Fig 1.
IBV prevents SGs formation in the majority of infected cells.
(A-C) Vero, H1299, and DF-1 cells were infected with IBV Beaudette strain at an MOI of 1 or mock infected. At the indicated time points, cells were subjected to immunostaining. Infected cells (red) were identified using a rabbit anti-N protein, SGs (green) with a (cross-reacting) mouse anti-G3BP1 and cell nuclei with DAPI (blue). The bar graphs showed the percentage of SGs positive cells to infected cells, which was counted in 20 random fields, presented as mean ± SD. (D and E) Vero and H1299 cells were infected with IBV as described in A-C. At 20 h.p.i., immunostaining was used to visualize the position of the structural components of SGs using anti-G3BP1 (red) and anti-TIAR (green) antibodies. The enlargement of the insets confirms their co-localization in cytoplasmic granules. The representative images of three independent experiments were shown. Scale bars: 10 μm.
Fig 2.
PKR and eIF2α phosphorylation status during IBV infection.
(A and B) Vero and H1299 cells were infected with IBV at a MOI of 1. Mock infection was included as negative control, poly I:C transfection for 6 h or 1 mM sodium arsenite for 30 min were included as positive control. Cells were harvested at the indicated time points and processed for western blot analysis using 10 μg total protein per lane. p-PKR, PKR, p-eIF2α, eIF2α, G3BP1, TIA-1, and IBV-N were detected with corresponding antibodies. β-actin was probed as a loading control. The representative images of two independent experiments were shown. The signals of protein bands were determined by Image J. The intensities of p-PKR or p-eIF2α were normalized to total PKR or total eIF2α. The ratio of p-PKR and p-eIF2α in IBV-infected cells, poly I:C-transfected cells, or sodium arsenite-treated cells, to mock-infected cells, were shown as p-PKR (+:-) or p-eIF2α (+:-).
Fig 3.
IBV abrogates eIF2α-dependent and -independent formation of SGs.
(A-C) H1299 and DF-1 cells were infected with IBV an MOI of 1. At 20 h.p.i., cells received heat shock treatment (50°C for 20 min), 1 mM sodium arsenite for 30 min, or 200 mM NaCl for 50 min, followed by immunostaining. Infected cells were detected with anti-N antibody (red), SGs with anti-G3BP1 (green) and cell nuclei with DAPI (blue). The representative images of three independent experiments were shown. Scale bars: 10 μm. (D) H1299 cells were mock-infected or infected with IBV and treated with heat shock or sodium arsenite as described in A and B. At 20 h.p.i., cell lysates (10 μg per lane) were subjected to western blotting analysis to detect p-eIF2α, eIF2α, IBV-N, and β-actin. The representative data of three independent experiments were shown. The signals of protein bands were determined by Image J. The intensities of p-eIF2α were normalized to total eIF2α. The ratio of p-eIF2α in IBV-infected cells, heat shock-treated cells, or sodium arsenite-treated cells, to mock-infected cells, were shown as p-eIF2α.
Fig 4.
IBV nsp15 inhibits the formation of SGs.
(A) Schematic diagram of the proteins encoded by IBV. (B) H1299 cells were transfected with plasmids encoding Flag-tagged IBV proteins or with vector PXJ40F. At 24 h post-transfection, cells received heat shock treatment at 50°C for 20 min. IBV proteins were stained with anti-Flag antibody (red) and SGs were detected with anti-G3BP1 (green). Cell nuclei were stained with DAPI (blue). The representative images of three independent experiments were shown. Scale bars: 10 μm.
Fig 5.
IBV nsp15 endoribonuclease activity is required for inhibition of eIF2α-dependent and -independent formation of SGs.
