Human Herpesvirus 6B Induces Cell Cycle Arrest Con
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病菌学杂志 2005年第3期
Department of Medical Microbiology and Immunology, University of Aarhus, Aarhus, Denmark
ABSTRACT
We studied the interactions between human herpesvirus 6B (HHV-6B) and its host cell. Productive infections of T-cell lines led to G1/S- and G2/M-phase arrest in the cell cycle concomitant with an increased level and enhanced DNA-binding activity of p53. More than 70% of HHV-6B-infected cells did not bind annexin V, indicating that the majority of cells were not undergoing apoptosis. HHV-6B infection induced Ser20 and Ser15 phosphorylation on p53, and the latter was inhibited by caffeine, an ataxia telangiectasia mutated kinase inhibitor. Thus, a productive HHV-6B infection suppresses T-cell proliferation concomitant with the phosphorylation and accumulation of p53.
TEXT
Human herpesvirus 6B (HHV-6B), the causative agent of exanthema subitum, infects virtually 100% of individuals in the Western world (8, 10). Using CD46 (23) and possibly unidentified coreceptors (7), the virus gains entrance to T cells and monocytes in the peripheral blood (5). It further spreads to other cells, including astrocytes within the central nervous system (9, 14). As with other herpesvirus infections, suppression of the immune system may lead to reactivation of the virus, with potentially severe clinical consequences. Despite this disease potential, little is known about the mechanism of HHV-6B interaction with T cells. HHV-6B was shown previously to shut off host cell DNA synthesis 65 h after infection (12). Prior to this, a marked increase in protein synthesis without subsequent cell division was observed (2), suggesting a disruption between cytoplasmic growth and cell cycle progression.
T cells (the MOLT 3 cell line) were infected with HHV-6B strain PL-1 at 400 50% tissue culture infective doses (TCID50s), as determined by the Reed-Muench method. In contrast to uninfected T cells, productively infected cells failed to increase in number (Fig. 1A). To evaluate the percentage of cells infected by HHV-6B, we stained T cells by immunofluorescence for the presence of p41, a nuclear protein that is essential for viral propagation (1). Approximately 100% of the cells displayed p41 nuclear expression 71 h after infection (Fig. 1B), and most of the cells showed clear signs of enlargement, which is indicative of increased protein synthesis (Fig. 1B, right panels). Since HHV-6B was previously reported to induce apoptosis (16, 17, 31), we further examined the viability of the cells. At first, no difference in membrane integrity could be detected between infected and uninfected cells by trypan blue dye exclusion tests (data not shown). Since one of the earliest indications of apoptosis is the translocation of phosphatidylserine from the inner to the outer plasma membrane, we further examined the binding of fluorescein isothiocyanate-conjugated annexin V, which has a high affinity for binding to phosphatidylserine. When tested during the first 7 days after infection, >90% of uninfected cells did not bind annexin V, as opposed to 70% of HHV-6B-infected cells (Fig. 1C). This demonstrated that about 20% of the HHV-6B-infected cells underwent virus-induced apoptosis, whereas the majority (70%) were arrested in the cell cycle without yet being committed to apoptosis. To estimate the proportion of cells that entered S phase after infection, we infected MOLT 3 cells with HHV-6B for 8 to 96 h and pulsed them for 1 h with 1 μCi of [3H]thymidine (Amersham Biosciences, H?rsholm, Denmark). In contrast to uninfected T cells, the HHV-6B-infected T cells stopped incorporating [3H]thymidine within the first 24 h of infection (Fig. 1D). Further kinetic analysis indicated a major drop in [3H]thymidine incorporation after approximately 10 h of infection (Fig. 1D, top). The stop in the cell cycle not only was seen when MOLT 3 cells were infected, but also was striking when a different T-cell line, SupT1, was productively infected with HHV-6B (Fig. 1D, open circles).
