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First published online 23 May 2006
doi: 10.1242/jcs.02974
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Research Article |
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
* Author for correspondence (e-mail: xlwang{at}bcm.tmc.edu)
Accepted 8 March 2006
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Key words: Human cytomegalovirus, p53, Endothelial dysfunction
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Introduction |
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; Kuerbitz et al., 1992), or apoptosis to eliminate cells with damaged genomes (Kuerbitz et al., 1992; Vogelstein et al., 2000). The main tumor suppressor activities of p53 in apoptosis are based on the transcription activation of pro-apoptotic target genes (Chao et al., 2000) or through suppression of anti-apoptotic genes (Murphy et al., 1999). The p53 protein has a short half-life and is usually expressed at low levels in unstressed cells. A delicate system, including ubiquitination and proteasomal degradation in the cytoplasm with Mdm2 as the main regulator of p53, keeps the levels of p53 protein low in unstressed cells (Haupt et al., 1997). The p53 protein is synthesized in the cytoplasm and is actively imported into the nucleus (Giannakakou et al., 2000) where it forms tetramers, which allow for DNA binding, thus making p53 transcriptionally active (McLure and Lee, 1998). Mutation of the p53 protein is the most common mechanism for inactivation of p53 DNA-binding function and can be identified in many different types of cancers (Ryan et al., 2001). However, aberrant sub-cellular localization of p53, such as in nuclear viral replication centers (Fortunato and Spector, 1998) or the cytoplasm (Takemoto et al., 2004), provides another inactivation pathway.
Human cytomegalovirus (HCMV) is one of the largest members of the herpesvirus family. It contains a double-stranded DNA genome of 229,354 bp, which encodes more than 200 different proteins. It is also one of the most common non-symptomatic pathogens in the adult population. HCMV can potentially infect all tissues and is capable of establishing a life-long latent infection after the primary infection. Endothelial cells are one of the major targets of HCMV infection where it potentially establishes latency (Sinzger et al., 1995). Following infection, HCMV gene expression occurs as a cascade in three stages designated as immediate early (IE), within the first 24 hours post infection (hpi); early, within 24-48 hpi; and late, after 48 hpi (Speir et al., 1994). Interestingly, HCMV infection in primary endothelial cells (Wang et al., 2000) and fibroblasts (Fortunato and Spector, 1998) is accompanied by an increase in p53 levels. Studies from this laboratory and others have also demonstrated that HCMV infection can cause severe endothelial dysfunction, e.g. dysregulated apoptosis (Kovacs et al., 1996; Reboredo et al., 2004; Shen et al., 2004) and hampered apoptosis in HeLa cells (Zhu et al., 1995), cancer cells (Michaelis et al., 2004) and fibroblasts (Reboredo et al., 2004). We previously reported that p53 is stabilized in the nucleus at the early stage of HCMV infection and becomes sequestrated in the cytoplasm at the later stage of the infection (Kovacs et al., 1996; Wang et al., 2001; Wang et al., 2000). The mechanism for this sequestration remains to be determined.
In the present study, we investigated p53 modulation and subcellular localization during the course of HCMV infection in human umbilical vein endothelial cells (HUVECs). For this purpose, we examined three possible hypotheses for the molecular events leading to cytoplasmic p53 sequestration. First, we examined the hypothesis that the increased cytoplasmic p53 could be due to an accelerated p53 nuclear export. Second, we investigated whether tethering within the cytoplasm could prevent p53 from entering the nucleus, thereby causing cytoplasmic sequestration. Third, whether delayed cytoplasmic degradation could cause cytoplasmic sequestration. Our findings suggest that the delayed p53 degradation is likely to be the predominant mechanism for cytoplasmic p53 sequestration in HCMV-infected HUVECs.
