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, 80 (5), 2257-66

Inhibition of the ATM/p53 Signal Transduction Pathway by Kaposi's Sarcoma-Associated Herpesvirus Interferon Regulatory Factor 1

Affiliations

Inhibition of the ATM/p53 Signal Transduction Pathway by Kaposi's Sarcoma-Associated Herpesvirus Interferon Regulatory Factor 1

Young C Shin et al. J Virol.

Abstract

Infected cells recognize viral replication as a DNA damage stress and elicit the ataxia telangiectasia-mutated (ATM)/p53-mediated DNA damage response signal transduction pathway as part of the host surveillance mechanisms, which ultimately induces the irreversible cell cycle arrest and apoptosis. Viruses have evolved a variety of mechanisms to counteract this host intracellular innate immunity. Kaposi's sarcoma-associated herpesvirus (KSHV) viral interferon regulatory factor 1 (vIRF1) interacts with the cellular p53 tumor suppressor through its central DNA binding domain, and this interaction inhibits transcriptional activation of p53. Here, we further demonstrate that KSHV vIRF1 downregulates the total p53 protein level by facilitating its proteasome-mediated degradation. Detailed biochemical study showed that vIRF1 interacted with cellular ATM kinase through its carboxyl-terminal transactivation domain and that this interaction blocked the activation of ATM kinase activity induced by DNA damage stress. As a consequence, vIRF1 expression greatly reduced the level of serine 15 phosphorylation of p53, resulting in an increase of p53 ubiquitination and thereby a decrease of its protein stability. These results indicate that KSHV vIRF1 comprehensively compromises an ATM/p53-mediated DNA damage response checkpoint by targeting both upstream ATM kinase and downstream p53 tumor suppressor, which might circumvent host growth surveillance and facilitate viral replication in infected cells.