To determine whether this observation only reflected a block in the G1/S phase, we stained uninfected and HHV-6B-infected MOLT 3 cells with 7-aminoactinomycin D (25) (Beckman Coulter, Marseille, France) after 24 and 48 h of infection. The distribution of cells within each phase of the cell cycle was analyzed by flow cytometry (Fig. 1E). Corroborating the annexin staining experiments, the flow cytometry analysis indicated a small fraction of cells undergoing HHV-6B-induced apoptosis (Fig. 1E, right panels). While the fraction of cells in S phase decreased after infection, the proportion of cells in G2/M was maintained. This suggested that HHV-6B infection induced a G1/S and a G2/M block in the cell cycle. These blocks were dependent on infectious virus, as UV inactivation of HHV-6B prior to infection abolished the inhibition of [3H]thymidine incorporation (Fig. 1F). This indicated that immediate early and early viral gene products were responsible for the cell cycle arrest.
The simultaneous arrest in G1/S and G2/M would be consistent with a p53-mediated effect (29). An important role of p53 is to maintain the integrity of the genome by inducing cell cycle arrest until damage has been repaired or by inducing apoptosis in cells constituting a risk for the organism (29). To further explore the mechanism of HHV-6B-induced cell cycle arrest, we therefore examined whether HHV-6B infection could induce p53 expression. Using an anti-human p53 antibody (Biosource, Nivelles, Belgium) for immunoblots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated whole-cell lysates from uninfected or HHV-6B-infected cells, we detected an increased level of p53 in HHV-6B-infected cells starting from 18 h postinfection (Fig. 2A). The amount increased with the time of infection, in contrast to the stable levels of ?-tubulin (anti-?-tubulin; Santa Cruz Biotechnology Inc., Santa Cruz, Calif.). Although the amount of p53 in uninfected cells varied slightly from experiment to experiment, it was always clearly upregulated by HHV-6B infection. In addition, the SupT1 cell line, which was arrested during cell cycle progression by HHV-6B infection, also upregulated the level of p53 protein (Fig. 2B). To directly address the induction of p53, we performed real-time reverse transcription-PCR analysis with the human equivalents of previously published murine p53 primers (4) essentially as described previously (21). A modest increase in the amount of p53 mRNA was detected in HHV-6B-infected cells, whereas no change in the expression of ?-actin was seen (data not shown). The normal cycling of p53 in a feedback loop with the p53-binding protein MDM2 indicates a tightly regulated transcription of p53 and a rapid turnover rate (19). However, since HHV-6B-infected T cells seemed to accumulate the p53 protein in an unregulated fashion, it is likely that the accumulation of p53 was a combination of an increase in p53 transcription and a decrease in p53 degradation.
Given their important consequences on the fate of the cell, p53 nuclear translocation and export are tightly regulated. To investigate whether HHV-6B-induced p53 was present in the nucleus, we prepared nuclear and cytoplasmic extracts from T cells (nuclear extract kit; Active Motif, Rixensart, Belgium) with and without HHV-6B infection for 48 h. As determined by immunoblotting, the majority of HHV-6B-induced p53 was located in the nucleus (Fig. 2C), but in contrast to the case for uninfected cells, a significant portion of p53 was found in the cytoplasm. Western blot analyses of nuclear (RCC1) and cytoplasmic (glyceraldehyde-3-phosphate dehydrogenase) marker proteins indicated that the nuclear fractions contained a minor cytoplasmic contamination, whereas the cytoplasmic fractions were highly pure and free from detectable nuclear RCC1 protein (data not shown). We therefore concluded that a significantly higher level of cytoplasmic p53 protein could be detected in HHV-6B-infected T cells than in uninfected cells. At the same time, p53 was easily detectable in the nuclei of HHV-6B-infected T cells, which was further confirmed by immunofluorescence staining (data not shown). The DNA-binding activity of p53 was studied by use of an enzyme-linked immunosorbent assay-based method (TransAM; Active Motif) (22) in which 96-well plates were coated with an oligonucleotide containing the p53 consensus binding site (5'-GGACATGCCGGGCATGTCC-3'). When the DNA-binding activity of p53 was quantified, a significant increase in p53 activity was observed as early as 12 h postinfection (Fig. 2D). Importantly, the increase in p53 DNA binding occurred at or before the cell cycle arrest.
