Seneca Valley Virus 3Cpro Cleaves Heterogeneous Nuclear Ribonucleoprotein K to Facilitate Viral Replication

Figures

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  Figure 1. SVV infection cleaves hnRNP K and induces hnRNP K degradation. (A,D) SVV-infected (MOI = 5) or mock-infected BHK-21 or PK-15 cells were extracted at 0, 3, 6, 9, 12, and 24 hpi, and protein samples were subjected to Western blotting with antibodies against hnRNP K, VP1, and β-actin. (B,E) Graphs of quantification analyzed with Image J, the level of hnRNP K band ratios normalized to β-actin. Statistical analysis using GraphPad Prism. Results are displayed as the mean ± standard deviation (SD) of three independent tests (***P < 0.001; NS, not significant). (C,F) SVV-infected (MOI = 5) or mock-infected BHK-21 cells were harvested at 0, 6, and 12 hpi and the mRNA levels of hnRNP K were quantified by qRT-PCR. Statistical analysis using GraphPad Prism. Results are displayed as the mean ± SD of three independent experiments (**P < 0.01; NS, not significant).

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  Figure 2. SVV infection induces relocalization of hnRNP K. (A) BHK-21 cells were infected with SVV (MOI = 5), at 0 and 12 hpi, cells were stained with antibodies against hnRNP K (red), VP1 monoclonal antibody (green), and DAPI (blue), captured under confocal microscopy. (B) SVV-infected (MOI = 5). BHK-21 cells were collected at 0, 3, 6, 9, and 12 hpi, cytoplasmic and nuclear fractions were extracted and subjected to Western blotting with antibodies against hnRNP K, VP1, histone H3, and β-actin. (C) BHK-21 cells were infected with SVV (MOI = 5), at 0 and 12 hpi, cells were stained with antibodies against hnRNP K (green), dsRNA monoclonal antibody (red), and DAPI (blue), captured under confocal microscopy. (D) SVV 5′UTR RNA was pulled down with hnRNP K from SVV-infected (MOI = 5) cell lysate. BHK-21 cells were infected with SVV for 12 h, the harvested cell lysates were immunoprecipitated with rabbit anti-hnRNP K, rabbit anti-HA, and without antibodies, then subjected to RT-PCR. SVV-infected cells and H2O used as a positive control and negative control for RT-PCR using SVV 5′UTR specific primers, respectively.

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  Figure 3. SVV 3Cpro cleaves hnRNP K. (A) BHK-21 cells were cotransfected with HA-hnRNP K and GFP-viral gene plasmids, the cells were collected by Western blotting analysis at 24 h post-transfection (hpt). (B) BHK-21 cells in six-well plates were cotransfected with HA-hnRNP K and GFP empty vector, GFP-3C, GFP-3C-H48A, GFP-3C-C160A, or GFP-3C-DM. The cell lysates were subjected to Western blotting at 24 hpt. (C) BHK-21 cells in six-well plates were transfected with GFP empty vector, GFP-3C, GFP-3C-H48A, GFP-3C-C160A, or GFP-3C-DM. The cell lysates were subjected to Western blotting at 24 hpt. (D) BHK-21 cells were transfected with GFP-3C (green) and GFP vector (green) and stained with antibodies against hnRNP K (red) and DAPI (blue), captured under confocal microscopy. (E) BHK-21 cells were single- or double- transfected with GFP-3C (green), GFP vector (green), HA-hnRNP K, or HA-hnRNP K (Q364A), stained with antibodies against HA (red) and DAPI (blue), captured under confocal microscopy. (F) Domain organization and engineering of truncation constructs of HA-hnRNP K. KH, K homology domain; KI, interactive domain. (G) BHK-21 cells were cotransfected with HA-hnRNP K and HA-hnRNP K (Q364A) with…

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  Figure 4. SVV 3Cpro meditated hnRNP K degradation via its enzyme activity, through the caspase pathway. (A) BHK-21 cells in six-well plates were cotransfected with 1 μg HA-hnRNP K and 0.1, 0.5, and 1 μg GFP-3C or empty vector, respectively. The cell lysates were subjected to Western blotting at 24 hpt. (B) The ratios of HA-hnRNP K normalized against β-actin of three independent experiments of (A) (*P < 0.05; ***P < 0.001). Graphs of quantification analyzed with Image J. Data are represented by mean ± SD. (C) The viability of BHK-21 cells was analyzed using CCK-8 assay after treatment with chemical reagents. Error bars indicate the mean ± SD of three independent experiments (NS, not significant). (D) BHK-21 cells in six-well plates were cotransfected with HA-hnRNP K and GFP empty vector, or GFP-3C. At 10 hpt, cells were treated with DMSO, Z-VAD-FMK (50 μM), MG132 (10 μM), and NH4Cl (10 mM). The cell lysates were subjected to Western blotting at 24 hpt. (E) The ratios of HA-hnRNP K normalized against β-actin of three independent experiments of (D) (*P < 0.05; ***P < 0.001). Graphs of quantification analyzed with Image J. Data are represented by mean ± SD. (F) BHK-21 cells in six-well plates…

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  Figure 5. The expression of hnRNP K is essential for SVV replication. (A) BHK-21 cells in six-well plates were mock-transfected or transfected with 20 pmol of hnRNP K-siRNA and negative control (NC)-siRNA for 48 h. The expression of hnRNP K was assessed by Western blotting. (B) The viability of si-RNA transfected BHK-21 cells was analyzed using CCK-8 assay. Error bars indicate the mean ± SD of three independent experiments (NS, not significant). (C) BHK-21 cells transfected with siRNA for 48 h were infected with SVV (MOI = 1), and the virus productions were assessed by TCID50. Error bars indicate the mean ± SD of three independent experiments (NS, not significant). (D) BHK-21 cells transfected with siRNA for 48 h were infected with SVV (MOI = 5), and the expression of VP1 was assessed by Western blotting. (E) The ratios of VP1 normalized against β-actin of three independent experiments of (C) (*P < 0.05; ***P < 0.001). Graphs of quantification analyzed with Image J. Data are represented by mean ± standard deviation (SD). (F) BHK-21 cells were transfected HA-hnRNP K (1–364), HA-hnRNP K (365–464), HA-hnRNP K, HA-hnRNP K (Q364A), and empty HA vector. At 24 hpt, the cells were infected with…