Department of Microbiology, Osaka University Medical School C1, 2-2 Yamada-Oka Suita, Osaka 565-0871, Japan
Correspondence
Koichi Yamanishi
yamanisi{at}micro.med.osaka-u.ac.jp
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ABSTRACT |
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Present address: Department of Microbiology, Jikei University, School of Medicine, 3-25-8 Nishi-shinbashi, Minato, Tokyo 105-8461, Japan.
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INTRODUCTION |
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Human herpesviruses (HHVs) are large, enveloped DNA viruses that carry a double-stranded DNA genome of approximately 120230 kbp. On the basis of a diverse collection of in vivo and in vitro biological properties, HHVs are divided into three subgroups: alpha, beta and gamma (McGeoch, 1989, 1990
; Roizmann et al., 1992
). Human herpesvirus 6 (HHV-6) is a ubiquitous betaherpesvirus that was first isolated in 1986 from the peripheral blood of patients with lymphoproliferative disorders (Salahuddin et al., 1986
) and acquired immunodeficiency syndrome (Josephs et al., 1986
; Tedder et al., 1987
). HHV-6 utilizes the cellular CD46 molecule as an entry receptor (Santoro et al., 1999
), predominantly infects and replicates in CD4+ lymphocytes (Lusso et al., 1988
; Takahashi et al., 1989
) and may establish latency in the monocyte/macrophage lineage (Kondo et al., 1991
).
Alterations in the level, function and localization of p53 by HHVs have been reported in a number of studies. Among the HHVs shown to affect p53 are EpsteinBarr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), which are gammaherpesviruses associated with tumorigenesis. Like small DNA tumour viruses, they encode proteins that target and inactivate p53: for EBV, this protein is BZLF1 (Zhang et al., 1994); for KSHV, these proteins are LANA (Friborg et al., 1999
), LANA2 (Rivas et al., 2001
), vIRF (Nakamura et al., 2001
; Seo et al., 2001
) and K-bZIP (Park et al., 2000
). Both herpes simplex virus (HSV) and human cytomegalovirus (HCMV), which represent alpha- and betaherpesviruses, respectively, stabilize p53 by their immediate-early (IE) gene products: infected cell protein zero (ICP0) for HSV (Hobbs & DeLuca, 1999
) and IE1 (Muganda et al., 1994
) and IE2 (Bonin & McDougall, 1997
; Muganda et al., 1994
; Speir et al., 1994
) for HCMV. In addition, the HCMV mtrII oncoprotein (UL111a) has also been reported to interact with p53 and down-regulate its transcriptional activity (Muralidhar et al., 1996
). For HHV-6, it has been reported that the transcriptional activity of p53 is repressed by its interaction with the DR7 protein in NIH3T3 cells transfected with the SalI-L fragment of the HHV-6 genome (Kashanchi et al., 1997
). However, the behaviour of p53 in the context of a viral infection remains to be investigated.
In this paper, we focused on changes in the level and localization of p53 caused by HHV-6 infection. We demonstrated that the p53 protein level increased in a variety of cell lines infected with HHV-6 and that this was due both to the increased synthesis of p53 and to its stabilization through deubiquitination. These changes were induced at a very early stage of infection and most likely involved the expression of viral IE genes. Confocal microscopy showed a cytoplasmic rather than nuclear localization of p53 and documented that p53 did not translocate to the nucleus even after UV irradiation. Furthermore, HHV-6-infected cells were resistant to UV-induced apoptosis, which was independent of viral IE proteins. Taken together, these data suggest the possibility that HHV-6 employs some mechanism that inhibits the nuclear localization of p53 following UV irradiation and the consequent apoptosis.
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METHODS |
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HHV-6A strain U1102 and HHV-6B strain HST were propagated in CBMCs until their cytopathic effects were maximal. The cells were lysed by freezing and thawing once. Cell debris was removed by centrifugation at 1500 g for 5 min. The supernatants were used for virus infections.
For the infection of cells with HHV-6, 2x106 cells were collected by centrifugation, resuspended in 1 ml of the virus stock described above and incubated at 37 °C for 1 h. Mock- or HHV-6-infected cells were cultured in the appropriate medium as described above.
