Table 1.
Primers and probes.
Figure 1.
(A) A549 cells were transfected with either pORF3-EGFP (ORF3) or pEGFP-N1 (GFP) for 48 h, stimulated with TNF-α (50 ng/ml) for 6 h, and then subjected to western blotting, using the untreated cells as a control (Contr). (B) Immunofluorescence laser confocal microscopy for the detection of p65. A549 cells were transfected with either pORF3-EGFP (ORF3) or pEGFP-N1 (GFP) for 48 h and then stimulated with TNF-α (50 ng/ml) for 6 h, with another untreated group regarded as the control. The distribution of p65 was examined using p65-specific monoclonal antibody (red). The nucleus was counterstained with DAPI and observed under a confocal laser scanning microscope (magnification: 100×). (C) TNF-α induced NF-κB DNA-binding activity in pORF3-expressing cells. After 6 h of TNF-α treatment, the nuclear extracts were used to determine the NF-κB activity by EMSA. NC, negative control; PC, positive control; ORF3, transfected with pORF3; GFP, transfected with empty pEGFP-N1; UN, untreated cells; CA, competition assay; MCA, mutant competitive assay. (D) Effects of pORF3 on TNF-α-induced NF-κB target genes by real-time RT-PCR. The relative expression of IL-1β, COX-2, and ICAM-1 was calculated following normalization to β-actin, and fold changes relative to the expression levels in untreated cells (control) are presented. (E) The levels of IL-1β, COX-2, and ICAM-1 secreted in the supernatant of the culture medium were detected by ELISA. The protein values used for the calculation were obtained from a corresponding standard curve. The data are presented as mean ± SD from three independent experiments (each performed in triplicate). *P<0.05, ΔP>0.05.
Figure 2.
A549 cells were transfected with 2 µg of either pORF3-EGFP (ORF3) or pEGFP-N1 empty vector (GFP), and untreated cells were used as the control. After 48 h, the cells were exposed to 50 ng/ml TNF-α for 6 h. The total proteins extracted from these cells were subjected to western blotting with anti-IKBα, anti-phosphorylated IKBα (P-IKBα), and anti-IKKβ antibodies. GAPDH was used as a loading control.
Figure 3.
ORF3 abrogated TNF-α-induced NF-κB signal via A20.
(A) A549 cells were transfected with pORF3-EGFP (ORF3) or an infectious cDNA clone of HEV (HEV), after 48 h, these cells were treated with 50 ng/ml TNF-α for 6 h. The relative mRNA expression levels of A20 and RIP1 were detected by real-time RT-PCR. Untreated cells served as a control. Relative values against β-actin were calculated, and fold changes relative to the control are provided. The results are representative of three independent experiments (each performed in triplicate). **P<0.01. (B) Western blot of A20 and RIP1. (C) Scrambled control (SC) or siRNA to A20 (siA20) were transfected into A549 cells, and cells were harvested after 24 h. Untreated cells were used as an empty vector control (UN). Total protein extracted from the harvested cells was used to evaluate A20 expression by western blot, using GAPDH as a loading control. (D) A549 cells were transiently transfected with pNF-κB-Luc, pretreated with or without pORF3, exposed to TNF-α for 6 h, and subjected to luciferase assay. The other two groups were exposed to siRNA against A20 (siA20) or scrambled siRNA (SC) for 24 h, respectively, before transfection with pNF-κB-Luc. The cells that were not transfected with pORF3 served as a control. Luciferase activity was normalized to β-galactosidase, and fold changes against control are provided. The results shown are representative of three independent experiments (each performed in triplicate). **P<0.01, *P<0.05, Δ P>0.05.
Figure 4.
UPR participated in the up-regulation of A20.
(A) Cells were treated with tunicamycin (TU; 2 µg/ml), pORF3, or were untreated (UN) for 48 h, and subjected to western blotting. (B) Spliced ATF6 was detected in A549 cells with or without pORF3 pretreated by western blotting. (C) A549 cells were pretreated with (+) or without (−) 250 µM AEBSF for 1 h and then transfected with pORF3 or not. Protein extracts were processed for western blotting by using A20 antibody. (D) Cells were co-transfected with pNF-κB-Luc and pORF3 for 48 h, and the other group that was transfected only with pNF-κB-Luc served as a control. Both groups were exposed to TNF-α for 6 h and subjected to a luciferase assay. Another group was exposed to AEBSF (250 µM) before co-transfection with pNF-κB-Luc and pORF3. Luciferase activity was normalized to β-galactosidase, and fold changes against the control are presented. The results are representative of three independent experiments (each performed in triplicate). *P<0.05.
Figure 5.
P2 domain played the inhibitory effect.
(A) Schematic illustration of expression vectors used in this study. The pEGFP-N1 vector (Clontech) carries the EGFP gene of a cytomegalovirus (CMV) promoter and multiple cloning sites. The plasmid of pORF3-EGFP containing the 123 amino-acid (aa) ORF3 lacking its stop codon was cloned upstream of the EGFP gene. This plasmid produces a 123-aa ORF3-EGFP fusion protein. Mutant plasmids of D1-EGFP (ΔD1) and D2-EGFP (ΔD2) were constructed by deleting (x) D1 and D2 domain, respectively. Mutant plasmids of P1-EGFP (ΔP1) and P2-EGFP (ΔP2) were constructed by changing proline (P) into alanine (A), respectively. (B) A549 cells were co-transfected with NF-κB reporter plasmid and pORF3 or one of the mutant plasmids for 48 h, exposed to TNF-α (50 ng/ml) for 6 h, and subjected to a luciferase assay. Luciferase activity was normalized to β-galactosidase, and fold change values were compared to the untreated cells (control). The results shown are representative of three independent experiments (each performed in triplicate). *P<0.05, **P<0.01.
Figure 6.
Putative mechanism underlying inhibition of NF-κB by pORF3.
The pORF3 activates transiently NF-κB and up-regulates A20 via the ATF6 pathway in the early phase, and causes degradation or inactivation of RIP1 in the late phase, leading to the blockade of the TNF-α-induced NF-κB signaling.