAMs and 3D4/21 cells were treated with purified pA137R protein and the transcription of multiple NF-κB-regulated genes was subsequently detected. We found that purified pA137R protein significantly induced the transcription of various proinflammatory genes in both PAMs and 3D4/21 cells (Fig. 3B and C). Furthermore, stimulation of PAMs with purified pA137R protein also induced the phosphorylation of p65 and IκBα (Fig. 3D). Additionally, PAMs were infected with the same amount (1.74 × 10⁸ genome copies) of ASFV-WT or ASFV-ΔA137R virions without outer envelope for 2 h, and then the mRNA level of IL-1β was measured. During this time period, the virus maintained its structural integrity while being endocytosed by its target cells (Weng et al., 2025). As shown in Fig. 3E, compared to ASFV-WT virions, ASFV-ΔA137R virions induced a lower level of IL-1β transcription. These results collectively suggest that pA137R protein localized on the capsid of ASFV virions is responsible for inducing the host NF-κB pathway.

Fig. 3.

ASFV pA137R protein activates NF-κB pathway. A HEK293T cells were transfected with pCAGGS-HA-TLR4 and NF-κB-Luc, TK-Renilla reporters along with increasing amounts of pCAGGS-HA-A137R. 24 h later, cells were lysed and fluorescence intensity was measured. B 3D4/21 cells were stimulated with 1 μg/mL of purified His-tagged A137R protein for 5 h. The mRNA levels of Il-1b, Tnf-α, Il-18 and Nlrp3 were measured by qPCR. C PAMs were stimulated with 1 μg/mL of purified His-tagged A137R protein for 5 h. The mRNA levels of Il-1b, Il-6, Tnf-α, Il-18 and Nlrp3 were measured by qPCR. D PAMs were stimulated with LPS for 12 h (20 μg/mL) or purified His-tagged A137R protein for 5 h (1 μg/mL). The levels of phosphorylated IκBα and p65 were detected by western blotting. E PAMs were stimulated with intracellular ASFV-WT or ASFV-ΔA137R virion without outer envelope at the same number of genome copies (1.74 × 10⁸) for 2 h. The mRNA level of Il-1β was measured by qPCR.

ASFV pA137R protein facilitates NLRP3 inflammasome assembly

We further investigated the impact of pA137R protein on the signal 2, namely the inflammasome pathway. HEK293T cells were transiently transfected with plasmids encoding NLRP3, ASC, caspase-1, pA137R protein, along with a pro-IL-1β-Gaussia luciferase (iGluc) reporter plasmid. Luciferase activity was then analyzed to evaluate inflammasome activation. As shown in Fig. 4A, expression of pA137R protein significantly promoted NLRP3-induced iGluc reporter activity. Consistent with this result, overexpression of pA137R protein facilitated NLRP3-induced ASC oligomerization in HEK293T cells (Fig. 4B). Moreover, Bimolecular Fluorescence Complementation (BiFC) analysis was used to investigate the effect of ASFV pA137R protein on NLRP3 oligomerization. The results showed that expression of pA137R protein markedly enhanced the formation of NLRP3-NEK7 specks, which indirectly demonstrates that pA137R protein promotes the assembly of the NLRP3 inflammasome (Fig. 4C).

Fig. 4.

ASFV pA137R protein facilitates NLRP3 inflammasome assembly. A HEK293T cells were transfected with plasmids expressing NLRP3 (12.5 ng per well), ASC (10 ng per well), caspase-1 (10 ng per well) along with iGluc reporter plasmid (100 ng per well). 24 h later, the supernatants were collected to detect the luciferase activity. B HEK293T cells were transfected with pCAGGS-HA-ASC, pCAGGS-Flag-NLRP3 and pCAGGS-Flag-A137R. 24 h later, cells were collected, washed with PBS three times and cross-linked with 2 mM DSS for 30 min. Thereafter, the oligomerization was assessed. C HEK293T cells were transfected with pCAGGS-Flag-NLRP3-VC, pCAGGS-Flag-NEK7 -VN along with pCAGGS-HA vector or pCAGGS-HA-A137R. 24 h later, cells were stimulated with 10 μM of Nigericin for 2 h. Then the cells were fixed, stained with mouse anti-Flag mAb and DAPI, and then observed by confocal microscopy. Scale bars, 20 μm.

