Fig 1.
IBV infection impairs nuclear translocation of transcription factors and suppresses transcription of antiviral genes and pro-inflammatory genes.
(A-D) Vero cells were infected with IBV at an MOI of 1, followed by transfection with poly(I:C) for 6 h, treatment with IFNβ for 40 min, treatment with TNFα for 30 min, or exposure to UV irradiation (1.92 J/cm2) for 20 min. Mock-infected cells served as the control group. Cells were harvested at the indicated time points and subjected to immunofluorescence analysis and Western blot analysis. Representative images from three independent experiments are shown, with scale bars indicating 10 μm. The fluorescence signals of corresponding proteins (IRF3, STAT1, STAT2, p65, and p38) in mock-infected cells and IBV-infected cells from three fields of view were quantified using ImageJ. The intensities of the fluorescence signals in the nucleus (black bars) and the cytoplasm (red bars) are presented in bar graphs. Error bars represent the standard deviation (SD). (E) DF-1 cells were infected with IBV at an MOI of 5 for 2 h, followed by transfection with poly(I:C) or treatment with TNFα for 6 h. Cells were harvested at 8 h.p.i. and subjected to qRT-PCR analysis. For detection of IFITM3 expression, DF-1 cells were infected with IBV at an MOI of 5, followed by treatment with IFNβ for 6 h at 6 h.p.i. Cells were harvested at 12 h.p.i. and subjected to qRT-PCR analysis. Mock-infected cells served as the control group. Error bars represent the SD of technical triplicates. Statistical significance levels are denoted as follows: ns (not significant), P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Fig 2.
IBV infection disrupts the NPC integrity and induces the phosphorylation of NUP62.
(A-B) Vero cells were infected with IBV or mock-infected, harvested at 6 h.p.i., 12 h.p.i., and 18 h.p.i., and subjected to immunofluorescence analysis. Representative images from three independent experiments are shown, with scale bars indicating 10 μm. The fluorescence signals of corresponding proteins (Nups, importins, and Ran) in mock-infected cells and IBV-infected cells from three fields of views were quantified using ImageJ. The intensities of the fluorescence signals in the nucleus (black bars) and the cytoplasm (red bars) are presented as bar graphs. Error bars represent the SD. Statistical significance levels are denoted as follows: ****P < 0.0001. (C) Vero and DF-1 cells were infected with IBV or mock-infected, harvested at the indicated time points, and subjected to western blot analysis. β-actin was detected as a loading control. Protein band signals were quantified using ImageJ, with the intensities of p-NUP62 normalized to total NUP62. The ratio of p-NUP62 in IBV-infected cells to mock-infected cells is presented as p-NUP62 (+:-).
Fig 3.
Screening of IBV proteins that induce mislocalization of FG-Nups and inhibit IFNβ-induced nuclear translocation of STAT1.
(A) Schematic diagram of proteins encoded by IBV. (B) Vero cells were transfected with plasmids encoding Flag-tagged IBV proteins or vector PXJ40. At 24 h post-transfection, cells were subjected to immunofluorescence staining for the detection of FG-Nups using mAb414 (left panel). In a parallel group, transfected cells were treated with IFNβ (1000 IU/mL) for 40 min before immunofluorescence staining for detection of STAT1 (right panel). Representative images from three independent experiments are shown, with scale bars indicating 10 μm. The fluorescence signals of FG-Nups and STAT1 in vector-transfected cells and IBV protein expressing cells from three fields of view were quantified using ImageJ. The intensities of the fluorescence signals in the nucleus (black bars) and the cytoplasm (red bars) are presented as bar graphs. Error bars represent the SD. Statistical significance levels are denoted as follows: ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Fig 4.
IBV N protein induces dislocation of Nups from the nuclear envelope to the cytoplasm, inhibits nuclear translocation of transcription factors, and suppresses the transcription of antiviral genes.
