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Journal of Virology, December 2003, p. 13214-13224, Vol. 77, No. 24
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.24.13214-13224.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Mechanisms Governing Maintenance of Cdk1/Cyclin B1 Kinase Activity in Cells Infected with Human Cytomegalovirus

Veronica Sanchez, Anita K. McElroy,{dagger} and Deborah H. Spector*

Molecular Biology Section and Center for Molecular Genetics, University of California, San Diego, La Jolla, California 92093-0366

Received 27 November 2002/ Accepted 4 September 2003


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ABSTRACT
 
Previous work has demonstrated dysregulation of key cell cycle components in human cytomegalovirus (HCMV)-infected human fibroblasts, resulting in cell cycle arrest (F. M. Jault, J.-M. Jault, F. Ruchti, E. A. Fortunato, C. L. Clark, J. Corbeil, D. D. Richman, and D. H. Spector, J. Virol. 69:6697-6704, 1995). The activation of the mitotic kinase Cdk1/cyclin B, which was detected as early as 8 h postinfection (p.i.) and maintained throughout the time course, was particularly interesting. To understand the mechanisms underlying the induction of this kinase activity, we have examined the pathways that regulate the activation of Cdk1/cyclin B1 complexes. The accumulation of the cyclin B1 subunit in HCMV-infected cells is the result of increased synthesis and reduced degradation of the protein. In addition, the catalytic subunit, Cdk1, accumulates in its active form in virus-infected cells. The decreased level of the Tyr15-phosphorylated form of Cdk1 in virus-infected fibroblasts is due in part to the down-regulation of the expression and activity of the Cdk1 inhibitory kinases Myt1 and Wee1. Increased degradation of Wee1 via the proteasome also accounts for its absence at 24 h p.i. At late times, we observed accumulation of the Cdc25 phosphatases that remove the inhibitory phosphates from Cdk1. Interestingly, biochemical fractionation studies revealed that the active form of Cdk1, a fraction of total cyclin B1, and the Cdc25 phosphatases reside predominantly in the cytoplasm of infected cells. Collectively, these data suggest that the maintenance of Cdk1/cyclin B1 activity observed in HCMV-infected cells can be explained by three mechanisms: the accumulation of cyclin B1, the inactivation of negative regulatory pathways for Cdk1, and the accumulation of positive factors that promote Cdk1 activity.


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INTRODUCTION
 
Human cytomegalovirus (HCMV), a betaherpesvirus, is a common pathogen and the leading viral cause of birth defects (46). The HCMV DNA genome is 230 kbp in length and carries approximately 150 open reading frames. Like other herpesviruses, viral gene expression is temporally regulated. Much work has described the complex host cell-virus interactions that control the expression of viral gene products. Infection with HCMV leads to the stimulation of signaling pathways and dysregulation of the cell cycle (for review, see reference 15). The binding of the virion to the cell surface activates mitogen-activated protein (MAP) kinase and phosphatidylinositol kinase pathways that contribute to the downstream activation of transcription factors, including NF-{kappa}B (8, 25, 26, 53). Other effects on cell activation require viral gene expression. For example, HCMV infection leads to sustained activation of the ERK kinases and downstream targets early in infection (47). In addition, several viral proteins reportedly alter cell cycle progression in transient expression systems (27, 37, 42).

We and others have also observed modification of many key factors that regulate the cell cycle. The cell cycle is the highly regulated process of preparation for cell division (for review, see reference 52). Quiescent, or G0, cells are stimulated to enter the cycle by growth signals. Once in G1, cells make the decision to commit to cell division. Entry into S phase is regulated by the cyclin-dependent kinase complex Cdk2/cyclin E. In S phase, the cell's replication machinery is activated and regulated by Cdk2 in a complex with cyclin A. After the DNA has been successfully duplicated, the cells enter G2 and then mitosis. Cell division is mediated by Cdk1/cyclin B complexes (for review, see reference 43). Cdk1 is also known as Cdc2 and maturation promoting factor. In complex with cyclin B1 or B2 in mammalian cells, it can phosphorylate many substrates, including other kinases (51), cytoskeletal components (44), proteins of the secretory pathway (35), and other cell cycle regulators (22). In fact, Cdk1 is required for the proper segregation of cellular material between daughter cells during cell division.

