Fig 1.
PRRSV-2 triggers inflammatory responses by activating NLRP3 inflammasome.
(A) PAMs infected with PRRSV-2 at an MOI of 0.05 for 12 h or 18 h, and gene transcription levels were evaluated by qRT-PCR. The relative expression changes were calculated by using 2^(-ΔΔCt) method. (B and C) The mature IL-1β (p17) levels in PAMs supernatants were assessed by ELISA and Western blot after PRRSV-2 infection at MOI of 0.05 for 12 or 18 hours (B), and at MOI of 0.01, 0.05, or 0.1 for 18 hours (C). The pro-IL-1β and GSDMD in cell lysates were analyzed by Western blot. (D) PAMs transfected with control siRNA or siRNA targeting NLRP3 (si-NLRP3-3) were infected with PRRSV-2 (MOI of 0.05) for 18 hours. The mature IL-1β (p17) levels in supernatants were measured by ELISA and Western blot, and pro-IL-1β and GSDMD in cell lysates were detected by Western blot. (E and F) PAMs were pre-treated with 50 μM MCC950 (E) or Z-YVAD-FMK (F) for 1 hour, then infected with PRRSV-2 at an MOI of 0.05 for 18 hours. The pro-IL-1β and GSDMD in lysates were analyzed by Western blot, and the mature IL-1β (p17) levels in supernatants were measured by ELISA and Western blot. (G) PAMs infected with PRRSV-2 (MOI of 0.05) for 18 hours or treated with LPS and Nigericin were analyzed by immunoprecipitation using an anti-NLRP3 antibody. (H and I) PAMs were infected with PRRSV-2 at an MOI of 0.05 for 18 h or treated with 100 ng/mL LPS for 8 h followed by 10 μM Nigericin for another 4 h as a positive control. ASC oligomerization was analyzed by cross-linking the cytosolic pellets and visualized by Western blot (H). ASC speck formation in mock, PRRSV-2-infected, and LPS/Nigericin-treated PAMs was observed by confocal microscopy (I, left). Percentages of ASC speck-positive cells are shown (I, right). NG, Nigericin; Sup, supernatant; WCL: whole cell lysate; Lys, lysate; Pel, pellets. For confocal microscopy assay, results were manually quantified from at least 30 randomly selected cells per condition per replicate using Image J. Scale bar: 10 μm. The protein levels were quantified by Image J and normalized to β-actin. The data are representative of results from three independent experiments. Error bars indicate the mean (± SD) of three repeats. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001; p > 0.05 stands for ns.
Fig 2.
PRRSV-2 nsp2 promotes the activation of NLRP3 inflammasome.
(A) HEK293T cells co-transfected with HA-tagged NLRP3, ASC, pro-CASP1, pro-IL-1β, and Flag-tagged PRRSV-2 protein plasmids or empty vector (pCAGGS) for 24 h were analyzed for IL-1β in supernatants by ELISA. (B) HEK293T cells co-transfected with HA-tagged NLRP3, ASC, pro-CASP1, pro-IL-1β, and varying concentrations of Flag-tagged nsp2 plasmids or pCAGGS for 24 h were analyzed for IL-1β in supernatants by ELISA and mature IL-1β (p17) in supernatants by Western blot. (C) HEK293T cells co-transfected with HA-tagged NLRP3, ASC, pro-CASP1, pro-IL-1β, and Flag-tagged nsp2 or pCAGGS. After 6 h, HEK293T cells were treated with 50 μM MCC950 for 18 h. Supernatants were analyzed for IL-1β by ELISA, and mature IL-1β (p17) by Western blot. (D) THP-1 cells infected with Lentivirus-CT or Lentivirus-nsp2, differentiated with PMA, then treated with either 1 μg/mL LPS for 12 h, 2 μM Nigericin for 1 h, or LPS (12 h) followed by Nigericin (1 h). Mature IL-1β (p17) in supernatants and GSDMD in lysates were analyzed by Western blot. (E) THP-1 cells were stably infected with Lentivirus-CT or Lentivirus-nsp2, differentiated with PMA. Then, THP-1 were transfected with control siRNA (si-NC) or siRNA targeting NLRP3 (si-NLRP3-1) 100 nM for 24 h, followed by stimulation with 1 μg/mL LPS for 12 h. Mature IL-1β (p17) in supernatants and GSDMD in lysates were determined by Western blot. Lenti, Lentivirus; CT, Control; NG, Nigericin; Sup, supernatant; Lys, lysate. The protein levels were quantified by Image J and normalized to β-actin. The data are representative of results from three independent experiments. Error bars indicate the mean (± SD) of three repeats. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001; p > 0.05 stands for ns.