ASFV pA137R protein interacts with NLRP3

We then investigated the mechanism of ASFV pA137R protein promoting the formation of NLRP3 inflammasome. To this end, we first investigated the potential interaction between pA137R protein and different components of the NLRP3 inflammasome. As shown in Fig. 5A, pA137R protein interacted with NLRP3 but not with ASC or caspase-1. Furthermore, pA137R protein also co-precipitated with endogenous NLRP3 in ASFV-infected PAMs, which is consistent with our previous result (Fig. 5B). In addition, GST-pulldown assay revealed a specific interaction between purified pA137R protein and HEK293T-expressed NLRP3 (Fig. 5C). Moreover, we found that pA137R protein and NLRP3 were colocalized in the cytoplasm of both HEK293T cells and PAMs (Fig. 5D and E). Additionally, we co-expressed pA137R protein, NLRP3 and ASC in HEK293T and found that they were obviously colocalized to form a “sphere-like” structure (Fig. 5F). This specific structure has also been reported in previous studies (Luo et al., 2017; Pan et al., 2021).

Fig. 5.

ASFV pA137R protein interacts with NLRP3. A HEK293T cells were transfected with pCAGGS-HA-NLRP3, pCAGGS-HA-ASC, pCAGGS-HA-caspase-1 along with pCAGGS-Flag-A137R. 24 h later, cells were lysed and co-IP assay was performed. B PAMs were infected with ASFV (MOI = 1). At 24 hpi, cells were lysed and IP assay was performed. C HEK293T cells were transfected with pCAGGS-Flag-NLRP3. 24 h later, cells were lysed and the lysates were mixed with purified GST or GST-tagged A137R protein. Then the interaction between pA137R protein and NLRP3 was analyzed by GST pull-down assay. D HEK293T cells were transfected with pCAGGS-Flag-NLRP3 and pCAGGS-HA-A137R, 24 h later, cells were fixed, incubated with mouse anti-Flag mAb, rabbit anti-HA pAb and DAPI and then observed by confocal microscopy. Scale bars, 20 μm. E PAMs were infected with ASFV (MOI = 1). At 24 hpi, cells were fixed, incubated with mouse anti-A137R pAb, rabbit anti-NLRP3 pAb and DAPI and then observed by confocal microscopy. Scale bars, 20 μm. F HEK293T cells were transfected with pCAGGS-Flag-NLRP3, pCAGGS-HA-ASC, pEGFP-vector or pEGFP-A137R. 24 h later, cells were fixed, incubated with mouse anti-Flag mAb, rabbit anti-HA pAb and DAPI and then observed by confocal microscopy. Scale bars, 20 μm.

NLRP3 contains three functionally distinct domains: nucleotide-binding domain (NACHT), leucine-rich-repeat domain (LRR), and pyrin domain (PYD). To map the specific domain of NLRP3 required for its interaction with pA137R protein, four plasmids encoding NLRP3 truncated mutants were constructed and co-IP assays were performed to assess their interaction with pA137R protein. The results showed that pA137R protein interacts with the NACHT and LRR domains of NLRP3, but not with the PYD domain (Fig. 6A and B). It has been reported that pA137R protein contains a gun-shaped N-terminal domain (NTD) and a flexible C-terminal domain (CTD) (Li et al., 2024). To detect which domain of pA137R protein is required for its interaction with NLRP3, we constructed plasmids expressing GFP-tagged pA137R protein NTD or CTD, followed by co-IP assay. As shown in Fig. 6C and D, NLRP3 interacts with the NTD but not the CTD of pA137R protein.

Fig. 6.

The NTD domain of pA137R protein interacts with the LRR-NACHT domain of NLRP3. A Schematic of full-length NLRP3 and its truncated mutants. B HEK293T cells were transfected with plasmids encoding full-length NLRP3 and its truncated mutants along with pCAGGS-Flag-A137R. 24 h later, cells were lysed and co-IP assay was performed. C Schematic representation of the pA137R protein construct. D HEK293T cells were transfected with pEGFP-vector, pEGFP-A137R or its CTD or NTD domain along with pCAGGS-Flag-NLRP3. 24 h later, cells were lysed and co-IP assay was performed.

Discussion

The inflammatory response is one of the key components of the host innate immune system. When intracellular pathogens enter their target cells, their various components, such as proteins or nucleic acids, act as inducers of the inflammatory response. During this process, infected cells undergo pyroptosis, leading to the release of intracellular pathogens, which are subsequently recognized by the host immune system.

The inflammatory response acts as a double-edged sword in antimicrobial defense: while it plays a beneficial role in eliminating pathogens, an excessive inflammatory response can cause significant tissue damage, leading to multi-organ dysfunction. ASFV infection induces a severe inflammatory response. Previous studies have shown that ASFV infection induces the exaggerated and prolonged release of proinflammatory cytokines in domestic pigs (Wang et al., 2020). Thus, it is imperative to identify the viral factors responsible for induction of the host inflammatory response, which is crucial for the development of an effective vaccine. In our study, we found that deletion of A137R gene impaired ASFV-induced IL-1β release, which indicated that pA137R protein is one of the key proinflammatory factors of ASFV.

