2457Research Article

IntroductionThe tumor suppressor protein p53 plays an important role inpreventing damaged or abnormal cells from becomingmalignant by inducing either G1/S cell cycle arrest to allowDNA repair (Kastan et al., 1992; Kuerbitz et al., 1992), orapoptosis to eliminate cells with damaged genomes (Kuerbitzet al., 1992; Vogelstein et al., 2000). The main tumorsuppressor activities of p53 in apoptosis are based on thetranscription activation of pro-apoptotic target genes (Chao etal., 2000) or through suppression of anti-apoptotic genes(Murphy et al., 1999). The p53 protein has a short half-life andis usually expressed at low levels in unstressed cells. A delicatesystem, including ubiquitination and proteasomal degradationin the cytoplasm with Mdm2 as the main regulator of p53,keeps the levels of p53 protein low in unstressed cells (Hauptet al., 1997). The p53 protein is synthesized in the cytoplasmand 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 commonmechanism for inactivation of p53 DNA-binding function andcan be identified in many different types of cancers (Ryan etal., 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 largestmembers of the herpesvirus family. It contains a double-stranded DNA genome of 229,354 bp, which encodes morethan 200 different proteins. It is also one of the most commonnon-symptomatic pathogens in the adult population. HCMVcan potentially infect all tissues and is capable of establishinga life-long latent infection after the primary infection.Endothelial cells are one of the major targets of HCMVinfection where it potentially establishes latency (Sinzger et al.,1995). Following infection, HCMV gene expression occurs asa 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) isaccompanied by an increase in p53 levels. Studies from thislaboratory and others have also demonstrated that HCMVinfection 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) andfibroblasts (Reboredo et al., 2004). We previously reported that

Human cytomegalovirus (HCMV) infection results inendothelial dysfunction, typically known as dysregulatedapoptosis, and aberrant expression and sub-cellularlocalization of p53, a tumor suppressor that accumulates atthe late stage of infection. In this study, we examined threehypotheses that could be responsible for HCMV-inducedcytoplasmic p53 accumulation at the later stage ofinfection: hyperactive nuclear export, cytoplasmic p53tethering and delayed p53 degradation. Leptomycin Btreatment, a nuclear export inhibitor, was unable to reducecytoplasmic p53, thereby eliminating the hyperactivenuclear export mechanism. The findings that nascent p53still entered nuclei after the nuclear export inhibitionindicated that cytoplasmic tethering may play a minor role.

Cytoplasmic p53 was still observed after the translationactivities were blocked by cycloheximide. There was morethan an eight-fold increase in the cytoplasmic p53 half-lifewith abnormal p53 ubiquitination. Taken together, theseresults suggest that delayed degradation could beresponsible for the cytoplasmic p53 accumulation. Thegeneral slow-down of the proteasomal activity and thedysregulated p53 ubiquitination process at the later stageof infection could contribute to the reduced cytoplasmicp53 degradation and might be relevant to dysregulatedendothelial apoptosis. The HCMV-induced changes in p53dynamics could contribute to endothelial dysfunction.

Key words: Human cytomegalovirus, p53, Endothelial dysfunction

Summary

Mechanisms for human cytomegalovirus-inducedcytoplasmic p53 sequestration in endothelial cellsBudi Utama1, Ying H. Shen1, Bradley M. Mitchell2, Irwan T. Makagiansar3, Yehua Gan1,Raveendran Muthuswamy1, Senthil Duraisamy1, David Martin4, Xinwen Wang1, Ming-Xiang Zhang1,Jing Wang1, Jian Wang1, Greg M. Vercellotti5, Wei Gu6 and Xing Li Wang1,*1Division of Cardiothoracic Surgery, Michael E. DeBakey Department of Surgery, 2Department of Ophthalmology and Department of MolecularVirology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA3Department of Developmental Neurobiology, The Burnham Institute, La Jolla, CA, USA4VRL Laboratories, San Antonio, TX, USA5Department of Medicine, University of Minnesota, Minneapolis, USA6Institute for Cancer Genetics, Columbia University, New York, USA*Author for correspondence (e-mail: xlwang@bcm.tmc.edu)

Accepted 8 March 2006Journal of Cell Science 119, 2457-2467 Published by The Company of Biologists 2006doi:10.1242/jcs.02974

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p53 is stabilized in the nucleus at the early stage of HCMVinfection and becomes sequestrated in the cytoplasm at thelater stage of the infection (Kovacs et al., 1996; Wang et al.,2001; Wang et al., 2000). The mechanism for this sequestrationremains to be determined.

In the present study, we investigated p53 modulation andsubcellular localization during the course of HCMV infectionin human umbilical vein endothelial cells (HUVECs). For thispurpose, we examined three possible hypotheses for themolecular events leading to cytoplasmic p53 sequestration.First, we examined the hypothesis that the increasedcytoplasmic p53 could be due to an accelerated p53 nuclearexport. Second, we investigated whether tethering within thecytoplasm could prevent p53 from entering the nucleus,thereby causing cytoplasmic sequestration. Third, whetherdelayed cytoplasmic degradation could cause cytoplasmicsequestration. Our findings suggest that the delayed p53degradation is likely to be the predominant mechanism forcytoplasmic p53 sequestration in HCMV-infected HUVECs.

