Swine NONO promotes IRF3-mediated antiviral immune response by Detecting PRRSV N protein

Figures

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  Fig 1. NONO inhibits PRRSV replication. (A) PAM-KNU cells were transfected with 10 pmol of control siRNA (siCtrl) or siRNA targeting NONO (siNONO) for 24 h. The mRNA levels of NONO were detected by real-time PCR (n = 3 independent experiment, **p < 0.01, bar indicates mean). The protein levels of NONO were detected by Western blotting with anti-NONO antibody. The same blot was incubated with β-actin antibody as a protein loading control (n = 3 independent experiment, one representative experiment is shown). (B-D) PAM-KNU cells were transfected with 10 pmol of siNONO or siCtrl for 24 h and then infected with HP-PRRSV SY0608 strain at an MOI of 1 (B). PAM-KNU cells were transfected with 1 μg of pXJ41-sNONO or pXJ41 for 24 h and infected with HP-PRRSV SY0608 strain at an MOI of 1 (C). PAM-KNU and sNONO KO-PAM-KNU cells were infected with HP-PRRSV SY0608 strain at an MOI of 1 (D). Culture supernatants were collected at indicated times and viral RNA was extracted from culture supernatants to analyze by real-time PCR. Virus titers in culture supernatants were measured by microtitration infectivity assay and calculated TCID50 using the Reed-Muench method (n = 3 independent experiment, *p <…

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  Fig 2. NONO positively regulates IFN-β signaling pathway. (A-C) PAM-KNU cells were transfected with 10 pmol of siNONO or siCtrl for 24 h and then treated with 1 μg/mL poly (I:C) for 6 h (A). PAM-KNU cells were transfected with 1 μg of pXJ41-sNONO or pXJ41 for 24 h and treated with 1 μg/mL poly (I:C) for 6 h (B). PAM-KNU and sNONO KO-PAM-KNU cells were treated with 1 μg/mL poly (I:C) for 6 h (C). Cells were harvested and total RNA was extracted using TRIzol reagent. The mRNA levels of IFN-β, ISG15 and Mx1 were analyzed by real-time PCR (n = 3 independent experiment, **p < 0.01, bar indicates mean).

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  Fig 3. NONO interacts with IRF3 and promotes IRF3-mediated IFN-β signaling pathway. (A) PAM-KNU cells were transfected with 6 μg of pXJ41-sNONO or pXJ41 for 24 h. Then NONO complex was pulled down by immunoprecipitation with anti-FLAG antibody. The precipitation was subjected to SDS-PAGE and stained with Coomassie Blue, followed by analysis using protein affinity purification mass spectrometry (AP-MS). (B) Significant IRF3 enriched in NONO immunoprecipitates by quantitative AP-MS (n = 3 independent experiments). (C) The sequence alignment between swine and human NONO. (D) HEK-293T or PAM-KNU cells were cotransfected with 3 μg of pXJ41-sNONO and 3 μg of pXJ41-sIRF3 or pXJ41 for 24 h, followed by co-IP with anti-FLAG antibody and immunoblot analysis with anti-FLAG, anti-MYC or anti-IRF3 antibody. Whole-cell lysis (WCL) was subjected to Western blotting using anti-FLAG, anti-MYC, anti-IRF3, or anti-β-actin antibody (n = 3 independent experiment, one representative experiment is shown). (E) PAM-KNU cells were transfected with 0.5 μg of pXJ41-sNONO and 0.5 μg of pXJ41-sIRF3 or pXJ41 for 24 h. The cells were harvested and total RNA was extracted by TRIzol reagent. The mRNA level of IFN-β was…

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  Fig 4. NONO interacts with PRRSV N protein. (A) Schematic representation of LC-NONO and LN-PRRSV proteins. (B) HEK-293T cells were cotransfected with 0.5 μg of LC-NONO and 0.5 μg of LN-PRRSV constructs for 24 h. Cells were harvested and analyzed for recombination Renilla luciferase reporter activity by luciferase assay system (n = 3 independent experiment, **p < 0.01, bar indicates mean). (C) HEK-293T cells were cotransfected with 3 μg of pXJ41-sNONO and 3 μg of pXJ41-N or pXJ41 for 24 h. Cells were harvested and subjected to co-IP with anti-FLAG or anti-HA antibody. Immunocomplexes were analyzed by Western blotting using anti-HA or anti-FLAG antibody. WCL was also subjected to Western blotting using anti-HA, anti-FLAG, or anti-β-actin antibody (n = 3 independent experiment, one representative experiment is shown). (D) PAM-KNU cells were cotransfected with 0.25 μg of pXJ41-sNONO and 0.25 μg of pXJ41-N or pXJ41 for 18 h. Indirect immunofluorescence (IFA) was performed using anti-HA antibody (green), anti-FLAG antibody (red), or DAPI (blue). Scale bar, 10 μm. (E) PAM-KNU cells were transfected with 6 μg of pXJ41-sNONO or pXJ41 for 18 h and then infected with PRRSV at an MOI of 1 for 12 h.…

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  Fig 5. PRRSV N associates with N-terminal domain of NONO. (A) Schematic representation of swine NONO structure. (B) HEK-293T cells were cotransfected with 3 μg of pXJ41-N and 3 μg of pXJ41-sNONO, pXJ41-sNONO-NTD or pXJ41-sNONO-CTD for 24 h. Cells were harvested and subjected to co-IP with anti-FLAG antibody. Immunocomplexes were analyzed by Western blotting using anti-HA antibody. WCL was also subjected to Western blotting using anti-HA, anti-FLAG, or anti-β-actin antibody (n = 3 independent experiment, one representative experiment is shown).