(A-C) H1299 cells were transfected with plasmids encoding IBV nsp15, nsp15-H223A, or nsp15-H238A, respectively. At 24 h post-transfection, cells received heat shock, sodium arsenite, or NaCl treatment. Nsp15, nsp15-H223A, and nsp15-H238A were detected with anti-Flag antibody (red) and G3BP1 was detected with anti-G3BP1 (green). Cell nuclei were stained with DAPI (blue). The representative images of three independent experiments were shown. Scale bars: 10 μm. (D) H1299 cells were transfected with plasmids encoding IBV nsp15 or with vector PXJ40F. At 24 h post-transfection, cells received heat shock or sodium arsenite treatment. Cell lysates (10 μg/lane) were subjected to Western blot analysis, to check the expression of Flag-nsp15 and to determine the levels of phospho-eIF2α, eIF2α, G3BP1, TIA-1, and β-actin. The representative data of two independent experiments were shown. The signals of protein bands were determined by Image J. The intensities of p-eIF2α were normalized to total eIF2α. The ratio of p-eIF2α in nsp15-transfected cells, heat shock-treated cells, soduim arsenite-treated cells, to vector PXJ40F-transfected cell, were shown as p-eIF2α.
Fig 6.
Nsp15s from PEDV, TGEV, SARS-CoV, SARS-CoV-2 harbor the conserved function to inhibit eIF2α-dependent and -independent formation of SGs, and the endoribonuclease activity is required.
LLC-PK1, ST or HeLa cells were transfected with plasmids encoding Flag-tagged nsp15s or their catalytic defective mutants (H-A) from PEDV (LLC-PK1 cells), TGEV (ST cells), SARS-CoV (HeLa cells), SARS-CoV-2 (Hela cells), respectively. At 24 h post-transfection, cells were received sodium arsenite or NaCl treatment. The cells expressing Flag-tagged nsp15s or catalytic defective mutants were detected with anti-Flag antibody (red) and the formation of SGs was detected with anti-G3BP1 (green). Cell nuclei were stained with DAPI (blue). The representative images of three independent experiments were shown. Scale bars: 10 μm.
Fig 7.
Nsp15s from IBV, PEDV, TGEV, SARS-CoV, SARS-CoV-2 harbor the conserved function to retain PABP1 in nucleus and endoribonuclease activity is required.
H1299, LLC-PK1, ST or HeLa cells were transfected with plasmids encoding Flag-tagged nsp15s or their catalytic defective mutants (H-A) from IBV (H1299 cells), PEDV (LLC-PK1 cells), TGEV (ST cells), SARS-CoV (HeLa cells), SARS-CoV-2 (Hela cells), respectively. At 24 h post-transfection, cells were received heat shock or sodium arsenite treatment. The cells expressing Flag-tagged nsp15s or catalytic defective mutants were detected with anti-Flag antibody (red) and PABP1 was detected with anti-PABP1 (green). Cell nuclei were stained with DAPI (blue). The representative images of three independent experiments were shown. Scale bars: 10 μm.
Fig 8.
Nsp15-defective recombinant virus rIBV-nsp15-H238A greatly induces canonical SGs.
(A) Schematic diagram of the nsp15 mutation site (H238-A) as described in the Material and methods, and the replication dynamics of IBV and rIBV-nsp15-H238A in Vero, H1299, and DF-1 cells. Cells were infected with IBV or rIBV-nsp15-H238A at an MOI of 1. Cell culture supernatants were collected at the indicated times at 4 h intervals and viral titers were determined by TCID50 in Vero cells. The dot graphs showed the growth kinetics of IBV and rIBV-nsp15-H238A of three independent determinations, presented as the mean ± SD. (B) Vero, H1299, and DF-1 cells were infected with IBV or rIBV-nsp15-H238A at a MOI of 1 for 20 h, followed by immunostaining. Infected cells were identified with anti-IBN-N (red) and the SGs were detected with anti-G3BP1 (green). Cell nuclei were stained with DAPI (blue). The bar graphs on the right panel showed the percentage of SGs positive cells to total infected cells, which were calculated over 20 random fields, presented as the mean ± SD. P values were calculated by Student’s test. ***, P < 0.001; ****, P < 0.0001. (C) H1299 cells were infected with rIBV-nsp15-H238A for 20 h and treated with 100 μg/ml of cycloheximide (CHX) or an equivalent volume of DMSO for 1 h, followed by immunostaining with anti-G3BP1 or anti-G3BP2 antibodies. The bar graphs showed the percentage of SGs positive cells to total cells, which were calculated over 20 random fields, presented as the mean ± SD. ****, P < 0.0001. The representative images of three independent experiments were shown. Scale bars: 10 μm.