ResultsHCMV-induced p53 nuclear accumulation at earlyinfection, followed by rapid nuclear exclusion andtemporary cytoplasmic sequestrationPreviously, we reported that nuclear p53 levels in HCMV-infected HUVECs were increased during the early stages ofinfection and p53 became sequestrated in the cytoplasm at thelater stages. However, the report was based on our observationsup to 3 days post-infection (dpi) only (Wang et al., 2001),which did not permit sufficient time for cell replication andsecondary infection. To better assess the mechanism of p53modulation and sub-cellular localization, a time-dependentstudy of the dynamic changes of p53 levels were performedafter a prolonged infection (13 dpi). We observed that duringthe first 3 dpi, p53 protein levels increased and predominantlylocalized in the nucleus, which was then followed by a rapid

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nuclear exclusion, and a concomitant increase in p53 in thecytoplasm at 3 dpi onward (Fig. 1). The later event coincidedwith the high expression of HCMV late proteins (data notshown). However, a low level of p53 was detectable byimmunofluorescence assays (IFA) in the nucleus of the infectedcells (Fig. 1, 5 dpi onward). The cytoplasmic p53 level wasstabilized between 4 and 6 dpi before its level started todecrease. This observation was verified by western blotting,which showed that HCMV infection in HUVECs induced agradual increase in the total amount of p53 approximately 3.5-fold over the steady-state levels during the first 3 dpi at amultiplicity of infection (MOI)=1.0 (Fig. 2A,B). The elevationstarted to appear after 1 dpi, while the HCMV immediate-earlyprotein expression in the nucleus was detected as early as 12hours post-infection (hpi; data not shown).

Increased nuclear p53 at early infection is due toaccumulationOur experiments further showed that the increased nuclear p53protein during the early stages (1-3 dpi) of the infection (Fig.2B1) was probably not due to an increased p53 transcription,since p53 mRNA was not significantly changed (Fig. 2B2). Ourfindings suggested that the increase in the p53 protein levelsduring early stage of infection in HUVECs was probablycaused by a stabilizing mechanism of p53 protein in thenucleus. This evidence corroborated a recent report whichsuggested that adenovirus IE1-72 promotes p53 nuclearaccumulation through an unknown mechanism (Castillo et al.,2005). Furthermore the initial sub-nuclear localization of p53during nuclear stabilization was restricted to discrete foci (datanot shown), which resembled the viral replication centerreported earlier in HCMV-infected fibroblasts (Fortunato andSpector, 1998). These discrete foci enlarged throughout theinfection to almost the entire nuclear region; but this could notbe detected once the nuclear exclusion of p53 had occurred.

Mdm2 is a p53-specific E3-ubiquitin ligase that

Fig. 1. p53 protein modulation and subcellular localization in HCMV strain VHL/E-infected HUVECs. Immunofluorescence assay of p53protein (red, Texas Red-X, upper panels), and merged images of p53 with nuclear HCMV immediate early (IE) proteins (green, FITC) andDAPI (blue, nuclear region; lower panels). p53 is mainly stabilized in the nucleus for the first 3 dpi (yellow arrows), followed by a rapid nuclearexclusion (3 dpi), which transfers virtually all of the p53 to the cytoplasm (3 dpi onward) as shown by red color in cytoplasmic regions (whitearrows) 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.

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2459p53 in HCMV-infected endothelial cells

ubiquitinates nuclear p53 and allows for nuclear export,followed by proteasome-dependent degradation in thecytoplasm (Fang et al., 2000). Low levels of Mdm2 ordisruption of the Mdm2-p53 interaction may cause the p53stabilization in nucleus. As shown in Fig. 2C1, Mdm2 proteinlevels were not markedly changed during the p53 nuclearstabilization period (1-3 dpi), although a significant increaseoccurred up to 11 dpi. The Mdm2 mRNA levels showed asimilar trend (Fig. 2C2) suggesting no significant perturbationoccurred at the Mdm2 transcription level during the nuclearp53 accumulation (1-3 dpi).

Rapid p53 nuclear exclusion coincided with an elevatedexpression of Crm1Nuclear export/import is crucial for most nuclear proteins tofunction 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 p53completes its functional requirements in the nucleus, theubiquitinated p53 monomer utilizes two leucine-rich nuclearexport signals (NES) allowing p53 to follow an active Crm1-RanGTP nuclear export pathway (Lohrum et al., 2001). Duringthe course of HCMV infection in HUVECs, we found thatCrm1 protein expression varied (Fig. 3), but appeared to fit intothe dynamic requirements of the p53 nuclear/cytoplasmic

Fig. 2. Expression of p53 and Mdm2 in HCMV-infected HUVECs. (A) Protein levels were semi-quantified using western blot analysis of thetotal cellular protein extracts from HCMV-infected HUVECs (MOI=1.0) during the first 13 dpi. Dynamic changes in protein levels of the totalp53, HCMV-immediate-early (IE) proteins (68/72 kDa) and Mdm2 were shown. (B,C) Graphs showing the corresponding mRNA and proteinlevels. The nucleus and cytoplasm bars show the proportional changes between the two subcellular compartments during the course ofinfection, based on IFA. All values have been normalized against corresponding actin level, and represent mean (± s.e.m.) of three differentbatches of HCMV-infected HUVECs (MOI=1.0). Each experiment was conducted in triplicate.