As a core component of the inflammatory signaling pathway, the NF-κB pathway regulates the expression of various inflammation-related cytokines. Different components of pathogens activate the NF-κB pathway through various PRRs. Previous studies showed that various viral proteins, such as the SARS-CoV-2 E protein, EBV dUTPase activate NF-κB pathway (Ariza et al., 2009; Zheng et al., 2021). In our study, deletion of A137R gene attenuated ASFV-induced expression of NF-κB-regulated genes and phosphorylation of p65 and IκBα. Additionally, intracellular virions of ASFV-ΔA137R induced lower level of IL-1β expression. Furthermore, purified pA137R protein induced the expression of various inflammatory-related genes and the phosphorylation of p65 and IκBα. These results confirm that pA137R protein is one of the triggers of the NF-κB pathway during ASFV infection. However, the PRRs responsible for pA137R protein recognition remain to be further identified.

Among different kinds of inflammasomes, NLRP3 inflammasome is unique owing to the diversity of its stimuli and the complexity of the underlying mechanisms of its activation. To date, numerous viral proteins have been identified as activators of the NLRP3 inflammasome by disrupting organelle function or intracellular ionic homeostasis (Ichinohe et al., 2010; Mao et al., 2024; Guo et al., 2025). In this study, we found that pA137R protein promotes NLRP3 inflammasome assembly by binding to LRR and NACHT domains of NLRP3. Consistent with our findings, previous studies have also identified various viral proteins that regulate NLRP3 inflammasome assembly via directly binding to these two domains of NLRP3 (Luo et al., 2017; Choudhury et al., 2022; Huang et al., 2023; Liu et al., 2025). However, they represent distinct impacts. In the resting state, NLRP3 oligomerizes into a cage-like structure, in which its NACHT domain is sequestered at the periphery of this oligomer. Upon activation, NLRP3 undergoes a series of conformational changes and, with the assistance of NEK7, assembles into the active NLRP3 inflammasome (Sharif et al., 2019). Thus, we speculate that the differences in the effects on the conformational changes or NLRP3–NEK7 interaction during NLRP3 inflammasome activation may be the fundamental cause underlying the diverse regulation of the NLRP3 inflammasome by these viral proteins. Nevertheless, a detailed dissection of protein-protein interaction sites is necessary to uncover the specific mechanisms responsible for these differential functional regulations.

Additionally, this study has several limitations. First, the purified His-A137R protein used in this study was produced in prokaryotic system. Although we employed an endotoxin-free purification protocol, the potential interference from residual LPS in the protein preparation cannot be completely ruled out. Additionally, most of the mechanistic experiments in this study were performed using overexpression systems in HEK293T cells, which may not fully recapitulate the physiological environment of ASFV-infected PAMs. Furthermore, due to limitations in laboratory conditions, we were unable to investigate the effect of A137R on ASFV infection-mediated endogenous NLRP3 inflammasome formation.

Conclusions

In summary, our findings identify the pA137R protein as a key factor that promotes ASFV-induced inflammation by activating the NF-κB pathway and modulating NLRP3 inflammasome assembly. These results uncover a direct molecular link between ASFV infection and host inflammatory signaling, highlighting a critical interface in ASFV pathogenesis. Future studies will be directed at elucidating the precise structural basis of NLRP3 conformation changes induced by pA137R protein, as well as defining the contribution of its pro-inflammatory activity to viral virulence in relevant animal models.

Materials and methods

Cells and viruses

As described previously, porcine alveolar macrophages (PAMs) were isolated from bronchoalveolar lavage from 4-week-old specific pathogen-free (SPF) pigs and maintained in RPMI-1640 medium (Li et al., 2015). HEK293T cells and 3D4/21 cells obtained from American Type Culture Collection (ATCC) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) and RPMI-1640 medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified incubator at 37 °C with 5% CO₂. ASFV HLJ/18 strain (GenBank accession number: MK333180) was isolated in PAMs from a pig farm during an ASF outbreak (Zhao et al., 2019) and propagated on porcine bone marrow cells. This strain served as the backbone for the generation of all subsequent gene-knockout mutants.