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), which did not permit sufficient time for cell replication and secondary infection. To better assess the mechanism of p53 modulation and sub-cellular localization, a time-dependent study of the dynamic changes of p53 levels were performed after a prolonged infection (13 dpi). We observed that during the first 3 dpi, p53 protein levels increased and predominantly localized in the nucleus, which was then followed by a rapid nuclear exclusion, and a concomitant increase in p53 in the cytoplasm at 3 dpi onward (Fig. 1). The later event coincided with the high expression of HCMV late proteins (data not shown). However, a low level of p53 was detectable by immunofluorescence assays (IFA) in the nucleus of the infected cells (Fig. 1, 5 dpi onward). The cytoplasmic p53 level was stabilized between 4 and 6 dpi before its level started to decrease. This observation was verified by western blotting, which showed that HCMV infection in HUVECs induced a gradual increase in the total amount of p53 approximately 3.5-fold over the steady-state levels during the first 3 dpi at a multiplicity of infection (MOI)=1.0 (Fig. 2A,B). The elevation started to appear after 1 dpi, while the HCMV immediate-early protein expression in the nucleus was detected as early as 12 hours post-infection (hpi; data not shown).
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). Furthermore the initial sub-nuclear localization of p53 during nuclear stabilization was restricted to discrete foci (data not shown), which resembled the viral replication center reported earlier in HCMV-infected fibroblasts (Fortunato and Spector, 1998). These discrete foci enlarged throughout the infection to almost the entire nuclear region; but this could not be detected once the nuclear exclusion of p53 had occurred.
Mdm2 is a p53-specific E3-ubiquitin ligase that ubiquitinates nuclear p53 and allows for nuclear export, followed by proteasome-dependent degradation in the cytoplasm (Fang et al., 2000). Low levels of Mdm2 or disruption of the Mdm2-p53 interaction may cause the p53 stabilization in nucleus. As shown in Fig. 2C1, Mdm2 protein levels were not markedly changed during the p53 nuclear stabilization period (1-3 dpi), although a significant increase occurred up to 11 dpi. The Mdm2 mRNA levels showed a similar trend (Fig. 2C2) suggesting no significant perturbation occurred at the Mdm2 transcription level during the nuclear p53 accumulation (1-3 dpi).
Rapid p53 nuclear exclusion coincided with an elevated expression of Crm1
Nuclear export/import is crucial for most nuclear proteins to function properly, including p53 (Middeler et al., 1997). Transport of p53 protein into the nucleus relies on nuclear localization signals (NLS) located at the C terminus. Once p53 completes its functional requirements in the nucleus, the ubiquitinated p53 monomer utilizes two leucine-rich nuclear export signals (NES) allowing p53 to follow an active Crm1-RanGTP nuclear export pathway (Lohrum et al., 2001). During the course of HCMV infection in HUVECs, we found that Crm1 protein expression varied (Fig. 3), but appeared to fit into the dynamic requirements of the p53 nuclear/cytoplasmic shuttling. An initial decrease of Crm1 was noticed during the first 3 dpi, followed by a marked increase, with peak levels detected at 5-7 dpi. After 7 dpi, the Crm1 levels gradually decreased to below the baseline level (0 dpi).
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). Cells were treated at 2, 3, 4 and 5 dpi for 18 hours before observation. As shown in Fig. 4, the nuclear export inhibitor LMB was able to abrogate the nuclear exclusion of the p53 if the cells were treated before the nuclear exclusion process started, between 3-4 dpi (Fig. 4, see arrow). This resulted in a reduced cytoplasmic p53 sequestration. However, when HUVECs were treated at 5 dpi, when p53 had already been completely exported out of nucleus, LMB was no longer effective in reducing the cytoplasmic p53 (Figs 3, 4). Furthermore, during the LMB treatment we were able to observe a significant increase in nuclear p53, presumably nascent p53, which was imported and trapped into the nucleus. This is in agreement with the fact that p53 has been continuously transcribed and translated during the course of infection (Fig. 2B1,B2).
Temporary cytoplasmic p53 sequestration after rapid nuclear exclusion was less likely to be caused by tethering or nuclear hyper-exportation
To investigate the mechanisms responsible for the cytoplasmic p53 sequestration after rapid nuclear exclusion during the later stages of HCMV-infection of HUVECs, we examined three hypotheses: (1) p53 is tethered in the cytoplasm, therefore, after being translated p53 is unable to enter the nucleus; (2) there is a hyperactive nuclear export of p53, as reported in neuroblastoma (Stommel et al., 1999); and (3) there is an extended half-life of cytoplasmic p53.