Fig. 3. Crm1 levels were transiently increased to facilitate rapidnuclear p53 exclusion in HCMV-infected HUVECs. Western blotanalysis (top) and graph of relative protein levels (bottom) showing asignificant increase in p53 levels. Crm1 protein levels were slightlydecreased when p53 was stabilized in the nucleus during the first 3dpi. A transient increase in Crm1 levels from 4-7 dpi coincided withthe nuclear p53 exclusion. This increase was followed by a decreasewith the time of infection to lower levels than before infection. Thereduction in Crm1 levels occurred at the same time as p53 wassequestrated in the cytoplasm, which is indicated by the broken bar atthe bottom of the graph. The experiments were carried out intriplicate using three different batches of HCMV-infected HUVECs(MOI=1.0).

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shuttling. An initial decrease of Crm1 was noticed during thefirst 3 dpi, followed by a marked increase, with peak levelsdetected at 5-7 dpi. After 7 dpi, the Crm1 levels graduallydecreased to below the baseline level (0 dpi).

To explore whether the rapid p53 nuclear exclusion is solelyCrm1 dependent, we treated the HCMV-infected HUVECswith 20 nM leptomycin-B (LMB), which inhibits the formationof the Crm1-NES complex blocking nuclear export (Freedmanand Levine, 1998). Cells were treated at 2, 3, 4 and 5 dpi for18 hours before observation. As shown in Fig. 4, the nuclearexport inhibitor LMB was able to abrogate the nuclearexclusion of the p53 if the cells were treated before the nuclearexclusion 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 hadalready been completely exported out of nucleus, LMB was nolonger effective in reducing the cytoplasmic p53 (Figs 3, 4).Furthermore, during the LMB treatment we were able toobserve a significant increase in nuclear p53, presumablynascent p53, which was imported and trapped into the nucleus.This is in agreement with the fact that p53 has beencontinuously transcribed and translated during the course ofinfection (Fig. 2B1,B2).

Temporary cytoplasmic p53 sequestration after rapidnuclear exclusion was less likely to be caused bytethering or nuclear hyper-exportationTo investigate the mechanisms responsible for the cytoplasmicp53 sequestration after rapid nuclear exclusion during the laterstages of HCMV-infection of HUVECs, we examined threehypotheses: (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 inneuroblastoma (Stommel et al., 1999); and (3) there is anextended half-life of cytoplasmic p53.

To distinguish between the first two possible mechanisms ofcytoplasmic p53 sequestration, we treated HUVECs at 7 dpiwith 10 nM LMB plus 40 �g/ml cycloheximide (CHX). LMBhas been reported to not induce the p53 stress response(Freedman and Levine, 1998). We selected 7 dpi because p53localized in the cytoplasm in most of the infected cells(MOI=1.0) at this stage and yet the p53 transcription andtranslation levels were still relatively the same as the earlystages of infection. If the cytoplasmic p53 in infected cells wasconstitutively tethered, then p53 protein should not remain inthe cytoplasm for 6 hours after CHX, since p53 half-life isnormally around 45-60 minutes. Furthermore, upon treatmentwith LMB, p53 should remain in the cytoplasm without anynascent p53 being trapped in the nucleus. If cytoplasmic p53sequestration is primarily due to hyperactive nuclear export,then treatment with LMB should trap p53 in the nucleus andcytoplasmic p53 levels should be diminished.

As shown in Fig. 5Af, after protein translation was blockedby CHX for 6 hours in 7 dpi HUVECs, there was no significantchange in cytoplasmic p53. In the meantime, the abundance ofnuclear p53 was lowered to similar levels as those in untreatedinfected cells (Fig. 5Aa). Analysis of the correspondingwestern 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 LMBtreatment for 6 hours, a significant increase of nuclear p53 wasdetectable (Fig. 5Ab) corresponding to an increase of total p53

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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 hoursbefore 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 stainedwith anti-p53 (red, Texas Red-X), anti-HCMV-IE proteins (green, FITC) and DAPI (blue) to counterstain the nuclei.

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protein of approximately 20% (Fig. 5B, infected cells). Theseresults indicate that nascent p53 was continuously transcribed(Fig. 2B2), translated, imported, and stabilized in the nucleusduring the period when p53 is sequestered in the cytoplasm atthe later stages of infection. Hence, we suggest that tetheringis unlikely to be the primary mechanism for cytoplasmic p53sequestration.

The accumulated p53 after LMB treatment during the laterstages of infection apparently came from nascent p53 and notfrom the shuttling p53. Using a modified protocol describedpreviously (Joseph et al., 2003), 7 dpi HUVECs were given acombined treatment of LMB (22 hours) and CHX (for the last6 hours of the 22 hours LMB treatment). LMB was added to

block nuclear export and CHX was added subsequently toblock nascent p53 synthesis to ensure that only the previouslystabilized pools of p53 was observed (Fig. 5Ad). We comparedthe combined treatment to LMB treatment alone for 6 and 22hours. As shown in Fig. 5A, the abundance of nuclear p53 afterthe LMB (22 hours)/CHX (6 hours) treatment was significantlylower 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 afterLMB treatment during the cytoplasmic p53 sequestration at thelater stages of HCMV infection was indeed nascent p53.

