Porcine parvovirus nonstructural protein NS1 activates NF-κB and it involves TLR2 signaling pathway

Figures

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  Fig. 1. PPV NS1 stimulates the production of IL-6 and TNF-α in a dose-dependent manner. The 293T cells were transfected with 4 μg of pEGFP-N1-NS1, mock- and 4 μg pEGFP-N1-transfected 293T cells were used as controls. The level of IL-6 (A) and TNF-α (B) at mRNA expression were detected by real-time reverse transcription polymerase chain reaction. The level of IL-6 (C) and TNF-α (D) protein expression were detected by enzyme-linked immunosorbent assay. (E and F) The 293T cells transfected with 1, 2, 4, and 6 μg of pEGFP-N1-NS1 were harvested after 12 h of transfection. All data are expressed as the mean ± SD of three independent experiments. Compared with the control group at the same time, there was a highly significant difference between groups (**p < 0.01), or significant difference between groups (*p < 0.05, Student's t-test). PPV, porcine parvovirus; IL, interleukin; TNF-α, tumor necrosis factor-alpha.

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  Fig. 2. NS1 induces the expression of IL-6 through NF-κB activation. To further evaluate the involvement of NF-κB in mediating the expression of IL-6, the effect of the NF-κB inhibitor, BAY 11-7082, on IL-6 was examined by real-time quantitative reverse transcription polymerase chain reaction (A) and enzyme-linked immunosorbent assay (B). The mock-transfected cells were used as the control. All experiments were performed in triplicate and repeated three times. IL, interleukin; NF-κB, nuclear factor kappa B.

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  Fig. 3. PPV NS1 protein induced NF-κB p65 nuclear translocation. An indirect immunofluorescence analysis was used to measure the cellular localization of NF-κB in PPV NS1-transfected 293T cells. The 293T cells were subjected to immunofluorescence staining for the detection of p65 subcellular localization using rabbit anti-p65 and FITC-conjugated secondary antibody (green). The nuclei were stained with DAPI (blue). PPV, porcine parvovirus; NF-κB, nuclear factor kappa B.

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  Fig. 4. PPV NS1 induced the phosphorylation of p65 after transfection. The activation of NF-κB was also confirmed after 293T cells were transfected with 4 μg pEGFP-N1-NS1 for 1, 3, 6, 12, 24, 36, and 48 h by real-time quantitative reverse transcription polymerase chain reaction (A) and western blotting (B). The expression of NS1 in 293T cells was confirmed at the mRNA level (C). To eliminate the effect of the empty vector on p65, we conducted an empty vector control test at the protein level (D). Compared with the control group at the same time, there was a highly significant difference between groups (**p < 0.01, Student's t-test). PPV, porcine parvovirus; NF-κB, nuclear factor kappa B.

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  Fig. 5. PPV NS1 induced IκBα degradation following transfection. The level of IκBα expression was confirmed following transfection with 4 μg of pEGFP-N1-NS1 at 1, 3, 6, 12, 24, 36, and 48 h by real-time quantitative reverse transcription polymerase chain reaction (A) and western blotting (B). The empty vector control test at the protein level was used to detect the expression of IκBα degradation (C). Compared with the control group at the same time, there was a highly significant difference between groups (**p < 0.01, Student's t-test). PPV, porcine parvovirus.

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  Fig. 6. The detection of NF-κB signaling pathway-related genes during pEGFP-N1-NS1 transfection. The expression of MyD88 (A), IRAK4 (D), TRAF6 (E), and TAK1 (F) mRNA in the 293T cells were tested after pEGFP-N1-NS1 transfection at different time points. The mock- and 4 μg pEGFP-N1-transfected 293T cells were used as the controls. The level of MyD88 protein expression was confirmed after transfection with 4 μg pEGFP-N1-NS1 by western blot (B) and the empty vector control test at the protein level were used to detect the expression of MyD88 (C). Compared with the control group at the same time, there was a highly significant difference between groups (**p < 0.01), or significant difference between groups (*p < 0.05, Student's t-test). NF-κB, nuclear factor kappa B.

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  Fig. 7. PPV NS1 protein induced the expression of TLR2 during infection. The expression of TLR2 mRNA (A) in 293T cells were tested following pEGFP-N1-NS1 transfection at different time points. Mock- and 4 μg pEGFP-N1-transfected 293T cells were used as the controls. The level of TLR2 protein expression was confirmed following transfection with 4 μg pEGFP-N1-NS1 by western blot (B) and an empty vector control test at the protein level was used to detect the level of TLR2 expression (C). The expression of TLR2 (D) and NF-κB (E) were both inhibited following TLR2 interference. Compared with the control group at the same time, there was a highly significant difference between groups (**p < 0.01), or significant difference between groups (*p < 0.05, Student's t-test). PPV, porcine parvovirus; TLR, toll-like receptor; NF-κB, nuclear factor kappa B.