Fig 3.
PRRSV-2 nsp2 interacts with NLRP3 protein.
(A) HEK293T cells co-transfected with Flag-tagged nsp2, empty vector (pCAGGS), HA-tagged NLRP3, ASC, or pro-CASP1 for 24 h were immunoprecipitated with an anti-Flag antibody. (B) HEK293T cells co-transfected with Flag-tagged NLRP3 and either empty vector (pCAGGS) or Myc-tagged nsp2 for 24 h were immunoprecipitated with an anti-Flag antibody. (C) HEK293T cells transfected with Flag-tagged nsp2 or empty vector (pCAGGS) and HA-tagged NLRP3 were visualized with confocal microscopy for DAPI (blue), nsp2 (green), and NLRP3 (red). (D) THP-1 cells were stably infected with Lentivirus-CT or Lntivirus-nsp2, differentiated with PMA, were stained for Flag-tagged nsp2 and NLRP3. Representative immunofluorescence images showing nsp2-Flag and NLRP3 co-localization were presented on the (D, left). The percentages of nsp2-Flag and NLRP3 co-localization containing cells were presented on the (D, right). WCL, whole cell lysate. For confocal microscopy assay, results were manually quantified from at least 30 randomly selected cells per condition per replicate using Image J. Scale bar: 10 μm. The data are representative of results from three independent experiments. Error bars indicate the mean (± SD) of three repeats. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001; p > 0.05 stands for ns.
Fig 4.
PRRSV-2 nsp2 TM domain is crucial for NLRP3 inflammasome activation.
(A) HEK293T cells were co-transfected with a plasmid encoding Myc-tagged nsp2, either alone or with a plasmid encoding Flag-tagged NLRP3 or its truncated mutants for 24 h. Cell lysates were immunoprecipitated using an anti-Flag antibody. (B) HEK293T cells were co-transfected with a plasmid encoding HA-tagged NLRP3, either alone or with a plasmid encoding Flag-tagged nsp2 or its truncated mutants for 24 h. Cell lysates were immunoprecipitated using an anti-Flag antibody. (C) PAMs were transfected with eGFP or eGFP-TM mRNA for 24 h. Confocal microscopy images show eGFP-TM and NLRP3 co-localization (C, left), with quantification of co-localized cells on (C, right). (D) HEK293T cells were co-transfected with plasmids encoding HA-tagged NLRP3, ASC, pro-CASP1, pro-IL-1β, and Flag-tagged nsp2 or its truncated mutants for 24 h. IL-1β levels in supernatants were measured by ELISA, and mature IL-1β (p17) in supernatants and protein expression were analyzed by Western blot. (E) PAMs were transfected with eGFP or eGFP-TM mRNA for 24 h, followed by treatment with 100 ng/mL LPS for 8 h and 10 μM Nigericin for 4 h. IL-1β levels in supernatants were measured by ELISA, and mature IL-1β (p17) in supernatants and protein expression were analyzed by Western blot. WCL, whole cell lysate; NG, Nigericin; Sup, supernatant; Lys, lysate. For confocal microscopy assay, results were manually quantified from at least 30 randomly selected cells per condition per replicate using Image J. Scale bar: 10 μm. The protein levels were quantified by Image J and normalized to β-actin. The data are representative of results from three independent experiments. Error bars indicate the mean (± SD) of three repeats. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001; p > 0.05 stands for ns.
Fig 5.
PRRSV-2 nsp2 facilitates NLRP3 inflammasome assembly.