Plasmids

To construct plasmids expressing Flag-NEK7 and HA-TLR4, total RNA was first extracted from PAMs. The cDNA corresponding to NEK7 and TLR4 was then amplified by RT-PCR and cloned into pCAGGS-Flag or pCAGGS-HA vector. The A137R gene was amplified from ASFV genomic DNA and then cloned into pCAGGS-Flag, pCAGGS-HA, pGEX-6P1, pET-21a and pEGFP-C1 vectors. Plasmids expressing GFP-tagged pA137R protein C-terminal domain and N-terminal domain were created based on the plasmid expressing pA137R protein. Plasmids expressing NLRP3, ASC, caspase-1 were constructed as described (Li et al., 2015). Plasmids expressing different truncated mutants of NLRP3 were created base on the plasmid expressing NLRP3. pCAGGS-Flag-NLRP3-VC and pCAGGS-Flag-NEK7-VN were constructed based on the plasmid expressing NLRP3 and NEK7.

Generation of ASFV-ΔA137R

ASFV-ΔA137R was generated by homologous recombination. Briefly, PAMs were transfected with a transfer vector harboring a cassette containing a p72 promoter and EGFP reporter gene along with a 1009 bp upstream homologous arm and a 1006 bp downstream homologous arm and then infected with ASFV HLJ/18. The mutant strain was identified by PCR and sequencing.

Antibodies and reagents

Rabbit anti-NLRP3 pAb was purchased from Bioss. Rabbit anti-ASC pAb was purchased from Proteintech. Rabbit anti-p65 mAb, anti-phospho-p65 (Ser536) mAb, anti-IκBα mAb and anti-phospho-IκBα (Ser32) were purchased from Cell Signaling Technology. Mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) pAb was purchased from Proteintech. Mouse anti-Flag M2 mAb, rabbit anti-HA pAb and anti-Flag pAb were purchased from Sigma-Aldrich. Rabbit anti-GFP pAb was purchased from Proteintech. The IRDye 800CW goat anti-rabbit IgG (H + L) and IRDye 800CW goat anti-mouse IgG (H + L) were purchased from LI-COR. Alexa Fluor 488 goat anti-Rabbit IgG (H + L), Alexa Fluor 594 goat anti-Mouse IgG and Alexa Fluor 633 goat anti-Rabbit IgG (H + L) were purchased from Thermo Fisher Scientific. Mouse anti-pA137R pAb and anti-p54 mAb was prepared by immunized mouse with purified recombinant pA137R and p54 protein. Porcine IL-1β ELISA Kit was purchased from RayBio. LPS and Nigericin were purchased from MCE. Dual Luciferase Reporter Assay System was purchased from Promega. Gaussia Luciferase Flash Assay Kit was purchased from Thermo Fisher Scientific. Disuccinimidyl suberate (DSS) was purchased from Thermo Fisher Scientific.

Protein expression and purification

To purify GST, GST- and His-tagged pA137R proteins, pGEX-6P1 vector, pGEX-6P1-A137R and pET-21a-A137R were transformed into BL21 (DE3). Transformed BL21 cells were grown in LB medium at 37 °C until the OD₆₀₀ reached 0.6–0.8. Protein expression was then induced by adding 0.5 mM (GST and GST-tagged A137R) or 1 mM (His-tagged A137R) isopropyl β-D-1-thiogalactopyranoside (IPTG), followed by incubation at 16 °C for 20 h. Cells were subsequently harvested by centrifugation and then lysed via ultrasonication, the recombinant protein was purified with Glutathione-Sepharose (Amersham Pharmacia Biotech) or a HisTrap HP 5 mL column (GE Healthcare).

Preparation of non-enveloped virions

Porcine bone marrow cells were infected with ASFV-WT or ASFV-ΔA137R at an MOI of 1. 24 h later, cell supernatants were discarded and the cells were washed with PBS three times. Then the cells were resuspended with PBS and lysed by repeated freeze-thaw cycles. Thereafter, centrifugation was performed to remove cell debris.

Quantitative PCR (qPCR)

For Relative Quantification PCR, total RNA was extracted by using RNAiso Plus reagent (Takara) and then reverse transcribed to cDNA by PrimeScript RT reagent kit (Takara). Gene expression levels were assessed by SYBR Green Premix detection system and the data were normalized to the level of β-actin expression. For Absolute Quantification PCR, ASFV genomic DNA was extracted using a QIAamp DNA minikit (Qiagen). Then the copy number of ASFV genomic DNA was measured by Taqman Premix detection system. Primers used in this study are listed in Supplementary Table S1.

Enzyme-linked immunosorbent assay (ELISA)

The level of IL-1β in cell culture supernatant was assessed by Porcine IL-1β ELISA Kit (RayBio) according to the manufacturer's instructions.

