Down-regulation of p53 by double-stranded RNA modulates the antiviral response

Abstract

p53 has been well characterized as a tumor suppressor gene, but its role in antiviral defense remains unclear. A recent report has demonstrated that p53 can be induced by interferons and is activated after vesicular stomatitis virus (VSV) infection. We observed that different nononcogenic viruses, including encephalomyocarditis virus (EMCV) and human parainfluenza virus type 3 (HPIV3), induced down-regulation of p53 in infected cells. Double-stranded RNA (dsRNA) and a mutant vaccinia virus lacking the dsRNA binding protein E3L can also induce this effect, indicating that dsRNA formed during viral infection is likely the trigger for down-regulation of p53. The mechanism of down-regulation of p53 by dsRNA relies on translation inhibition mediated by the PKR and RNase L pathways. In the absence of p53, the replication of both EMCV and HPIV3 was retarded, whereas, conversely, VSV replication was enhanced. Cell cycle analysis indicated that wild-type (WT) but not p53 knockout (KO) fibroblasts undergo an early-G(1) arrest following dsRNA treatment. Moreover, in WT cells the onset of dsRNA-induced apoptosis begins after p53 levels are down-regulated, whereas p53 KO cells, which lack the early-G(1) arrest, rapidly undergo apoptosis. Hence, our data suggest that the down-regulation of p53 facilitates apoptosis, thereby limiting viral replication.

FIG. 1.

Down-regulation of p53 in response to viral infection. (A) HT1080 cells were infected at the indicated multiplicity of infection (MOI) (in PFU/cell) with EMCV, HPIV3, or VSV for 16 h. (B) HepG2 cells were infected with either RSV at 0.001 tissue culture infective dose/cell, Sendai virus at 100 hemagglutination units/ml, VSV at 0.1 PFU/cell, EMCV at 0.1 and 0.01 PFU/cell, or VV or the VV E3L mutant (VVE3L) at 0.1 PFU/cell for 16 h. Whole-cell extracts were collected and analyzed by Western blotting with specific antibodies.

FIG. 2.

Viral replication in the absence of p53. (A and B) Stable pools of HT1080 cells expressing shRNA against p53 (shRNA-p53) or GFP (shRNA-GFP) were transduced with a lentivirus expressing GFP (+) or an empty-vector control (−). (A) Whole-cell extracts were prepared and subjected to Western blot analysis. (B) Fluorescence originating from GFP was measured, normalized to total protein, and displayed on a graph. (C) The shRNA-expressing cells were infected with 0.001 PFU/cell of EMCV, HPIV3, or VSV. (D) At 24 h p.i. for EMCV and VSV, and at 48 h p.i. for HPIV3, the supernatant was collected, and the virus yield was determined and plotted as a proportion of the yield obtained from control shRNA-GFP cells. (E) Primary MEFs derived from WT (+/+) or p53 KO (−/−) mice were transduced with a lentivirus expressing human p53 or GFP for 48 h, and expression was analyzed by Western blotting with or without transfection of 2 μg/ml of poly(I:C) for 8 h. (F) Primary WT or p53 KO MEFs were either left untreated or transduced with a lentivirus expressing GFP or human p53 at increasing concentrations (1× or 5×) for 24 h. The cells were subsequently infected with 0.001 PFU/cell of EMCV. At 24 h p.i., the supernatant was collected, and the virus yield for each treatment was determined and plotted as a proportion of the yield obtained from WT cells.

FIG. 3.