To distinguish between the first two possible mechanisms of cytoplasmic p53 sequestration, we treated HUVECs at 7 dpi with 10 nM LMB plus 40 µg/ml cycloheximide (CHX). LMB has been reported to not induce the p53 stress response (Freedman and Levine, 1998). We selected 7 dpi because p53 localized in the cytoplasm in most of the infected cells (MOI=1.0) at this stage and yet the p53 transcription and translation levels were still relatively the same as the early stages of infection. If the cytoplasmic p53 in infected cells was constitutively tethered, then p53 protein should not remain in the cytoplasm for 6 hours after CHX, since p53 half-life is normally around 45-60 minutes. Furthermore, upon treatment with LMB, p53 should remain in the cytoplasm without any nascent p53 being trapped in the nucleus. If cytoplasmic p53 sequestration is primarily due to hyperactive nuclear export, then treatment with LMB should trap p53 in the nucleus and cytoplasmic p53 levels should be diminished.
As shown in Fig. 5Af, after protein translation was blocked by CHX for 6 hours in 7 dpi HUVECs, there was no significant change in cytoplasmic p53. In the meantime, the abundance of nuclear p53 was lowered to similar levels as those in untreated infected cells (Fig. 5Aa). Analysis of the corresponding western blot analysis from total cell lysate using NIH Image J (Fig. 5B, infected cells) confirmed an approximately 15% decrease of the total p53 protein. Furthermore, after LMB treatment for 6 hours, a significant increase of nuclear p53 was detectable (Fig. 5Ab) corresponding to an increase of total p53 protein of approximately 20% (Fig. 5B, infected cells). These results indicate that nascent p53 was continuously transcribed (Fig. 2B2), translated, imported, and stabilized in the nucleus during the period when p53 is sequestered in the cytoplasm at the later stages of infection. Hence, we suggest that tethering is unlikely to be the primary mechanism for cytoplasmic p53 sequestration.
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The accumulated p53 after LMB treatment during the later stages of infection apparently came from nascent p53 and not from the shuttling p53. Using a modified protocol described previously (Joseph et al., 2003), 7 dpi HUVECs were given a combined treatment of LMB (22 hours) and CHX (for the last 6 hours of the 22 hours LMB treatment). LMB was added to block nuclear export and CHX was added subsequently to block nascent p53 synthesis to ensure that only the previously stabilized pools of p53 was observed (Fig. 5Ad). We compared the combined treatment to LMB treatment alone for 6 and 22 hours. As shown in Fig. 5A, the abundance of nuclear p53 after the LMB (22 hours)/CHX (6 hours) treatment was significantly lower than after the LMB (6 or 22 hours) treatment alone. These were equivalent to approximately 
8% and 10% decrease in the total p53 protein level, respectively (Fig. 5B). These findings indicate that the trapped p53 in the nucleus after LMB treatment during the cytoplasmic p53 sequestration at the later stages of HCMV infection was indeed nascent p53.
We then examined the possibility of hyperactive nuclear export during temporary cytoplasmic p53 sequestration after rapid nuclear exclusion. After LMB treatment for 6 or 22 hours, no gross changes in cytoplasmic p53 were observed (Fig. 5Ab,c). By contrast, nuclear p53 staining was significantly increased. These findings indicate that hyperactive nuclear export was unlikely to be responsible for cytoplasmic p53 sequestration after rapid nuclear exclusion of p53. To show that LMB was still pharmacologically active after the delayed incubation (22 hours), we transferred LMB-containing medium, after 6 and 22 hours of treatments, to fresh 7 dpi cells. As indicated by the increased p53 levels, the 6-and 22-hour old LMB-containing media still actively inhibited nuclear export in untreated 7 dpi infected cells after 6 hours of incubation (Fig. 5C).
Temporary cytoplasmic p53 sequestration had an extended half-life
We then examined whether the cytoplasmic p53 sequestration at the later stage of infection was caused by an extended p53 half-life. As shown in Fig. 6A,B, from the time-frame observation of uninfected and infected-cells after protein translation inhibitor CHX treatment, 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 (7 dpi onward).
