The B169L protein of African swine fever virus functions as a viroporin that activates the calcium-mediated inflammasome

Figures

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  Fig 1. The B169L protein (pB169L) of ASFV activates the NLRP3 inflammasome. (A) Schematic of pro-interleukin 1beta (pro-IL-1β)-Gaussia luciferase (iGLuc) reporter. (B) Screening for ASFV structural proteins that activate the NLRP3 inflammasome. HEK293T cells were cotransfected with the iGLuc-based NLRP3 inflammasome system (300 ng of pFlag-pro-IL-1β-Gluc (pFlag-iGluc), 10 ng of pFlag-pro-caspase-1, 10 ng of pFlag-ASC, and 60 ng of pFlag-NLRP3) and the plasmids expressing the structural proteins of ASFV or the empty vector pCAGGS, and the luciferase activity in the supernatants were examined at 24 hours posttransfection (hpt). (C and D) pB169L activates the inflammasome in a dose-dependent manner. HEK293T cells were transfected with increasing doses of pFlag-B169L or pCAGGS as well as the NLRP3-dependent inflammasome reporter as described above, and the luciferase activity of the supernatants and the intracellular protein expression levels were evaluated at 24 hpt. (E) pB169L promotes ASC oligomerization. HEK293T cells were cotransfected with pHA-ASC and pFlag-B169L or pCAGGS. At 24 hpt, the cells were incubated with 200 mM dioctanoyl di-succinimidyl ester (DSS) (final concentration: 4…

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  Fig 2. The B169L protein (pB169L) of ASFV activates inflammasome through driving ion levels. (A) Ca2+ inhibitors inhibit the activation of the NLRP3 inflammasome induced by pB169L. HEK293T cells were cotransfected with the iGLuc-based NLRP3 inflammasome system (300 ng of pFlag-pro-IL-1β-Gluc (pFlag-iGluc), 10 ng of pFlag-pro-caspase-1, 10 ng of pFlag-ASC, and 60 ng of pFlag-NLRP3) and the plasmids expressing the structural proteins of ASFV or the empty vector pCAGGS. At 12 hours posttransfection (hpt), the cells were treated with either EIPA, amiloride, verapamil, nifedipine, 4-AP, or glibenclamide for 12 hours. The supernatant luciferase activity was examined at 24 hpt. (B) pB169L dose-dependent promotes intracellular calcium ion increase. pFlag-B169L was transfected into HEK293T cells at different doses (50, 100, 150, or 200ng), and Flou-4 was added 24 hours later which were examined using a Fluo-4 Flux analysis kit. (C) The Ca2+-chelating agent BAPTA inhibits the activation of the NLRP3 inflammasome induced by pB169L. HEK293T cells were transfected with pFlag-B169L or pCAGGS as well as the NLRP3-dependent inflammasome reporter as described above. At 12 hpt, the cells were treated…

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  Fig 3. The B169L protein (pB169L) of ASFV exhibits the characteristics of viroporins. (A) A schematic diagram of the pB169L domains. The figure was generated by using the online software IBS 2.0 (https://www.ibs.renlab.org/). (B) Predicted multimeric structure of pB169L generated by AlphaFold2. (C and D) The oligomeric state of pB169L. HEK293T cells were cotransfected with pFlag-B169L and pEGFP-B169L (2 μg/well). At 24 hours posttransfection (hpt), the cells were lysed, and the resulting whole cell lysates (WCLs) were subjected to coimmunoprecipitation (co-IP) assay using the anti-Flag antibodies, followed by Western blotting analysis. as above (C). HEK293T cells were transfected with pFlag-B169L or pCAGGS. At 24 hpt, the cells were lysed, and the resulting WCLs were analyzed by native-PAGE as above (D). (E–G) pB169L inhibits the growth of Escherichia coli. The E. coli BL21(DE3) pLys cells transformed with pGEX-B169L, pGEX-B169L-ΔTM, or pGEX-E301R were grown to an OD value of 0.4 to 0.6 at 600 nm (OD600nm). Subsequently, the bacterial was divided into two equal portions and subjected to induction with 0.8 mM IPTG at 16°C or without IPTG induction under the same conditions. Cell growth…