Consistent with an enhanced DNA-binding activity, HHV-6B-infected cells (48 h) and, to a much lesser degree, mock-treated cells (48 h) displayed p53 phosphorylation on Ser15 (anti-p53 pS15; Biosource), a phosphorylation site that is associated with reduced MDM2 binding and increased p53 stability (30). Importantly, only the HHV-6B-infected cells showed a mobility shift for phosphorylated p53, probably indicating phosphorylation at multiple sites (Fig. 3). Recently, vesicular stomatitis virus and herpes simplex virus were reported to induce ataxia telangiectasia mutated (ATM)-dependent phosphorylation of p53 on Ser18 (corresponding to human Ser15) in mouse embryonic fibroblasts, as phosphorylation was significantly reduced in fibroblasts deficient in the Atm gene (27). It was suggested that the viral induction of p53 in the presence of alpha/beta interferon (IFN-/?) enhanced the apoptotic response in virally infected cells. T cells, however, are a minor source of IFN-/?. Nevertheless, the presence of Ser15 phosphorylation in infected T cells would be consistent with an HHV-6B-induced activation of ATM. To further address this point, we attempted to inhibit the ATM kinase activity by using caffeine (3, 24). In uninfected cells, the presence of 5 mM caffeine had no impact on the level of p53 or p53 phosphorylated on Ser20, whereas the low level of p53 with Ser15 phosphorylation was significantly reduced (Fig. 3). In HHV-6B-infected T cells, preincubation with 5 mM caffeine 30 min prior to infection caused a reduction in the amount of mobility-shifted Ser15-phosphorylated p53, suggesting an HHV-6B-mediated induction of ATM kinase activity (Fig. 3). The finding of virus-induced ATM-mediated phosphorylation of p53 by a completely different virus (vesicular stomatitis virus) (27) suggests that a more general mechanism of virus-mediated p53 phosphorylation remains to be elucidated.
Our studies demonstrated that a productive infection of MOLT 3 cells with HHV-6B leads to cell cycle arrest concomitant with the virus-mediated phosphorylation and accumulation of p53. Surprisingly, we observed that p21, a known p53-induced mediator of cell cycle arrest, was not upregulated in HHV-6B-infected T cells, nor was PUMA, a p53-induced protein involved in apoptosis. Supporting this observation, reverse transcription-PCR analysis indicated that p21 mRNA was not upregulated in HHV-6B-infected cells (unpublished observations). This was in accordance with the results of a recent paper, in which HHV-6A-infected cord blood mononuclear cells were shown to accumulate in G2/M in the absence of p21 and 14-3-3 accumulation (11). The DR7 gene product from HHV-6A may inhibit p53-induced transcription by interacting with its DNA-binding domain (18). Whether the corresponding gene product from HHV-6B explains the lack of p21 induction is unclear, since we did detect increased p53 DNA-binding activity. Indeed, p53-dependent apoptosis in the absence of transcriptional activation of p53 target genes has been reported (6), suggesting the possibility of p53-mediated effects in growth arrest induction that are separate from transcriptional activation. Thus, the potential role of p53 in the cell cycle growth inhibition of HHV-6B-infected T cells remains to be further defined.
Previously, HHV-6B was shown to induce apoptosis (16, 17, 31). However, our findings are not in conflict with those of an initial report (17) in which apoptosis was found to be enhanced by extracellular factors (tumor necrosis factor alpha) and predominantly present in uninfected but not productively infected cells. The report of IFN-/? priming of virus-infected cells for apoptosis by a p53-dependent mechanism is a further example of the potential influence of the extracellular environment (27). In contrast to the results of experiments performed on transformed cells (our data and reference 17), apoptosis was shown to occur in primary cells such as cord blood mononuclear cells and blood cells from infants with HHV-6 infections (16, 31). It is likely that differentiation and cell type-specific factors are important for the decision to undergo apoptosis. Indeed, thymocytes and immature T cells go through apoptosis as a part of negative selection if p53 increases and no survival signals are delivered (30). Posttranslational modifications of p53 may determine whether apoptosis or growth arrest is induced. Based on data from mice, it has been suggested that Chk2 regulates p53 to promote apoptosis whereas ATM favors cell cycle arrest (15). HHV-6B infection induced p53 phosphorylation on Ser15, a site that is known to be phosphorylated by ATM. Importantly, this was seen as a major, slightly retarded band on a sodium dodecyl sulfate gel, with a minor band present below the major band. HHV-6B infection also induced the phosphorylation of p53 on Ser20, a site that is phosphorylated by Chk2. However, if Ser20-phosphorylated p53 was also phosphorylated on Ser15, it would be expected to migrate with the same slightly retarded mobility as that identified on the Ser15-p53 Western blot. Since this was not the case, we speculate that the Ser15-phosphorylated p53 was primarily derived from arrested cells and that the Ser20-phosphorylated p53 was derived from apoptotic cells.