Because it plays such a crucial role in cell division, Cdk1 activity is tightly regulated (see Fig. 9A) (43). First, the Cdk1 catalytic subunit is regulated by phosphorylation. Inhibitory phosphates are added to Thr14 and Tyr15 by two kinases, Wee1 and Myt1 (7, 19, 33, 34, 39, 41, 56, 58). These phosphates are removed by members of a family of dual-specificity protein phosphatases known as Cdc25 (29). Cdc25B is an S/G2 phosphatase that is thought to play the role of starter phosphatase by removing the phosphate groups at Thr14 and Tyr15 and initially activating Cdk1 (31). Cdk1/cyclin B can then phosphorylate and activate Cdc25C, thus beginning a feedback loop that amplifies Cdk1/cyclin B activity and the signal for cell division (22). Cdk1 is also phosphorylated at Thr161 by the Cdk-activating kinase CAK, or Cdk7 (20).



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  		FIG. 9. Model for activation of Cdk1/cyclin B1 complexes in HCMV-infected cells. (A) The addition of inhibitory phosphates to the catalytic subunit, Cdk1, is mediated by Myt1 and Wee1 kinases. Myt1 is inhibited by phosphorylation mediated by p90Rsk1, which itself is activated by the ERK kinases. The removal of the Cdk1 inhibitory phosphates is catalyzed by Cdc25B, an S/G2 phase phosphatase, and Cdc25C, a G2/M phosphatase. Cdc25B initially activates Cdk1/cyclin B1 complexes, which in turn activate Cdc25C. Cdc25C amplifies the activation of Cdk1/cyclin B1 complexes during mitosis. In HCMV-infected cells, Myt1 and Wee1 expression is reduced while the Cdc25 phosphatases accumulate. This results in the accumulation of the nonphosphorylated (Thr14/Tyr15), active form of Cdk1 in HCMV-infected cells. (B) APC regulates the degradation of cyclin B1 and Tome-1. Tome-1 acts in concert with the SCF complex to promote degradation of Wee1. In HCMV-infected cells, we observed accumulation of cyclin B1 and degradation of Wee1. These results are consistent with down-regulation of the APC in virus-infected cells.

The activity of Cdk1/cyclin B complexes is also regulated by the availability of the cyclin subunit. Cyclin B expression fluctuates throughout the cell cycle. During S phase, cyclin B mRNA and protein begin to accumulate. These levels become maximal at G2/M. As the cells pass through mitosis, cyclin B is ubiquitinated and degraded by the anaphase-promoting complex (APC) (for review, see reference 18). This degradation continues through G1. Substrate specificity of Cdk1 complexes is dependent upon association with either cyclin B1 or B2 (13). Cyclin B1 shuttles between the nucleus and cytoplasm during interphase but is targeted to the nucleus during M phase. Cyclin B2 is associated with the Golgi apparatus during interphase and mitosis. Therefore, Cdk1 activity is regulated by the availability of the cyclin subunit in terms of expression and cellular localization. The pathways described above are simplified as more proteins are discovered and shown to function in the regulation of mitosis.

Human fibroblasts infected with HCMV in the G0/G1 phase of the cycle do not replicate their DNA; viral gene expression prevails and cells become blocked in a pseudo-G1 state, with high levels of cyclin E-associated kinase activity (9, 12, 24, 36, 49). The pocket proteins that regulate transcription in complex with E2Fs in a cell cycle-dependent manner become phosphorylated but are not degraded (24, 38). The tumor suppressor protein p53 is also stabilized in HCMV-infected cells and is sequestered in viral replication centers (16, 24). Expression of the proto-oncogenes fos, jun, and myc is observed as well as expression of key enzymes necessary for cellular DNA replication (6, 12, 21, 23). In contrast, cyclin A and its associated kinase activity are suppressed (24, 49). Notably, cyclin B1 and its associated kinase activity are induced and maintained at high levels in HCMV-infected cells (24, 49).

In this report, we describe the mechanisms by which high levels of Cdk1/cyclin B1 activity are sustained in HCMV-infected human fibroblasts. We found that induction of cyclin B1 in virus-infected cells occurs at early times in the infection and that early viral gene expression is required for the high levels of accumulation. This accumulation is the result of both increased levels of synthesis and reduced degradation of cyclin B1. In addition, we detected down-regulation of both the expression and activity of the Myt1 and Wee1 inhibitory kinases in HCMV-infected cells. The absence of Wee1 at 24 h postinfection (p.i.) is due to increased degradation via the proteasome. Accumulation of the Cdc25 phosphatases was also detected. By immunofluorescence, we observed that cyclin B1 is distributed in both the nucleus and cytoplasm. We also found that activated Cdk1 is localized predominantly to the cytoplasm; however, our biochemical and immunofluorescence data suggest that pools of Cdk1 exist in the nucleus.


