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First published online 23 May 2006
doi: 10.1242/jcs.02974

Journal of Cell Science 119, 2457-2467 (2006)
Published by The Company of Biologists 2006
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Mechanisms for human cytomegalovirus-induced cytoplasmic p53 sequestration in endothelial cells

B. Utama1, Y. H. Shen1, B. M. Mitchell2, I. T. Makagiansar3, Y. Gan1, R. Muthuswamy1, S. Duraisamy1, D. Martin4, X. Wang1, M.-X. Zhang1, J. Wang1, J. Wang1, G. M. Vercellotti5, W. Gu6 and X. Li Wang1,*

1 Division of Cardiothoracic Surgery, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX 77030, USA
2 Department of Ophthalmology and Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA
3 Department of Developmental Neurobiology, The Burnham Institute, La Jolla, CA, USA
4 VRL Laboratories, San Antonio, TX, USA
5 Department of Medicine, University of Minnesota, Minneapolis, USA
6 Institute for Cancer Genetics, Columbia University, New York, USA


Figure 1
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  		Fig. 1. p53 protein modulation and subcellular localization in HCMV strain VHL/E-infected HUVECs. Immunofluorescence assay of p53 protein (red, Texas Red-X, upper panels), and merged images of p53 with nuclear HCMV immediate early (IE) proteins (green, FITC) and DAPI (blue, nuclear region; lower panels). p53 is mainly stabilized in the nucleus for the first 3 dpi (yellow arrows), followed by a rapid nuclear exclusion (3 dpi), which transfers virtually all of the p53 to the cytoplasm (3 dpi onward) as shown by red color in cytoplasmic regions (white arrows) adjacent to the colocalized IE (green) and nuclei (blue). Temporary cytoplasmic p53 sequestration would generally last for 48-68 hours. Bars below the pictures indicate the three stages of HCMV infection.

 

Figure 2
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  		Fig. 2. Expression of p53 and Mdm2 in HCMV-infected HUVECs. (A) Protein levels were semi-quantified using western blot analysis of the total cellular protein extracts from HCMV-infected HUVECs (MOI=1.0) during the first 13 dpi. Dynamic changes in protein levels of the total p53, HCMV-immediate-early (IE) proteins (68/72 kDa) and Mdm2 were shown. (B,C) Graphs showing the corresponding mRNA and protein levels. The nucleus and cytoplasm bars show the proportional changes between the two subcellular compartments during the course of infection, based on IFA. All values have been normalized against corresponding actin level, and represent mean (± s.e.m.) of three different batches of HCMV-infected HUVECs (MOI=1.0). Each experiment was conducted in triplicate.

 

Figure 3
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  		Fig. 3. Crm1 levels were transiently increased to facilitate rapid nuclear p53 exclusion in HCMV-infected HUVECs. Western blot analysis (top) and graph of relative protein levels (bottom) showing a significant increase in p53 levels. Crm1 protein levels were slightly decreased when p53 was stabilized in the nucleus during the first 3 dpi. A transient increase in Crm1 levels from 4-7 dpi coincided with the nuclear p53 exclusion. This increase was followed by a decrease with the time of infection to lower levels than before infection. The reduction in Crm1 levels occurred at the same time as p53 was sequestrated in the cytoplasm, which is indicated by the broken bar at the bottom of the graph. The experiments were carried out in triplicate using three different batches of HCMV-infected HUVECs (MOI=1.0).

 

Figure 4
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  		Fig. 4. Effect of LMB on nuclear p53 exclusion in HCMV-infected HUVECs. The infected cells were treated with 20 nM LMB for 18 hours before observation. LMB was able to abrogate the nuclear exclusion when the cells were treated before the onset of nuclear-p53 exclusion (arrows, 4 dpi). Once p53 was completely exported out of nucleus (5 dpi), LMB was unable to inhibit nuclear exclusion. Cells were stained with anti-p53 (red, Texas Red-X), anti-HCMV-IE proteins (green, FITC) and DAPI (blue) to counterstain the nuclei.