(A-B) Vero cells were transfected with either the vector PXJ40 or a plasmid encoding IBV N protein. At 24 h post-transfection, cells were harvested and subjected to immunofluorescence analysis. (C) Vero cells were transfected with either vector PXJ40 or a plasmid encoding IBV N protein. At 18 h post-transfection, cells were further treated with poly(I:C) for 6 h. In parallel experimental groups, cells were transfected with the vector PXJ40 or a plasmid encoding IBV N protein for 24 h, followed by treatment with IFNβ, TNFα, or UV irradiation, respectively. Cells were then harvested and subjected to immunofluorescence analysis. (A-C) Representative images from three independent experiments are shown, with scale bars indicating 10 μm. The fluorescence signals of corresponding proteins (Nups, importin β1, Ran and transcription factors) in vector-transfected cells and N expressing cells from three fields of view were quantified using ImageJ. The intensities of the fluorescence signals in the nucleus (black bars) and the cytoplasm (red bars) are presented as bar graphs. Error bars represent the SD. The relative fluorescence intensities of the IBV N protein and importin α1 were quantified using ImageJ. The quantitative data on the relative signal intensities and distributions of these two proteins are presented in the graph shown in the bottom panel of (B). Statistical significance levels are denoted as follows: ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (D) DF-1 cells were transfected with the vector PXJ40 or a plasmid encoding IBV N protein. At 24 h post-transfection, cells were transfected with poly(I:C) or treated with IFNβ or TNFα for 12 h, followed by qRT-PCR analysis. Error bars represent the SD of technical triplicates. Statistical significance: ****P < 0.0001.
Fig 5.
Disruption of nucleocytoplasmic trafficking, inhibition of nuclear translocation of transcription factors, and suppression of antiviral genes transcription by diverse coronaviruses N proteins.
(A-C) Vero cells were transfected with plasmids encoding Flag-tagged N proteins from different genera of coronaviruses, while vector PXJ40 served as a control. Cells were harvested 24 h post-transfection and subjected to immunofluorescence analysis. (D-E) Vero cells were transfected with plasmids encoding N proteins or vector PXJ40. At specific time points after transfection, cells were treated with poly(I:C), IFNβ, TNFα, or UV irradiation, respectively, followed by immunofluorescence analysis. (F) Vero cells were transfected with plasmids encoding N proteins or vector PXJ40, together with plasmid encoding tandem GFP-GFP. Cells were harvested 24 h post-transfection and subjected to immunofluorescence analysis. (A-F) Representative images from three independent experiments are shown, with scale bars indicating 10 μm. The fluorescence signals of corresponding proteins in vector-transfected cells and N expressing cells from three fields of view were quantified using ImageJ. The intensities of the fluorescence signals in the nucleus (black bars) and the cytoplasm (red bars) are presented as bar graphs. Error bars represent the SD. The relative fluorescence intensities of the N protein and importin α1 were quantified using ImageJ. The quantitative data on the relative signal intensities and distributions of these two proteins are presented in the graph shown in the bottom panel of (C). Statistical significance levels are denoted as follows: ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (G) HEK-293T cells were transfected with plasmids encoding N proteins or vector PXJ40. At 24 h post-transfection, cells were treated with poly(I:C), IFNβ, or TNFα. Cells were harvested at 12 h post-treatment, and the transcription levels of IFNβ, IFITM3, or IL-8 were detected using qRT-PCR analysis.
Fig 6.
Bioinformatic analysis of IBV N-protein interactome.
Plasmid encoding IBV N protein was transfected into HEK-293T cells for 30 h. Whole-cell lysates were immunoprecipitated using anti-Flag antibody, and three independent immunoprecipitated samples were subjected to LC-MS/MS analysis. PXJ40-transfected cell lysates served as negative control to eliminate nonspecific binding proteins. (A) Heatmap illustrating the enrichment of Gene Ontology (GO) annotations within the IBV N-protein interactome. (B) Heatmap displaying the enrichment of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in the IBV N-protein interactome. (C) IBV N protein interacts with several cellular proteins involved in nucleocytoplasmic transport.
Fig 7.
Diverse coronaviruses N proteins interact with RACK1.