Antibodies.
p53 was detected by Western blotting with a monoclonal antibody (mAb) (DO-1) (Santa Cruz Biotechnology) at a 1 : 2000 dilution and by indirect immunofluorescence with a goat polyclonal antibody (FL-393) (Santa Cruz Biotechnology) used at a 1 : 200 dilution. mAbs against Rb (G3-245) (BD Biosciences) and -tubulin (B-5-1-2) (Sigma) were used at a 1 : 500 and 1 : 10 000 dilution, respectively.
To detect HHV-6B early and late antigens, the mAbs OHV-2, against the viral DNA replication compartment, and OHV-3, against glycoprotein H (gH), were used at a 1 : 100 dilution. For viral IE antigens, rabbit polyclonal antisera specific to HHV-6B IE1 and IE2 were used at a 1 : 1000 dilution. The mAbs OHV-2 (Mori et al., 2000) and OHV-3 (Takeda et al., 1997
) and the rabbit polyclonal antiserum against IE1 (Mori et al., 2002
) have been previously described. We expressed part of the HHV-6 U86 ORF in bacteria as a glutathione S-transferase fusion protein and raised U86-specific antiserum in rabbits to characterize the IE2 protein. U27 mAb was generated by immunizing mice with a recombinant U27 protein according to the method described elsewhere (Mori et al., 2002
). An immunogen was prepared as follows. A PCR product generated with the primers U27C334F (5'-AGTCGAATTCTTTGAAAGTTTTATGAACATCATC-3') and U27C1129R (5'-AGTCGTCGACTATCTCTGTCTCTTAGGATTGGAGC-3') was digested with EcoRI and SalI and inserted into a pMAL-c2 vector (New England Biolabs) at these sites. A maltose-binding protein fusion protein, MBPU27, expressed in Escherichia coli, was purified with amylose resin (New England Biolabs) and used for immunization. In the same way, we also generated U95 mAb using the primers U95C1180F (5'-AGTCGAATTCCAGGGAAAAGTTGTTTCTTCGAC-3') and U95C2061R (5'-AGTCGTCGACTACTGCTGAAGTCCGATCTTCATGAC-3').
Western blot analysis.
Cells were resuspended in Laemmli reducing sample buffer (LRSB) (50 mM Tris, pH 6·8, 2 % SDS, 10 % glycerol, 50 mM dithiothreitol, 0·5 % bromophenol blue) at a concentration of 104 cells (µl buffer)-1, then boiled for 5 min. Equal volumes of whole-cell lysate were loaded on to SDS-polyacrylamide gels, fractionated by electrophoresis and Western blotted on to PVDF membranes (Bio-Rad). The blots were blocked with 3 % skimmed milk in Tris-buffered saline (TBS) overnight at 4 °C, washed twice briefly with TBST (TBS containing 0·02 % Tween 20) and incubated for 1 h at room temperature with primary antibody in TBS containing 3 % BSA. The blots were then washed three times with TBST, incubated for 1 h at room temperature with a 1 : 2500 dilution of the appropriate secondary antibody conjugated to horseradish peroxidase (Amersham-Pharmacia) in TBS containing 3 % skimmed milk, washed three times with TBST and developed using an ECL detection kit (Amersham-Pharmacia) according to the manufacturer's instruction. The images on the blot were directly quantified by densitometry using a FluorChem IS-8000 imaging system (Alpha Innotech).
To detect the ubiquitinated forms of p53 according to a previously described method (Ling et al., 2003; Xirodimas et al., 2001
; Zaika et al., 1999
), Molt-3 cells infected with HHV-6B strain HST were treated with 0·5 µM MG132 (Peptide Institute), an inhibitor of the 26S proteasome, for the indicated 6 h periods, harvested after treatment and subjected to Western blot analysis.
Plasmids and transient transfection.
The HHV-6 U95 expression plasmid, pcDNA-U95, was generated as described previously (Takemoto et al., 2001) and the HHV-6 IE1 cDNA clone was obtained in the same way as U95. Briefly, the PstI-digested fragment from pSTY03 (Isegawa et al., 1999
) was 5'-end-labelled with [
-32P]ATP and used as a probe to screen the IE1 cDNA clone from a cDNA library of HHV-6 strain HST-infected cells (Mori et al., 1998
). The full-length IE1 cDNA clone whose sequence had been determined was excised from the library vector as a NotI fragment and inserted into pCEP4 at a NotI site. The HCMV major IEs (MIEs) expression plasmid, pSV2-neo-IE1/2, was a generous gift from Tsuguya Murayama (Kagoshima University).