dsRNA treatment induces down-regulation of p53 without changing the half-life of the protein. (A) HT1080 cells were transfected with 2 μg/ml of poly(I:C), and whole-cell extracts were prepared at different times and analyzed with specific antibodies as indicated. (B) Inhibition of dsRNA-induced apoptosis does not block down-regulation of p53. HT1080 cells were transfected with 2 μg/ml of poly(I:C) and treated with increasing concentrations of Z-VAD-FMK, an irreversible caspase inhibitor, for 8 h. Whole-cell extracts were collected and analyzed by Western blotting with specific antibodies. (C and D) To determine of the half-life of p53 protein in HT1080 cells, the cells were treated with 5 μg/ml of cycloheximide with or without 2 μg/ml of poly(I:C) for the indicated times. (C) Whole-cell lysates were extracted and subjected to Western blot analysis. (D) The ratio of p53 to β-actin was calculated at each time point and plotted on a graph. (E) HT1080 cells were grown in a medium without methionine for 2 h before being pulsed with [³⁵S]methionine. After 30 min of incubation with [³⁵S]methionine, the cells were put back into a normal medium with or without 2 μg/ml of poly(I:C). Extracts were collected at the indicated times and subjected to IP using an antibody against p53 (FL-393). The IP was separated by electrophoresis in SDS-PAGE, transferred to a nylon membrane, and exposed to a phosphorimager screen. The intensity corresponding to p53 protein was quantified using ImageQuant software. (F) HT1080 cells were transfected with 2 μg/ml of poly(I:C) for 8 h in the presence of increasing concentrations of the proteasome inhibitor MG132. Whole-cell extracts were prepared and analyzed by Western blotting. (G) HT1080 cells were transfected with 2 μg/ml of poly(I:C) for the indicated times in the presence of 5 μM of MG132. WCL were prepared and subjected to IP with antibodies against p53 as described above. The IP was separated by electrophoresis in SDS-PAGE, transferred to a nylon membrane, and analyzed by Western blotting (WB) with the indicated antibodies. The heavy chain (IgG-HC) of the antibody used for the IP was used as a control. (H) WCL controls for the IP were also directly analyzed by Western blotting with the indicated antibodies.

FIG. 4.

dsRNA induces down-regulation of p53 through activation of PKR and RNase L pathways. HT1080 cells were transfected with 2 μg/ml of poly(I:C) for different times (A and B) or at different concentrations of poly(I:C) for 8 h (C and D). The cells were then washed and incubated for 15 min in a medium containing [³⁵S]methionine. Whole-cell extracts were collected, and proteins were precipitated using TCA. Incorporation of [³⁵S]methionine into newly synthesized proteins was measured by scintillation counting and normalized to that of the control cells (A and C). Alternatively, the whole-cell extracts were analyzed by Western blotting with specific antibodies (B and D). (E) HT1080 cells were transfected with 2 μg/ml of poly(I:C) and treated with increasing concentrations of 2-aminopurine for 8 h. Whole-cell extracts were collected and analyzed by Western blotting. (F) HT1080 cells were transfected with 2 μg/ml of poly(I:C) for the indicated times, and total RNA was extracted and subjected to Northern blot analysis using the p53 cDNA as a probe. The 28S rRNA is shown as a control. (G and H) Primary MEFs derived from WT, PKR single-KO, RNase L single-KO, or PKR/RNase L DKO mice were transfected with 2 μg/ml of poly(I:C) for 8 h (G) or for the indicated times (H). Whole-cell lysates were prepared and analyzed by Western blotting. The p53/β-actin ratio is also given for each lane. (I) WT and DKO cells were transfected with 2 μg/ml of poly(I:C). After the indicated times, cells were washed and incubated for 15 min in a medium containing [³⁵S]methionine. Whole-cell extracts were collected, proteins were precipitated using TCA, and incorporation of [³⁵S]methionine into newly synthesized proteins was measured by scintillation counting and normalized to that of the control cells.

FIG. 4.

dsRNA induces down-regulation of p53 through activation of PKR and RNase L pathways. HT1080 cells were transfected with 2 μg/ml of poly(I:C) for different times (A and B) or at different concentrations of poly(I:C) for 8 h (C and D). The cells were then washed and incubated for 15 min in a medium containing [³⁵S]methionine. Whole-cell extracts were collected, and proteins were precipitated using TCA. Incorporation of [³⁵S]methionine into newly synthesized proteins was measured by scintillation counting and normalized to that of the control cells (A and C). Alternatively, the whole-cell extracts were analyzed by Western blotting with specific antibodies (B and D). (E) HT1080 cells were transfected with 2 μg/ml of poly(I:C) and treated with increasing concentrations of 2-aminopurine for 8 h. Whole-cell extracts were collected and analyzed by Western blotting. (F) HT1080 cells were transfected with 2 μg/ml of poly(I:C) for the indicated times, and total RNA was extracted and subjected to Northern blot analysis using the p53 cDNA as a probe. The 28S rRNA is shown as a control. (G and H) Primary MEFs derived from WT, PKR single-KO, RNase L single-KO, or PKR/RNase L DKO mice were transfected with 2 μg/ml of poly(I:C) for 8 h (G) or for the indicated times (H). Whole-cell lysates were prepared and analyzed by Western blotting. The p53/β-actin ratio is also given for each lane. (I) WT and DKO cells were transfected with 2 μg/ml of poly(I:C). After the indicated times, cells were washed and incubated for 15 min in a medium containing [³⁵S]methionine. Whole-cell extracts were collected, proteins were precipitated using TCA, and incorporation of [³⁵S]methionine into newly synthesized proteins was measured by scintillation counting and normalized to that of the control cells.