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MATERIALS AND METHODS
 
Cells and virus. Human foreskin fibroblasts (HFF) were passaged and maintained in minimum essential medium (MEM)-Earle's medium containing 10% fetal bovine serum and supplemented with glutamine, penicillin, streptomycin, and amphotericin as previously described (38). Cells were grown to confluence and allowed to arrest for 3 days prior to trypsinization and replating at a lower density (49). Cells were infected at a multiplicity of infection (MOI) of 5 with the Towne strain of HCMV at the time of release from confluence (G0 infection) or were mock infected with conditioned medium. For experiments performed in the absence of serum, confluence-synchronized cells were replated at a lower density in serum-free MEM-Earle's medium supplemented with antibiotics. HFF were infected with serum-free virus (47) at an MOI of 5 at 2 days postplating, and cultures were maintained in serum-free medium throughout the duration of the experiment.

Western blotting. Confluence-synchronized HFF were infected at a MOI of 5 as described above. At the time points indicated, cells were trypsinized, counted, and frozen. Pellets were lysed in reducing sample buffer (RSB) (50 mM Tris [pH 6.8], 2% sodium dodecyl sulfate, 10% glycerol, 5% 2-mercaptoethanol, 25 mM sodium fluoride, 1 mM sodium orthovanadate, 5 mM ß-glycerophosphate, 1 mM phenylmethylsulfonyl fluoride, 50 µM leupeptin, and 100 µM pepstatin A) containing bromophenol blue and then were sonicated and boiled for 5 min. Approximately 7.5 x 104 cells per lane were loaded onto polyacrylamide gels as previously described (50). Proteins were transferred to nitrocellulose. The following antibodies were used to probe filters: anti-cyclin B1 (BD Biosciences), anti-IE1/2 and anti-UL44 (the Goodwin Research Institute), anti-Cdk1 and anti-phospho-Tyr15 Cdk1 (Santa Cruz Biotechnology, Inc.), anti-Rsk1 (Santa Cruz Biotechnology, Inc.), anti-phospho-Ser381 Rsk1 (Cell Signaling Technology), anti-Wee1 (Santa Cruz Biotechnology, Inc.), anti-Cdc25B (Oncogene Research Products), anti-Cdc25C (Santa Cruz Biotechnology, Inc.), and anti-ß actin (Sigma, St. Louis, Mo.). The antibody against G6PD was a generous gift from Rod Nakayama (University of California, Irvine). These primary antibodies were diluted in BLOTTO (5% nonfat milk and 0.05% Tween 20 in Tris-buffered saline [pH 7.4]). Antibodies against Myt1 were a generous gift from Robert Booher (Onyx Pharmaceuticals) and were diluted in BLOTTO containing 15% nonfat milk and 0.1% Tween 20. Horseradish peroxidase-conjugated secondary antibodies were purchased from Calbiochem and diluted 1:1,000 to 1:10,000. SuperSignal chemiluminescent reagents were purchased from Pierce and used per the manufacturer's instructions.

Inhibition of the proteasome by MG132. G0-arrested cells were released from confluence and simultaneously infected with HCMV Towne at an MOI of 5 or mock infected with conditioned medium as described above. At the time points indicated, mock- and virus-infected cultures were treated with 2.5 or 10 µM MG132 (Calbiochem) dissolved in dimethyl sulfoxide (DMSO) or were treated with DMSO (control) for 3 h prior to harvesting of cells. Lysates were prepared in RSB as described above, and samples were processed for Western blotting.

Cycloheximide block and release experiments. Cycloheximide block and release experiments were performed as described by McElroy et al. (38). Briefly, G0-synchronized HFF were released from confluence and replated at a lower density. Cycloheximide (Sigma) was added to a final concentration of 100 µg/ml at 1 h postplating. After this 1-h pretreatment, cells were mock infected or infected with HCMV Towne at an MOI of 5 in the presence or absence of 100 µg of cycloheximide per ml. The cells were washed three times at 3 h p.i. to remove the cycloheximide block and were refed with medium with or without cycloheximide. At the designated time intervals, actinomycin D (Sigma) was added to a final concentration of 20 µg/ml. All samples were harvested at 18 h p.i. Cell pellets were lysed in RSB and processed for Western blotting.