MT-4 cells (2x106) were transfected with 1 µg of viral IE gene expression plasmids using lipofectamine plus (Invitrogen) according to the manufacturers instructions and subjected to Western blotting and an immunofluorescence assay (IFA) 36 h post-transfection.
Pulsechase and immunoprecipitation experiments.
Molt-3 cells (3x107 cells) were either mock infected or infected with HHV-6B strain HST, then pulsed at 24 h post-infection (p.i.) with 100 µCi [35S]methionine ml-1 for 1 h, washed twice with serum-free medium and cultured in medium containing unlabelled L-methionine for 0, 2, 4, 6, 8 or 16 h of chase. The cells were lysed in RIPA buffer (10 mM Tris, pH 7·4, 1 % NP-40, 0·1 % SDS, 0·1 % sodium deoxycholate, 0·15 M NaCl, 1 mM EDTA and 2 µg each of leupeptin, aprotinin and pepstatin ml-1) for 1 h at 4 °C, then spun in a centrifuge. The supernatants of the cell lysates were pre-incubated with protein GSepharose, which had previously been blocked with 0·1 % FCS to reduce non-specific binding of proteins to Sepharose beads. Subsequently, the supernatants were incubated with anti-p53 or -tubulin mAb-bound protein GSepharose at 4 °C overnight. The Sepharose beads were rinsed four times with RIPA buffer, resuspended in LRSB and boiled for 5 min. The immunoprecipitated proteins were separated by 10 % SDS-PAGE and treated with Enlight (Mo Bi Tec). The gel was dried and exposed to a BAS-III imaging plate (Fujifilm). The autoradiogram was imaged and quantified with a BAS 2000 II Bio-imaging analyser (Fujix). The percentage stability was calculated as the value relative to that of each protein chased for 0 h, which was defined as 100 %.
Indirect immunofluorescence assay.
Cells were plated on glass coverslips, fixed in cold acetone for 5 min and incubated for 1 h at room temperature with the primary antibody in PBS containing 3 % BSA. The coverslips were then washed with PBST for 5 min, followed by a wash with PBS for 5 min and incubated for 30 min at room temperature with the appropriate secondary antibody labelled with fluorescein isothiocyanate (FITC) or tetramethyl rhodamine isothiocyanate (TRITC) (Dako). After washing as above, the coverslips were mounted in glycerol and examined by fluorescence microscopy. The confocal images were captured using a Zeiss LSM410 confocal microscope and software provided by the manufacturer.
Exposure of viruses and cells to UV irradiation.
To inactivate HHV-6B strain HST by UV light, 1 ml of an HST stock was plated in a 6 cm diameter dish and irradiated at 2500 J m-2. To confirm the inactivation of the virus, an IFA detected no IE1 gene expression at 24 h p.i., indicating that there was no virus survival.
To induce DNA damage and subsequent p53-dependent apoptosis in Molt-3 cells infected with strain HST, cells at 6 or 24 h p.i. were concentrated to 2x106 cells ml-1 in culture medium, irradiated at 60 J m-2 and cultured at a concentration of 4x105 cells ml-1 for 12 h. The induction of apoptosis was analysed by the detection of DNA fragmentation following electrophoresis, a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) assay and annexin V staining to visualize phosphatidylserine externalized in the early apoptotic phase.
DNA extraction and electrophoresis.
Cells (5x105) exposed to UV irradiation were collected, resuspended in 200 µl of PBS(-) containing 0·5 % SDS and 500 µg proteinase K ml-1 and incubated for 1 h at 55 °C. RNase A (50 µg) was added and incubation at 55 °C was continued for 1 h. Samples were mixed with 300 µl sodium iodide solution (6 M NaI, 26 mM Tris, pH 8·0, 13 mM EDTA, 0·5 % sodium-N-lauroylsarcosinate) containing 3 µl Ethachin mate (Nippongene) and incubated for 15 min at 60 °C. DNA was precipitated with 2-propanol, dried and resuspended in TE (10 mM Tris, pH 8·0, 1 mM EDTA). The extracted DNA was subjected to electrophoresis in a 2 % agarose gel and visualized by UV light after staining with ethidium bromide.
