Classical Swine Fever Virus NproAntagonizes IRF3 To Prevent Interferon-Independent TLR3- and RIG-I-Mediated Apoptosis

Figures

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  FIG 1. Npro antagonizes poly(I·C)- and Sendai virus-mediated apoptosis in PK-15 cells. (A) Twelve-well plates were seeded with PK-15 cells or PK-15 cells stably expressing His-Npro or (B) PK-15 cells stably expressing EGFP (*) or EGFP-Npro (**) and treated with poly(I·C) or SeV. Eighteen hours posttreatment, whole-cell lysates were prepared and analyzed by Western blotting using polyclonal Abs recognizing ISG15, Npro, or GFP and MAbs recognizing cleaved caspase-3, Mx1, or RIG-I as indicated. (C) PK-15 cells, PK-15 cells stably expressing His-Npro, or PK-15 cells infected (MOI of 0.5) with CSFV Alfort for 24 h were treated with STS. Eight hours posttreatment, whole-cell lysates were prepared and analyzed by Western blotting using a polyclonal Ab recognizing Npro and a MAb recognizing cleaved caspase-3 as indicated. (A to C) A MAb recognizing γ-tubulin was used to determine relative protein concentrations. Experiments were repeated on at least two separate occasions.

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  FIG 2. Type I IFN amplifies poly(I·C)-mediated apoptosis but is not essential for its induction. (A) Schematic representation of RXT inhibition of JAK/STAT-mediated IFN response. (B) Twelve-well plates were seeded with PK-15 cells and treated with poly(I·C) or SeV in the presence or absence of JAK-STAT inhibitor RXT. Eighteen hours posttreatment, whole-cell lysates were prepared and analyzed by Western blotting using a polyclonal Ab recognizing ISG15 or MAbs recognizing cleaved caspase-3, Mx1, RIG-I, or GFP as indicated. A MAb recognizing γ-tubulin was used to determine relative protein concentrations. (C) Twelve-well plates were seeded with PK-15 and SK6 cells and treated with increasing concentrations of porcine IFN-α in the presence or absence of poly(I·C). Eighteen hours posttreatment, whole-cell lysates were prepared and analyzed by Western blotting as in panel A. Experiments were repeated on at least two separate occasions.

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  FIG 3. Poly(I·C) and SeV induce apoptosis through TLR3/IRF3 and RIG-I/IRF3 signaling pathways, respectively. (A to F) Twelve-well plates were seeded with WT PK-15 and knockout PK-15 cell lines (TLR3−/−, RIG-I−/−, and IRF3−/−) and treated with poly(I·C) (A, C, and E) or SeV (B, D, and F) in the presence or absence of RXT. Eighteen hours posttreatment, whole-cell lysates were harvested and analyzed by Western blotting using a polyclonal Ab recognizing ISG15 and MAbs recognizing cleaved caspase-3, Mx1, or RIG-I, as indicated. A MAb recognizing γ-tubulin was used to determine relative protein concentrations. Experiments were repeated on at least two separate occasions.

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  FIG 4. Bax directly mediates apoptosis in a manner that relies upon transcription-independent functions of IRF3. (A) Yeast strains cotransformed with plasmids expressing the indicated proteins fused to either the GAL4 DNA-binding domain (in pGBKT7) or activation domain (in pGADT7) were cultured on dropout media to identify interactions. Cotransfection of plasmids encoding Npro, BaxΔC, or IRF3 with the reciprocal plasmid vector (pGBKT7 or pGADT7) served as negative interaction controls. Cotransfection of the large T antigen (T) and p53 or T and lamin served as positive and negative system controls. (B and C) Twelve-well plates were seeded with WT and Bax−/− PK-15 cell lines and treated with poly(I·C) or SeV. Eighteen hours posttreatment, whole-cell lysates were (B) imaged and then (C) harvested and analyzed by Western blotting using polyclonal Abs recognizing ISG15 or Bax and MAbs recognizingcleaved caspase-3 or Mx1. (D) Alignments of porcine, human, and murine IRF3 protein sequences implicated in transcriptional activity (turquoise) were performed in MEGA7. Mutations (pink) were designed in porcine IRF3 (poIRF3) to generate N-terminal FLAG-tagged WT and S1 mutant (S394A S396A) poIRF3…

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  FIG 5. Npro blocks poly(I·C)- and Sendai virus-mediated mitochondrial localization of proapoptotic Bax. (A) WT, IRF3−/−, and His-Npro-expressing PK-15 cells were treated with poly(I·C) or SeV in the presence of 100 µM caspase inhibitor Z-VAD(OMe)-FMK (Bachem). Eighteen hours posttreatment, cells were treated with MitoTracker and analyzed by immunofluorescence using a polyclonal Ab recognizing Bax. Nuclei are stained blue with DAPI. Scale bars represent 45 μm. (B) Immunofluorescence images of single cells were collected from the experiment detailed in panel A. Scale bars represent 20 μm. (C and D) Whole-cell lysates prepared from replicate samples of panel A were analyzed by Western blotting using polyclonal Abs recognizing Bax or Npro and a MAb recognizing cleaved caspase-3. A MAb recognizing γ-tubulin was used to determine relative protein concentrations. Experiments were repeated on at least two separate occasions.

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  FIG 6. Antagonism of SeV-mediated apoptosis in CSFV-infected cells is dependent on the expression of Npro. (A) WT PK-15 and SK6 cells were infected (MOI of 0.2) with CSFV Alfort, CSFV Brescia, rCSFV Alfort, or ΔNpro rCSFV Alfort as indicated for 72 h and then treated with SeV. Eighteen hours posttreatment, cells were treated with MitoTracker and analyzed by immunofluorescence staining using a polyclonal Ab recognizing Bax and a MAb recognizing CSFV E2. Nuclei are stained blue with DAPI. (B and C) The percentage of cells displaying Bax localization to the mitochondria following each treatment was then quantified. ***, P < 0.001. Experiments were repeated on at least two separate occasions.

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  FIG 7. Model of TLR3- and RIG-I-mediated apoptosis and its antagonism by CSFV Npro. Upon stimulation with poly(I·C) and SeV, TLR3 and RIG-I initiate apoptosis in an IRF3-dependent manner, independent of its functions as a transcription factor and characterized by mitochondrial relocalization of Bax and activation of caspase-3. IRF3 also triggers induction of IFN-β and IFN-dependent and -independent upregulation of ISGs which might amplify the TLR3/IRF3 signaling axis. CSFV Npro (purple), apoptotic signaling (red), IFN signaling (blue), and uncertain or inferred (?) pathways are indicated.