(A and B) THP-1 cells stably infected with Lentivirus-CT or Lentivirus-nsp2, differentiated with PMA, followed by stimulation with 1 μg/mL LPS or culture media for 12 h. Cell lysates were immunoprecipitated with an anti-NLRP3 antibody (A). ASC oligomerization was assessed by cross-linking cytosolic pellets and analyzed by Western blot (B). (C) THP-1 cells stably infected with Lentivirus-CT or Lentivirus-nsp2, differentiated with PMA, then stained with anti-Flag, anti-NLRP3, and anti-ASC antibodies. Confocal microscopy images showing co-localization of nsp2-Flag, NLRP3, and ASC were presented (C, left). Gray values for each channel were measured by ImageJ (C, middle), and percentages of co-localizing cells were shown (C, right). (D) PAMs were transfected with eGFP or eGFP-TM mRNA for 24 h. Confocal images showing eGFP-TM, NLRP3, and ASC co-localization were presented (D, left). Gray values were measured by ImageJ (D, middle), and percentages of co-localizing cells were shown (D, right). (E) PAMs were infected with PRRSV-2 at MOI of 0.05 for 18 h. Confocal images showing co-localization of nsp2, NLRP3, and ASC were shown (E, left). Gray values were measured by ImageJ (E, middle), and percentages of co-localizing cells were displayed (E, right). WCL, whole cell lysate; Lys, lysate; Pel, pellets. For confocal microscopy assay, results were manually quantified from at least 30 randomly selected cells per condition per replicate using Image J. Scale bar: 10 μm. The protein levels were quantified by Image J and normalized to β-actin. The data are representative of results from three independent experiments. Error bars indicate the mean (± SD) of three repeats. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001; p > 0.05 stands for ns.
Fig 6.
IKKβ is essential for nsp2-driven NLRP3 inflammasome activation.
(A) THP-1 cells stably infected with Lentivirus-CT or Lentivirus-nsp2, differentiated with PMA, then transfected with control siRNA (si-NC) or NEK7-targeting siRNA (si-NEK7-3) at 100 nM for 24 h, followed by stimulation with 1 μg/mL LPS or culture medium for 12 h. Mature IL-1β (p17) in supernatants and GSDMD in lysates were assessed by Western blot. (B and C) THP-1 cells stably infected with Lentivirus-CT or Lentivirus-nsp2, differentiated with PMA, then transfected with control siRNA (si-NC) or IKKβ-targeting siRNA (si-IKKβ-2) at 100 nM for 24 h, followed by stimulation with 1 μg/mL LPS or culture medium for 12 h. Mature IL-1β (p17) in supernatants and GSDMD in lysates were assessed by Western blot (B). ASC oligomerization was assessed by cross-linking cytosolic pellets and visualized by Western blot (C). (D) HEK293T cells were co-transfected with HA-tagged IKKβ and either empty vector (pCAGGS), Flag-tagged nsp2 or NLRP3. Cell lysates were immunoprecipitated with an anti-HA antibody. (E) HEK293T cells were co-transfected with Flag-tagged NLRP3 and either empty vector (pCAGGS) or HA-tagged IKKβ. Cell lysates were immunoprecipitated with an anti-Flag antibody. (F) THP-1 cells stably infected with Lentivirus-CT or Lentivirus-nsp2, differentiated with PMA, followed by stimulation with 1 μg/mL LPS or culture medium for 12 h. Cell lysates were immunoprecipitated with an anti-NLRP3 antibody. (G) HEK293T cells were transfected with IKKβ-HA plasmid alone or with NLRP3-Flag or its truncated mutants. At 24 hpt, cell lysates were immunoprecipitated using an anti-Flag antibody. (H) HEK293T cells were transfected with NLRP3-HA plasmid alone or with IKKβ-Flag or its truncated mutants. At 24 hpt, cell lysates were immunoprecipitated using an anti-Flag antibody. WCL, whole cell lysate; Sup, supernatant; Lys, lysate; Pel, pellets. The protein levels were quantified by Image J and normalized to β-actin. The data are representative of results from three independent experiments.
Fig 7.
PRRSV-2 nsp2 protein promotes NLRP3 translocation to dTGN and oligomerization through IKKβ.