TUNEL assay.
Cells (106) exposed to UV irradiation were collected after 12 h and subjected to the TUNEL assay according to the manufacturer's instructions (in situ cell death detection kit; Boehringer) except for the fixation step (70 % ethanol at -20 °C for 1 h). Samples were analysed on a FACSCalibur (CELLQuest software; BD Biosciences).
Annexin V staining.
Cells (106) exposed to UV irradiation were collected after 6 h and subjected to annexin V staining, according to the manufacturer's instructions (Annexin V-FITC apoptosis detection kit; BioVision). Samples were analysed on a FACSCalibur.
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RESULTS |
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The p53 level increases at the initial stage of HHV-6 infection
To determine the time point of the increase in p53 level, total cell extracts from HST-infected Molt-3 cells harvested at 0, 4, 8, 16, 24, 48 and 72 h p.i. were analysed by Western blotting. In these HST-infected cells, the p53 level began to increase by 4 h p.i., reached a maximum at 48 h p.i. and remained high until 72 h p.i. (Fig. 2A and B). In contrast, the level and hyperphosphorylated form of another tumour suppressor protein, Rb, was constant throughout the course of the HST infection.
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These observations suggest the involvement of one or more IE genes in the elevation of p53 levels in HHV-6-infected cells.
HHV-6 infection induces the stabilization and synthesis of p53
p53 function is controlled through several mechanisms, one of the most effective being the regulation of protein stability. Numerous studies have shown the involvement of multiple pathways in p53 stabilization. To test whether the elevation of p53 in HHV-6-infected cells is dependent on the regulation of the protein's stability, we performed a pulsechase radiolabelling experiment in Molt-3 cells infected with strain HST, at 24 h p.i. p53 protein pulse-labelled with [35S]methionine was extracted and immunoprecipitated with DO-1-conjugated protein GSepharose beads after chase periods of 0, 2, 4, 6, 8 and 16 h. As shown in Fig. 3(A) and (B), a slight increase in the amount of p53 was observed in both mock- and HHV-6-infected cells after a 2 h chase, but from the 4 h chase to the 16 h chase, the stability of the p53 protein in the HHV-6-infected cells was approximately twofold higher than in the mock-infected cells at every time point. In contrast, the stability of
-tubulin showed no signifi<1?show=[to]>cant difference between mock- and HHV-6-infected cells. These results indicated that the stability of p53 increases in HHV-6-infected cells. In addition, HHV-6-infected cells that were pulse-labelled for 1 h (and chased for 0 h) exhibited a 1·4-fold higher amount of p53 protein than mock-infected cells, indicating that the synthesis of p53 protein was enhanced in the HHV-6-infected cells. These results suggest that the elevation of p53 in HHV-6-infected cells is due to increases in both its stability and synthesis.
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Finally, we confirmed the prevention of apoptosis by HHV-6 infection using annexin V staining. The levels of apoptosis (percentage of annexin-V-positive and propidium iodide-negative cells) of mock- and HST-infected Molt-3 cells at 30 h p.i. were only 2·87 % and 3·55 %, respectively, without any treatment (Fig. 6C). In contrast, the level of apoptosis of the mock-infected cells caused by UV irradiation (60 J m-2) had increased to 29·62 % by 6 h post-irradiation; that of the HST-infected cells at 24 h p.i. was only 9·41 %, demonstrating that the HST-infected cells are indeed resistant to UV-induced apoptosis. Taken together, these findings imply that HHV-6 protects infected cells against the p53-dependent apoptosis induced by UV irradiation by inhibiting p53 from translocating to the nucleus.
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DISCUSSION |
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In the present study, we demonstrated that the p53 level increased in Molt-3 cells soon after infection (4 h p.i.) with HHV-6 strain HST, but did not increase in Molt-3 cells infected with UV-inactivated virus, suggesting the possibility that viral IE gene expression is involved. In addition, a pulsechase experiment revealed that the increase in p53 level was due to increases in both the synthesis and the stabilization of the protein. When cells were radiolabelled for 1 h, the labelled p53 protein in HST-infected Molt-3 cells was higher by 1·4-fold after a 0 h chase and also higher by twofold at every time point examined after a 4 h chase than in mock-infected cells, indicating that both synthesis and stabilization of p53 were upregulated in the infected cells. The extremely small amount of protein (Fig. 3A, 16 h chase in mock-infected cells) may be due to loss of material as a result of cell death by the time of the 16 h chase. It is reported that HCMV stabilizes p53 but does not increase its synthesis (Fortunato & Spector, 1998) and that the p53 increase caused by HSV-1 ICP0 is independent of the transcription of p53 (Hobbs & DeLuca, 1999
). In this study, we showed that HHV-6 has the same effect on the regulation of p53, i.e. stabilization, as other herpesviruses, but also a different effect an increase in p53 synthesis.
