Seneca Valley virus 3C pro degrades heterogeneous nuclear ribonucleoprotein A1 to facilitate viral replication

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  Figure 1.. SVV infection induces hnRNP A1 degradation. (a, d) BHK-21cells and PK-15 cells after SVV infection were collected at 0, 3, 6, 9, and 12 hpi, and 0, 12, and 24 hpi, respectively. The levels of cellular hnRNP A1 were analyzed by Western blotting. (b, e) The expression of cellular hnRNP A1 normalized against β-actin. (c, f) BHK-21 and PK-15 cells were infected with SVV, respectively. Infected cells were harvested, and the transcriptional levels of hnRNP A1 were calculated by qRT-PCR. Expression of hnRNP A1 was normalized to the actin mRNA level. GraphPad Prism was used for statistical analysis. The data are represented as the mean ± SD from three independent experiments (*P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant)

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  Figure 2.. SVV infection induces translocation of hnRNP A1 to the cytoplasm from the nucleus. (a) At 12 h after SVV infection, the cultured BHK-21 cells were stained with hnRNP A1 antibody (red), SVV VP1 antibody (green), and DAPI (blue), then examined by confocal microscopy. (b) Statistical analysis for the percentage redistribution of hnRNP A1 after SVV infection. GraphPad Prism was used for statistical analysis, and the data was expressed as mean ± SD from three independent experiments. (***P < 0.001). (c) At 6 h after SVV infection, the cultured BHK-21 cells were stained with the hnRNP A1 antibody (green), SVV VP1 antibody (green), dsRNA antibody (red), and DAPI (blue), then examined by confocal microscopy. (d) Cell cytoplasmic and nuclear components were extracted from SVV-infected cells. Samples were detected by Western blotting with antibodies against hnRNP A1, VP1, histone H3, and β-actin. (e) The relative gray intensity of cytoplasmic hnRNP A1 was normalized against total hnRNP A1 and quantified by ImageJ; GraphPad Prism was used for statistical analysis, and the data was expressed as mean ±SD from three independent experiments. (***P < 0.001)

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  Figure 3.. SVV 3C was responsible for hnRNP A1 degradation and translocation. (a) Western blot analysis of BHK-21 cells cotransfected with GFP-tagged SVV protein-expressing plasmids and HA-hnRNP A1. (b) Rations to β-actin of three independent experiments (*P < 0.05; ***P < 0.001). Image J was employed to quantify the protein levels. (c) Fluorescence for transfection of plasmids expressing GFP (green) and GFP-3C(green), endogenous hnRNP A1 (red) was stained with the hnRNP A1 antibody, and DAPI (blue) in BHK-21 cells, and then examined by confocal microscopy. (d) Fluorescence to analyze transfection of plasmids expressing GFP (green), GFP-3C (green), and HA hnRNP A1 were stained with the HA-tagged antibody (red), and DAPI (blue) in BHK-21 cells, and then examined by confocal microscope

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  Figure 4.. Enzyme activity of 3C was essential for hnRNP A1 degradation. (a) BHK-21 cells were cotransfected with HA-hnRNP A (1 μg) and an increasing amount of GFP-3C (0.1, 1, 2 μg) or empty vector (1 μg), respectively. The cell lysates were analyzed at 24 h post-transfection using Western blot. (b) The ratios to β-actin of three independent experiments of (A) (*P < 0.05; ***P < 0.001). Image J was employed to quantify the protein levels. (c) BHK-21 cells were transfected for 24 h with GFP-3C-WT, GFP-3C-H48A, GFP-3C-C160A, GFP-3C-DM, or empty vector, respectively. The cell lysates were analyzed at 24 h post-transfection using Western blot. (d) The ratios to β-actin of three independent experiments of (C) (*P < 0.05; ***P < 0.001). Image J was employed to quantify the protein levels. (e) The viability of BHK-21 cells after treatment with chemical reagents was tested by CCK-8 assay (NS, not significant). (f) BHK-21 cells were cotransfected with HA-hnRNP A1 and GFP-3C or empty vector, respectively. Cells were treated with DMSO, Z-VAD-FMK (50 μM), MG132 (10 μM), and NH4Cl (10 mM). The cell lysates were analyzed at 24 h post-transfection using Western blot. (g) The ratios to β-actin of three…

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  Figure 5.. Downregulation of the expression of hnRNP A1 inhibits SVV replication in BHK-21 cells. (a) The silencing efficiency of hnRNP A1 in BHK-21 cells was measured by Western blot at 48 h after siRNA transfection at a concentration of 20 pM, and with no siRNA transfection. (b) The viability of siRNA-transfected BHK-21 cells and normal BHK-21 cells, tested using the CCK-8 assay (NS, not significant). The viability was analyzed using GraphPad Prism, and data were represented as the mean ± SD of three independent experiments (NS, no significant). (c) Western blotting analyzes the levels of VP1 protein and hnRNP A1 in siRNA-transfected BHK-21 cells after SVV infection. (d) The relative gray intensity for VP1 normalized against β-actin was quantified using ImageJ. The results are reported as the mean ± SD of three independent experiments (***P < 0.001; NS, not significant). (e) siRNA transfected and non-siRNA transfected BHK-21 cells were infected with SVV; viral titers in the supernatants at 6 and 12 h post-infection were determined by TCID50 assay. The virus titer was examined using GraphPad Prism, and data were reported as mean ± SD (***P < 0.001; NS, not significant)

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  Figure 6.. Ectopic expression of hnRNP A1 enhanced SVV replication in BHK-21 cells. (a) Fluorescence of rescued lentiviruses in HEK-293 FT transfected cells and lentivirus-transduced BHK-21 cells expressing hnRNP A1-GFP and GFP. (b) Cell viability was examined by CCK-8 assay in lentiviruses-transduced BHK-21 cells (NS, not significant). (c) Viral titers (tested by TCID50 assay) in the supernatants of SVV infected cells after 6 and 12 hpi (*P < 0.05; **P < 0.01; NS, not significant). (d) Western blot analysis of VP1, hnRNP A1-GFP, GFP, and β-actin in lysates collected from SVV-infected cells at 0, 6, and 12 h post-infection, and using anti-VP1 and anti-GFP antibodies. (e) The relative gray intensity for VP1 normalized against β-actin was quantified using ImageJ. The results are reported as the mean ± SD from three independent experiments. (***P < 0.001; NS, not significant)