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. 2015 Jan 30;290(5):3172-82.
doi: 10.1074/jbc.M114.619890. Epub 2014 Dec 10.

"V体育安卓版" p53 degradation by a coronavirus papain-like protease suppresses type I interferon signaling

Lin Yuan  1 , Zhongbin Chen  2 , Shanshan Song  3 , Shan Wang  3 , Chunyan Tian  3 , Guichun Xing  3 , Xiaojuan Chen  2 , Zhi-Xiong Xiao  4 , Fuchu He  3 , Lingqiang Zhang  5
Affiliations

p53 degradation by a coronavirus papain-like protease suppresses type I interferon signaling

Lin Yuan et al. J Biol Chem. .

Abstract

Infection by human coronaviruses is usually characterized by rampant viral replication and severe immunopathology in host cells. Recently, the coronavirus papain-like proteases (PLPs) have been identified as suppressors of the innate immune response. However, the molecular mechanism of this inhibition remains unclear VSports手机版. Here, we provide evidence that PLP2, a catalytic domain of the nonstructural protein 3 of human coronavirus NL63 (HCoV-NL63), deubiquitinates and stabilizes the cellular oncoprotein MDM2 and induces the proteasomal degradation of p53. Meanwhile, we identify IRF7 (interferon regulatory factor 7) as a bona fide target gene of p53 to mediate the p53-directed production of type I interferon and the innate immune response. By promoting p53 degradation, PLP2 inhibits the p53-mediated antiviral response and apoptosis to ensure viral growth in infected cells. Thus, our study reveals that coronavirus engages PLPs to escape from the innate antiviral response of the host by inhibiting p53-IRF7-IFNβ signaling. .

Keywords: Coronavirus; Innate Immunity; Interferon; Mouse Double Minute 2 Homolog (MDM2); PLP2; Virus; p53 V体育安卓版. .