We then examined the possibility of hyperactive nuclear

Fig. 5. The temporary cytoplasmicp53 sequestration (7 dpi) wasprobably not caused by cytoplasmictethering or the hyperactive nuclearexport. HCMV-infected cells weretreated with LMB, CHX or acombination of the two drugs.Incubation times are indicated inhours. All immunofluorescenceimages (A) were taken with theidentical conditions, level ofinfections, IFA protocol, and equalexposure/digital picture processingconditions. Cells were stained asfollows: anti-p53 (red, Texas Red-X),cytoplasmic anti-HCMV late proteins(green, FITC) and DAPI (blue). Thecorresponding western blot analysis(B) of the mock- and HCMV-infectedcell extracts that were isolated fromthe identically infected cells treatedwith a single or a combination of thetwo drugs reconfirmed the IFA resultsshown in A. Numbers below the lanesare relative p53 protein levels andhave been normalized againstcorresponding actin bands. (C) Thepharmacological activity of LMB, 6and 22 hours after use for the infectedHUVECs treatment, was examined infresh HUVECs. The same nuclearp53 accumulation pattern as those ofthe direct LMB treatment (data notshown) and increased p53 levelconfirmed that LMB remainedpharmacologically active even 22hours after incubation with theinfected HUVECs.

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export during temporary cytoplasmic p53 sequestration afterrapid nuclear exclusion. After LMB treatment for 6 or 22hours, no gross changes in cytoplasmic p53 were observed(Fig. 5Ab,c). By contrast, nuclear p53 staining wassignificantly increased. These findings indicate thathyperactive nuclear export was unlikely to be responsible forcytoplasmic p53 sequestration after rapid nuclear exclusion ofp53. To show that LMB was still pharmacologically active afterthe delayed incubation (22 hours), we transferred LMB-containing medium, after 6 and 22 hours of treatments, to fresh7 dpi cells. As indicated by the increased p53 levels, the 6- and

22-hour old LMB-containing media still actively inhibitednuclear export in untreated 7 dpi infected cells after 6 hours ofincubation (Fig. 5C).

Temporary cytoplasmic p53 sequestration had anextended half-lifeWe then examined whether the cytoplasmic p53 sequestrationat the later stage of infection was caused by an extended p53half-life. As shown in Fig. 6A,B, from the time-frameobservation of uninfected and infected-cells after proteintranslation inhibitor CHX treatment, p53 half-life was

markedly extended from the basal 45-60minutes in uninfected HUVECs (mainlynuclear p53 at 0 dpi) to more than 8hours of mainly cytoplasmic p53 at thelater time of the infection (7 dpionward).

p53 protein ubiquitination in HCMV-infected HUVECsTo investigate the interaction betweenMdm2 and p53 ubiquitination inHCMV-infected HUVECs, we isolatedthe protein extract using Laemmli-SDSsample buffer (see Materials andMethods) from equal numbers ofinfected cells in six-well plates treatedwith/without the proteasomal inhibitorMG132 at different time points ofinfections. The protein extracts wereseparated using 12% PAGE, transferredto membrane and probed with anti-p53(fl393) rabbit polyclonal antibodies.

In cells received no MG132treatment, the band of mono-ubiqitinated p53 (larger than the p53monomer with an estimated molecularmass of ~60 kDa) was faint and barelydetectable (Fig. 7A). By contrast, cellstreated with 10 �M MG132 for 6 hours(Shibata et al., 2002) showed multiple

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Fig. 6. Cytoplasmic sequestrated p53had delayed half-life. (A) Western blotanalysis of cell extracts after cellswere treated with the proteintranslation inhibitor CHX. Bands werevisualized using anti-p53 (DO-1) andanti-I�B�. The densities werenormalized against the correspondingactin levels. (B) Graphs show thecomparison of the p53 half-lifebetween mock- and HCMV-infectedHUVECs. p53 half-life was markedlyextended from the basal 45-60 minutesin uninfected HUVECs (mainlynuclear p53 at 0 dpi) to more than 8hours of mainly cytoplasmic p53 at thelater time of the infection (B).

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2463p53 in HCMV-infected endothelial cells

bands of ubiquitinated p53 (Fig. 7B). They included a distinctsingle 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 intensityfor 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 ofMG132, the ubiquitinated p53 may have been rapidly degradedin the cytoplasm. When proteasomal activity was inhibited byMG132, however, the ubiquitinated p53 was preserved/stabilized and became detectable.

Our experiments further showed that the p53 ubiquitinationpatterns were not significantly different in cells with or withoutMG132 treatment at the very early stage of the infection (1dpi), in which p53 was mostly present within nucleus. From 3dpi onward, concomitant with the p53 nuclear exclusion andcytoplasmic p53 sequestration, the levels of ubiquitinated-p53changed dynamically (Fig. 7B). Whereas the single (p53-Ub(1))and double (p53-Ub(2)) mono-ubiquitinated p53 increasedsignificantly towards the later stage of infection, the multiplemono-ubiqutinated p53 (p53-Ub(n)) apparently decreasedduring the same period.