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In cells received no MG132 treatment, the band of mono-ubiqitinated p53 (larger than the p53 monomer with an estimated molecular mass of 60 kDa) was faint and barely detectable (Fig. 7A). By contrast, cells treated with 10 µM MG132 for 6 hours (Shibata et al., 2002) showed multiple bands of ubiquitinated p53 (Fig. 7B). They included a distinct single band representing the single mono-ubiquitinated p53 (p53-Ub(1) at 60 kDa), a double mono-ubiquitinated p53 (p53-Ub(2) at 75 kDa) and several multiple mono-ubiquitinated p53 (p53-Ub(n)) bands of higher molecular masses. The intensity for the band corresponding to single mono-ubiquitinated p53 (p53-Ub(1)) was significantly increased after 3 dpi in HCMV-infected cells. The results suggest that in the absence of MG132, the ubiquitinated p53 may have been rapidly degraded in the cytoplasm. When proteasomal activity was inhibited by MG132, however, the ubiquitinated p53 was preserved/stabilized and became detectable.
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Our experiments further showed that the p53 ubiquitination patterns were not significantly different in cells with or without MG132 treatment at the very early stage of the infection (1 dpi), in which p53 was mostly present within nucleus. From 3 dpi onward, concomitant with the p53 nuclear exclusion and cytoplasmic p53 sequestration, the levels of ubiquitinated-p53 changed dynamically (Fig. 7B). Whereas the single (p53-Ub(1)) and double (p53-Ub(2)) mono-ubiquitinated p53 increased significantly towards the later stage of infection, the multiple mono-ubiqutinated p53 (p53-Ub(n)) apparently decreased during the same period.
It was of interest to see whether the dynamic changes of p53, its ubiquitinated forms, and Mdm2 proteins during HCMV infection after MG132 treatment may affect its subcellular localization and expression pattern. To explore this issue, IFA analysis was utilized to observe the MG132 treated/untreated HCMV-infected cells (MOI=0.3) at 5 dpi using indirectly fluorescence-labeled anti-p53 (fl-393) rabbit pAb and anti-Mdm2 mAb. As shown in Fig. 8, MG132 treatment had no effect on the subcellular localization of p53. Only a slightly stronger signal was observed from the MG132-treated cells in comparison to the untreated cells. However, it was important to observe the effects of MG132 on Mdm2 subcellular localization and levels as well. As shown in Fig. 8, during the early stages of infection or in non-infected cells, relatively low levels of Mdm2 were observed mainly in the nuclear region (labeled with stars). The Mdm2 levels increased dramatically in the nucleus with the progression of infection (white arrows), and during the onset of p53 nuclear exclusion (white arrows with label `a') in both the nucleus and cytoplasm. When the p53 protein was temporarily sequestered in the cytoplasm at the later stage of infection (shown by the presence of the owl eye or the enlarged kidney shape of infected nuclei indicated by the yellow arrow, Fig. 8), the levels of Mdm2 were significantly reduced in the cytoplasm and in most areas of the nucleus. This observation confirmed our previous results of dynamic changes of Mdm2 during the HCMV infection, using western blot analysis (Fig. 2).
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We further explored the correlation between Crm1 expression and the dynamics of p53 subcellular localization. The Crm1 protein level at 0 dpi was relatively higher than at 3 dpi, possibly because of the need for active nuclear/cytoplasmic shuttling of p53 in unstressed cells (Fig. 3). The modest decrease of Crm1 levels during the first 3 dpi, which coincided with the accumulation of p53 in nucleus, could result in the reduced p53 nuclear export. The markedly increased Crm1 levels at 5-7 dpi possibly allowed the rapid nuclear exclusion of p53. When p53 was temporarily sequestered and later degraded in the cytoplasm, Crm1 apparently was no longer functionally required, therefore gradually decreased. These findings suggest that the changes in Crm1 levels may respond to the dynamics of p53 subcellular localization rather than the steady state p53 levels. As p53 is not the only protein using the active Crm1-RanGTP nuclear export pathway (Fornerod et al., 1997), we speculate that a specific mechanism, based on the p53 subcellular localization and/or HCMV proteins expressed, may regulate Crm1 expression or they may work in cooperation to meet the needs for a dynamic nuclear p53 export during the HCMV infection.