(A and B) THP-1 cells stably infected with Lentivirus-CT or Lentivirus-nsp2, differentiated with PMA, pre-treated with 2.5 μM TPCA-1 or DMSO for 1 h, and then stimulated with 1 μg/mL LPS or medium for 12 h. Confocal images showing nsp2, NLRP3, and TGN46 co-localization are presented (A, left), with gray values measured by ImageJ (A, middle) and co-localization percentages shown (A, right). Cell lysates were analyzed by SDD-AGE and SDS-PAGE (B). Lenti, Lentivirus; CT, Control. For confocal microscopy assay, results were manually quantified from at least 30 randomly selected cells per condition per replicate using Image J. Scale bar: 10 μm. The protein levels were quantified by Image J and normalized to β-actin. The data are representative of results from three independent experiments. Error bars indicate the mean (± SD) of three repeats. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001; p > 0.05 stands for ns.
Fig 8.
PRRSV-2 infection triggers inflammatory responses via IKKβ-dependent NLRP3 translocation to dTGN.
(A) PAMs infected with PRRSV-2 (MOI of 0.05) for 18 h were immunoprecipitated with an anti-NLRP3 antibody. (B) PAMs transfected with control siRNA or siRNA targeting IKKβ (si-IKKβ-2) were infected with PRRSV-2 (MOI of 0.05) for 18 hours. IL-1β levels in supernatants were measured by ELISA, and mature IL-1β and GSDMD were detected by Western blot. (C and D) PAMs pre-treated with 2.5 μM TPCA-1 or DMSO for 1 h were infected with PRRSV-2 (MOI of 0.05). After 6 hpi, the medium was replaced with TPCA-1-free medium for another 6 h. Representative immunofluorescence images showing nsp2, NLRP3, and TGN46 co-localization were presented on (C, left). The gray value of each channel was measured by ImageJ software (C, right). The percentages of cells with NLRP3 and TGN46 co-localization were displayed on (C, bottom). Cell lysates were analyzed by SDD-AGE and SDS-PAGE (D). WCL, whole cell lysate; Lys, lysate; Sup, supernatant. For confocal microscopy assay, results were manually quantified from at least 30 randomly selected cells per condition per replicate using Image J. Scale bar: 10 μm. The protein levels were quantified by Image J and normalized to β-actin. The data are representative of results from three independent experiments. Error bars indicate the mean (± SD) of three repeats. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001; p > 0.05 stands for ns.
Fig 9.
IKKβ-dependent translocation of NLRP3 to the dTGN is essential for virus-induced inflammation.
(A and B) THP-1 cells were differentiated into macrophages using 100 ng/mL PMA. The cells were then transfected with either control siRNA (si-NC) or IKKβ-targeting siRNA (si-IKKβ-2) at a concentration of 100 nM for 24 hours, followed by infection with PRV (MOI of 5) (A) or EMCV (MOI of 10) for 12 hours (B). Western blot analysis was performed to detect mature IL-1β and GSDMD. (C and D) THP-1 cells differentiated into macrophages with 100 ng/mL PMA were pre-treated with either 2.5 μM TPCA-1 or DMSO for 1 hour before being infected with PRV (MOI of 5) (C) or EMCV (MOI of 10) for 12 hours (D). Representative immunofluorescence images captured via confocal microscopy showing co-localization of NLRP3 and TGN46 are displayed on the left. Gray values for each channel were quantified using ImageJ software (middle), and the percentage of cells with NLRP3 and TGN46 co-localization is shown on the right. Lys, lysate; Sup, supernatant. For confocal microscopy assay, results were manually quantified from at least 30 randomly selected cells per condition per replicate using Image J. Scale bar: 10 μm. The protein levels were quantified by Image J and normalized to β-actin. The data are representative of results from three independent experiments. Error bars indicate the mean (± SD) of three repeats. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001; p > 0.05 stands for ns.
Fig 10.
Schematic model of PRRSV-2 nsp2 protein activates NLRP3 inflammasome through IKKβ-dependent trans-Golgi network translocation.
PRRSV-2 infection triggers inflammatory responses through the NLRP3 inflammasome. PRRSV-2 nsp2 interacts with NLRP3, facilitating the recruitment of IKKβ to NLRP3. This interaction ultimately promotes NLRP3 translocation to the dTGN and enhances its oligomerization in an IKKβ-dependent manner. Oligomerized NLRP3 further recruits ASC and pro-Caspase-1, forming the inflammasome complex and activating Caspase-1. Activated Caspase-1 then cleaves pro-IL-1β and GSDMD, triggering pyroptosis and the release of IL-1β, which ultimately results in pulmonary inflammatory injury. This picture was created by Figdraw (www.figdraw.com).