Luciferase reporter gene assay

HEK293T cells were plated in 48-well plates and co-transfected with indicated plasmids along with NF-κB (100 ng per well) and TK-Renilla (5 ng per well) reporter plasmids. 24 h later, cells were lysed and fluorescence intensity was measured using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's Instructions. Data were normalized to the transfection efficiency by dividing the firefly luciferase activity by the Renilla luciferase activity.

iGluc-based inflammasome reconstruction system

According to previous research (Song et al., 2020), HEK293T cells were plated in 48-well plates at 5 × 10⁴ per well and then co-transfected with plasmids expressing NLRP3 (12.5 ng per well), caspase-1 (10 ng per well), ASC (10 ng per well) and pA137R protein (200 ng per well), along with iGluc reporter plasmid (100 ng per well). 24 h later, cell culture supernatants were collected and the fluorescence intensity was measured using a Gaussia Luciferase Flash Assay Kit (Thermo Fisher Scientific).

Confocal microscopy and co-localization analysis

HEK293T cells were transfected or co-transfected with indicated plasmids, 24 h later, cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.3% Triton X-100 for 20 min. Cells were incubated with indicated primary and secondary antibodies after a 2-h block with 5% BSA and then were visualized using a Zeiss LSM-880 laser scanning fluorescence microscope (Carl Zeiss AG, Oberkochen, Germany) under a 63× oil objective.

PAMs were infected with ASFV for 24 h (MOI = 1). All subsequent steps were performed as described above.

Bimolecular Fluorescence Complementation (BiFC) assay

HEK293T cells were co-transfected with pCAGGS-Flag-NLRP3-VC, pCAGGS-Flag-NEK7-VN along with pCAGGS-HA vector or pCAGGS-HA-A137R. After 24 h, cells were fixed, incubated with primary and secondary antibodies and visualized using a Zeiss LSM-880 laser scanning fluorescence microscope (Carl Zeiss AG, Oberkochen, Germany) under a 63× oil objective.

Co-IP assay and Western blot analysis

Cells (HEK293T or PAMs) were harvested and washed with PBS once and then were lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM MgCl₂, 1 mM EDTA, 1% Triton X-100 and 10% glycerol) containing 1 mM PMSF and 1 × protease inhibitor cocktail (Basel) for 30 min. Then the supernatant was collected and incubated with anti-flag (M2) beads (Sigma-Aldrich) or added indicated antibodies and protein A + G Agarose (MCE) at 4 °C for 8 h. Then the beads were washed for 3 times with lysis buffer. For Western blot analysis, the samples were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinyl difluoride (PVDF) membranes (Sigma Aldrich). Then the membranes were incubated with primary and secondary antibodies. Finally, the membranes were visualized by the Odyssey Dual Color Infrared Fluorescence Imaging System (LI-COR).

GST-pulldown assay

HEK293T cells were transfected with pCAGGS-Flag-NLRP3. 24 h later, cells were lysed. Then 10 μg of purified GST or GST-tagged A137R protein was added to the lysates and incubated for 8 h. After that, Glutathione Agarose (MCE) was added and incubated for another 8 h. Finally, beads were collected, washed for 3 times with lysis buffer, and then Western blot analysis was performed.

Statistical analysis

Statistical significance was determined using the unpaired two-tailed Student's t-test or one-way ANOVA with the SPSS 16.0 software package (version 16.0, SPSS, Inc., Chicago, IL, USA). All data were expressed as the mean ± standard deviation (SD). ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001.

Data availability

All the data generated during the current study are included in manuscript and supplemental materials.

Ethics statements

In accordance with biosafety guidelines, all experiments involving live ASFV was performed in the enhanced biosafety level 3 (P3) facilities in the Harbin Veterinary Research Institute (HVRI) of the Chinese Academy of Agricultural Sciences (CAAS) approved by the Ministry of Agriculture and Rural Affairs. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of the People's Republic of China.

Author contributions

Yi Zeng: conceptualization, data curation, formal analysis, investigation, methodology, validation, visualization, writing-original draft. Xiaohong Liu: methodology, writing-review & editing. Siqi Dong: writing-review & editing. Hanyu Wu: writing-review & editing. Changjiang Weng: funding acquisition, project administration, resources, supervision, writing-review & editing. Li Huang: conceptualization, funding acquisition, project administration, writing-original draft.

Conflict of interest

The authors have no conflict of interest. Prof. Changjiang Weng is editorial board member for Virologica Sinica and was not involved in the editorial review or the decision to publish this article.

Appendix A. Supplementary data

The following is the Supplementary data to this article.