We next addressed the state of ubiquitination of p53 and observed the reduced level of ubiquitinated p53 in HST-infected Molt-3 cells soon after infection (Fig. 4). Little has been reported to date on the possibility that viral infection causes the stabilization of cellular proteins through deubiquitination, whereas there are many reports of viruses promoting the ubiquitination and subsequent degradation of specific cellular proteins to optimize the conditions of the cell for its replication. Recently, p53 was shown to interact directly with herpesvirus-associated ubiquitin-specific protease (HAUSP). HAUSP is known to interact with HSV-1 ICP0, to co-localize with the PML nuclear body and to stabilize p53 through its intrinsic deubiquitinating enzyme activity (Li et al., 2002
). It is possible that herpesviruses exploit cellular enzymes such as the ubiquitin-specific processing protease (UBP) to stabilize specific cellular proteins that are required for efficient virus replication, but no evidence currently exists to support this scenario.
Normally, p53 is activated in response to various genotoxic stresses, including UV irradiation, and accumulates in the nucleus, as shown in Fig. 5(B, a). However, in HHV-6-infected cells, the cytoplasmic localization of p53 was unchanged, even when the infected cells were irradiated with UV light at 24 h p.i. (Fig. 5B, b and c). This result led us to hypothesize that a viral protein may directly bind p53 and inhibit its nuclear localization. One possible candidate for preventing p53-dependent apoptosis is DR7, which has been reported to bind to and inactivate p53 (Kashanchi et al., 1997
).
More intriguing is that the localization of p53 in cells infected with HHV-6 is quite different from that seen in cells infected with other HHVs. During productive infection for all the subgroups of HHVs, namely HSV (Wilcock & Lane, 1991; Zhong & Hayward, 1997
), HCMV (Fortunato & Spector, 1998
) and KSHV (Katano et al., 2001
), p53 has been reported to co-localize with virus replication compartments in the nucleus. The recruitment of p53 into these virus replication foci leads us to speculate that these viruses exploit p53 for their efficient replication, but no evidence currently exists to support this assumption. Although there are reports that HCMV sequesters p53 in the cytoplasm in infected multinucleated giant cells, p53 has also been shown to co-localize with IE84 protein in the nucleus in single nuclear non-giant cells that are thought to be in the early phase of infection (Kovacs et al., 1996
; Wang et al., 2001
). In contrast, in HHV-6-infected cells, p53 predominantly accumulated in the cytoplasm and co-localized with gH, but not with the early nuclear antigen labelled by OHV-2, which is a putative component of the virus replication compartment. In addition, there is no evidence that p53 accumulates in the nucleus at any stage of HHV-6 infection (data not shown). If p53 plays some positive role in the replication of other HHVs, it will be of interest to find out whether HHV-6 employs a replication mechanism that does not require the recruitment of p53, unlike other herpesviruses.
Here we have demonstrated that the p53-dependent apoptosis induced by UV irradiation was blocked at 24 h p.i. (Fig. 6). To determine whether UV-induced apoptosis in Molt-3 cells was actually mediated by p53 activation, the number of cells that were positive for the nuclear accumulation of p53 was counted in three fields. Approximately 43 % of the cells were positive for nuclear accumulation of p53 (data not shown), indicating that UV irradiation induces p53-dependent apoptosis in Molt-3 cells, when their 55 % TUNEL-positive labelling is taken into account (Fig. 6B
).
Whereas the elevation of the p53 level occurred at the IE stage, the block of apoptosis was not observed until the late stage, indicating that these two effects of HHV-6 infection on p53 are carried out by different viral proteins.
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ACKNOWLEDGEMENTS |
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Received 3 September 2003;
accepted 2 December 2003.