FIG. 4.

dsRNA induces down-regulation of p53 through activation of PKR and RNase L pathways. HT1080 cells were transfected with 2 μg/ml of poly(I:C) for different times (A and B) or at different concentrations of poly(I:C) for 8 h (C and D). The cells were then washed and incubated for 15 min in a medium containing [³⁵S]methionine. Whole-cell extracts were collected, and proteins were precipitated using TCA. Incorporation of [³⁵S]methionine into newly synthesized proteins was measured by scintillation counting and normalized to that of the control cells (A and C). Alternatively, the whole-cell extracts were analyzed by Western blotting with specific antibodies (B and D). (E) HT1080 cells were transfected with 2 μg/ml of poly(I:C) and treated with increasing concentrations of 2-aminopurine for 8 h. Whole-cell extracts were collected and analyzed by Western blotting. (F) HT1080 cells were transfected with 2 μg/ml of poly(I:C) for the indicated times, and total RNA was extracted and subjected to Northern blot analysis using the p53 cDNA as a probe. The 28S rRNA is shown as a control. (G and H) Primary MEFs derived from WT, PKR single-KO, RNase L single-KO, or PKR/RNase L DKO mice were transfected with 2 μg/ml of poly(I:C) for 8 h (G) or for the indicated times (H). Whole-cell lysates were prepared and analyzed by Western blotting. The p53/β-actin ratio is also given for each lane. (I) WT and DKO cells were transfected with 2 μg/ml of poly(I:C). After the indicated times, cells were washed and incubated for 15 min in a medium containing [³⁵S]methionine. Whole-cell extracts were collected, proteins were precipitated using TCA, and incorporation of [³⁵S]methionine into newly synthesized proteins was measured by scintillation counting and normalized to that of the control cells.

FIG. 4.

dsRNA induces down-regulation of p53 through activation of PKR and RNase L pathways. HT1080 cells were transfected with 2 μg/ml of poly(I:C) for different times (A and B) or at different concentrations of poly(I:C) for 8 h (C and D). The cells were then washed and incubated for 15 min in a medium containing [³⁵S]methionine. Whole-cell extracts were collected, and proteins were precipitated using TCA. Incorporation of [³⁵S]methionine into newly synthesized proteins was measured by scintillation counting and normalized to that of the control cells (A and C). Alternatively, the whole-cell extracts were analyzed by Western blotting with specific antibodies (B and D). (E) HT1080 cells were transfected with 2 μg/ml of poly(I:C) and treated with increasing concentrations of 2-aminopurine for 8 h. Whole-cell extracts were collected and analyzed by Western blotting. (F) HT1080 cells were transfected with 2 μg/ml of poly(I:C) for the indicated times, and total RNA was extracted and subjected to Northern blot analysis using the p53 cDNA as a probe. The 28S rRNA is shown as a control. (G and H) Primary MEFs derived from WT, PKR single-KO, RNase L single-KO, or PKR/RNase L DKO mice were transfected with 2 μg/ml of poly(I:C) for 8 h (G) or for the indicated times (H). Whole-cell lysates were prepared and analyzed by Western blotting. The p53/β-actin ratio is also given for each lane. (I) WT and DKO cells were transfected with 2 μg/ml of poly(I:C). After the indicated times, cells were washed and incubated for 15 min in a medium containing [³⁵S]methionine. Whole-cell extracts were collected, proteins were precipitated using TCA, and incorporation of [³⁵S]methionine into newly synthesized proteins was measured by scintillation counting and normalized to that of the control cells.