HHV-6B-infected MOLT 3 cells were arrested during the cell cycle, and only a minority underwent apoptosis during the first week after infection. Arresting cells, rather than destroying them, may be an evolutionarily developed advantage for a virus that arose at about the same time as vertebrates. HHV-6B is a relatively slowly growing virus which does not produce high titers, and from a biological point of view, it will favor survival and will hide from the immune system. Keeping productively infected cells alive may help in achieving this goal, whereas apoptotic cells are taken up by dendritic cells for cross-presentation and necrotic cells initiate immune reactions by the liberation of viral proteins.
HHV-6B is a betaherpesvirus related to human cytomegalovirus (HCMV). Infection by HCMV may also lead to cell cycle arrest (20). Indeed, various HCMV-encoded proteins have been shown to mediate this block. Importantly, the UL36 gene product inhibits caspase activation (26) and the UL37 gene product interferes with the mitochondrion-induced activation of apoptosis (13). Thus, it will be important to determine if HHV-6B has developed similar mechanisms for the prevention of apoptosis in cell cycle-arrested T cells.
ADDENDUM Since the submission of our work, a paper on p53 and HHV-6 infection was published by Takemoto et al. (28). In this paper, p53 was shown to be induced and stabilized following HHV-6 infection concomitant with a block in apoptosis induction. These authors found that p53 is sequestered in the cytoplasm, and they suggest that this may be part of the mechanism that protects infected cells from apoptosis.
ACKNOWLEDGMENTS
We thank P. Lusso for the PL-1 strain of HHV-6B, Z. Berneman for cell lines, S. R. Paludan for ?-actin primers, and B. Vandahl and S. Birkelund for help with the electronic retrieval of immunofluorescence data.
This work was supported by grants from the University of Aarhus, the Danish Multiple Sclerosis Society, Leo Nielsen and Karen Margrethe Nielsen's Foundation, and the King Christian the Xth Foundation.
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ABSTRACT
We studied the interactions between human herpesvirus 6B (HHV-6B) and its host cell. Productive infections of T-cell lines led to G1/S- and G2/M-phase arrest in the cell cycle concomitant with an increased level and enhanced DNA-binding activity of p53. More than 70% of HHV-6B-infected cells did not bind annexin V, indicating that the majority of cells were not undergoing apoptosis. HHV-6B infection induced Ser20 and Ser15 phosphorylation on p53, and the latter was inhibited by caffeine, an ataxia telangiectasia mutated kinase inhibitor. Thus, a productive HHV-6B infection suppresses T-cell proliferation concomitant with the phosphorylation and accumulation of p53.
TEXT
Human herpesvirus 6B (HHV-6B), the causative agent of exanthema subitum, infects virtually 100% of individuals in the Western world (8, 10). Using CD46 (23) and possibly unidentified coreceptors (7), the virus gains entrance to T cells and monocytes in the peripheral blood (5). It further spreads to other cells, including astrocytes within the central nervous system (9, 14). As with other herpesvirus infections, suppression of the immune system may lead to reactivation of the virus, with potentially severe clinical consequences. Despite this disease potential, little is known about the mechanism of HHV-6B interaction with T cells. HHV-6B was shown previously to shut off host cell DNA synthesis 65 h after infection (12). Prior to this, a marked increase in protein synthesis without subsequent cell division was observed (2), suggesting a disruption between cytoplasmic growth and cell cycle progression.