It was of interest to see whether the dynamic changes of p53,

its ubiquitinated forms, and Mdm2 proteins during HCMVinfection after MG132 treatment may affect its subcellularlocalization and expression pattern. To explore this issue, IFAanalysis was utilized to observe the MG132 treated/untreatedHCMV-infected cells (MOI=0.3) at 5 dpi using indirectlyfluorescence-labeled anti-p53 (fl-393) rabbit pAb and anti-Mdm2 mAb. As shown in Fig. 8, MG132 treatment had noeffect on the subcellular localization of p53. Only a slightlystronger signal was observed from the MG132-treated cells incomparison to the untreated cells. However, it was importantto observe the effects of MG132 on Mdm2 subcellularlocalization and levels as well. As shown in Fig. 8, during theearly stages of infection or in non-infected cells, relatively lowlevels of Mdm2 were observed mainly in the nuclear region(labeled with stars). The Mdm2 levels increased dramaticallyin the nucleus with the progression of infection (white arrows),and during the onset of p53 nuclear exclusion (white arrowswith label ‘a’) in both the nucleus and cytoplasm. When thep53 protein was temporarily sequestered in the cytoplasm atthe later stage of infection (shown by the presence of the owleye or the enlarged kidney shape of infected nuclei indicatedby the yellow arrow, Fig. 8), the levels of Mdm2 weresignificantly reduced in the cytoplasm and in most areas of thenucleus. This observation confirmed our previous results ofdynamic changes of Mdm2 during the HCMV infection, usingwestern blot analysis (Fig. 2).

DiscussionIn the present study, we show that during the early stages ofthe HCMV infection of HUVECs, p53 was transientlystabilized in the nucleus with p53 levels increasing toapproximately 3.5 fold in the first 3 dpi (Fig. 1 and Fig. 2A,B).The increased p53 levels during the early stage of infection (<3dpi) were apparently due to nuclear stabilization, as nosignificant changes in p53 transcription were detected (Fig.2B2). Following this early nuclear accumulation, a rapidnuclear exclusion of p53 occurred between 3-4 dpi, whichapparently was facilitated by transiently increased Crm1 levels.We showed that this rapid nuclear exclusion was Crm1dependent since LMB halted or reduced the nuclear exclusionof p53 and its subsequent cytoplasmic sequestration. However,once p53 was completely exported out of nucleus, Crm1inhibition was no longer effective in reducing the p53 exclusionfrom the nucleus (Fig. 4, 5 dpi untreated/treated LMB panels).

We further explored the correlation between Crm1expression and the dynamics of p53 subcellular localization.The Crm1 protein level at 0 dpi was relatively higher than at 3dpi, possibly because of the need for active nuclear/cytoplasmic shuttling of p53 in unstressed cells (Fig. 3). Themodest decrease of Crm1 levels during the first 3 dpi, whichcoincided with the accumulation of p53 in nucleus, could resultin the reduced p53 nuclear export. The markedly increasedCrm1 levels at 5-7 dpi possibly allowed the rapid nuclearexclusion of p53. When p53 was temporarily sequestered andlater degraded in the cytoplasm, Crm1 apparently was nolonger functionally required, therefore gradually decreased.These findings suggest that the changes in Crm1 levels mayrespond to the dynamics of p53 subcellular localization ratherthan the steady state p53 levels. As p53 is not the only proteinusing the active Crm1-RanGTP nuclear export pathway(Fornerod et al., 1997), we speculate that a specific mechanism,

Fig. 7. The time-dependent changes in ubiquitinated p53 pattern inHCMV-infected HUVECs. Western blot with anti-p53 (fl-393)polyclonal antibody was employed to demonstrate the ubiquitinatedp53 pattern in uninfected (0 dpi) and HCMV-infected (1-11 dpi)HUVECs with (B) or without (A) 10 �M MG132 (proteasomeinhibitor) treatment. In the MG132-treated cells, mono-ubiquitinatedp53 (p53-Ub(1) at ~60 kDa) significantly increased towards the laterstages of infection (3 dpi onwards). The increased levels wereobserved also for the corresponding double mono-ubiquitinatedbands (p53-Ub(2) at ~75 kDa). By contrast, the multiple mono-ubiquitinated p53 bands (p53-Ub(n)) of higher molecular massestended to decrease towards the later stages of infection (3 dpionwards). Molecular masses are given on the left in kDa.

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based on the p53 subcellular localization and/or HCMVproteins expressed, may regulate Crm1 expression or they maywork in cooperation to meet the needs for a dynamic nuclearp53 export during the HCMV infection.

The rapid nuclear exclusion was concomitant with theincrease in cytoplasmic p53. By contrast to normal unstressedcells in which ubiquitinated p53 was degraded quickly in thecytoplasm, we identified a temporarily cytoplasmic p53sequestration in HUVECs during the later stages of HCMVinfection. We found that the cytoplasmic localization of p53was not in a punctate pattern or as a p53 cytoplasmic body asfrequently identified in cells infected with adenovirus Type-12E1B-oncoprotein (Zhao and Liao, 2003), or as finely dispersedconstitutive cytoplasmic p53 as observed in cells productivelyinfected 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 inmonomeric/dimeric forms (data not shown). This findingindicates that the cytoplasmic p53 detected during the laterstage of infection had been ubiquitinated in the nucleus,allowing conformational changes or disruption of tetramer p53and subsequent nuclear export (Gu et al., 2001). Our series ofexperiments (Figs 4, 5) clearly indicated that the temporarycytoplasmic p53 sequestration during the later stage ofinfection after rapid nuclear exclusion (4 dpi onward) was notdue to hyperactive nuclear export.

