The role of IBV PL1pro in virus replication and suppression of host innate immune responses

Figures

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  Fig. 1. Multiple sequence alignments of PL1pro from different IBV isolates

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  Fig. 2. Recovery of rIBV, rIBV-ΔPL1pro, and rIBV-ΔPL1pro-N from cells electroporated with in vitro-synthesized full-length transcripts. (a) A schematic of the genome in nsp3 of rIBV, rIBV-ΔPL1pro, and rIBV-ΔPL1pro-N. (b) In vitro transcription of the full-length transcripts of rIBV, rIBV-ΔPL1pro, and rIBV-ΔPL1pro-N. Lanes 2–6,8–12,14–18: The five cDNA fragments covering IBV sequences from plasmids pKTO-IBV-A (pKTO-IBV-ΔPL1pro, pKTO-IBV-ΔPL1pro-N), pGEM-IBV-B, pXL-IBV-C, pGEM-IBV-D, and pGEM-IBV-E, respectively; Lanes 20–22: In vitro assembly of the five fragments into a full-length cDNA of rIBV, rIBV-ΔPL1pro, and rIBV-ΔPL1pro-N; Lanes 24–26: Generation of the full-length in vitro transcripts of rIBV, rIBV-ΔPL1pro, and rIBV-ΔPL1pro-N; Lanes 1, 7, 13, 19 and 23: DNA markers. The full-length gels are in Additional file 1: Supplemental Fig. 2. (c) CPE in Vero cells electroporated with in vitro-synthesized transcripts derived from the assembled full-length clones of rIBV, rIBV-ΔPL1pro, and rIBV-ΔPL1pro-N. Scale bars: 50 μm

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  Fig. 3. Analysis of the growth properties of rIBV, rIBV-ΔPL1pro, and rIBV-ΔPL1pro-N. (a) Plaque formation in rIBV, rIBV-ΔPL1pro, and rIBV-ΔPL1pro-N infected Vero cells. Three viral stock supernatants were serially diluted and inoculated onto the Vero cells for plaque assays, the dilution of the viral stock is indicated. The original images of virus plaque formation are in Additional file 1: Supplemental Fig. 3a. (b) CPE formation in rIBV, rIBV-ΔPL1pro, and rIBV-ΔPL1pro-N infected H1299 cells. Scale bars: 50 μm. (c) Growth kinetics of the recombinant viruses in Vero cells and H1299 cells. The raw data is presented in Additional file 2. (d) Western blot analysis IBV N protein expression. The expression of IBV N protein in H1299 cells infected with rIBV and rIBV-ΔPL1pro-N at a MOI of 0.5 was assessed by Western blot at the indicated times post-infection, respectively, and β-actin was used as an internal control from the same experiment. The full-length blots are in Additional file 1: Supplemental Fig. 3b. Numbers below the image was IBV N/β-actin ratios of band optical density values from Image J software

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  Fig. 4. The N-terminal region of PL1pro confers resistance to treatment of IBV with type I IFN and regulates the production of IFN-β. (a) Indirect immunofluorescence assay. H1299 cells were prestimulated with the indicated concentrations of IFN-α (50, 100, 150 ng/ml) for 2 h and subsequently infected with rIBV and rIBV-ΔPL1pro-N at an MOI of 0.5. At 18 h post-infection, cells were fixed and stained for IBV-N(green). Scale bars: 50 μm. (b) Western blot analysis IBV N protein expression. H1299 cells were prestimulated with IFN-α for 2 h and subsequently infected with rIBV and rIBV-ΔPL1pro-N at an MOI of 0.5. At 18 h post-infection, the expression of IBV N protein was assessed by Western blot. The full-length blots are in Additional file 1: Supplemental Fig. 4a. (c) Absence of the N-terminal region of PL1pro upregulates the expression of IFN-β. H1299 cells were infected with rIBV and rIBV-ΔPL1pro-N at MOI of 0.5 and 1, respectively, Western blot was used to detect the expression of IFN-β at the indicated times post-infection, respectively. The full-length blots are in Additional file 1: Supplemental Fig. 4b

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  Fig. 5. Absence of the N-terminal region of PL1pro promotes phosphorylation of STAT1 and nuclear expression of p-STAT1. (a) The absence of the N-terminal region of PL1pro upregulates the expression of p-STAT1. H1299 cells were infected with rIBV and rIBV-ΔPL1pro-N at MOI of 0.5 and 1, respectively, then stimulated with IFN-α (100ng/ml) at 6 h post-infection. Western blot was used to detect the expression of STAT1 and p-STAT1 at the indicated times post-infection, respectively. The full-length blots are in Additional file 1: Supplemental Fig. 5. (b) Immunostaining against p-STAT1 in nuclei of H1299 cells. H1299 cells were infected with rIBV and rIBV-ΔPL1pro-N at a MOI of 0.5. At the indicated times post-infection, cells were fixed and stained for p-STAT1 (green). The histogram shows the nuclear expression ratio of p-STAT1. Data were presented as mean ± SD (N = 3). *P < 0.05. Scale bars: 50 μm

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  Fig. 6. Absence of the N-terminal region of PL1pro up-regulates the expressions of IFIT family members during later stages of infection. (a) mRNA expressions of ISGs in rIBV and rIBV-ΔPL1pro-N infected cells. H1299 cells were stimulated with IFN-α (100ng/ml) at 6 h post-infection, and qRT-PCR was performed to analyze the mRNA expressions of IFIT1 and IFIT2. Data were presented as mean ± SD (N = 3). *P < 0.05. The raw data is presented in Additional file 2. (b) Protein expressions of ISGs in rIBV and rIBV-ΔPL1pro-N infected cells. H1299 cells were stimulated with IFN-α (100ng/ml) at 6 h post-infection, Western blot was performed to analyze the protein expressions of IFIT1 and IFIT2. The full-length blots are in Additional file 1: Supplemental Fig. 6

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  Fig. 7. Overexpression of IBV PL1pro-N downregulates the expressions of IFN-β, p-STAT1, IFIT1, and IFIT2 in cells. H1299 cells were overexpressed with IBV PL1pro-N, then infected with rIBV. Western blot was performed to analyze IFN-β, p-STAT1, IFIT1, and IFIT2 protein expressions. The full-length blots are in Additional file 1: Supplemental Fig. 7

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  Fig. 8. The working model of IBV PL1proN on virus replication and innate immune response