The rapid nuclear exclusion was concomitant with the increase in cytoplasmic p53. By contrast to normal unstressed cells in which ubiquitinated p53 was degraded quickly in the cytoplasm, we identified a temporarily cytoplasmic p53 sequestration in HUVECs during the later stages of HCMV infection. We found that the cytoplasmic localization of p53 was not in a punctate pattern or as a p53 cytoplasmic body as frequently identified in cells infected with adenovirus Type-12 E1B-oncoprotein (Zhao and Liao, 2003), or as finely dispersed constitutive cytoplasmic p53 as observed in cells productively infected by human herpesvirus-6 (Takemoto et al., 2004). Furthermore, using anti-p53 pAb421 (Ostermeyer et al., 1996), we showed that the cytoplasmic sequestered p53 was either in monomeric/dimeric forms (data not shown). This finding indicates that the cytoplasmic p53 detected during the later stage of infection had been ubiquitinated in the nucleus, allowing conformational changes or disruption of tetramer p53 and subsequent nuclear export (Gu et al., 2001). Our series of experiments (Figs 4, 5) clearly indicated that the temporary cytoplasmic p53 sequestration during the later stage of infection after rapid nuclear exclusion (4 dpi onward) was not due to hyperactive nuclear export.
There were no significant changes in cytoplasmic p53 when protein translation was blocked at 7 dpi with CHX treatment (6 hours) alone (Fig. 5Af). We further showed that nascent p53 was trapped in the nucleus of 7 dpi HUVECs when cells were treated with either LMB/CHX or LMB alone (Fig. 5A,B). All the evidence suggests that a tethering mechanism would play, at best, a minor role in cytoplasmic p53 sequestration.
With both hyperactive nuclear export and the tethering mechanisms being unlikely to contribute to the cytoplasmic p53 sequestration in HCMV-infected HUVECs, our findings suggest that extended p53 half-life may be the responsible mechanism. Several possibilities for extended p53 half-life in HCMV-infected HUVECs have been investigated here. We show that the lower proteasomal degradation activity after HCMV infection in endothelial cells may contribute to some extent, as also shown in adenovirus E1A-infected cells, in which inhibition of proteasomal activities by targeting the proteasomal regulatory subunit S2, extended p53 half-life and increased the level of p53 without inhibiting the p53 ubiquitination process (Turnell et al., 2000; Zhang et al., 2004). However, this may not play a primary role here, since the half-life of I
B
protein increased only modestly from an average of 3-4 hours to 5-6 hours during the course of infection. While the IB half-life extension stayed relatively the same after the infection, p53 half-life became more extended toward the later stage of infection, during which p53 protein was mainly present in the form of cytoplasmic p53 (Fig. 6). We propose that the significant extension of the cytoplasmic p53 half-life is likely to be caused by the dysregulated p53 ubiquitination process.
We have demonstrated a different p53 ubiquitination patterns between the early stage (mostly nuclear p53) and the late stage of infection (mostly cytoplasmic p53), which were observed after the inhibition of proteasomal degradation by MG132. We identified that when the majority of p53 was sequestered in the nucleus (1 dpi), the ubiquitinated p53 pattern remained relatively unchanged (Fig. 7B) amid the slight increase of the total p53 levels. This suggests that there was no significant changes in the p53 ubiquitination pattern during the very early days of infection in comparison to the cells before infection, although the accumulation of both p53 and Mdm2 could be observed in the nucleus. A significant increase of the single mono-ubiquitinated p53 (60 kDa) was demonstrable by the time of the p53 nuclear exclusion (>3 dpi), whereas the increased levels of double mono-ubiquitinated p53 (75 kDa) were observed at 7 dpi onward. By contrast, multiple-ubiquitinated p53 at higher molecular masses tended to decrease toward the later stage of infection after a brief spike at 3 dpi (Fig. 7B).
