Grass carp reovirus VP56 and VP35 induce formation of viral inclusion bodies for replication

Figures

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  Figure 1. GCRV-II infection induces VIBs formation (A) GCO cells were infected with GCRV-II, fixed at 6, 18, 24, and 48 hpi, and subjected to immunofluorescence using confocal microscopy with an anti-NS38 polyclonal antibody. (B) GCO cells were transfected with pEGFP-N1 (control) or pEGFP-NS38. At 24 h post transfection, GCO cells transfected with pEGFP-NS38 were infected with or without GCRV-II. The cells were fixed and stained with DAPI. The samples were detected using confocal microscope. White squares in the upper right corner indicate the magnified area shown in the corresponding areas. Scale bar: 10 μm.

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  Figure 2. GCRV-II NS38 interacts with VP1, VP56, and VP35 (A) 293T cells were transfected with pEGFP-NS38, together with pFlag-VP1, pFlag-VP2, pFlag-VP3, pFlag-NS79, pFlag-VP5, pFlag-VP4, pFlag-VP56, pFlag-VP41, pFlag-VP6, pFlag-VP35, or p3XFlag-CMV-14 (control). The cells were collected at 24 h post transfection, followed by Co-IP assay with anti-Flag antibody. Then the immunoprecipitates and cell lysates were analyzed with anti-Flag and anti-EGFP antibodies. (B) 293T cells were transfected with pEGFP-NS38, together with pDsRed-VP1, pDsRed-VP56, or pDsRed-VP35. At 24 h post transfection, the cells were fixed and stained with DAPI. The samples were detected using the confocal microscope. Scale bar: 10 μm.

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  Figure 3. VP56 and VP35 induce VIBs formation (A) GCO cells were transfected with pEGFP-NS38, together with pDsRed-VP1, pDsRed-VP56, or pDsRed-VP35. At 24 h post transfection, the cells were fixed and stained with DAPI. The samples were detected using confocal microscope. (B) GCO cells were transfected with pDsRed-VP1, pDsRed-VP56, or pDsRed-VP35. At 24 h post transfection, the cells were fixed and stained with DAPI. The samples were detected using confocal microscope. Scale bar: 10 μm.

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  Figure 4. GCRV VP56 interacts with VP1, NS79, VP5, NS38, and VP35 (A) 293T cells were transfected with pEGFP-VP56, together with pFlag-VP1, pFlag-VP2, pFlag-VP3, pFlag-NS79, pFlag-VP5, pFlag-VP4, pFlag-VP41, pFlag-VP6, pFlag-NS38, pFlag-VP35, or p3XFlag-CMV-14(control). The cells were collected at 24 h post transfection, followed by Co-IP assay with anti-Flag antibody. Then the immunoprecipitates and cell lysates were analyzed with anti-Flag and anti-EGFP antibodies. (B) 293T cells were transfected with pEGFP-VP56, together with pDsRed-VP1, pDsRed-NS79, pDsRed-VP5, pDsRed-NS38, or pDsRed-VP35. At 24 h post transfection, the cells were fixed and stained with DAPI. The samples were detected using confocal microscope. Scale bar: 10 μm.

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  Figure 5. GCRV VP35 interacts with VP1, VP2, VP3, VP4, VP56, and NS38 (A) 293T cells were transfected with pEGFP-VP35, together with pFlag-VP1, pFlag-VP2, pFlag-VP3, pFlag-NS79, pFlag-VP5, pFlag-VP4, pFlag-VP56, pFlag-VP41, pFlag-VP6, pFlag-NS38, or p3XFlag-CMV-14(control). The cells were collected at 24 h post transfection, followed by Co-IP assay with anti-Flag antibody. Then the immunoprecipitates and cell lysates were analyzed with anti-Flag and anti-EGFP antibodies. (B) 293T cells were transfected with pEGFP-VP35, together with pDsRed-VP1, pDsRed-VP2, pDsRed-VP3, pDsRed-VP4, pDsRed-VP56, or pDsRed-NS38. At 24 h post transfection, the cells were fixed and stained with DAPI. The samples were detected using confocal microscope. Scale bar: 10 μm.

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  Figure 6. VP56 and VP35 recruit viral proteins to the VIBs (A) GCO cells were transfected with pEGFP-VP56, together with pDsRed-VP1, pDsRed-NS79, pDsRed-VP5, pDsRed-NS38, or pDsRed-VP35. At 24 h post transfection, the cells were fixed and stained with DAPI. The samples were detected using confocal microscope. (B) GCO cells were transfected with pEGFP-VP35, together with pDsRed-VP1, pDsRed-VP2, pDsRed-VP3, pDsRed-VP4, pDsRed-VP56, or pDsRed-NS38. At 24 h post transfection, the cells were fixed and counterstained with DAPI. The samples were detected using confocal microscope. Scale bar: 10 μm.

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  Figure 7. N-terminal domain of VP56 is responsible for the VIBs formation and interaction with the recruited viral proteins (A) The schematic shows the plasmids expressing different VP56 truncated mutants. (B) GCO cells were transfected with pEGFP-VP56-N, pEGFP-VP56-M, or pEGFP-VP56-C. At 24 h post transfection, the cells were fixed and stained with DAPI. The samples were detected using confocal microscope. Scale bar: 10 μm. (C) 293T cells were transfected with pEGFP-VP56-N, together with pMyc-VP56-N, pFlag-VP1, pFlag-NS79, pFlag-VP5, pFlag-NS38, or pFlag-VP35. The cells were collected at 24 h post transfection, followed by Co-IP assay with anti-Myc or anti-Flag antibody. Then the immunoprecipitates and cell lysates were analyzed with anti-EGFP, anti-Myc, and anti-Flag antibodies.

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  Figure 8. Middle domain of VP35 is responsible for the VIBs formation and interaction with the recruited viral proteins (A) The schematic shows the plasmids expressing different VP35 truncated mutants. (B) GCO cells were transfected with pEGFP-VP35-N, pEGFP-VP35-M, or pEGFP-VP35-C. At 24 h post transfection, the cells were fixed and stained with DAPI. The samples were detected using confocal microscope. Scale bar: 10 μm. (C) 293T cells were transfected with pEGFP-VP35-M, together with pMyc-VP35-M, pFlag-VP1, pFlag-VP2, pFlag-VP3, pFlag-VP4, pFlag-VP56, or pFlag-NS38. The cells were collected at 24 h post transfection, followed by Co-IP assay with anti-Myc or anti-Flag antibody. Then the immunoprecipitates and cell lysates were analyzed with anti-EGFP, anti-Myc, and anti-Flag antibodies.

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  Figure 9. Newly synthesized viral RNAs are localized to the VIBs in GCRV-II infected GCO cells (A) GCO cells were transfected with BrUTP after treatment with or without ActD for 30 min. At 1 h post transfection, the cells were fixed and stained with an anti-BrU antibody. (B) At 15 h post of GCRV-II infection, the cells were treated with ActD for 30 min and then transfected with or without BrUTP in presence of ActD for 1 h, followed by staining with anti-BrU antibody and polyclonal antibodies against VP56 and VP35. DAPI staining was applied to detect the cell nucleus. Scale bar: 10 μm.