Fig 9.
Nsp15-defective recombinant virus rIBV-nsp15-H238A strongly activates PKR by promoting dsRNA accumulation and eventually stimulates IFN response.
(A) H1299 cells were mock-infected or infected with IBV or rIBV-nsp15-H238A of 1 MOI for 20 h. Cells were lysed for western blotting analysis to detect the level of p-PKR, PKR, p-eIF2α, eIF2α, IBV-S, IBV-M, IBV-N, and β-actin. Figures are representative of two independent experiments. The signals of protein bands were determined by Image J. The intensities of p-PKR or p-eIF2α were normalized to total PKR or eIF2α. The intensities of IBV-S, IBV-M, IBV-N were normalized to β-actin. The ratio of p-PKR and p-eIF2α in IBV infected cells to mock infected cells were shown as p-PKR and p-eIF2α, the ratio of IBV-S, IBV-N, and IBV-N of rIBV-nsp15-H238A to IBV were shown as IBV-S, IBV-M, and IBV-N. (B) H1299 and DF-1 cells were infected with IBV or rIBV-nsp15-H238A for 20 h, respectively. Total RNA was extracted and subjected to quantitative RT-PCR to determine the transcription of IFN-β. The bar graphs showed the relative mRNA level of IFN-β of three independent experiments, presented as mean ± SD. P values were calculated by Student’s test. **, P < 0.01; ***, P < 0.001. (C) H1299 cells were infected with IBV or rIBV-nsp15-H238A for 20 h, followed by immunostaining. DsRNA (red) was detected with J2 antibody and G3BP1 or IBV-N (green) were determined with corresponding antibodies. The representative images of three independent experiments were shown. Bar graph showed the quantification of dsRNA levels by using Image J. ***, P < 0.001. Scale bars: 10 μm. (D) H1299 and DF-1 cells were infected with IBV or rIBV-nsp15-H238A for 20 h, respectively. Total RNA was extracted and 2 μg RNA was spotted onto a Hybond-N+ membrane. After UV-crosslink, the membrane was subjected to dsRNA-specific antibody J2 incubation. The representative blots of two independent experiments were shown. The signals of dots were determined by Image J. The ratio of dsRNA in IBV infected- or rIBV-nsp15-H238A-infected cells to mock-infected cells were shown.
Fig 10.
Depletion of SGs scaffold proteins reduces rIBV-nsp15-H238A-triggered IRF3-IFN-β signaling.
(A) H1299 G3BP 1/2 positive cells or H1299-G3BP1/2-/- cells were treated with sodium arsenite or infected with rIBV-nsp15-H238A for 20 h, followed by immunostaining with anti-G3BP1 (red) and anti-TIAR (green). The images are representative of three independent experiments. Scale bars: 10 μm. (B) H1299 G3BP1/2 positive cells and H1299-G3BP1/2-/- cells were mock-infected or infected with rIBV-nsp15-H238A for 20 h. Cell lysates were analyzed by western blot to detect G3BP1, G3BP2, p-PKR, PKR, p-TBK1, TBK1, p-IRF3, IRF3, p-p65, p65, IBV-S, IBV-M, IBV-N, and β-actin. The figures are representative of two independent experiments. The signals of protein bands were determined by Image J. The intensities of p-PKR, p-TBK1, p-IRF3, p-p65 were normalized to total PKR, TBK1, IRF3, p65, and the intensities of IBV-S, IBV-M, IBV-N were normalized to β-actin. The ratio of p-PKR, p-TBK1, p-IRF3, p-p65 in H1299-G3BP1/2-/- cells or rIBV-nsp15-H238A-infected cells to H1299 G3BP1/2 positive cells was were shown. The ratio of IBV-S, IBV-M, IBV-N in H1299-G3BP1/2-/- cells to those in H1299 G3BP1/2 positive cells were shown. (C) H1299 G3BP1/2 positive cells and H1299-G3BP1/2-/- cells were inoculated with rIBV-nsp15-H238A for 20 h and the induction of IFN-β and IFIT-1 was quantified by quantitative RT-PCR. The levels of IFN-β or IFIT-1 from three independent experiments were shown in the bar graphs, presented as mean ± SD. P values were calculated by Student’s test. ***, P < 0.001; ****, P < 0.0001. (D) The supernatant from rIBV-nsp15-H238A-infected H1299 G3BP1/2 positive cells and H1299-G3BP1/2-/- cells was collected at 20 h.p.i. and virus titers were measured by TCID50 assay. The bar graphs present the virus titers from three independent determinations, shown as mean ± SD. **, P < 0.01.