 

Figure 5
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  		Fig. 5. The temporary cytoplasmic p53 sequestration (7 dpi) was probably not caused by cytoplasmic tethering or the hyperactive nuclear export. HCMV-infected cells were treated with LMB, CHX or a combination of the two drugs. Incubation times are indicated in hours. All immunofluorescence images (A) were taken with the identical conditions, level of infections, IFA protocol, and equal exposure/digital picture processing conditions. Cells were stained as follows: anti-p53 (red, Texas Red-X), cytoplasmic anti-HCMV late proteins (green, FITC) and DAPI (blue). The corresponding western blot analysis (B) of the mock- and HCMV-infected cell extracts that were isolated from the identically infected cells treated with a single or a combination of the two drugs reconfirmed the IFA results shown in A. Numbers below the lanes are relative p53 protein levels and have been normalized against corresponding actin bands. (C) The pharmacological activity of LMB, 6 and 22 hours after use for the infected HUVECs treatment, was examined in fresh HUVECs. The same nuclear p53 accumulation pattern as those of the direct LMB treatment (data not shown) and increased p53 level confirmed that LMB remained pharmacologically active even 22 hours after incubation with the infected HUVECs.

 

Figure 6
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  		Fig. 6. Cytoplasmic sequestrated p53 had delayed half-life. (A) Western blot analysis of cell extracts after cells were treated with the protein translation inhibitor CHX. Bands were visualized using anti-p53 (DO-1) and anti-I{kappa}B{alpha}. The densities were normalized against the corresponding actin levels. (B) Graphs show the comparison of the p53 half-life between mock- and HCMV-infected HUVECs. p53 half-life was markedly extended from the basal 45-60 minutes in uninfected HUVECs (mainly nuclear p53 at 0 dpi) to more than 8 hours of mainly cytoplasmic p53 at the later time of the infection (B).

 

Figure 7
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  		Fig. 7. The time-dependent changes in ubiquitinated p53 pattern in HCMV-infected HUVECs. Western blot with anti-p53 (fl-393) polyclonal antibody was employed to demonstrate the ubiquitinated p53 pattern in uninfected (0 dpi) and HCMV-infected (1-11 dpi) HUVECs with (B) or without (A) 10 µM MG132 (proteasome inhibitor) treatment. In the MG132-treated cells, mono-ubiquitinated p53 (p53-Ub(1) at ~60 kDa) significantly increased towards the later stages of infection (3 dpi onwards). The increased levels were observed also for the corresponding double mono-ubiquitinated bands (p53-Ub(2) at ~75 kDa). By contrast, the multiple mono-ubiquitinated p53 bands (p53-Ub(n)) of higher molecular masses tended to decrease towards the later stages of infection (3 dpi onwards). Molecular masses are given on the left in kDa.

 

Figure 8
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  		Fig. 8. MG132 treatment did not cause any significant changes in p53 and Mdm2 patterns or subcellular localization in HCMV-infected HUVECs. HCMV-infected cells (5 dpi at MOI=0.3) were treated with or without MG132 for 6 hours before IFA. All IFA images were taken with the identical cells conditions, level of infections, IFA protocol, and equal exposure/digital picture processing conditions. Cells were stained red by the anti-p53-Texas Red-X, green by anti-Mdm2-FITC and blue by DAPI for nuclear counterstaining. MG132 treatment had no effect on the subcellular localization of p53. However, a slight increase of both p53 and Mdm2 levels can be observed after MG132 treatment compared to cells without treatments. Stars indicate the uninfected cells, which have relatively low levels of p53 and Mdm2 in the nuclear region. Mdm2 and p53 levels were significantly increased, mainly in the nuclear region of the infected cells at very early stage of post-infection (white arrows). During the period of p53 nuclear exclusion (white arrows `a') the p53 and Mdm2 levels were elevated mainly in the nuclear and also in parts of the cytoplasmic regions. At the later stages of infection, cytoplasmic p53 sequestration was observed coincidentally with the lower levels of Mdm2 (yellow arrows).

 

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© The Company of Biologists Ltd 2006