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  Fig 4. The B169L protein (pB169L) of ASFV serves as calcium-permeable cation channels. (A) Schematic diagram of single channel electrophysiological recording by bilayer lipid membrane system. The figure was generated by using the online software BioRender (https://app.biorender.com/). (B) Testing buffers used in the single channel recording experiments. (C) Single-channel current recording of pB169L, pB169L-ΔTM, and pB169L-TM1 at the indicated potentials and 500 mM: 50 mM KCl solutions. Proteins were added to the cis side. “C” represents closed, and “O” represents open. Current histograms of the traces were shown in the right. (D) Linear current-voltage (I-V curve) of pB169L and pB169L-TM1 in 500 mM: 50 mM KCl solution (n ≥ 3). (E) Single-channel current recording of pB169L at the indicated potentials and asymmetric NaCl solutions. Proteins were added to the cis side. “C” represents closed, and “O” represents open. Current histograms of the traces were shown in the right. (F) Single-channel current recording of pB169L at the indicated potentials and asymmetric Na+/Ca2+ mixed solutions. Proteins were added to the cis side. “C” represents closed, and “O” represents open. Current…

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  Fig 5. The dominant-negative B169L protein (pB169L) reduces its channel activity and attenuates inflammasome activation. (A) iGLuc luciferase activity analysis of pB169L domains. HEK293T cells were cotransfected with pFlag-B169L, pFlag-B169L-ΔTM, -ΔTM1, or -ΔTM2 in the presence of the iGLuc-based NLRP3 inflammasome system, and the supernatants were collected to determine luciferase activity at 24 hours posttransfection (hpt). (B) Oligomerization analysis of pB169L domains. HEK293T cells were transfected with 2 µg of the indicated plasmids for 24 hours, followed by DSS cross-linking assay using 4 µM DSS (final concentration: 4 μM) at 4°C for 30 minutes. Protein oligomerization was analyzed by Western blotting. (C) Calcium imaging analysis of pB169L domains. Representative average traces of Ca2+ image of HEK293 cells transfected with pCAGGS, pFlag-B169L, pFlag-B169L-ΔTM1, pFlag-B169L-ΔTM2, or pFlag-B169L-ΔTM. HEK293 cells were transfected with indicated plasmids. Fura-2 AM was used to monitor ER calcium storage. Ionomycin and Thapsigargin were added at the time indicated by the arrow. (D) Scatter plot of ionomycin and thapsigargin (Tg) induced ratio change of cells transfected with…

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  Fig 6. The B169L protein (pB169L) is essential for IL-1β production induced by ASFV infection. (A) Effectiveness of siRNAs targeting the ASFV B169L gene. PAMs were transfected with siB169Ls and infected with ASFV (multiplicity of infection (MOI) = 0.1) at 8 hours posttransfection (hpt). At 2 hours postinfection (hpi), the supernatants were discarded and replaced with fresh medium. The transcription level of B169L was quantified by reverse transcription-quantitative PCR (RT-qPCR) at 24 hpi. (B–G) Effects of ASFV B169L gene knockdown on proinflammatory cytokines. PAMs (106 cells/well) were transfected with siRNA-2. At 8 hpt, the cells were infected with ASFV (MOI = 0.1). The transcription levels of B169L, IL-1β, NLRP3, and IL-18 were quantified by RT-qPCR at 24 hpi. The supernatants were collected and the secretion of IL-1β (F) and IL-18 (G) was tested by enzyme-linked immunosorbent assay (ELISA). (H–M) Impact of overexpression pB169L in WSL cells on proinflammatory cytokines. WSL cells were transfected with pFlag-B169L or pCAGGS. At 24 hpt, the transcriptional of IL-1β, and NLRP3 were analyzed by RT-qPCR. The supernatants were detected by a porcine IL-1β/IL-18 ELISA kit. (N–P) Effects of…

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  Fig 7. Schematic diagram of the ASFV pB169L-induced activation of NLRP3 inflammasome. pB169L localizes to the ER to promote calcium release and activate the NLRP3 inflammasome. The P29A and K55A mutation reduces calcium efflux and weakens NLRP3 inflammasome activation, while the K57A mutation disrupts B169L oligomerization and impairs NLRP3 inflammasome activation. The figure was generated by using the online software BioRender (https://app.biorender.com/).