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Figures

FIGURE 1.
FIGURE 1.
PLP2 deubiquitinates and stabilizes MDM2. A, PLP2 interacts with MDM2 in vivo. Coimmunoprecipitation (IP) of PLP2 and exogenous MDM2 from p53+/+ HCT116 cells and whole cell lysates (Ly) were tested by HA and V5 antibodies. B, p53+/+ HCT116 cells transfected with the indicated plasmids were subject to immunoprecipitation with anti-V5 antibodies. The lysates and immunoprecipitates were analyzed. C, p53+/+ HCT116 cells transfected with the indicated plasmids were subjected to immunoprecipitation with anti-Myc antibodies. The lysates and immunoprecipitates were analyzed. D, MDM2 ubiquitination (Ub) is inhibited by coexpression of PLP2 in vivo. p53+/+ HCT116 cells were transfected with HA-ubiquitin, MDM2 and V5-PLP2, and ubiquitinated MDM2 was precipitated, followed by immunoblotting with anti-HA. E, p53+/+ HCT116 cells transfected with the indicated constructs were treated with MG132 for 8 h before harvest. Smurf1 was immunoprecipitated with anti-Flag and immunoblotted with anti-HA. F and G, PLP2 expression increases the steady-state level of endogenous (F) and exogenous (G) MDM2. HCT116 cells were transfected with increasing amounts of V5-PLP2. After 48 h, total lysates were immunoblotted to detect the expression of MDM2. H, half-life analysis of MDM2 in the presence of overexpressed PLP2. CHX, cycloheximide. I, subcellular localization of MDM2. MCF7 cells were fixed and stained with anti-MDM2 antibodies before visualization. J, regulation of the subcellular localization of MDM2 by PLP2. MCF7 cells transfected with the V5-PLP2 constructs were fixed and stained with anti-V5 and anti-MDM2 antibodies before visualization. K, deubiquitinase activity was required for PLP2 to increase MDM2 stability. Protein expression analysis was performed by Western blotting with the indicated antibodies for cells transfected with WT PLP2 (lane 2), the C1678A mutant (lane 3), the H1836A mutant (lane 4), and the D1849A mutant (lane 5).
FIGURE 2.
FIGURE 2.
PLP2 increases p53 degradation through the ubiquitin-proteasome pathway. A, PLP2 expression decreases the steady-state level of p53. p53+/+ HCT116 cells were transfected with increasing amounts of V5-PLP2. After 48 h, total lysates were immunoblotted to detect the expression of p53. B, PLP2-triggered p53 down-regulation is blocked by treatment with the proteasome inhibitor MG132 (20 μm, 8 h). C, half-life analysis of p53 in the presence of overexpressed PLP2. Quantitative analysis was performed by measuring the integrated optical density. CHX, cycloheximide. D, p53−/− HCT116 cells were transfected with the indicated constructs. After 48 h, protein expression analysis was performed by Western blotting with the indicated antibodies, as shown. E, subcellular localization of p53. MCF7 cells were fixed and stained as indicated. F, MCF7 cells transfected with the V5-PLP2 constructs were treated with MG132 for 4 h to avoid the p53 degradation. Forty-eight hours later, the cells were fixed and stained with the indicated antibodies before visualization. G and H, PLP2 inhibits p53 activity. p53 activity in HCT116 cells was measured using a pG13L luciferase reporter gene assay, and the expression of PLP2 was determined by Western blot analysis. Representative results of three independent reporter assay experiments are shown. The data are shown as the means ± S.D. (n = 3). I, p53−/−MDM2−/− MEF cells transfected with the indicated constructs. The whole cell lysate was subjected to Western blot with indicated antibody. J, deubiquitinase activity was required for PLP2 to increase p53 degradation. Protein expression analysis was performed by Western blotting with the indicated antibodies for cells transfected with WT PLP2 (lane 2), the C1678A mutant (lane 3), the H1836A mutant (lane 4), and the D1849A mutant (lane 5). The differences are statistically significant (**, p value < 0.001). K, MEF cells were treated with poly(I·C) or transfected with V5-nsp3 (SARS-CoV), and the whole cell lysate was subjected to Western blot with indicated antibody. L, Vero cells were infected with PEDV, and the whole cell lysate was subjected to Western blot with indicated antibody. M protein is necessary for the assembly of virus, which is an indicator of viral successful infection.
FIGURE 3.
FIGURE 3.
PLP2 blocks type I interferon signaling by targeting the p53 pathway. A, PLP2 inhibits IFN-β luciferase activity. The transcription activity of IFN-β in HCT116 cells was measured by using an IFN-β luciferase reporter gene assay. Representative results of three independent reporter assay experiments are shown. The data are shown as the means ± S.D. (n = 3). B, PLP2 inhibits the transcription of the IFN-β gene. p53+/+ HCT116 cells and p53−/− HCT116 cells were transfected with V5-PLP2 or treated with poly(I·C), and IFN-β mRNA levels were analyzed by qPCR. The data are shown as the means ± S.D. (n = 3). C, p53-deficient H1299 cells were transfected with the indicated plasmids, and IFN-β mRNA levels were analyzed by qPCR. The data are shown as the means ± S.D. (n = 