Since Mdm2 is responsible for different forms of p53 ubiquitination, we postulate that the p53 sequestration at the later stage of HCMV infection in HUVECs could be due to the dynamics of Mdm2 levels. It has been reported that Mdm2 alone at low levels, catalyzes mono-ubiquitination, which is adequate for p53 nuclear export. At a high concentration, however, Mdm2 induces poly-ubiquitination and facilitates degradation (Li et al., 2003). Therefore, the ubiquitination outcome is determined by the Mdm2:p53 ratio. The low ratios will result in p53 mono-ubiquitination, whereas the high ratios (3.6 and above) will poly-ubiquitinate p53 (Li et al., 2003). Our results showed that during the nuclear p53 accumulation (<3 dpi), Mdm2 also accumulated in the nucleus (Fig. 8). However, during the same period of the first 3 dpi the Mdm2 levels increased only modestly ( twofold) in comparison to a highly increased p53 levels (3.5 fold, Fig. 2B1,C1). We propose that the Mdm2 levels during the very early days of infection (<3 dpi) might not be sufficient to induce a proper poly-ubiquitination of the disproportionately large amount of p53. Alternatively, the p53 poly-ubiquitination process could also be inhibited by the increased p53 phosphorylation at serine 15 or 20, as we reported previously (Shen et al., 2004). Phosphorylation of these positions is known to hinder Mdm2-p53 interaction and subsequent ubiquitination/degradation (Shieh et al., 1997).
As shown in Figs 2 and 8, Mdm2 reached a high level at 3 dpi. It appears that Mdm2 levels at this stage were sufficient to mono-ubiquitinate p53 for nuclear export, but not for p53 poly-ubiquitination and degradation. However, we cannot exclude the possibility that the HCMV proteins expressed, in addition to Mdm2 activities, may help to export p53 out of nucleus. This is suggested by the fact that adenovirus E4orf6 and E1B55K (Blanchette et al., 2004) and HSV-1 regulatory protein ICP0 (Boutell and Everett, 2003) have been reported to be able to interact and ubiquitinate p53 for subsequent nuclear export and proteasomal degradation. It has also been shown that human papillomaviruses (HPV) type 18 E6 can poly-ubiquitinate p53, independent of Mdm2, for nuclear proteasomal degradation or for a nuclear export to cytoplasm through the Crm1 nuclear export pathway for subsequent cytoplasmic proteasomal degradation (Stewart et al., 2005). In addition, HCMV proteins US2 and US11 are known to catalyze the dislocation and transfer of MHC class I heavy chains from the endoplasmic reticulum for ubiquitination and degradation (Shamu et al., 2001; Shamu et al., 1999; Wiertz et al., 1996), providing additional evidence that viral proteins can influence protein trafficking within the cell for ubiquitination and proteasomal degradation.
Our results have further shown that at 3 dpi onwards the single and double mono-ubiquitinated p53 levels were significantly increased whereas the multiple mono-ubiquitinated p53, of higher molecular mass, tended to decrease. It is interesting that the dynamics of p53 ubiquitination forms all coincided with the cytoplasmic p53 sequestration. It is tempting to hypothesize that the increased presence of mono-ubiquitinated cytoplasmic p53 could be related to increased endothelial apoptosis after 4 dpi of HCMV infection, as we previously reported (Shen et al., 2004). The onset of apoptosis was also coincidental with the cytoplasmic p53 sequestration as detailed above. Previous studies have shown that p53 can trigger apoptosis in the cytoplasm by direct interaction with anti-apoptotic Bcl-2 proteins in mitochondria, which liberates Bax and triggers apoptosis (Chipuk et al., 2004; Erster et al., 2004; Mihara et al., 2003). We speculate that this mechanism might be applied to the sequestrated cytoplasmic p53 in HCMV-infected HUVECs. This speculation is supported by the fact that mono-ubiquitination also helps proteins to traffic to a proper destination at subcellular level, i.e. single mono-ubiquitinated p53 to mitochondria. A study is currently underway to investigate this hypothesis.
The decreased levels of the higher molecular mass multiple mono-ubiquitinated products at the later stage of infection could also be the result of the presence of the deubiquitinating enzymes such as herpes virus-associated ubiquitin-specific protease (HAUSP). HAUSP directly deubiquinates and stabilizes p53 (Li et al., 2002), and Mdm2 can act a the substrate for HAUSP under certain physiological conditions, which in turn control the p53 levels (Cummins and Vogelstein, 2004). Whether levels of HAUSP change during the later stages of HCMV infection remains to be determined. It is clear that in addition to a general slow-down of the proteasomal activity (Turnell et al., 2000; Zhang et al., 2004) an inadequate p53 poly-ubiquitination at the later stage of HCMV infection could contribute to reduced cytoplasmic p53 degradation, hence the cytoplasmic p53 sequestration.