FIG. 4.

dsRNA induces down-regulation of p53 through activation of PKR and RNase L pathways. HT1080 cells were transfected with 2 μg/ml of poly(I:C) for different times (A and B) or at different concentrations of poly(I:C) for 8 h (C and D). The cells were then washed and incubated for 15 min in a medium containing [³⁵S]methionine. Whole-cell extracts were collected, and proteins were precipitated using TCA. Incorporation of [³⁵S]methionine into newly synthesized proteins was measured by scintillation counting and normalized to that of the control cells (A and C). Alternatively, the whole-cell extracts were analyzed by Western blotting with specific antibodies (B and D). (E) HT1080 cells were transfected with 2 μg/ml of poly(I:C) and treated with increasing concentrations of 2-aminopurine for 8 h. Whole-cell extracts were collected and analyzed by Western blotting. (F) HT1080 cells were transfected with 2 μg/ml of poly(I:C) for the indicated times, and total RNA was extracted and subjected to Northern blot analysis using the p53 cDNA as a probe. The 28S rRNA is shown as a control. (G and H) Primary MEFs derived from WT, PKR single-KO, RNase L single-KO, or PKR/RNase L DKO mice were transfected with 2 μg/ml of poly(I:C) for 8 h (G) or for the indicated times (H). Whole-cell lysates were prepared and analyzed by Western blotting. The p53/β-actin ratio is also given for each lane. (I) WT and DKO cells were transfected with 2 μg/ml of poly(I:C). After the indicated times, cells were washed and incubated for 15 min in a medium containing [³⁵S]methionine. Whole-cell extracts were collected, proteins were precipitated using TCA, and incorporation of [³⁵S]methionine into newly synthesized proteins was measured by scintillation counting and normalized to that of the control cells.

FIG. 4.

dsRNA induces down-regulation of p53 through activation of PKR and RNase L pathways. HT1080 cells were transfected with 2 μg/ml of poly(I:C) for different times (A and B) or at different concentrations of poly(I:C) for 8 h (C and D). The cells were then washed and incubated for 15 min in a medium containing [³⁵S]methionine. Whole-cell extracts were collected, and proteins were precipitated using TCA. Incorporation of [³⁵S]methionine into newly synthesized proteins was measured by scintillation counting and normalized to that of the control cells (A and C). Alternatively, the whole-cell extracts were analyzed by Western blotting with specific antibodies (B and D). (E) HT1080 cells were transfected with 2 μg/ml of poly(I:C) and treated with increasing concentrations of 2-aminopurine for 8 h. Whole-cell extracts were collected and analyzed by Western blotting. (F) HT1080 cells were transfected with 2 μg/ml of poly(I:C) for the indicated times, and total RNA was extracted and subjected to Northern blot analysis using the p53 cDNA as a probe. The 28S rRNA is shown as a control. (G and H) Primary MEFs derived from WT, PKR single-KO, RNase L single-KO, or PKR/RNase L DKO mice were transfected with 2 μg/ml of poly(I:C) for 8 h (G) or for the indicated times (H). Whole-cell lysates were prepared and analyzed by Western blotting. The p53/β-actin ratio is also given for each lane. (I) WT and DKO cells were transfected with 2 μg/ml of poly(I:C). After the indicated times, cells were washed and incubated for 15 min in a medium containing [³⁵S]methionine. Whole-cell extracts were collected, proteins were precipitated using TCA, and incorporation of [³⁵S]methionine into newly synthesized proteins was measured by scintillation counting and normalized to that of the control cells.

FIG. 5.

Role of p53 in apoptosis induced by dsRNA. Primary cultures of WT and p53 KO mice were transfected with 2 μg/ml of poly(I:C) for the indicated times. Cells were fixed, stained with propidium iodide, and analyzed by flow cytometry for DNA content. (A) The percentage of cells in G₁ was divided by the percentage in G₂, the value obtained was normalized to that for the untreated control, and the numbers were plotted on a graph. (B) The percentage of sub-G₁ cells at each time point after treatment, indicative of apoptosis, was also plotted on a graph. (C) Primary cultures of MEFs derived from WT and p53 KO mice were transfected with 2 μg/ml of poly(I:C) or treated with a combination of either 10 ng/ml of TNF and 1 μg/ml cycloheximide or 100 μg/ml of untransfected poly(I:C) and 1 μg/ml cycloheximide. After 16 h, cells were fixed and stained for TUNEL, and the percentages of TUNEL-positive cells were plotted on a graph. (D) WCL from WT and p53 KO MEFs, untreated or transfected with poly(I:C), were prepared and subjected to Western blot analysis. (E) Primary WT, PKR KO, RNase L KO, and PKR/RNase L DKO MEFs were transfected for 16 h with different concentrations of poly(I:C), and caspase 3/7 activity was measured.