T cells (the MOLT 3 cell line) were infected with HHV-6B strain PL-1 at 400 50% tissue culture infective doses (TCID50s), as determined by the Reed-Muench method. In contrast to uninfected T cells, productively infected cells failed to increase in number (Fig. 1A). To evaluate the percentage of cells infected by HHV-6B, we stained T cells by immunofluorescence for the presence of p41, a nuclear protein that is essential for viral propagation (1). Approximately 100% of the cells displayed p41 nuclear expression 71 h after infection (Fig. 1B), and most of the cells showed clear signs of enlargement, which is indicative of increased protein synthesis (Fig. 1B, right panels). Since HHV-6B was previously reported to induce apoptosis (16, 17, 31), we further examined the viability of the cells. At first, no difference in membrane integrity could be detected between infected and uninfected cells by trypan blue dye exclusion tests (data not shown). Since one of the earliest indications of apoptosis is the translocation of phosphatidylserine from the inner to the outer plasma membrane, we further examined the binding of fluorescein isothiocyanate-conjugated annexin V, which has a high affinity for binding to phosphatidylserine. When tested during the first 7 days after infection, >90% of uninfected cells did not bind annexin V, as opposed to 70% of HHV-6B-infected cells (Fig. 1C). This demonstrated that about 20% of the HHV-6B-infected cells underwent virus-induced apoptosis, whereas the majority (70%) were arrested in the cell cycle without yet being committed to apoptosis. To estimate the proportion of cells that entered S phase after infection, we infected MOLT 3 cells with HHV-6B for 8 to 96 h and pulsed them for 1 h with 1 μCi of [3H]thymidine (Amersham Biosciences, H?rsholm, Denmark). In contrast to uninfected T cells, the HHV-6B-infected T cells stopped incorporating [3H]thymidine within the first 24 h of infection (Fig. 1D). Further kinetic analysis indicated a major drop in [3H]thymidine incorporation after approximately 10 h of infection (Fig. 1D, top). The stop in the cell cycle not only was seen when MOLT 3 cells were infected, but also was striking when a different T-cell line, SupT1, was productively infected with HHV-6B (Fig. 1D, open circles).
To determine whether this observation only reflected a block in the G1/S phase, we stained uninfected and HHV-6B-infected MOLT 3 cells with 7-aminoactinomycin D (25) (Beckman Coulter, Marseille, France) after 24 and 48 h of infection. The distribution of cells within each phase of the cell cycle was analyzed by flow cytometry (Fig. 1E). Corroborating the annexin staining experiments, the flow cytometry analysis indicated a small fraction of cells undergoing HHV-6B-induced apoptosis (Fig. 1E, right panels). While the fraction of cells in S phase decreased after infection, the proportion of cells in G2/M was maintained. This suggested that HHV-6B infection induced a G1/S and a G2/M block in the cell cycle. These blocks were dependent on infectious virus, as UV inactivation of HHV-6B prior to infection abolished the inhibition of [3H]thymidine incorporation (Fig. 1F). This indicated that immediate early and early viral gene products were responsible for the cell cycle arrest.
The simultaneous arrest in G1/S and G2/M would be consistent with a p53-mediated effect (29). An important role of p53 is to maintain the integrity of the genome by inducing cell cycle arrest until damage has been repaired or by inducing apoptosis in cells constituting a risk for the organism (29). To further explore the mechanism of HHV-6B-induced cell cycle arrest, we therefore examined whether HHV-6B infection could induce p53 expression. Using an anti-human p53 antibody (Biosource, Nivelles, Belgium) for immunoblots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated whole-cell lysates from uninfected or HHV-6B-infected cells, we detected an increased level of p53 in HHV-6B-infected cells starting from 18 h postinfection (Fig. 2A). The amount increased with the time of infection, in contrast to the stable levels of ?-tubulin (anti-?-tubulin; Santa Cruz Biotechnology Inc., Santa Cruz, Calif.). Although the amount of p53 in uninfected cells varied slightly from experiment to experiment, it was always clearly upregulated by HHV-6B infection. In addition, the SupT1 cell line, which was arrested during cell cycle progression by HHV-6B infection, also upregulated the level of p53 protein (Fig. 2B). To directly address the induction of p53, we performed real-time reverse transcription-PCR analysis with the human equivalents of previously published murine p53 primers (4) essentially as described previously (21). A modest increase in the amount of p53 mRNA was detected in HHV-6B-infected cells, whereas no change in the expression of ?-actin was seen (data not shown). The normal cycling of p53 in a feedback loop with the p53-binding protein MDM2 indicates a tightly regulated transcription of p53 and a rapid turnover rate (19). However, since HHV-6B-infected T cells seemed to accumulate the p53 protein in an unregulated fashion, it is likely that the accumulation of p53 was a combination of an increase in p53 transcription and a decrease in p53 degradation.