In summary, our study shows, for the first time, in HCMV-infected endothelial cells that p53 cytoplasmic sequestration, which is a possible mechanism for HCMV-induced endothelial dysfunction, is caused by the increase of p53 half-life and an abnormal p53 ubiquitination pattern and degradation. It is not clear, however, whether these changes in p53 protein are directly caused by HCMV gene products or the host cell proteins triggered by the HCMV infection. Using the modern tools of protein-protein interactions, studies are currently under way in our laboratory to examine the roles of individual HCMV proteins in p53 nucleocytoplasmic trafficking. Our findings, nevertheless, put forward yet another potential system facilitating complicated interactions between viruses and cells to set a favorable environment for viral survival and/or replication, which would simultaneously result in endothelial dysfunction.
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; Kovacs et al., 1996). Cells were grown in culture flasks, pre-coated with sterile 0.5% gelatin, at 37°C in a humidified 5% CO2 in F12K medium (VitaCell-ATTC, Manassas, VA, USA) containing 20% fetal bovine serum (Invitrogen Corp., Carlsbad, CA), penicillin (100 IU/ml) and streptomycin (100 µg/ml; Cellgro Mediatech, Inc., Herndon, VA), 100 µg/ml sodium heparin (Acros, NJ, USA), and 30 µg/ml endothelial cell growth supplement (Sigma). Cells up to passage eight were used in the experiments unless otherwise stated.
Virus preparation
Three strains of HCMV [AD169 (ATCC #VR-538), Towne (ATCC #VR-977) and VHL/E (kindly provided by Jim Waldman, Ohio State University, OH, USA)] were used in the early study. For preparation of a high titer of virus stocks and to preserve the natural endothelial cytopathogenicity of the original isolate, all strains were propagated in HUVECs as described previously (Kahl et al., 2000). However, as the VHL/E strain was the strain that caused the highest permissive HCMV infection in HUVECs, we used this strain throughout the study. Sub-confluent monolayer of HUVECs were infected with multiplicity of infection MOI=0.01 pfu/cell, harvested at 100% cytopathic effect (CPE), and kept in a 1:1 mixture of medium and sucrose phosphate buffer (with a sucrose concentration of 188 mM) at -80°C until used. Virus titer was determined with rapid quantization using a monoclonal antibody to the major immediate-early (IE) viral protein as reported previously (Chou and Scott, 1988; Waldman et al., 1989).
Infection of HUVECs with HCMV
The fully confluent non-synchronized HUVEC monolayers were infected with HCMV at approximately MOI=1.0 (unless otherwise stated) and incubated at 37°C for 1-2 hours for virus absorption. The monolayers were then washed three times with pre-warmed Dulbecco phosphate-buffered saline (PBS), before fresh complete medium was added. Cells were cultured at 37°C in a CO2 incubator. Only attached cells were harvested at various days post-infection (dpi).
Antibodies and chemicals
The following antibodies were obtained from various commercial sources: anti-human Mdm2 (IF2) monoclonal antibodies (mAb; Zymed, San Francisco, CA, USA); anti-human Mdm2 (SMP14) mAb (BD Bioscience), anti-Crm1 mAb and anti-IB pAb (BD Pharmingen); anti-p53 (DO-1) mAb, anti-p53 (fl-393) polyclonal antibodies (pAb), anti-actin pAb (Sigma); Texas Red-X conjugated goat anti-rabbit antibodies (Molecular Probes); anti-HCMV-immediate early antigen mAb, anti-HCMV-late antigen mAb and FITC goat anti-mouse (Chemicon); horseradish peroxidase-conjugated goat anti-rabbit IgG and horseradish peroxidase-conjugated goat anti-mouse IgG (Jackson Immuno-Research); mouse and rabbit IgG (Santa Cruz). All chemicals were purchased from Sigma except paraformaldehyde solution (16%), which was from Electron Microscopy Sci., Washington, PA, and protein A/G-Plus agarose beads, which were from Santa Cruz. Trizol was from Invitrogen. RNase-free DNase I was from Promega, and the iScript cDNA synthesis kit and SYBR Green I Supermix kit were from Bio-Rad. Oligonucleotides were purchased from IDT Inc., Houston, TX, USA.