There were no significant changes in cytoplasmic p53 whenprotein translation was blocked at 7 dpi with CHX treatment(6 hours) alone (Fig. 5Af). We further showed that nascent p53was trapped in the nucleus of 7 dpi HUVECs when cells were

treated with either LMB/CHX or LMB alone (Fig. 5A,B). Allthe evidence suggests that a tethering mechanism would play,at best, a minor role in cytoplasmic p53 sequestration.

With both hyperactive nuclear export and the tetheringmechanisms being unlikely to contribute to the cytoplasmicp53 sequestration in HCMV-infected HUVECs, our findingssuggest that extended p53 half-life may be the responsiblemechanism. Several possibilities for extended p53 half-life inHCMV-infected HUVECs have been investigated here. Weshow that the lower proteasomal degradation activity afterHCMV infection in endothelial cells may contribute to someextent, as also shown in adenovirus E1A-infected cells, inwhich inhibition of proteasomal activities by targeting theproteasomal regulatory subunit S2, extended p53 half-life andincreased the level of p53 without inhibiting the p53ubiquitination 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 averageof 3-4 hours to 5-6 hours during the course of infection. Whilethe I�B� half-life extension stayed relatively the same after theinfection, p53 half-life became more extended toward the laterstage of infection, during which p53 protein was mainlypresent in the form of cytoplasmic p53 (Fig. 6). We proposethat the significant extension of the cytoplasmic p53 half-lifeis likely to be caused by the dysregulated p53 ubiquitinationprocess.

We have demonstrated a different p53 ubiquitinationpatterns between the early stage (mostly nuclear p53) and thelate stage of infection (mostly cytoplasmic p53), which wereobserved after the inhibition of proteasomal degradation by

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Fig. 8. MG132 treatment did not cause any significant changes in p53 and Mdm2 patterns or subcellular localization in HCMV-infectedHUVECs. HCMV-infected cells (5 dpi at MOI=0.3) were treated with or without MG132 for 6 hours before IFA. All IFA images were takenwith the identical cells conditions, level of infections, IFA protocol, and equal exposure/digital picture processing conditions. Cells were stainedred by the anti-p53-Texas Red-X, green by anti-Mdm2-FITC and blue by DAPI for nuclear counterstaining. MG132 treatment had no effect onthe subcellular localization of p53. However, a slight increase of both p53 and Mdm2 levels can be observed after MG132 treatment comparedto cells without treatments. Stars indicate the uninfected cells, which have relatively low levels of p53 and Mdm2 in the nuclear region. Mdm2and p53 levels were significantly increased, mainly in the nuclear region of the infected cells at very early stage of post-infection (whitearrows). During the period of p53 nuclear exclusion (white arrows ‘a’) the p53 and Mdm2 levels were elevated mainly in the nuclear and also inparts of the cytoplasmic regions. At the later stages of infection, cytoplasmic p53 sequestration was observed coincidentally with the lowerlevels of Mdm2 (yellow arrows).

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MG132. We identified that when the majority of p53 wassequestered in the nucleus (1 dpi), the ubiquitinated p53 patternremained relatively unchanged (Fig. 7B) amid the slightincrease of the total p53 levels. This suggests that there was nosignificant changes in the p53 ubiquitination pattern during thevery early days of infection in comparison to the cells beforeinfection, although the accumulation of both p53 and Mdm2could be observed in the nucleus. A significant increase of thesingle mono-ubiquitinated p53 (~60 kDa) was demonstrable bythe time of the p53 nuclear exclusion (>3 dpi), whereas theincreased levels of double mono-ubiquitinated p53 (~75 kDa)were observed at 7 dpi onward. By contrast, multiple-ubiquitinated p53 at higher molecular masses tended todecrease toward the later stage of infection after a brief spikeat 3 dpi (Fig. 7B).