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  Fig 6. NONO interacts with N-terminal domain of PRRSV N. (A) Schematic representation of PRRSV N structure. (B) HEK-293T cells were cotransfected with 3 μg of pXJ41-sNONO and 3 μg of pXJ41-N, pXJ41-N-NTD or pXJ41-N-CTD for 24 h. Cells were harvested and subjected to co-IP with anti-HA antibody. Immunocomplexes were analyzed by Western blotting using anti-FLAG antibody. WCL was also subjected to Western blotting using anti-HA, anti-FLAG, or anti-β-actin antibody (n = 3 independent experiment, one representative experiment is shown). (C) HEK-293T cells were cotransfected with 3 μg of pXJ41-sNONO and 3 μg of pXJ41-N(NLS) or pXJ41 for 24 h. Cells were harvested and subjected to co-IP with anti-HA antibody. Immunocomplexes were analyzed by Western blotting using anti-FLAG antibody. WCL was also subjected to Western blotting using anti-HA, anti-FLAG, or anti-β-actin antibody (n = 3 independent experiment, one representative experiment is shown). (D) PAM-KNU cells were cotransfected with 0.25 μg of pXJ41-sNONO and 0.25 μg of pXJ41-N(NLS) or pXJ41 for 18 h. Indirect immunofluorescence (IFA) was performed using anti-HA antibody (green), anti-FLAG antibody (red), or DAPI (blue). Scale bar, 10 μm.…

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  Fig 7. PRRSV N positively regulates IFN-β signaling pathway by interaction with NONO. (A) PAM-KNU cells were cotransfected with 1 μg of pXJ41-sNONO and 1 μg of pXJ41-N or pXJ41 for 24 h and treated with 1 μg/mL poly (I:C) for 6 h. Cells were harvested and total RNA was extracted. The mRNA level of IFN-β was analyzed using real-time PCR (n = 3 independent experiment, *p < 0.05, **p < 0.01, bar indicates mean). (B-C) HEK-293T cells were cotransfected with 0.3 μg of pXJ41-sNONO and 0.3 μg of pXJ41-N or pXJ41, along with 0.3 μg of pIFN-β-Luc or pPRDI-III-Luc and 0.03 μg of pRL-TK luciferase reporter for 24 h, and then treated with 1 μg/mL poly (I:C) for 6 h. Cells were harvested and IFN-β promoter (B) or PRD I-III promoter (C) activity was analyzed by a dual-luciferase reporter assay (n = 3 independent experiment, *p < 0.05, **p < 0.01, bar indicates mean).

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  Fig 8. NONO promotes IRF3-induced IFN-β expression through binding to PRRSV N protein. (A) PAM-KNU cells were transfected with 0.5 μg of pXJ41-N or pXJ41 for 24 h, along with 0.5 μg of pXJ41-sRIG-I, pXJ41-sMAVS, pXJ41-sTBK1, or pXJ41-sIRF3. Cells were harvested and total RNA was extracted. The mRNA level of IFN-β was analyzed using real-time PCR (n = 3 independent experiment, *p < 0.05, **p < 0.01, bar indicates mean). Whole-cell lysates were immunoblotted with anti-MYC or anti-HA antibody. The same blot was incubated with β-actin antibody as a protein loading control (n = 3 independent experiment, one representative experiment is shown). (B) PAM-KNU cells were transfected with 0.3 μg of pXJ41-N or pXJ41 together with 0.3 μg of pXJ41-sRIG-I, pXJ41-sMAVS, pXJ41-sTBK1, or pXJ41-sIRF3 with 0.3 μg of pIFN-β-Luc and 0.03 μg of pRL-TK luciferase reporter for 24 h. The activation of IFN-β promoter was detected using a dual-luciferase reporter assay (n = 3 independent experiment, *p < 0.05, **p < 0.01, bar indicates mean). Whole-cell lysates were immunoblotted with anti-MYC or anti-HA antibody. The same blot was incubated with β-actin antibody as a protein loading control (n = 3 independent…

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  Fig 9. NONO enhances phosphorylation of IRF3 and interaction with IRF3 by binding to PRRSV N. (A-B) HEK-293T cells were cotransfected with 1 μg of pXJ41-sNONO and pXJ41-N, pXJ41-N(NLS), or pXJ41 for 24 h, along with 1 μg/mL poly (I:C) treated for 6 h (A) or 1 μg of pXJ41-hTBK1 transfected (B). Cell lysates were analyzed by Western blotting using antibody against p-IRF3, IRF3, HA, FLAG, or β-actin (n = 3 independent experiment, one representative experiment is shown). The band intensities of p-IRF3 (long exposure) are shown as the relative protein expression levels, normalized with β-actin (n = 3 independent experiment, *p < 0.05, **p < 0.01, "ns" stands for not statistically significant, bar indicates mean). (C) HEK-293T cells were cotransfected with 2 μg of pXJ41-sNONO and 2 μg of pXJ41-N, pXJ41-N(NLS), or pXJ41 for 24 h, along with 2 μg of pXJ41-sIRF3. Cells were harvested and subjected to co-IP with anti-FLAG antibody. Immunocomplexes were analyzed by Western blotting using anti-MYC, anti-FLAG or anti-HA antibody. WCL was also subjected to Western blotting using anti-FLAG, anti-HA, anti-MYC, or anti-β-actin antibody (n = 3 independent experiment, one representative experiment is…

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  Fig 10. The working model for NONO positively regulating IRF3-mediated IFN-β signaling pathway by detection of PRRSV N.