Figures

FIG. 1.
FIG. 1.
Downregulation of p53 protein amount by vIRF1. (A) vIRF1 expression deceases the endogenous p53 protein amount. TRExBJAB-cDNA5, TRExBJAB-vIRF1, TRExBCBL1-cDNA5, and TRExBCBL1-vIRF1 cells were treated with doxycycline (Doxy) for the indicated times, and their cell lysates were used for immunoblotting with anti-p53, anti-vIRF (myc), and antitubulin antibodies. (B) RNase protection assay. TRExBCBL1-cDNA5 and TRExBCBL1-vIRF1 cells were treated with doxycycline for the indicated times, their total RNA was isolated by phenol-chloroform extraction, and contaminated DNA was removed by DNase I treatment. Thirty micrograms of total RNA was then subjected to RPA using an RPA kit from BD PharMingen (San Diego, CA) according to the manufacturer's recommendation. (C) Reduction of p53 protein stability by vIRF1 expression. TRExBJAB-cDNA5, TRExBJAB-vIRF1, TRExBCBL1-cDNA5, and TRExBCBL1-vIRF1 cells were stimulated with doxycycline for 24 h, followed by treatment with cycloheximide (50 μg/ml) for the indicated times. Whole-cell lysates were used for immunoblotting with anti-p53 and antitubulin antibodies. The half-life of p53 was calculated by the formula described in Materials and Methods.
FIG. 2.
FIG. 2.
Enhanced ubiquitination of p53 induced by vIRF1. (A) TRExBJAB-cDNA5 (C) and TRExBJAB-vIRF1 (V) cells were treated with doxycycline for 24 h and further incubated in the presence (+) or absence (−) of MG132 for 4 h. Whole-cell lysates were used for immunoblotting with anti-p53 and antitubulin antibodies. The polyvinylidene difluoride nitrocellulose membrane was exposed for a short time (middle panel) to detect the unmodified p53 or for a long time (top panel) to detect the ubiquitin-modified p53. The monoubiquitinated and polyubiquitinated p53 are marked with * and **, respectively. (B) Mdm2 is necessary for vIRF1-induced degradation of p53. Expression vectors containing the Flag-tagged wild-type p53, p53(14/19) mutant, or p53(22/23) mutant were transfected into stable p53−/− MEF or p53−/− mdm2−/− MEF expressing control-puro (C) or vIRF1-puro (V). At 48 h posttransfection, cell lysates were used for immunoblotting with anti-FLAG and antitubulin antibodies.
FIG. 3.
FIG. 3.
vIRF1 inhibits the Ser15 phosphorylation of p53. HCT116 cells (HCT116-puro [C] and HCT116-vIRF1 cells [V]) and TRExBJAB cells (TRExBJAB-cDNA5 [C] and TRExBJAB-vIRF1 cells [V]) were treated with or without MG132 for 4 h. TRExBJAB cells were treated with doxycycline for 24 h prior to MG132 treatments. Whole-cell lysates were then used for immunoblotting with anti-Ser15 phospho-specific p53, anti-p53, and antitubulin antibodies.
FIG. 4.
FIG. 4.
vIRF1 interacts with and inhibits ATM kinase. (A) vIRF1 suppresses ATM and Chk2 activation. TRExBCBL1-cDNA5 and TRExBCBL1-vIRF1 cells were incubated with doxycycline for 24 h, followed by treatment with or without etoposide for 4 h. Cell lysates were then used for immunoblotting with anti-Ser1981 phospho-specific ATM, anti-ATM, anti-phospho-specific Chk2, anti-Chk2, anti-Cdc25C, anti-p53, anti-vIRF1 (myc), and antitubulin antibodies. (B) The carboxyl-terminal transcriptional domain of vIRF1 interacts with ATM. pEF-IRES-vIRF1 wt or pEF-IRES-vIRF1 mutants were transfected into 293T cells. Cells were lysed at 48 h posttransfection with lysis buffer, and whole-cell lysates (WCL) were used for immunoprecipitation (IP) with an anti-ATM antibody, followed by immunoblotting (IB) with anti-Myc antibody (bottom right panel). WCL were also used for immunoblotting with an anti-myc antibody to demonstrate the expression of vIRF1 wt and mutant proteins (bottom left panel). (C) Interaction of full-length vIRF1 with ATM in KSHV-infected BABCL1 cells. TRExBCBL1-vIRF1 cells were treated with doxycycline for 48 h and lysed with lysis buffer. WCL were then used for immunoprecipitation with anti-ATM and anti-V5 antibodies, followed by immunoblotting with an anti-myc antibody. WCL were also used for immunoblotting with an anti-myc antibody to demonstrate the myc-tagged vIRF1 expression.
FIG. 5.
FIG. 5.
vIRF1 interaction with ATM is necessary to block ATM activation. (A) Correlation between vIRF1 interaction and ATM inhibition. TRExBCBL1-cDNA5 (C), TRExBCBL1-vIRF1 (V), TRExBCBL1-vIRF1 mt1, TRExBCBL1-vIRF1 mt2, TRExBCBL1 mt4, and TRExBCBL1-vIRF1 mt5 cells were incubated with doxycycline for 24 h, followed by treatment with or without etoposide for 4 h. Each vIRF1 mutant except vIRF1 mt1 was as described in Fig. 4. vIRF1 mt1 contained amino-terminal amino acids 1 to 80. Cell lysates were used for immunoblotting with anti-Ser1981 phospho-specific ATM, anti-Ser15 phospho-specific p53, anti-p53, and antitubulin antibodies. (B) Enhanced ubiquitination of p53 in vIRF1-expressing HCT116 cells. HCT116-puro (C) and HCT116-vIRF1 (V) cells were treated with etoposide for 4 h, and their lysates were used for immunoblotting with anti-p53 and antitubulin antibodies. The monoubiquitinated p53 is marked with an asterisk.
FIG. 6.
FIG. 6.
vIRF1 inhibits ATM activation and γH2AX induction (A) and enhances p53 cytoplasmic localization (B). (A) TRExBCBL1-cDNA5 and TRExBCBL1-vIRF1 cells were incubated with doxycycline for 24 h, treated with or without etoposide (5 μM) for 3 h, fixed with paraformaldehyde, and reacted with anti-Ser1981 phospho-specific ATM (αp-ATM) or anti-γH2AX antibody (green). Cells were stained with Topro-3 (blue) to show the nucleus and then subjected to confocal microscopy. (B) vIRF1 facilitates the cytoplasmic localization of p53. TRExBCBL1-cDNA and TRExBCBL1-vIRF1 cells were treated with doxycycline (Doxy) for 24 h and then further incubated in the presence or absence of 50 nM leptomycin (LMB) for 3 h. These cells were fixed, reacted with anti-p53 (green) and anti-vIRF1 (red) antibodies, and subjected to confocal microscopy.

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