Fig 11.
Depletion of SGs scaffold proteins reduces poly I:C-induced IRF3-IFN-β signaling.
(A) H1299 G3BP1/2 positive cells and H1299-G3BP1/2-/- cells were transfected with poly I:C (1 μg/ml) for 6 h. The levels of p-PKR, PKR, G3BP1, G3BP2, p-TBK1, TBK1, p-IRF3, IRF3, and actin were determined by Western blot analysis. Images are representative of two independent experiments. The signals of protein bands were determined by Image J. The intensities of p-PKR, p-TBK1, p-IRF3 were normalized to total PKR, TBK1, IRF3. The ratio of p-PKR, p-TBK1, p-IRF3 in poly I:C transfected cells or H1299-G3BP1/2-/- cells to H1299 G3BP1/2 positive cells was were shown. (B) H1299 G3BP1/2 positive cells and H1299-G3BP1/2-/- cells were transfected with poly I:C. The nuclear translocation of IRF3 was examined by immunostaining. Scale bars: 10 μm. Images are representative of three independent experiments. The bar graphs on the right panel indicate the percentage of nuclear IRF3 positive cells to total cells, which were calculated by 20 random fields, presented as mean ± SD. ***, P < 0.001. (C) H1299 G3BP1/2 positive cells and H1299-G3BP1/2-/- cells were transfected with poly I:C. The induction of IFN-β and IFIT-1 was quantified by quantitative RT-PCR. The bar graphs show relative expression levels of IFN-β or IFIT-1 from three independent experiments, presented as mean ± SD. ***, P < 0.001; ****, P < 0.0001.
Fig 12.
PRRs and innate immunity signaling intermediates aggregate to IBV-induced SGs.
(A and B) H1299 cells were mock-infected or infected with IBV followed by immunostaining at 20 h.p.i.. Anti-G3BP1 (red) was used to monitor SGs formation, and PKR, MDA5, TLR3, MAVS, TRAF3, TRAF6, TBK1, IKKε (green) were detected with corresponding antibodies. Cell nuclei were stained with DAPI (blue). The Pearson’s correlation coefficient was measured by using Image J. Scale bars, 10 μm. Images are representative of three independent experiments.
Fig 13.
The working model of inhibition of anti-viral SG formation by coronavirus nsp15.
Coronavirus genome replication and mRNA transcription produce negative strand RNA and dsRNA which contain poly(U) sequence. Nsp15 functions to cleave poly(U) negative strand RNA or dsRNA, reducing their accumulation. This prevents the activation of PKR and the formation of SGs. In parallel, nsp15 also targets to host factors, probably by cleaving host mRNA, or by interfering with host mRNA processing/nuclear export. This, in turns, prevents SGs assembly or promotes their disassembly. Defective nsp15 endoribonuclease activity in coronavirus, results in the accumulation of viral dsRNA, activation of PKR, and subsequent formation of SGs. The aggregation of PRRs and signaling intermediates to SGs facilitates the signaling transduction and IRF3 activation, finally inducing the expression of IFN-β. Production of IFN-β in turn, effectively limits coronavirus replication.