Given their important consequences on the fate of the cell, p53 nuclear translocation and export are tightly regulated. To investigate whether HHV-6B-induced p53 was present in the nucleus, we prepared nuclear and cytoplasmic extracts from T cells (nuclear extract kit; Active Motif, Rixensart, Belgium) with and without HHV-6B infection for 48 h. As determined by immunoblotting, the majority of HHV-6B-induced p53 was located in the nucleus (Fig. 2C), but in contrast to the case for uninfected cells, a significant portion of p53 was found in the cytoplasm. Western blot analyses of nuclear (RCC1) and cytoplasmic (glyceraldehyde-3-phosphate dehydrogenase) marker proteins indicated that the nuclear fractions contained a minor cytoplasmic contamination, whereas the cytoplasmic fractions were highly pure and free from detectable nuclear RCC1 protein (data not shown). We therefore concluded that a significantly higher level of cytoplasmic p53 protein could be detected in HHV-6B-infected T cells than in uninfected cells. At the same time, p53 was easily detectable in the nuclei of HHV-6B-infected T cells, which was further confirmed by immunofluorescence staining (data not shown). The DNA-binding activity of p53 was studied by use of an enzyme-linked immunosorbent assay-based method (TransAM; Active Motif) (22) in which 96-well plates were coated with an oligonucleotide containing the p53 consensus binding site (5'-GGACATGCCGGGCATGTCC-3'). When the DNA-binding activity of p53 was quantified, a significant increase in p53 activity was observed as early as 12 h postinfection (Fig. 2D). Importantly, the increase in p53 DNA binding occurred at or before the cell cycle arrest.
Consistent with an enhanced DNA-binding activity, HHV-6B-infected cells (48 h) and, to a much lesser degree, mock-treated cells (48 h) displayed p53 phosphorylation on Ser15 (anti-p53 pS15; Biosource), a phosphorylation site that is associated with reduced MDM2 binding and increased p53 stability (30). Importantly, only the HHV-6B-infected cells showed a mobility shift for phosphorylated p53, probably indicating phosphorylation at multiple sites (Fig. 3). Recently, vesicular stomatitis virus and herpes simplex virus were reported to induce ataxia telangiectasia mutated (ATM)-dependent phosphorylation of p53 on Ser18 (corresponding to human Ser15) in mouse embryonic fibroblasts, as phosphorylation was significantly reduced in fibroblasts deficient in the Atm gene (27). It was suggested that the viral induction of p53 in the presence of alpha/beta interferon (IFN-/?) enhanced the apoptotic response in virally infected cells. T cells, however, are a minor source of IFN-/?. Nevertheless, the presence of Ser15 phosphorylation in infected T cells would be consistent with an HHV-6B-induced activation of ATM. To further address this point, we attempted to inhibit the ATM kinase activity by using caffeine (3, 24). In uninfected cells, the presence of 5 mM caffeine had no impact on the level of p53 or p53 phosphorylated on Ser20, whereas the low level of p53 with Ser15 phosphorylation was significantly reduced (Fig. 3). In HHV-6B-infected T cells, preincubation with 5 mM caffeine 30 min prior to infection caused a reduction in the amount of mobility-shifted Ser15-phosphorylated p53, suggesting an HHV-6B-mediated induction of ATM kinase activity (Fig. 3). The finding of virus-induced ATM-mediated phosphorylation of p53 by a completely different virus (vesicular stomatitis virus) (27) suggests that a more general mechanism of virus-mediated p53 phosphorylation remains to be elucidated.
Our studies demonstrated that a productive infection of MOLT 3 cells with HHV-6B leads to cell cycle arrest concomitant with the virus-mediated phosphorylation and accumulation of p53. Surprisingly, we observed that p21, a known p53-induced mediator of cell cycle arrest, was not upregulated in HHV-6B-infected T cells, nor was PUMA, a p53-induced protein involved in apoptosis. Supporting this observation, reverse transcription-PCR analysis indicated that p21 mRNA was not upregulated in HHV-6B-infected cells (unpublished observations). This was in accordance with the results of a recent paper, in which HHV-6A-infected cord blood mononuclear cells were shown to accumulate in G2/M in the absence of p21 and 14-3-3 accumulation (11). The DR7 gene product from HHV-6A may inhibit p53-induced transcription by interacting with its DNA-binding domain (18). Whether the corresponding gene product from HHV-6B explains the lack of p21 induction is unclear, since we did detect increased p53 DNA-binding activity. Indeed, p53-dependent apoptosis in the absence of transcriptional activation of p53 target genes has been reported (6), suggesting the possibility of p53-mediated effects in growth arrest induction that are separate from transcriptional activation. Thus, the potential role of p53 in the cell cycle growth inhibition of HHV-6B-infected T cells remains to be further defined.
