Herpes simplex virus type 1 infection induces the stabilization of p53 in a USP7- and ATM-independent manner

Abstract

The major oncoprotein p53 regulates several cellular antiproliferation pathways that can be triggered in response to a variety of cellular stresses, including viral infection. The stabilization of p53 is a key factor in the ability of cells to initiate an efficient transcriptional response after cellular stress. Here we present data demonstrating that herpes simplex virus type 1 (HSV-1) infection of HFFF-2 cells, a low-passage-number nontransformed human primary cell line, results in the stabilization of p53. This process required viral immediate-early gene expression but occurred independently of the viral regulatory protein ICP0 and viral DNA replication. No specific viral protein could be identified as being solely responsible for the effect, which appears to be a cellular response to developing HSV-1 infections. HSV-1 infection also induced the phosphorylation of p53 at residues Ser15 and Ser20, which have previously been implicated in its stabilization in response to DNA damage. However, an HSV-1 infection of ATM(-/-) cells, which lack a kinase implicated in these phosphorylation events, did not lead to the phosphorylation of p53 at these residues, but nonetheless p53 was stabilized. We also show that the wild-type p53 expressed by osteosarcoma U2OS cells can be stabilized in response to DNA damage induced by UV irradiation, but not in response to HSV-1 infection. These data suggest that multiple cellular mechanisms are initiated to stabilize p53 during an HSV-1 infection. These mechanisms occur independently of ICP0 and its ability to sequester USP7 and may differ from those initiated in response to DNA damage.

FIG. 1.

HSV-1 infection results in stabilization of p53. (A) HFFF-2 cells were either mock infected or infected with HSV-1 (strain 17+) at an MOI of 1 PFU/cell for the indicated times. (B) Histogram representing the relative increases in p53 levels during HSV-1 infection of HFFF-2 cells. Densitometry analysis was performed on p53 Western blots from a series of experiments, and the results are expressed as relative changes in intensity (arbitrary units). Error bars represent the standard errors for three independent experiments. (C) HFFF-2 cells were either mock infected or infected with HSV-1 for 8 h at an MOI of 1 PFU/cell. The cells were then either harvested directly in SDS-PAGE loading buffer or treated with CHX (final concentration, 10 μg/ml) for an additional 10 h before being harvested. The cell extracts were analyzed by Western blotting with monoclonal antibodies that recognize ICP0, p53, and actin and with phospho-specific p53-Ser15 and p53-Ser20 rabbit polyclonal antibodies, as indicated. Note that in this and subsequent figures, the relative intensities of bands of the different proteins in the vertical columns vary according to trivial factors and cannot therefore be used for quantitative purposes (for example, for comparing the apparent absolute levels of p53 to actin).

FIG. 2.

HSV-1 infection decreases rate of p53 degradation. (A) HFFF-2 cells were pulse labeled (as described in Materials and Methods) prior to being mock infected or infected with HSV-1 (strain 17+) at an MOI of 10 PFU/cell. The cellular levels of metabolically labeled p53 were determined by immunoprecipitation and SDS-PAGE analysis (as described in Materials and Methods). (B) Histogram representing quantification of metabolically labeled p53 from mock-infected or HSV-1-infected HFFF-2 cells (as described for panel A) by phosphorimaging analysis. Relative intensity values for both mock-infected and HSV-1 17+-infected cells are given as percentages of the total amounts of metabolically labeled p53 at time zero.

FIG. 3.

p53 stabilization requires IE gene expression but not viral DNA replication. HFFF-2 cells were either mock infected or infected with HSV-1 (strain 17+) at an MOI of 1 PFU/cell in the presence or absence (+ and −, respectively) of CHX (final concentration, 10 μg/ml) (A) or ACG (final concentration, 10 μg/ml) (B).

FIG. 4.

ICP0 is not required for phosphorylation and stabilization of p53 during HSV-1 infection. HFFF-2 cells were either mock infected or infected with HSV-1 (strain 17+) and the indicated mutants. (A) The mutants used were HSV-1 IE-1 mutants that expressed no ICP0 (dl1403/ΔICP0) or had a deletion in the RING finger domain of ICP0 (FXE) and two ICP0 USP7-negative binding mutants, M1 (a substitution mutant carrying the mutations 623R to L and 624K to I in ICP0) and D12 (a deletion mutant lacking amino acids 594 to 633 of ICP0). These mutants were used at an MOI of 1 PFU/cell. (B) The mutant used was dl1403 (ΔICP0), which was added at various MOIs (as indicated). At 8 h p.i., the cells were either harvested or treated with CHX, as designated (final concentration, 10 μg/ml), for an additional 10 h before being harvested. The cell extracts were then analyzed by Western blotting with anti-ICP0, -ICP4, -p53, and -actin monoclonal antibodies and a phospho-specific p53-Ser15 rabbit polyclonal antibody.

FIG. 5.

Efficient phosphorylation of p53 at Ser15 requires ICP4 expression and its DNA binding activity, but not ICP27 expression. HFFF-2 cells were either mock infected or infected with HSV-1 (strain 17+) and an HSV-1 mutant that fails to express ICP4 (in1411/ΔICP4) (A), a temperature-sensitive ICP4 mutant (tsK) (B), or an HSV-1 mutant that fails to express ICP27 (17+27-pR19lacZ/ΔICP27) (C) at an MOI of 1 PFU/cell. The cells were incubated at 37°C, unless stated otherwise, and were harvested at the indicated time points. The cell extracts were then analyzed by Western blotting with the appropriate anti-ICP0, -ICP4, -ICP27, -p53, and -actin monoclonal antibodies and a phospho-specific p53-Ser15 rabbit polyclonal antibody.

FIG. 6.

ATM is required for HSV-1-induced phosphorylation of p53 at Ser15 and Ser20 but is not essential for p53 stabilization. (A) HFFF-2 cells were pretreated with various concentrations (as indicated) of wortmannin (Wort) 1 h prior to infection. The cells were subsequently mock infected or infected with HSV-1 (strain 17+) at an MOI of 1 PFU/cell for 8 h before being harvested. (B) AT^−/− or AT^+/+ cells were either mock infected or infected with HSV-1 (strain 17+) for 8 h before being harvested or treated with CHX (final concentration, 10 μg/ml), as indicated, for an additional 10 h prior to harvesting. The cell extracts were then analyzed by Western blotting with anti-ICP0, -p53, and -actin monoclonal antibodies and phospho-specific p53-Ser15 and p53-Ser20 rabbit polyclonal antibodies.

FIG. 7.

HSV-1 infection of U2OS cells fails to induce stabilization of p53. (A) U2OS and HFFF-2 cells were mock infected or infected with either HSV-1 (strain 17+) or an HSV-1 IE-1 mutant that fails to express ICP0 (dl1403/ΔICP0) at an MOI of 1 PFU/cell for 8 h before being harvested. (B) U2OS cells were infected as described above or were UV irradiated and harvested at 8 h posttreatment or treated with CHX (final concentration, 10 μg/ml), as indicated, for an additional 10 h prior to being harvested. The cell extracts were then analyzed by Western blotting with anti-ICP0, -ICP4, -p53, and -actin monoclonal antibodies and a phospho-specific p53-Ser15 rabbit polyclonal antibody.