Porcine reproductive and respiratory syndrome virus induces interleukin-15 through the NF-κB signaling pathway

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) mainly infects macrophages/dendritic cells and modulates cytokine expression in these cells. Interleukin-15 (IL-15) is a pleiotropic cytokine involved in wide range of biological activities. It has been shown to be essential for the generation, activation, and proliferation of NK and NKT cells and for the survival and activation of CD8(+) effector and memory T cells. In this study, we discovered that PRRSV infection upregulated IL-15 production at both the mRNA and protein levels in porcine alveolar macrophages (PAMs), blood monocyte-derived macrophages (BMo), and monocyte-derived dendritic cells (DCs). We subsequently demonstrated that the NF-κB signaling pathway was essential for PRRSV infection-induced IL-15 production. First, addition of an NF-κB inhibitor drastically reduced PRRSV infection-induced IL-15 production. We then found that NF-κB was indeed activated upon PRRSV infection, as evidenced by IκB phosphorylation and degradation. Moreover, we revealed an NF-κB binding motif in the cloned porcine IL-15 (pIL-15) promoter, deletion of which abrogated the pIL-15 promoter activity in PRRSV-infected alveolar macrophages. In addition, we demonstrated that PRRSV nucleocapsid (N) protein had the ability to induce IL-15 production in porcine alveolar macrophage cell line CRL2843 by transient transfection, which was mediated by its multiple motifs, and it also activated NF-κB. These data indicated that PRRSV infection-induced IL-15 production was likely through PRRSV N protein-mediated NF-κB activation. Our findings provide new insights into the molecular mechanisms underling the IL-15 production induced by PRRSV infection.

Fig 1

IL-15 is upregulated in PRRSV-infected porcine alveolar macrophages (PAMs), blood monocyte-derived macrophages (BMo), and blood monocyte-derived dendritic cells (DCs). (A and B) PAMs were inoculated with medium alone, PRRSV (CH-1a strain), or heat-inactivated PRRSV at a multiplicity of infection (MOI) of 1.0. (C and D) BMo were inoculated with medium alone or PRRSV at an MOI of 1.0. (E and F) DCs were inoculated with medium alone or PRRSV at an MOI of 1.0. Total RNA was extracted from cell lysates at 6, 12, and 24 h postinoculation. IL-15 mRNA was quantified by real-time PCR (A, C, and E), and results were normalized to GAPDH and expressed as fold induction over medium alone at 6 h postinoculation. Supernatants were harvested at 6, 12, and 24 h postinoculation, and levels of IL-15 (pg/ml) released were determined by ELISA (B, D, F). Mean ± standard error of the mean (SEM) from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.005 (for live virus compared to medium control); #, P < 0.05; ##, P < 0.01 (for live PRRSV compared to UV inactivated). Statistical analysis was performed by Student's t test.

Fig 2

Cloning, sequence analysis, and characterization of porcine IL-15 promoter. (A) Cloning and sequence analysis of the 1,100-bp porcine IL-15 gene 5′-flanking region. The putative transcription factor binding sites are shown in boxes; positions of the putative regulatory motifs relative to the translation initiation site ATG are shown (denoted as +1). (B) Schematic representation of the porcine IL-15 promoter and promoter deletion mutants inserted into pGL3 basic luciferase vectors: −1023/77-luc, −579/77-luc, −372/77-luc, and −172/77-luc porcine IL-15 promoter vectors. The relative lengths and positions of the 5′-ends of these fragments are indicated. (C) The −1023/77-luc, −579/77-luc, −372/77-luc, −172/77-luc porcine IL-15 promoter vectors and pGL3 empty vector were transfected into Marc-145 cells. Twenty-four hours later, cells were inoculated with PRRSV (MOI = 3.0) or medium; cells were harvested to determine luciferase activity 24 h postinfection. Data are mean ± SEM from three independent experiments. *, P < 0.05; **, P < 0.01; as determined by Student's t test.

Fig 3

PRRSV N protein induces IL-15 production. (A) PRRSV structural protein expression vectors, including ORF2, ORF3, ORF4, ORF5, ORF6, ORF7, and pcDNA 3.1 vector control, were cotransfected into CRL2843 with pIL-15 −372/77 promoter pGL3 vector. Forty-eight hours later, cells were harvested for luciferase activity detection. Poly(I·C) treatment was set up as the positive control. (B and C) PRRSV structural protein expression vectors were transfected into CRL2843 cells. Twenty-four hours later, total RNA was extracted from cell lysates. IL-15 mRNA was quantified by real-time PCR (B), and results were normalized to GAPDH and expressed as fold induction over pcDNA vector control. (C) Supernatants were harvested 24 h after transfection, and levels of IL-15 (pg/ml) released were determined by ELISA. Data are mean ± SEM from three independent experiments. *, P < 0.05; **, P < 0.01; as determined by Student's t test.