Since Mdm2 is responsible for different forms of p53ubiquitination, we postulate that the p53 sequestration at thelater stage of HCMV infection in HUVECs could be due to thedynamics of Mdm2 levels. It has been reported that Mdm2alone at low levels, catalyzes mono-ubiquitination, which isadequate for p53 nuclear export. At a high concentration,however, Mdm2 induces poly-ubiquitination and facilitatesdegradation (Li et al., 2003). Therefore, the ubiquitinationoutcome is determined by the Mdm2:p53 ratio. The low ratioswill result in p53 mono-ubiquitination, whereas the high ratios(3.6 and above) will poly-ubiquitinate p53 (Li et al., 2003). Ourresults showed that during the nuclear p53 accumulation (<3dpi), Mdm2 also accumulated in the nucleus (Fig. 8). However,during the same period of the first 3 dpi the Mdm2 levelsincreased only modestly (~ twofold) in comparison to a highlyincreased p53 levels (~3.5 fold, Fig. 2B1,C1). We propose thatthe Mdm2 levels during the very early days of infection (<3dpi) might not be sufficient to induce a proper poly-ubiquitination of the disproportionately large amount of p53.Alternatively, the p53 poly-ubiquitination process could alsobe inhibited by the increased p53 phosphorylation at serine15 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 3dpi. It appears that Mdm2 levels at this stage were sufficientto mono-ubiquitinate p53 for nuclear export, but not for p53poly-ubiquitination and degradation. However, we cannotexclude the possibility that the HCMV proteins expressed, inaddition to Mdm2 activities, may help to export p53 out ofnucleus. This is suggested by the fact that adenovirus E4orf6and E1B55K (Blanchette et al., 2004) and HSV-1 regulatoryprotein ICP0 (Boutell and Everett, 2003) have been reportedto be able to interact and ubiquitinate p53 for subsequentnuclear export and proteasomal degradation. It has also beenshown that human papillomaviruses (HPV) type 18 E6 canpoly-ubiquitinate p53, independent of Mdm2, for nuclearproteasomal degradation or for a nuclear export to cytoplasmthrough the Crm1 nuclear export pathway for subsequentcytoplasmic proteasomal degradation (Stewart et al., 2005). Inaddition, HCMV proteins US2 and US11 are known to catalyzethe dislocation and transfer of MHC class I heavy chains fromthe 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 andproteasomal degradation.

Our results have further shown that at 3 dpi onwards thesingle and double mono-ubiquitinated p53 levels weresignificantly increased whereas the multiple mono-ubiquitinated p53, of higher molecular mass, tended todecrease. It is interesting that the dynamics of p53ubiquitination forms all coincided with the cytoplasmic p53sequestration. It is tempting to hypothesize that the increasedpresence of mono-ubiquitinated cytoplasmic p53 could berelated to increased endothelial apoptosis after 4 dpi of HCMVinfection, as we previously reported (Shen et al., 2004). Theonset of apoptosis was also coincidental with the cytoplasmicp53 sequestration as detailed above. Previous studies haveshown that p53 can trigger apoptosis in the cytoplasm by directinteraction 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 thismechanism might be applied to the sequestrated cytoplasmicp53 in HCMV-infected HUVECs. This speculation issupported by the fact that mono-ubiquitination also helpsproteins to traffic to a proper destination at subcellular level,i.e. single mono-ubiquitinated p53 to mitochondria. A study iscurrently underway to investigate this hypothesis.

The decreased levels of the higher molecular mass multiplemono-ubiquitinated products at the later stage of infectioncould also be the result of the presence of the deubiquitinatingenzymes such as herpes virus-associated ubiquitin-specificprotease (HAUSP). HAUSP directly deubiquinates andstabilizes p53 (Li et al., 2002), and Mdm2 can act a thesubstrate for HAUSP under certain physiological conditions,which in turn control the p53 levels (Cummins and Vogelstein,2004). Whether levels of HAUSP change during the laterstages of HCMV infection remains to be determined. It is clearthat in addition to a general slow-down of the proteasomalactivity (Turnell et al., 2000; Zhang et al., 2004) an inadequatep53 poly-ubiquitination at the later stage of HCMV infectioncould 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 endothelialdysfunction, is caused by the increase of p53 half-life and anabnormal p53 ubiquitination pattern and degradation. It is notclear, however, whether these changes in p53 protein aredirectly caused by HCMV gene products or the host cellproteins triggered by the HCMV infection. Using the moderntools of protein-protein interactions, studies are currently underway in our laboratory to examine the roles of individualHCMV proteins in p53 nucleocytoplasmic trafficking. Ourfindings, nevertheless, put forward yet another potential systemfacilitating complicated interactions between viruses and cellsto set a favorable environment for viral survival and/orreplication, which would simultaneously result in endothelialdysfunction.

Materials and MethodsEndothelial cell culturePrimary HUVECs were isolated from human umbilical cord as described previously(Jaffe et al., 1973; 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 F12Kmedium (VitaCell-ATTC, Manassas, VA, USA) containing 20% fetal bovine serum

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(Invitrogen Corp., Carlsbad, CA), penicillin (100 IU/ml) and streptomycin (100�g/ml; Cellgro Mediatech, Inc., Herndon, VA), 100 �g/ml sodium heparin (Acros,NJ, USA), and 30 �g/ml endothelial cell growth supplement (Sigma). Cells up topassage eight were used in the experiments unless otherwise stated.

Virus preparationThree strains of HCMV [AD169 (ATCC #VR-538), Towne (ATCC #VR-977) andVHL/E (kindly provided by Jim Waldman, Ohio State University, OH, USA)] wereused in the early study. For preparation of a high titer of virus stocks and to preservethe natural endothelial cytopathogenicity of the original isolate, all strains werepropagated in HUVECs as described previously (Kahl et al., 2000). However, as theVHL/E strain was the strain that caused the highest permissive HCMV infection inHUVECs, we used this strain throughout the study. Sub-confluent monolayer ofHUVECs were infected with multiplicity of infection MOI=0.01 pfu/cell, harvestedat 100% cytopathic effect (CPE), and kept in a 1:1 mixture of medium and sucrosephosphate buffer (with a sucrose concentration of 188 mM) at –80°C until used.Virus titer was determined with rapid quantization using a monoclonal antibody tothe major immediate-early (IE) viral protein as reported previously (Chou and Scott,1988; Waldman et al., 1989).