(A) HEK-293T cells were co-transfected with plasmids encoding Flag-IBV N and HA-RACK1. Co-transfections with Flag-IBV-N and PCMV-HA, or PXJ40 and HA-RACK1 were performed as control groups. Cell lysates were immunoprecipitated using anti-Flag or anti-HA antibodies, followed by immunoblot analysis. (B) DF-1 cells were infected with IBV at an MOI of 1. At 12 h.p.i., cell lysates were immunoprecipitated using anti-IBV N antibody, followed by immunoblot analysis. (C) Vero cells were infected with IBV at an MOI of 1 or mock-infected. Immunostaining was performed at 6 h.p.i., 12 h.p.i., and 18 h.p.i. Representative images from three independent experiments are shown. Scale bars: 10 μm. The fluorescence signals of RACK1 in mock-infected cells and IBV-infected cells from three fields of view were quantified using ImageJ. The intensities of the RACK1 fluorescence signals in the nucleus (black bars) and the cytoplasm (red bars) are presented as bar graphs. Error bars represent the SD. Statistical significance levels are denoted as follows: ns, P > 0.05; *P < 0.05. (D) HEK-293T cells were co-transfected with plasmids encoding Flag-tagged N proteins from different coronaviruses and HA-RACK1. Co-transfection of PXJ40 with plasmid encoding HA-RACK1 served as a control. HEK-293T cell lysates were immunoprecipitated using anti-Flag antibody, followed by immunoblot analysis.
Fig 8.
Implication of PKCα/β activity in the cytoplasmic distribution of Nups.
(A-C) Vero cells were transfected with plasmids encoding Flag-tagged N proteins from different coronaviruses or PXJ40. After 24 h post-transfection, cells were treated with Enzastaurin (1 μM) or DMSO for 1 h, followed by immunostaining to assess the subcellular distribution of N, PKCα/β, and FG-Nups/NUP62/NUP153. (D) Plasmid encoding HA-tagged PKCα or PKCβ was co-transfected with PXJ40, or co-transfected with plasmid encoding Flag-tagged IBV N for 24 h, followed by immunostaining to evaluate the subcellular localization distribution of PKCα, PKCβ, N, FG-Nups, NUP62, and NUP153. Representative images from three independent experiments are shown. Scale bars: 10 μm. The fluorescence signals of Nups and PKC in vector-transfected cells and N expressing cells from three fields of view were quantified using ImageJ. The intensities of the fluorescence signals in the nucleus (black bars) and the cytoplasm (red bars) are presented as bar graphs. Error bars represent the SD. Statistical significance levels are denoted as follows: ns, P > 0.05; ****P < 0.0001.
Fig 9.
Role of PKCα/β activity in NUP62 phosphorylation, the cytoplasmic distribution of FG-Nups, suppression of the antiviral response, and virus replication.
(A) Vero or DF-1 cells were infected with IBV at an MOI of 1 or mock-infected. Cells were harvested at 6, 12, and 18 h.p.i. and subjected to Western blot analysis using corresponding antibodies. The intensities of p-PKCα, p-PKCβ, and p-NUP62 bands were normalized to total PKCα/β or NUP62, respectively. The ratio of p-PKCα, p-PKCβ, and p-NUP62 in IBV-infected cells to those in mock-infected cells is denoted as p-PKCα (+:-), p-PKCβ (+:-), and p-NUP62 (+:-). (B) Vero and DF-1 cells were infected with IBV or mock-infected and subjected to immunostaining at indicated time point. (C) Vero cells were infected with IBV or mock-infected. At 2 h.p.i., cells were treated with DMSO or 1μM Enzastaurin for 4 h. The subcellular localization of the indicated proteins was analyzed with immunostaining at 6 h.p.i.. (B-C) Representative images from three independent experiments are shown, with scale bars indicating 10 μm. The fluorescence signals of PKCα/β and Nups in mock-infected and IBV-infected Vero cells were quantified from three fields of view using ImageJ. The intensities of the fluorescence signals in the