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. 2005 Sep;79(17):11467-75.
doi: 10.1128/JVI.79.17.11467-11475.2005.

Human cytomegalovirus IE1-72 activates ataxia telangiectasia mutated kinase and a p53/p21-mediated growth arrest response

Jonathan P Castillo  1 , Fiona M Frame, Harry A Rogoff, Mary T Pickering, Andrew D Yurochko, Timothy F Kowalik
Affiliations

Human cytomegalovirus IE1-72 activates ataxia telangiectasia mutated kinase and a p53/p21-mediated growth arrest response

Jonathan P Castillo et al. J Virol. 2005 Sep.

Abstract

Human cytomegalovirus (HCMV) encodes several proteins that can modulate components of the cell cycle machinery. The UL123 gene product, IE1-72, binds the Rb-related, p107 protein and relieves its repression of E2F-responsive promoters; however, it is unable to induce quiescent cells to enter S phase in wild-type (p53(+/+)) cells. IE1-72 also induces p53 accumulation through an unknown mechanism. We present here evidence suggesting that IE1-72 may activate the p53 pathway by increasing the levels of p19(Arf) and by inducing the phosphorylation of p53 at Ser15. Phosphorylation of this residue by IE1-72 expression alone or HCMV infection is found to be dependent on the ataxia-telangiectasia mutated kinase. IE2-86 expression leads to p53 phosphorylation and may contribute to this phenotype in HCMV-infected cells. We also found that IE1-72 promotes p53 nuclear accumulation by abrogating p53 nuclear shuttling. These events result in the stimulation of p53 activity, leading to a p53- and p21-dependent inhibition of cell cycle progression from G(1) to S phase in cells transiently expressing IE1-72. Thus, like many of the small DNA tumor viruses, the first protein expressed upon HCMV infection activates a p53 response by the host cell.