3). D, deubiquitinase activity is critical for PLP2 to block type I interferon signaling. IFN-β mRNA levels were detected by qPCR from cells transfected with WT PLP2 (lane 3), the C1678A mutant (lane 4), the H1836A mutant (lane 5), or the H1849A mutant (lane 6). E, PLP2 inhibits the transcription of the IRF9 gene in a p53-dependent manner. IRF9 mRNA levels in p53+/+ and p53−/− HCT116 cells transfected with or without V5-PLP2 were determined by qPCR. The data are shown as the means ± S.D. (n = 3). F, PLP2 inhibits the transcription of the IRF7 gene in a p53-dependent manner. IRF7 mRNA levels in p53+/+ and p53−/− HCT116 cells transfected with or without V5-PLP2 were determined by qPCR. The data are shown as the means ± S.D. (n = 3). G, PLP2 inhibits the transcription of the IFN-β gene promoted by LPS. p53+/+ HCT116 cells were transfected with V5-PLP2 or treated with LPS, and IFN-β mRNA levels were analyzed by qPCR. The data are shown as the means ± S.D. (n = 3). H, LPS does not affect the transcription of the P53 gene. p53+/+ HCT116 cells were transfected with V5-PLP2 or treated with LPS, and p53 mRNA levels were analyzed by qPCR. The data are shown as the means ± S.D. (n = 3). I, MEF cells were treated with poly(I·C) or transfected with V5-nsp3 (SARS-CoV), and IRF7 mRNA levels were analyzed by qPCR. The data are shown as the means ± S.D. (n = 3). J, Vero cells were infected with PEDV, and IRF7 mRNA levels were analyzed by qPCR. The data are shown as the means ± S.D. (n = 3). The differences are statistically significant (**, p value < 0.001). NS, not significant (p value > 0.05). Con, control.
FIGURE 4.
FIGURE 4.
PLP2 inhibits the p53-dependent apoptosis. A, PLP2 expression decreases the steady-state level of Puma. p53−/− MEF cells were transfected with Flag-p53 and increasing amounts of V5-PLP2. After 48 h, total lysates were immunoblotted to detect the expression of Puma and p53. B, poly(I:C) increases the transcription of the P53 gene. p53 mRNA levels were analyzed by qPCR. The data are shown as the means ± S.D. (n = 3). C and D, overexpression of PLP2 decreases p53-dependent apoptosis. Apoptosis in p53+/+ and p53−/− HCT116 cells was determined by staining with annexin V. The data are shown as the means ± S.D. (n = 3). The differences are statistically significant (**, p value < 0.001). NS, not significant (p value > 0.05).
FIGURE 5.
FIGURE 5.
p53 transactivates IRF7 to regulate IFN-β. A, siRNA-mediated knockdown of endogenous IRF9. Protein expression analysis was performed by Western blotting with the indicated antibodies. B and C, the transcription of the IRF7 gene (B) and IFN-β gene (C) is not entirely inhibited in p53+/+HCT116 cells by siRNA-mediated knockdown of endogenous IRF9. D, p53 expression results in the increasing induction of IRF7 mRNA in p53 WT MEF cells compared with p53 knock-out MEF cells. E, putative p53 binding sites located in the 3′-trailer region of the IRF7 gene. F, this genomic region was cloned into the pGL3 firefly luciferase reporter vector (IRF7-p53BS luc). H1299 cells were cotransfected with IRF7-p53BS luc or p53 vectors and a Renilla luciferase construct to control for transfection efficiency and assessed for dual luciferase activity. G, ChIP assays were performed in U2OS cells that were transfected with or without poly(I·C) using control IgG or anti-p53 antibodies, and RT-PCR was performed for the indicated IRF7 3′-trailer regions. H, the binding activity of p53 to oligonucleotides containing p53-binding sites from the 3′-trailer region of IRF7 was determined by EMSA. The differences are statistically significant (**, p value<0.001). IP, immunoprecipitation.
FIGURE 6.
FIGURE 6.
PLP2 positively supports viral replication. A and B, replication of SeV (A) and the change in the IFN-β mRNA level analyzed by qPCR (B) in p53 WT and knock-out MEF cells transfected with PLP2 expression or control vectors. Cells were infected at an multiplicity of infection of 100 pfu/cell and harvested at various hours, as indicated. C, moderation of cytopathogenicity of SeV for cells at 24 and 48 h p.i. under single cycle growth conditions. Infections were initiated with SeV at an multiplicity of infection of 100 pfu/cell. p.i., post-infection. Arrows, fused cells; arrowheads, cytopathogenicity. D, the change in the IFN-β mRNA level analyzed by qPCR in p53 WT and knock-out MEF cells transfected with PLpro expression or control vectors. Cells were infected at an multiplicity of infection of 100 pfu/cell and harvested at indicated hours.
FIGURE 7.
FIGURE 7.
Proposed model for the regulation of p53-dependent apoptosis and the type I IFN response by PLP2. At the early stage of infection, p53 transactivates the IRF9 and IRF7 genes. At the advanced stage of infection, viral nucleic acids in the cells encode viral proteins, including PLP2, which decreases the stability and transcriptional activity of p53 by increasing the MDM2-mediated ubiquitination and nuclear export of p53 and, in turn, strongly inhibits p53-mediated IFN-β production and apoptosis while promoting viral growth in cells.
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