Infection of HUVECs with HCMVThe fully confluent non-synchronized HUVEC monolayers were infected withHCMV at approximately MOI=1.0 (unless otherwise stated) and incubated at 37°Cfor 1-2 hours for virus absorption. The monolayers were then washed three timeswith pre-warmed Dulbecco phosphate-buffered saline (PBS), before fresh completemedium was added. Cells were cultured at 37°C in a CO2 incubator. Only attachedcells were harvested at various days post-infection (dpi).

Antibodies and chemicalsThe 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 andanti-I�B� pAb (BD Pharmingen); anti-p53 (DO-1) mAb, anti-p53 (fl-393)polyclonal antibodies (pAb), anti-actin pAb (Sigma); Texas Red-X conjugated goatanti-rabbit antibodies (Molecular Probes); anti-HCMV-immediate early antigenmAb, 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 paraformaldehydesolution (16%), which was from Electron Microscopy Sci., Washington, PA, andprotein A/G-Plus agarose beads, which were from Santa Cruz. Trizol was fromInvitrogen. RNase-free DNase I was from Promega, and the iScript cDNA synthesiskit and SYBR Green I Supermix kit were from Bio-Rad. Oligonucleotides werepurchased from IDT Inc., Houston, TX, USA.

Western blot analysisHUVECs 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 aconcentration of 103 cells/�l LSB, mixed and boiled for 5 minutes. Equal volumesof whole cell lysate were loaded on to 10% or 12% SDS-polyacrylamide gels (forPAGE), 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 (1� PBS containing 0.05% Tween 20). The membranewas incubated with the primary antibody in 3% non-fat powdered milk in PBST at4°C overnight, washed extensively with PBST, blocked with 5% non-fat powderedmilk 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 SuperSignalWest Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA) according tothe manufacturer’s instructions.

Real-time quantitative RT-PCRTotal RNA isolation, cDNA synthesis, designed primers and real-time quantitativeRT-PCR were performed as previously reported (Gan et al., 2005). The primers wereas follows. Human p53, sense: 5�-CCGCAGTCAGATCCTAGCG-3�, antisense:5�-AATCATCCATTGCTTGGGACG-3�; human �-actin: sense 5�-CTGGAACGG-TGAAGGTGACA-3�, antisense: 5�-AAGGGACTTCCTGTAACAATGCA-3�;human Mdm2: sense: 5�-ACCTCCCAACCAACTCAGTTC-3�, antisense: 5�-AGTGCAAATGAGCCATTGATCT-3�.

Immunofluorescent staining and microscopyFor immunofluorescence assays (IFA), cells were grown on glass coverslips andinfected with HCMV. After infection, cells were briefly washed with PBS, fixedwith 4% paraformaldehyde for 30 minutes and permeabilized with PBS containing0.5% Triton X-100 for 8 minutes. Cells were then washed three times with PBS,blocked with 5% BSA, and incubated with primary antibodies. After overnightincubation at 4°C, the cells were washed extensively with PBS and blocked with5% 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 inPBS was used for blocking nonspecific binding sites and for dilution of primaryand secondary antibodies. The DNA dye 4�6� diamidino-2-phenylindoledihydrochloride (DAPI) was added at a concentration of 0.1 �g/ml and incubatedfor 15 minutes to counterstain double-stranded DNA in nuclei. The slides wereexamined with a Leica DMLS epifluorescence microscope equipped with a LeicaDC 100 digital camera and the data were analyzed with Image-Pro Plus V4.5software (Media Cybernetics Inc.).

For IFA analysis of the cells treated with MG132, cells grown on the cover slipswere infected at an MOI=0.3. Two hours after infection, fresh medium was addedwithout removing the initial virus inoculums to allow subsequent infection. Thisprovided cells at different stages of infection. The IFA analysis was performed at 5dpi.

Determination of p53 half-lifeThe mock and HCMV-infected HUVECs grown in six-well plate were treated with40 �g/ml of protein translation inhibitor, cycloheximide (CHX). Cell extracts wereisolated from individual wells at 0, 1, 2, 3 and 5 hours post infection. The p53 steadystate levels were determined by western blot analysis as mentioned above. Imageswere digitally acquired using an HP ScanJet 5200C Scanner (Hewlett-Packard) andquantified using NIH ImageJ Software analysis (NIH). The levels of p53 werenormalized against the corresponding actin level.

p53 ubiquitination analysisMock-infected and HCMV-infected HUVECs grown in six-well plates at varioustime points were treated with or without 10 �M MG132 (proteasomal inhibitor) for6 hours before being harvested by washing twice with warm PBS. Cells were lysedusing LSB supplemented with 10 mM iodoacetamide at a concentration of 103

cells/�l LSB, mixed and boiled for 10 minutes. Equal volumes of whole cell extractwere then loaded on to 10% or 12% SDS-polyacrylamide gels (PAGE), transferredto PVDF membranes and subjected to western blot analysis as detailed above usinganti-p53 (fl-393) pAb.

The study was supported by an NIH grant R01-HL071608; XingLi Wang is an AHA Established Investigator (AHA0400031). Wethank James W. Waldman for donating HCMV strain VHL/E, JaredBurks for critically reviewing the manuscript, and Lin Zhang fortechnical assistance.

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