Modulation of p53 cellular function and cell death by African swine fever virus

Abstract

Modulation of the activity of tumor suppressor p53 is a key event in the replication of many viruses. We have studied the function of p53 in African swine fever virus (ASFV) infection by determining the expression and activity of this transcription factor in infected cells. p53 levels are increased at early times of infection and are maintained throughout the infectious cycle. The protein is transcriptionally active, stabilized by phosphorylation, and localized in the nucleus. p53 induces the expression of p21 and Mdm2. Strikingly, these two proteins are located at the cytoplasmic virus factories. The retention of Mdm2 at the factory may represent a viral mechanism to prevent p53 inactivation by the protein. The expression of apoptotic proteins, such as Bax or active caspase-3, is also increased following ASFV infection, although the increase in caspase-3 does not appear to be, at least exclusively, p53 dependent. Bax probably plays a role in the induction of apoptosis in the infected cells, as suggested by the release of cytochrome c from the mitochondria. The significance of p21 induction and localization is discussed in relation to the shutoff of cellular DNA synthesis that is observed in ASFV-infected cells.

FIG. 1.

ASFV inhibition of cellular DNA and protein synthesis is concomitant with the expression of cellular proteins that control the cell cycle. (A) Cellular DNA synthesis was determined as described in Materials and Methods. The radioactivity incorporated at the indicated times of infection into nuclear fractions from 2 × 10⁶ cells (the number of cells in the culture at the time of infection) was measured. Protein synthesis was determined by measuring the incorporation of [³⁵S]methionine into acid-insoluble material from 2 × 10⁶ cells. For both DNA and protein synthesis, the ratio between ASFV-infected and mock-infected cultures is represented in the graph. TCA, trichloroacetic acid. (B) Cultures of Vero cells (5 × 10⁵) were mock infected (M) or infected with ASFV and labeled at different times after infection with 200 μCi of [³⁵S]methionine-cysteine/ml in cysteine-methionine-free medium for 2 h. The cells were resuspended in 250 μl of TNT buffer, and cell extracts (10 μl) were analyzed by SDS-PAGE. Proteins were detected by fluorography. (C) Kinetics of p53, p21, Bax, and PCNA expression analyzed by Western blotting in mock-infected (M) or ASFV-infected Vero cells. Times after infection are indicated above the lanes. (D) The p53 transcriptional activity was analyzed in mock-infected or ASFV-infected Vero cells by transient transfection of specific plasmids containing the luciferase gene under the control of p53-binding motif or of p21 or Mdm2 promoters, as indicated in Materials and Methods. The transfected cells were incubated overnight and then infected with BA71V at a multiplicity of infection of 5 PFU/cell. At the indicated times after infection, cell extracts were prepared, and the luciferase activity was measured in a luminometer. RLU, relative light units.

FIG. 2.

Subcellular localization and phosphorylation of p53 during ASFV infection. (A) Mock-infected or ASFV-infected Vero cells were labeled with Mitotracker (red) and anti-p53 antibody (green) and then examined by confocal microscopy. Shown are images corresponding to one of three independent experiments performed. (B) Nuclear extracts (30 μg) from mock-infected or ASFV-infected Vero cells were subjected to Western blot analysis using an anti-p53 (αp53) antibody (left) or anti-p-p53 antibodies that recognize phosphorylated p53 in Ser392 (center) or in Ser15 (right).

FIG. 3.

Subcellular localization of p21, Mdm2, and PCNA. (A) Mock-infected or ASFV-infected Vero cells were labeled with Mitotracker (red) and anti-p21, anti-Mdm2, or anti-PCNA antibodies (green) and then examined by confocal microscopy. Shown are images corresponding to one of three independent experiments performed. (B) ASFV-infected Vero cells were stained at 13 h p.i. with DAPI (blue) and anti-p21 or anti-Mdm2 antibodies (green) and examined by fluorescence microscopy. The arrows point to virus factories identified by DAPI staining.

FIG. 4.

ASFV-induced cytochrome c release and expression of p53 and caspase-3 activation under different infection conditions. (A) Mock-infected or ASFV-infected Vero cells were labeled with Mitotracker (red) and anti-cytochrome c (αCit-C) antibody (green) and then examined by confocal microscopy. Colocalization of mitochondria and cytochrome c yields a yellow signal. Shown are images corresponding to one of two independent experiments performed. (B) Extracts from mock-infected cells (M), ASFV-infected cells (V), cells incubated with UV-inactivated virus (UVV), cells mock infected (CHX/M) or infected (CHX/V) in the presence of CHX (10 μg/ml), and cells mock infected (CAR/M) or infected (CAR/V) in the presence of CAR (40 μg/ml) were analyzed by Western blotting using specific antibodies against p53 (α p53) or caspase-3 (α casp.3). A representative experiment is shown. (C) Luciferase activity in Vero cells transfected with the p53RE-Luc reporter plasmid. Twelve hours after transfection, the cells were mock infected or infected under the conditions described for panel B, and cell extracts were prepared and analyzed for p53 transcriptional activity. The results of a representative experiment are shown. a.u., arbitrary units.