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. 2013 Mar;87(5):2463-74.
doi: 10.1128/JVI.01926-12. Epub 2012 Dec 12.

Human cytomegalovirus pUL29/28 and pUL38 repression of p53-regulated p21CIP1 and caspase 1 promoters during infection

John P Savaryn  1 , Justin M Reitsma, Tarin M Bigley, Brian D Halligan, Zhikang Qian, Dong Yu, Scott S Terhune
Affiliations

Human cytomegalovirus pUL29/28 and pUL38 repression of p53-regulated p21CIP1 and caspase 1 promoters during infection

John P Savaryn et al. J Virol. 2013 Mar.

Abstract

During infection by human cytomegalovirus (HCMV), the tumor suppressor protein p53, which promotes efficient viral gene expression, is stabilized. However, the expression of numerous p53-responsive cellular genes is not upregulated. The molecular mechanism used to manipulate the transcriptional activity of p53 during infection remains unclear. The HCMV proteins IE1, IE2, pUL44, and pUL84 likely contribute to the regulation of p53. In this study, we used a discovery-based approach to identify the protein targets of the HCMV protein pUL29/28 during infection. Previous studies have demonstrated that pUL29/28 regulates viral gene expression by interacting with the chromatin remodeling complex NuRD. Here, we observed that pUL29/28 also associates with p53, an additional deacetylase complex, and several HCMV proteins, including pUL38. We confirmed the interaction between p53 and pUL29/28 in both the presence and absence of infection. HCMV pUL29/28 with pUL38 altered the activity of the 53-regulatable p21CIP1 promoter. During infection, pUL29/28 and pUL38 contributed to the inhibition of p21CIP1 as well as caspase 1 expression. The expression of several other p53-regulating genes was not altered. Infection using a UL29-deficient virus resulted in increased p53 binding and histone H3 acetylation at the responsive promoters. Furthermore, expression of pUL29/28 and its interacting partner pUL38 contributed to an increase in the steady-state protein levels of p53. This study identified two additional HCMV proteins, pUL29/28 and pUL38, which participate in the complex regulation of p53 transcriptional activity during infection.

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Figures

Fig 1
Fig 1
HCMV pUL29/28 and pUL38 interact with cellular p53. (A) Fibroblasts were infected at an MOI of 6 with ADwt (wt) or ADin28FLAG-C (in28FLAG-C). Immunoprecipitation was performed at 24 hpi from whole-cell lysates using an antibody against p53 or the HA epitope. Western blotting was performed using antibodies directed against the indicated proteins. (B) U2OS cells stably expressing pUL38 (U2OS-pUL38) were transfected with p53FLAG and either empty pCGN, pUL29/28HA, or pUL29HA. Immunoprecipitation was performed at 48 h posttransfection using whole-cell lysates and an antibody against the FLAG epitope. Western blot analysis was completed using antibodies against the indicated proteins.
Fig 2
Fig 2
Expression of HCMV pUL29/28 and pUL38 alters p53-regulatable gene expression in the absence of infection. (A) Control U2OS or U2OS-pUL38 (+pUL38) were transfected with a plasmid containing the p21CIP1 promoter driving luciferase along with 1.0, 10.0, and 100.0 ng of either empty pCGN or pUL29/28 plasmid DNA. Relative luciferase activity was measured at 48 h after transfection. The data are means for two replicate samples ± standard deviations. Statistical analysis was performed using Student's t test (**, P ≤ 0.01). (B) The U2OS and U2OS-pUL38 (+pUL38) cells were either transfected with empty vector or pUL29/28, or treated with nutlin-3 for 24 h. Cells were harvested 48 h posttransfection. Western blot analysis was performed using antibodies against the indicated proteins. (C) U2OS cells were transfected with pUL29/28HA expression vector. At 40 h posttransfection, the cells were treated with or without nocodazole to synchronize the population. Cells were stained using antibody against the HA epitope and propidium iodide, and DNA content was measured by flow cytometry. The data are displayed as percentages of cells in each phase of cell cycle and are the means from two biological experiments ± standard errors of the means. Statistical analysis was performed using Student's t test (*, P ≤ 0.05; **, P ≤ 0.01).
Fig 3
Fig 3
Disruption of pUL29/28 during infection results in elevated expression of a subset of p53-regulatable genes. (A) Fibroblasts were either mock infected or infected with ADwt or ADdel29 at an MOI of 6 and harvested at the indicated times postinfection. Relative RNA was measured by qRT-PCR for the indicated genes and normalized to cellular GAPDH levels. The data are the means for two technical replicates ± standard deviations, with the results being validated in a separate independent biological replicate. Statistical analysis was performed using Student's t test (*, P ≤ 0.05; **, P ≤ 0.01). (B) Experiments were completed as described above, but no statistical difference in expression were observed.
Fig 4
Fig 4
pUL29/28 influences p21CIP1 and CASP1 expression during infection. (A) Fibroblasts were infected with ADwt or ADdel29 at an MOI of 6 and harvested at the indicated times postinfection. Relative RNA was measured using qRT-PCR. (B) Samples were collected at the indicated times postinfection and processed for Western blot analysis using antibodies against the indicated proteins. (C) For nutrient stress, fibroblasts were serum starved for 24 h and infected with ADwt or ADdel29 at an MOI of 6 in 7% FBS, 25 mM glucose (hi) for 1 h. Cells were then washed and replenished with either normal medium (hi) or medium containing 5.56 mM glucose and no serum (lo). Western blot analysis was performed using whole-cell lysates harvested at the indicated times postinfection as well as from mock-infected samples and antibodies against the indicated proteins.
Fig 5
Fig 5
HCMV pUL38 contributes to the regulation of p53 during infection. (A) Fibroblasts were either mock infected or infected with ADwt or ADdel38 at an MOI of 5. At 18 hpi, RNA was collected, and qRT-PCR was used to measure relative RNA levels normalized to GAPDH for the indicated genes. (B) Fibroblasts were either mock infected or infected with ADwt or ADdel38 and evaluated by Western blotting at 24 and 48 hpi using the indicated antibodies.
Fig 6
Fig 6
pUL29/28 influences p53 and histone H3 K9 acetyl binding to responsive promoters. (A) Fibroblasts were mock infected, treated with 10 μM nutlin-3 for 18 h, or infected with ADwt or ADdel29 at an MOI of 6 for 18 h. Chromatin immunoprecipitation (ChIP) was performed using an antibody against pan-p53 or control IgG. Quantitative PCR was used to determine the relative output DNA levels normalized to input. The data are means from four replicate experiments ± standard deviations and are presented as percentage of input. The PUMA data set represents two experiments. (B) ChIP analysis was performed as described above but using an antibody against histone 3 lysine 9 acetylation. The data are means for four replicates ± standard deviation and are presented as percentages of input. Statistical analysis was performed using Student's t test (*, P ≤ 0.05).
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