Previously, HHV-6B was shown to induce apoptosis (16, 17, 31). However, our findings are not in conflict with those of an initial report (17) in which apoptosis was found to be enhanced by extracellular factors (tumor necrosis factor alpha) and predominantly present in uninfected but not productively infected cells. The report of IFN-/? priming of virus-infected cells for apoptosis by a p53-dependent mechanism is a further example of the potential influence of the extracellular environment (27). In contrast to the results of experiments performed on transformed cells (our data and reference 17), apoptosis was shown to occur in primary cells such as cord blood mononuclear cells and blood cells from infants with HHV-6 infections (16, 31). It is likely that differentiation and cell type-specific factors are important for the decision to undergo apoptosis. Indeed, thymocytes and immature T cells go through apoptosis as a part of negative selection if p53 increases and no survival signals are delivered (30). Posttranslational modifications of p53 may determine whether apoptosis or growth arrest is induced. Based on data from mice, it has been suggested that Chk2 regulates p53 to promote apoptosis whereas ATM favors cell cycle arrest (15). HHV-6B infection induced p53 phosphorylation on Ser15, a site that is known to be phosphorylated by ATM. Importantly, this was seen as a major, slightly retarded band on a sodium dodecyl sulfate gel, with a minor band present below the major band. HHV-6B infection also induced the phosphorylation of p53 on Ser20, a site that is phosphorylated by Chk2. However, if Ser20-phosphorylated p53 was also phosphorylated on Ser15, it would be expected to migrate with the same slightly retarded mobility as that identified on the Ser15-p53 Western blot. Since this was not the case, we speculate that the Ser15-phosphorylated p53 was primarily derived from arrested cells and that the Ser20-phosphorylated p53 was derived from apoptotic cells.
HHV-6B-infected MOLT 3 cells were arrested during the cell cycle, and only a minority underwent apoptosis during the first week after infection. Arresting cells, rather than destroying them, may be an evolutionarily developed advantage for a virus that arose at about the same time as vertebrates. HHV-6B is a relatively slowly growing virus which does not produce high titers, and from a biological point of view, it will favor survival and will hide from the immune system. Keeping productively infected cells alive may help in achieving this goal, whereas apoptotic cells are taken up by dendritic cells for cross-presentation and necrotic cells initiate immune reactions by the liberation of viral proteins.
HHV-6B is a betaherpesvirus related to human cytomegalovirus (HCMV). Infection by HCMV may also lead to cell cycle arrest (20). Indeed, various HCMV-encoded proteins have been shown to mediate this block. Importantly, the UL36 gene product inhibits caspase activation (26) and the UL37 gene product interferes with the mitochondrion-induced activation of apoptosis (13). Thus, it will be important to determine if HHV-6B has developed similar mechanisms for the prevention of apoptosis in cell cycle-arrested T cells.
ADDENDUM Since the submission of our work, a paper on p53 and HHV-6 infection was published by Takemoto et al. (28). In this paper, p53 was shown to be induced and stabilized following HHV-6 infection concomitant with a block in apoptosis induction. These authors found that p53 is sequestered in the cytoplasm, and they suggest that this may be part of the mechanism that protects infected cells from apoptosis.
ACKNOWLEDGMENTS
We thank P. Lusso for the PL-1 strain of HHV-6B, Z. Berneman for cell lines, S. R. Paludan for ?-actin primers, and B. Vandahl and S. Birkelund for help with the electronic retrieval of immunofluorescence data.
This work was supported by grants from the University of Aarhus, the Danish Multiple Sclerosis Society, Leo Nielsen and Karen Margrethe Nielsen's Foundation, and the King Christian the Xth Foundation.
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