Western blot analysis
HUVECs were lysed in Laemmli SDS sample buffer (LSB: 50 mM Tris, pH 6-8, 2% SDS, 10% glycerol, 50 mM dithiothreitol, 0.5% bromophenol blue) at a concentration of 103 cells/µl LSB, mixed and boiled for 5 minutes. Equal volumes of whole cell lysate were loaded on to 10% or 12% SDS-polyacrylamide gels (for PAGE), fractionated by electrophoresis and transferred to PVDF membranes (Amersham Bioscience, Piscataway, NJ, USA). The blots were blocked in 5% non-fat powdered milk in PBST (1x PBS containing 0.05% Tween 20). The membrane was incubated with the primary antibody in 3% non-fat powdered milk in PBST at 4°C overnight, washed extensively with PBST, blocked with 5% non-fat powdered milk in PBST and then incubated with appropriate secondary anti-rabbit or anti-mouse horseradish peroxidase-labeled antibodies for 1 hour at room temperature. After three times final washes with PBST, bands were visualized with SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA) according to the manufacturer's instructions.
Real-time quantitative RT-PCR
Total RNA isolation, cDNA synthesis, designed primers and real-time quantitative RT-PCR were performed as previously reported (Gan et al., 2005). The primers were as follows. Human p53, sense: 5'-CCGCAGTCAGATCCTAGCG-3', antisense: 5'-AATCATCCATTGCTTGGGACG-3'; human ß-actin: sense 5'-CTGGAACGGTGAAGGTGACA-3', antisense: 5'-AAGGGACTTCCTGTAACAATGCA-3'; human Mdm2: sense: 5'-ACCTCCCAACCAACTCAGTTC-3', antisense: 5'-AGTGCAAATGAGCCATTGATCT-3'.
Immunofluorescent staining and microscopy
For immunofluorescence assays (IFA), cells were grown on glass coverslips and infected with HCMV. After infection, cells were briefly washed with PBS, fixed with 4% paraformaldehyde for 30 minutes and permeabilized with PBS containing 0.5% Triton X-100 for 8 minutes. Cells were then washed three times with PBS, blocked with 5% BSA, and incubated with primary antibodies. After overnight incubation at 4°C, the cells were washed extensively with PBS and blocked with 5% goat serum in PBS and then incubated with secondary goat anti-rabbit or anti-mouse antibodies labeled with FITC or Texas Red-X. Five percent goat serum in PBS was used for blocking nonspecific binding sites and for dilution of primary and secondary antibodies. The DNA dye 4'6' diamidino-2-phenylindole dihydrochloride (DAPI) was added at a concentration of 0.1 µg/ml and incubated for 15 minutes to counterstain double-stranded DNA in nuclei. The slides were examined with a Leica DMLS epifluorescence microscope equipped with a Leica DC 100 digital camera and the data were analyzed with Image-Pro Plus V4.5 software (Media Cybernetics Inc.).
For IFA analysis of the cells treated with MG132, cells grown on the cover slips were infected at an MOI=0.3. Two hours after infection, fresh medium was added without removing the initial virus inoculums to allow subsequent infection. This provided cells at different stages of infection. The IFA analysis was performed at 5 dpi.
Determination of p53 half-life
The mock and HCMV-infected HUVECs grown in six-well plate were treated with 40 µg/ml of protein translation inhibitor, cycloheximide (CHX). Cell extracts were isolated from individual wells at 0, 1, 2, 3 and 5 hours post infection. The p53 steady state levels were determined by western blot analysis as mentioned above. Images were digitally acquired using an HP ScanJet 5200C Scanner (Hewlett-Packard) and quantified using NIH ImageJ Software analysis (NIH). The levels of p53 were normalized against the corresponding actin level.
p53 ubiquitination analysis
Mock-infected and HCMV-infected HUVECs grown in six-well plates at various time points were treated with or without 10 µM MG132 (proteasomal inhibitor) for 6 hours before being harvested by washing twice with warm PBS. Cells were lysed using LSB supplemented with 10 mM iodoacetamide at a concentration of 103 cells/µl LSB, mixed and boiled for 10 minutes. Equal volumes of whole cell extract were then loaded on to 10% or 12% SDS-polyacrylamide gels (PAGE), transferred to PVDF membranes and subjected to western blot analysis as detailed above using anti-p53 (fl-393) pAb.
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