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. 2005 Jul;79(14):8802-11.
doi: 10.1128/JVI.79.14.8802-8811.2005.

Influenza virus infection increases p53 activity: role of p53 in cell death and viral replication

Elizabeth Turpin  1 , Kimberly Luke, Jeremy Jones, Terrence Tumpey, Kouacou Konan, Stacey Schultz-Cherry
Affiliations

Influenza virus infection increases p53 activity: role of p53 in cell death and viral replication

Elizabeth Turpin et al. J Virol. 2005 Jul.

Abstract

The induction of apoptotic cell death is a hallmark of influenza virus infection. Although a variety of cellular and viral proteins have been implicated in this process, to date no conserved cellular pathway has been identified. In this study, we report that the tumor suppressor protein p53 is essential for the induction of cell death in influenza virus-infected cells. In primary human lung cells, influenza virus increased p53 protein levels. This was also noted in the human lung cell line A549, along with the up-regulation of p53-dependent gene transcription. Reduction of p53 activity in A549 cells inhibited influenza virus-induced cell death as measured by trypan blue exclusion and caspase activity. These findings were not cell type specific. Influenza virus-induced cell death was absent in mouse embryo fibroblasts isolated from p53 knockout mice, which was not the case in wild-type mouse embryo fibroblasts, suggesting that p53 is a common cellular pathway leading to influenza virus-induced cell death. Surprisingly, inhibiting p53 activity led to elevated virus replication. Mechanistically, this may be due to the decrease in interferon signaling in p53-deficient cells, suggesting that functional p53 is involved in the interferon response to influenza infection. To our knowledge, these are the first studies demonstrating that p53 is involved in influenza virus-induced cell death and that inhibiting p53 leads to increased viral titers, potentially through modulation of the interferon response.

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Figures

FIG. 1.
FIG. 1.
p53 activity increases during influenza infection. Equal protein concentrations of mock- (treated with media alone) and WSN-infected (MOI 2) human primary lung cell lysates (A), mock- and PR8-infected (MOI 2) A549 lysates (B), and heat-inactivated (HI) PR8-treated A549 lysates (C) were analyzed for p53 levels by Western blotting. (D) A549 cells transfected with the p53 reporter (PG13-Luc) construct or control (MG15-Luc) construct were mock or PR8 infected (MOI 2), and samples were collected at 24 hpi. The luciferase activity of lysates was standardized for equal protein concentration and expressed as the n-fold increase in luciferase activity in infected cells over that in mock-infected controls. Error bars represent the standard errors of the means, and the asterisk indicates samples that are significant (P < 0.05) as determined by ANOVA.
FIG. 2.
FIG. 2.
Nuclear accumulation and serine phosphorylation of p53. (A) A549 cells were treated with media alone (mock) or infected with PR8 (MOI 2); total RNA was isolated at 1, 3, 5, 8, and 24 hpi, and RT-PCR was performed for p53 or β-actin. (B) A549 cells, treated with media alone (mock) or PR8 infected (MOI 2), were double-stained for virus antigen (NS) and p53 at 12 hpi and examined by fluorescent microscopy. The nucleus was visualized by DAPI. Magnification, ×42. (C) Cellular lysates from mock- or PR8-infected A549 cells at 24 hpi were immunoprecipitated with a phospho-Ser15-specific p53 antibody followed by binding to protein G Sepharose beads. Equal protein levels of total or immunoprecipitated lysates were analyzed by probing Western blots with a p53 antibody (DO-1).
FIG. 3.
FIG. 3.
Inhibition of p53 decreases influenza virus-induced cell death. (A) Parental A549 cells or A549 cells stably expressing a p53 dominant-negative mutant (clones 3 and 6) were transfected with the p53 reporter (PG13-Luc) or the control (MG15-Luc) construct. Cells were mock or PR8 infected (MOI 2), and the luciferase activities of lysates were measured. Results were standardized for equal protein concentration and are expressed as the n-fold increases in activity in PR8-infected cells over those in mock-infected controls. (B) Cell viabilities of mock- or PR8-infected (MOI 2) parental A549 cells or dominant-negative p53 mutants (clones 3 and 6) were determined by trypan blue exclusion. Error bars represent the standard errors of the means, and asterisks indicate samples that are significant (P < 0.05) as determined by ANOVA.
FIG. 4.
FIG. 4.
p53 is essential for influenza virus-induced cell death. (A) Cell viabilities of mock and infected MEF p53+/+ and p53−/− cells were determined by trypan blue exclusion. (B) MEF p53−/− cells were transfected with pCMV-p53 (+ p53) or mock transfected (MEM), and treatment with MEM alone (mock) or with infection (PR8) followed. Viabilities were measured by trypan blue exclusion. (C) Caspase 3/7 activity was measured in mock or infected MEF p53+/+ and p53−/− cells. Caspase activity was normalized to equal cell numbers and is graphed as the n-fold increase in infected cells over that in mock-infected cells. All error bars represent the standard errors of the means, and asterisks indicate samples that are significant (P < 0.05) as determined by ANOVA.
FIG. 5.
FIG. 5.
p53 inhibition increases viral titers. Confluent cultures of parental A549 cells, i.e., A549 p53 dominant-negative clones 3 and 6 (A) or MEF p53−/− and MEF p53+/+ cells (B) were infected with PR8 at an MOI of 0.05. At various times postinfection, supernatants were collected and viral titers determined by TCID50 on MDCK cells. (C) Confluent cultures of parental A549 cells and p53 dominant-negative clones 3 and 6 were infected with PR8 at an MOI of 2. At various times postinfection, supernatants were collected and viral titers determined by use of the TCID50 for MDCK cells. Asterisks indicate samples that are significant (P < 0.05) as determined by ANOVA.
FIG. 6.
FIG. 6.
Inhibition of p53 decreases IFN activity. Parental A549 cells or p53 dominant-negative clones 3 and 6 were transfected with the IFN reporter (ISRE-Luc) or control (CIS-Luc) and then mock or PR8 infected. Luciferase activity was measured in cell lysates at 12 hpi. Results were normalized to equal cell numbers and are expressed as the n-fold increase in activity in infected cells over that in mock-infected controls. Error bars represent the standard errors of the means, and asterisks indicate samples that are significant (P < 0.05) as determined by ANOVA.
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