Basal Level p53 Suppresses Antiviral Immunity Against Foot-And-Mouth Disease Virus

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  Figure 1. p53 protein level was dynamically regulated during foot-and-mouth disease virus (FMDV) infection partly through MDM2-dependent proteasome pathway. (A) p53 level was dynamically regulated during FMDV infection. BHK-21 cells, seeded in 60-mm dishes, were either mock-treated or infected with FMDV (multiplicity of infection (MOI) = 0.1) and harvested at the time points indicated. Western blotting (WB) was performed to detect p53 and VP1. (B) MG132 treatment increased the protein abundance of p53. BHK-21 cells were seeded in 6-well plates and were respectively treated with MG132 at a gradient concentration from 100 nM to 50 µM for 2 h. Samples were collected and then WB was performed to detect p53 expression. (C) Treatment with MDM2-specific inhibitor increased the p53 protein level. BHK-21 cells were plated in 60-mm dishes and then treated with MDM2 inhibitor NVP-CGM097 and Nutlin-3a at different concentrations (200 nM to 600 nM) for 12 h, and then the expression level of p53 was measured by WB. (D) Pre-treatment with MDM2 inhibitor compromised the reductive effect of FMDV infection on p53. BHK-21 cells were respectively pre-treated with 600 nM of the MDM2 inhibitor NVP-CGM097 and…

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  Figure 2. p53 knockdown reduced FMDV replication while its overexpression had no effect in BHK-21 cells. (A) Knockdown of p53 by short hairpin RNA (shRNA). BHK-21 cells were respectively transfected with plasmid expressing shRNA targeting p53 or a scramble shRNA as control (shCtrl). Cells were collected at 24 h and 36 h after transfection, and p53 level was detected by WB. (B) and (C) p53 knockdown reduced FMDV replication. BHK-21 cells were seeded in 6-well plates and were transfected with shRNA plasmid (1 and 2) to knockdown p53. At 24 h after transfection, Cells were infected with FMDV (MOI = 0.1) and collected at 8 h post infection (hpi) for (B) or at the indicated time points for (C). WB was used to detect the expression of VP1 and p53 at protein level in (B), while RT-qPCR was used to detect the relative level of VP1 mRNA in (C). (D) Optimization of p53 overexpression. BHK-21 cells were transfected with HA-p53 (H1 and H2), Flag-p53 (F1 and F2), or empty plasmid as control (Ctrl). p53 expression was detected by WB at 36 h and 48 h after transfection. (E) and (F) p53 overexpression had no significant effect on FMDV replication. BHK-21 cells were transfected with p53 overexpression…

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  Figure 3. p53 knockout inhibited FMDV replication in BHK-21 and PK-15 cells. (A–C). p53 knockout inhibited FMDV replication in BHK-21 cells. Wild-type (WT) BHK-21 and the verified p53 knockout (KO) cells were seeded in 60-mm plates. Cells were either mock-treated or infected with FMDV (MOI = 0.1). At the indicated time points, samples were collected for WB (A) and RT-qPCR (B) to detect VP1 at protein and mRNA level respectively. For (C), different cell lines were seeded in T25 cell culture flask and were then infected with 1ml of FMDV (TCID50 = 10−6). After serial passages, virus titer was determined using TCID50 method at the 5th passage. (D–F) p53 knockout inhibited FMDV replication in PK-15 cells. The experiments were performed exactly the same as in (A–C), except the cell lines used were in PK-15 background. (D) is the WB result showing VP1 protein level, (E) is the RT-qPCR result showing VP1 mRNA level, and (F) shows virus titer at the 5th passage. For A and D, the experiments were performed at least twice and similar results were obtained. For B and E, the experiments were performed in triplicates and the bars are SD. β-actin was used as the internal control for both WB and RT-qPCR.…

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  Figure 4. Knockout of p53 enhanced the expression of immune-related genes. (A) Knockout of p53 in PK-15 enhanced the expression of IFNB-1 and virus sensors upon FMDV infection. WT PK-15 and two p53 knockout cell lines were seeded in 60-mm dishes and were then infected with FMDV (MOI = 0.1). At the indicated time points, cells were collected for RT-qPCR to detect the expression level of IFNB-1, MDA-5, RIG-I and TLR3. (B) Expression of IFNB-1 and several cytokines was significantly increased in the primary peritoneal macrophages isolated from p53-KO mice. Primary peritoneal macrophages were respectively isolated from WT and p53-KO mice and cultured in 12-well plates. Cells were then infected with FMDV (MOI = 0.1) and harvested at the indicated time points. RT-qPCR was used to detect the relative expression of IFNB-1 and the other cytokines. The experiments were performed in triplicates and the bars are SD. ** indicates significant differences at p < 0.01 according to Student’s t test. β-actin was used as the internal control.

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  Figure 5. Effects of p53 knockout on FMDV replication and pathogenicity in mice. (A) FMDV-induced death was delayed in p53-KO mice. Four-week-old WT and p53-KO mice were intraperitoneally injected with FMDV (100 LD50), and the survival rate was calculated each day. The experiment was repeated twice and a similar result was obtained. n = 10 for each experiment. (B) FMDV RNA was reduced in the hearts, spleens and muscles of p53-KO mice. The adult mice were challenged with FMDV using the same method as described in (A). Mice were euthanized at 3 days post-infection (dpi) and their hearts, spleens, and thigh muscles were sampled for RNA extraction. RT-qPCR was performed to quantify the relative level of VP1 mRNA. The experiment was performed in triplicates and the bars are SD. (C) FMDV titer was reduced in the hearts, spleens, and muscles of p53-KO mice. Samples were collected the same as in (B), and the virus titer was determined using TCID50 method. n = 3. The lines represent average values. (D) Histopathological changes in the hearts, spleens, and muscles of p53-KO mice were alleviated. Samples were collected the same as in (B) and were immediately fixed with 4% paraformaldehyde solution…