Fig 4

PRRSV N protein middle region involved in pIL-15 activation. (A) Schematic diagram represents the PRRSV N protein serial deletion mutant constructs. (B and C) The pIL-15 −1023/77 promoter (B) and the pIL-15 −372/77 promoter (C) were cotransfected into CRL2843 cells with PRRSV N protein deletion mutant vectors or pcDNA3.1 empty vector. Forty-eight hours later, cells were harvested for luciferase activity detection. Poly(I·C) treatment for 8 h was set up as positive control; the pGL3 empty vector cotransfected with pcDNA3.1⁺ vector was the negative control. Data are mean ± SEM from three independent experiments. *, P < 0.05; **, P < 0.01; as determined by Student's t test.

Fig 5

Effects of signal transduction inhibitors on IL-15 expression induced by PRRSV infection or PRRSV N protein. (A and B) PAMs were pretreated with inhibitors of PKC (GF-109203X [GF]), NF-κB (BAY11-7082 [BAY]), MEK (PD98059 [PD]), PI3K (LY294002 [LY]), MAPK (SB202190 [SB]), or DMSO control for 1 h, and then cells were inoculated with or without PRRSV (MOI = 1.0). Twenty-four hours later, total RNAs were extracted for detecting IL-15 mRNA by real-time PCR (A), and cell culture supernatants were harvested for testing IL-15 protein by ELISA (B). (C) Marc-145 cells were pretreated with NF-κB inhibitor (BAY11-7082 [BAY]) or DMSO control for 1 h and then were transfected with pIL-15 −372/77-luc pGL3 vector or pGL3-luc basic control vector. Twenty-four hours later, cells were infected with PRRSV and cells were harvested for luciferase activity detection 24 h after PRRSV infection. (D) CRL2843 cells were pretreated with NF-κB inhibitor (BAY11-7082 [BAY]) or medium control for 1 h. Then, pIL-15 −372/77-luc pGL3 vector or pGL3-luc basic control vector was cotransfected with PRRSV N expression vector into CRL2843 cells. Cells were harvested for luciferase activity detection 48 h later. Data are mean ± SEM from three independent experiments. Inhibitors were present at the end of the experiments. *, P < 0.05; as determined by Student's t test.

Fig 6

Activation of NF-κB signaling pathway by PRRSV and PRRSV N protein. (A) PAMs were inoculated with PRRSV (MOI = 1.0), and cells were harvested at 0.5, 3, 6, 12, and 24 h postinfection. (B) CRL2843 cells were transfected with different amounts of N protein expression vector, and cells were harvested 24 h later. Cells were treated with poly(I·C) for 3 h as a positive control. Harvested cells were lysed for Western blotting to examine levels of IκB-α, p-IκB-α, N protein, and β-actin. (C to E) NF-κB p65 and PRRSV N protein localizations were determined by confocal microscopy. CRL2843 cells were transfected with pcDNA3.1 control vector (C) or transfected with PRRSV N protein expression vector (D and E) for 24 h. NF-κB p65 (green) and PRRSV N protein (red) were then detected by indirect immunofluorescence staining by antibodies against p65 and PRRSV N protein. Nucleus (blue) was stained with DAPI.

Fig 7

NF-κB binding motif in the pIL-15 −372/77 promoter region is essential for IL-15 production. (A) Schematic diagram represents pIL-15 promoter deletion mutant constructs. (B) Marc-145 cells were transfected with pIL-15 −372/77 promoter, −372/77(ΔNF-κB)-luc, −372/77(ΔIRF-E)-luc, −372/77(ΔNF-κB/ΔIRF-E)-luc, or pGL3 empty vector. Twenty-four hours later, cells were inoculated with PRRSV (MOI = 3.0) or medium control and then harvested for luciferase activity detection 24 h postinfection. (C) pIL-15 −372/77 promoter, −372/77(ΔNF-κB)-luc, −372/77(ΔIRF-E)-luc, −372/77(ΔNF-κB/ΔIRF-E)-luc, or pGL3 basic control vector was cotransfected into CRL2843 cells with PRRSV N protein or pcDNA3.1 control. Cells were harvested for luciferase activity detection 48 h later. (D) pIL-15 −1023/77 promoter, −1023/77(ΔNF-κB)-luc, or pGL3 basic control vector was cotransfected into CRL2843 cells with PRRSV N protein or pcDNA3.1 control. Cells were harvested for luciferase activity detection 48 h later. Data are mean ± SEM from three independent experiments. *, P < 0.05; as determined by Student's t test.