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Figures

FIG. 1.
FIG. 1.
Expression of HCMV IE1-72 results in p53 and p19Arf accumulation. (A to C) Immunoblot detection of p53 and p19Arf. (A) Immunoblot analyses were performed with an anti-p53 polyclonal antibody on whole-cell extracts from WT MEFs infected with either AdIE1-72 or control virus. Cells were harvested at the indicated times postinfection. An extract from p53−/− MEFs was included as a negative control for p53 detection. (B) Whole-cell extracts from REF52 cells infected with either AdIE1-72 or control virus were probed for endogenous p19Arf by immunoblotting with an anti-p19Arf polyclonal antibody. An extract from p19Arf−/− MEFs was included as a negative control for p19Arf detection. (C) Immunoblot analysis for p53 was performed on whole-cell extracts from p19Arf−/− MEFs infected with either AdIE1-72 or a control virus. Cells were harvested at the indicated times postinfection. An extract from uninfected p53−/− MEFs was included as a negative control for p53 detection. An extract from UV-treated (60J/m2) p19Arf−/− MEFs was included as a positive control for p53 accumulation.
FIG. 2.
FIG. 2.
IE1-72 expression and HCMV infection increase the levels of phosphorylated p53. Immunoblot analyses for the phospho-Ser18 form of p53 were performed on whole-cell extracts from WT MEFs (A) and p19Arf−/− MEFs (B) infected with either AdIE1-72 or a control virus. (C) Detection of total and modified forms of p53 in HCMV or AdIE1-72-infected cells. HEL fibroblasts were infected with HCMV, AdIE1-72, or a control virus and whole-cell extracts derived 24 hpi. Phosphorylated forms of p53 at Ser15 or Ser20 were detected by using antibodies specific for each modification. IE1-72 levels were detected with a monoclonal antibody.
FIG. 3.
FIG. 3.
Phosphorylation of p53 at Ser15 after IE1-72 expression requires activated ATM. (A) Immunoblot analysis for total and phospho-Ser15 form of p53 in response to caffeine treatment. HEL fibroblasts were infected with either AdIE1-72 or a control virus and cultured in the presence of increasing doses of caffeine for the 6 h prior to harvesting at 24 hpi. (B) Immunoblot analyses for total and phospho-Ser15 p53 in human cells. Cultures of dermal fibroblasts isolated from an AT patient or an age- and gender-matched normal donor (WT) were infected with either AdIE1-72 or a control virus, and whole-cell extracts were harvested at 24 hpi. (C) Immunoblot analyses for total and phospho-Ser1981 ATM. Whole-cell extracts from HCMV, AdIE1-72 or control virus-infected HEL fibroblasts were generated at 24 hpi.
FIG. 4.
FIG. 4.
IE protein expression leads to ATM activation and p53 phosphorylation. (A) Immunoblot analysis for p53 in human cells. HEL fibroblasts were infected with AdIE2-86 or control virus and whole-cell extracts derived at 24 hpi. (B) Immunoblot detection of ATM and p53 forms in HEL fibroblasts. Cells were mock-infected or infected with HCMV or CR208 and harvested at 24 hpi.
FIG. 5.
FIG. 5.
IE1-72 expression induces p53 nuclear retention. (A) Immunofluorescent detection of p53 shuttling. SAOS-2 cells were coinfected with Adp53 and either AdIE1-72 or AdCon. At 2 h after infection, cells were replated with Mdm2−/−/p53−/− MEFs and cultured overnight at 37°C. The cells were then fused together by treatment with polyethylene glycol and de novo protein synthesis was inhibited with cycloheximide. Cells were fixed 1 h after fusing. Immunofluorescent detection of human Ku-86 (red) and p53 (green) was performed to identify the human-mouse heterokaryons and p53 localization, respectively. (B and C) Quantitation of p53 shuttling. Human-mouse heterokaryons (n = 20) were identified in each experiment, and the detection of p53 in the mouse (Ku-86-negative) nuclei was scored as positive for p53 shuttling. The averaged results from two experiments are shown in panel B. The averaged results from three experiments that include infections of AdCon and the calculated standard errors are depicted in panel C. Numbers in parentheses indicate the MOIs of the recombinant adenovirus used.
FIG. 6.
FIG. 6.
IE1-72 expression induces an accumulation of p21 levels in a p53-dependent manner. (A) Analysis of transcriptional activation of a p21 promoter-reporter. HEL fibroblasts were cotransfected with a p21-promoter luciferase construct, WAF-1-Luc, and either an IE1-72 cDNA expressing plasmid, pcDNA3-IE1-72, or a control plasmid, pcDNA3. A Renilla luciferase plasmid was also cotransfected and served as a normalization control for transfection efficiency in our reporter assays. Samples were harvested at 24 h posttransfection, and the luciferase activity was analyzed as described in the text. The results are depicted as fold induction of p21 promoter activity relative to the control plasmid transfected cells. Error bars indicate the standard error of the mean. (B) Immunoblot analysis for p21. Whole-cell extracts were harvested at 24 hpi from WT MEFs infected with either AdIE1-72 or a control virus. (C and D) Detection of p21 expression by immunohistochemistry. (C) REF52 cells were infected with either AdIE1-72 or a control virus and immunohistochemically stained for p21 protein at 24 and 48 hpi. Cells infected with Adp53 (MOI = 20) served as a positive control for p21 induction. A minimum of 300 cells was scored for each condition. The results are shown as the percentage of cells within the population and represent the average number of p21-positive cells from three separate analyses. Error bars indicate the standard error of the mean. (D) Number of p21-positive cells within populations of p53−/− MEFs infected with AdIE1-72, a control virus, or Adp53. The result from a representative experiment is shown.
FIG. 7.
FIG. 7.
IE1-72 expression induces quiescent cells to enter S phase in the absence of p21. Serum-starved, early-passage WT and p21−/− MEFs were infected with AdIE1-72 or a control virus and maintained under low-serum conditions. At 24 hpi, MEFs were harvested, and DNA was stained with propidium iodide. Flow cytometry analysis was performed, and the percentage of cells in S phase was scored. The averaged results from two separate experiments are represented as histograms for each cell type.
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