nucleus (black bars) and the cytoplasm (red bars) are presented as bar graphs, with error bars representing the SD. The fluorescence intensities of IBV N protein and PKCα/β in mock-infected and IBV-infected DF-1 cells were quantified using ImageJ. The relative distributions of these proteins are presented in the graph in the right panel of (B). (D-F) DF-1 cells were infected with IBV or mock-infected, followed by treatment with DMSO or 1 μM Enzastaurin at 2 h.p.i. At 18 h.p.i., Western blot analysis was performed using the indicated antibodies (D). RT-PCR was conducted with primers targeting IBV gene 1 and gene N to measure the levels of IBV genome and transcripts (E). Additionally, the culture medium was collected and subjected to a plaque assay to determine IBV titers (F). (G-I) DF-1 cells were transfected with siRNA targeting PKCα/β or non-target control siRNA (NC) for 48 h, followed by IBV infection at 12 h.p.i. and 18 h.p.i. Western blot analysis was performed to detect the indicated proteins (G). RT-PCR was conducted with primers specific to IBV gene 1 and gene N to measure the levels of IBV genome and transcripts (H). The culture medium was collected and analyzed using a plaque assay to determine IBV titers (I). The ratio of p-PKCα, p-PKCβ, p-NUP62, and N protein levels in Enzastaurin-treated or siPKCα/β-transfected cells compared to those in DMSO-treated or NC-transfected cells is denoted as p-PKCα (+:-), p-PKCβ (+:-), NUP62 (+:-), and N (+:-) (D and G). (J) DF-1 cells were transfected with siRNA targeting PKCα/β or siRNA (NC) for 48 h, followed by IBV infection at an MOI of 5. The infected cells were then treated with poly(I:C) or TNFα at 2 h.p.i. for 6 h, or with IFNβ at 6 h.p.i. for 6 h. qRT-PCR was conducted to measure the transcription levels of IFNβ, IFITM3, and IL-8. For panel E, H, and J, error bars represent the SD of technical triplicates. Statistical significance levels are denoted as follows: ns (not significant), P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Fig 10.
Role of RACK1 in maintaining phosphorylation of PKCα, PKCβ, and NUP62, suppressing antiviral gene expression, and promoting IBV infection.
(A-D) DF-1 cells were transfected with RACK1 siRNA or control siRNA (NC), followed by IBV infection or mock infection at 48 h post-transfection. Cells were harvested at the indicated time points. Western blot analysis was performed to examine the phosphorylation and expression of the indicated proteins (A). qRT-PCR was conducted using primers targeting IBV gene 1 or the N gene to assess the levels of IBV genome and transcripts (B). The culture medium was collected and subjected to a plaque assay to measure the released virus titer (C). qRT-PCR was also used to measure the mRNA levels of IFNβ, IFITM3, and IL-8 (D). (E) DF-1 cells were transfected with RACK1 siRNA or NC for 48 h, followed by IBV infection for 2 h and subsequent transfection with poly(I:C), or treatment with TNFα, or IFNβ, respectively. Cells were harvested and subjected to qRT-PCR analysis to measure the mRNA levels of IFNβ, IFITM3, and IL-8. For panel A, the intensities of RACK1, p-PKCα, p-PKCβ, p-NUP62, and IBV N bands were normalized to β-actin, PKCα/β, NUP62, and β-actin, respectively. The ratio of these protein signals in RACK1 siRNA-transfected cells compared to NC-transfected cells is denoted as RACK1 (+:-), p-PKCα (+:-), p-PKCβ (+:-), p-NUP62 (+:-), and IBV N (+:-). For panels D and E, the value for mock-infected cells is set to 1. For panels B, D, and E, error bars represent the SD of technical triplicates. Statistical significance levels are denoted as follows: ns, P > 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Fig 11.
Interaction between IBV N, p-PKCα, and RACK1, and their role in suppression of innate immune response.
(A) DF-1 cells were transfected with Flag-tagged IBV N or vector PXJ40 for 24 h. Cells were then subjected to Co-IP using anti-Flag antibody, followed by Western blot analysis. (B) Plasmids encoding HA-RACK1 and Flag-tagged IBV N, or HA-RACK1 and PXJ40, were co-transfected into DF-1 cells for 24 h. Cell lysates were subjected to Co-IP using anti-HA antibody, followed by Western blot analysis. (C) DF-1 cells or Vero cells were infected with IBV for 12 h and subjected to Co-IP using anti-IBV N antibody. The interactions of IBV N, p-PKCα, p-PKCβ, and RACK1 were detected by Western blot analysis. (D) HEK-293T cells were transfected with plasmids encoding Flag-tagged N proteins from eleven coronaviruses or PXJ40 for 24 h. Cell lysates were subjected to Co-IP with an anti-Flag antibody, followed by immunoblotting with the indicated antibodies. (E) siRACK1 or siRNA (NC) was transfected into DF-1 cells for 36 h, followed by transfection of plasmid encoding Flag-tagged IBV N or vector PXJ40 for 24 h. Cell lysates were subjected to Co-IP using anti-Flag antibody, followed by Western blot analysis. (F) siRNA targeting RACK1, PKCα/β, or siRNA (NC) was transfected into DF-1 cells for 36 h, followed by transfection of plasmid encoding Flag-tagged IBV N or vector PXJ40. At 24 h post-transfection, cells were stimulated with poly(I:C), IFNβ, or TNFα. Cells were harvested 12 h post-stimulation, and the transcription levels of IFNβ, IFITM3, and IL-8 were measured by qRT-PCR analysis. The group transfected with vector PXJ40 without stimulation was set as 1. Error bars represent the SD of technical triplicates. Statistical significance levels are denoted as follows: **P < 0.01; ***P < 0.001; ****P < 0.0001.
Fig 12.
Significance of cytoplasmic localization of IBV N protein in disrupting the nuclear envelope localization of Nups and suppressing antiviral gene expression.
(A) Schematic representation of truncated mutants of the IBV N protein. (B) Vero cells were transfected with plasmids encoding Flag-tagged IBV N, ΔNTD, ΔSR, ΔCTD, ΔNES, or PXJ40. At 18 h post-transfection, cells were treated with poly (I:C), while treatment with IFNβ or TNFα was initiated at 24 h post-transfection. Subsequently, cells were subjected to immunofluorescence analysis. (C-D) Vero cells were transfected with plasmids encoding Flag-tagged IBV N, ΔNTD, ΔSR, ΔCTD, ΔNES, or PXJ40. At 24 h post-transfection, cells were subjected to immunofluorescence analysis. (B-D) Representative images from three independent experiments are shown, with scale bars indicating 10 μm. The fluorescence signals of corresponding proteins in in vector-transfected and N expressing cells from three fields of view were quantified using ImageJ. The intensities of the fluorescence signals in the nucleus (black bars) and the cytoplasm (red bars) are presented as bar graphs, with error bars representing the SD. (E) DF-1 cells were transfected with plasmids encoding Flag-tagged IBV N or the truncated mutants, or PXJ40. At 24 h post-transfection, cell lysates were subjected to Co-IP with anti-Flag antibody and subsequently immunoblotted with corresponding antibodies. (F) DF-1 cells were transfected with plasmids encoding Flag-tagged IBV N, its truncated mutants, or vector PXJ40. At 24 h post-transfection, cells were treated with poly(I:C), IFNβ, or TNFα for 12 h. Cells were harvested, and the levels of IFNβ, IFITM3, and IL-8 were determined using qRT-PCR analysis. Error bars represent the SD of technical triplicates. Statistical significance is indicated as follows: ns, P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.
Fig 13.
A working model illustrating how coronavirus N protein disrupts the nuclear transport system to inhibit the nuclear translocation of transcription factors and subsequent gene expression.
In the left panel, the canonical nuclear import pathway is described, where the NLS of cargo proteins (e.g., transcription factors) is recognized by nuclear import receptors such as importin α, which forms a complex with importin β. This import complex translocates through the NPC by interacting with FG-Nups. Upon entering the nucleoplasm, importin β binds to Ran-GTP, leading to the disassembly of the import complex and the release of the cargo. Importin β, bound to Ran-GTP, is then transported back to the cytoplasm, while importin α is recycled by the cellular apoptosis susceptibility (CAS) protein (also known as exportin 2). Hydrolysis of GTP by Ran releases importin β for the next round of import. In the right panel, during coronavirus infection, the N protein interacts with RACK1 and recruits p-PKCα to form a ternary N-RACK1-p-PKCα complex. This complex phosphorylates NUP62, causing its cytoplasmic dispersion along with other Nups. As a result, the NPC becomes unable to transport cargo into the nucleus, thereby inhibiting the nuclear translocation of transcription factors such as IRF3, STAT1/STAT2, and p65. This inhibition subsequently blocks the expression of downstream antiviral genes. This figure was created using Biorender (BioRender, Toronto, ON, Canada).