Immunofluorescence. Confluence-synchronized HFF were seeded on glass coverslips and infected in G0 with HCMV Towne at an MOI of 5. At the time points indicated, coverslips were washed twice in phosphate-buffered saline (PBS) and fixed in ice-cold methanol for 10 min. Immunofluorescence staining was done as previously described (50). Fixed cells were blocked in 10% normal goat serum (NGS) in PBS for 20 min at room temperature (RT) prior to incubation with primary antibodies. Antibodies were diluted in PBS containing 10% NGS as follows: anti-Cdk1 antibody (Santa Cruz Biotechnology, Inc.), 1:50; and anti-cyclin B1 antibody (BD Biosciences), 1:50. After a 30-min incubation at RT, coverslips were washed three times with PBS, for 3 min per wash. Coverslips were then incubated with fluorescein isothiocyanate- or tetramethyl rhodamine isocyanate-conjugated donkey anti-mouse isotype-specific secondary antibodies in PBS containing 10% NGS and Hoechst stain for 30 min at RT. Coverslips were washed three times in PBS, for 3 min per wash, prior to mounting onto slides with SlowFade Light (Molecular Probes) mounting medium to prevent photobleaching. Images were captured by using MetaMorph Software (Universal Imaging Corporation, Downingtown, Pa.) and were processed with Adobe Photoshop.

Biochemical fractionation. Confluence-synchronized HFF cells were infected with HCMV Towne at an MOI of 5. At 24 and 48 h p.i., cells were digested with trypsin and counted. Fractionation was conducted as previously described (55). Cell pellets were resuspended on ice in isotonic lysis buffer to a concentration of 106 cells/50 µl. Digitonin was added to samples to a final concentration of 0.2 mg/ml. Samples were incubated on ice for 15 min with frequent vortexing. Nuclei were pelleted by spinning at 3,000 rpm (700 x g) for 5 min. The supernatant (fraction 1) was carefully removed from the pellet, which was washed two times with isotonic lysis buffer. Fraction 1 was further clarified by centrifugation at 5,000 rpm for 10 min. The clarified supernatant was collected and represents the soluble cytosolic fraction (C). The nuclear pellet was resuspended in 50 µl of isotonic lysis buffer and layered on 50 µl of 37% sucrose. Nuclei were spun through the cushion at 5,000 rpm (2,000 x g) for 10 min. The supernatant was collected and the sucrose was carefully removed from the pellet. The pellet was washed once with isotonic lysis buffer. The pellet was then extracted with digitonin (2 mg/ml) in isotonic lysis buffer for 5 min on ice with frequent vortexing. Insoluble material was pelleted at 3,000 rpm for 5 min. The supernatant was collected and the pellet was washed twice in isotonic lysis buffer. The pellet represents the detergent-resistant fraction containing predominantly the nuclei and other insoluble material (N). The fractions were mixed with an equal volume of 2x RSB to make 1x RSB. Approximately equal cell numbers were loaded onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels.

In vitro kinase assays. In vitro kinase assays were performed as previously described (24). Cell pellets containing 3 x 105 cells were solubilized on ice in lysis buffer (10 mM Tris [pH 7.4], 5 mM EDTA, 130 mM NaCl, 10 mM NaH2PO4, 1 mM dithiothreitol, 1% Triton X-100, 25 mM sodium fluoride, 1 mM sodium orthovanadate, 5 mM ß-glycerophosphate, 1 mM phenylmethylsulfonyl fluoride, 50 µM leupeptin, and 100 µM pepstatin A) for 30 min. Clarified supernatants were precleared with protein G beads (Santa Cruz Biotechnology, Inc.) for 1 h. Ten micrograms of antibody against cyclin B1 (clone GNS1; Lab Vision) coupled to protein G beads was incubated with lysates for 4 h at 4°C. Immunoprecipitates were washed two times with lysis buffer and split into two samples, one for Western blotting and the other for kinase assays. Immunoprecipitates were washed once in kinase assay buffer (20 mM HEPES [pH 7.4], 10 mM MgCl2). In vitro kinase assays were performed at RT for 30 min in kinase assay buffer containing 1 mM dithiothreitol, 10 µM ATP, 5 µg of histone H1 (Upstate USA, Inc., Charlottesville, Va.), and 2 µCi of [32P]ATP. Reactions were stopped by adding an equal volume of 2x RSB. Samples were resolved in SDS-12% PAGE gels. For Western blots, samples were resolved on SDS-10% PAGE gels and processed as described above. Horseradish peroxidase-linked isotype-specific secondary antibodies (Southern Biotechnology Associates, Inc., Birmingham, Ala.) were used for blots probed with anti-Cdk1 antibody to prevent detection of light chains of immunoglobulin.


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RESULTS
 
Steady-state levels of cyclin B1 are maintained in HCMV-infected cells in the absence of serum. Previous work from our laboratory described the induction of cyclin B1 and its associated kinase activity in human fibroblasts infected with HCMV (24). Those experiments were performed in cells that were previously synchronized by serum starvation and given serum stimulation at the time of infection. In order to determine if the effect on cyclin B1 accumulation was a direct effect of viral infection or a result of serum stimulation, we assessed the expression of cyclin B1 in cells infected with HCMV in the absence of serum. Cells were grown to confluence and allowed to arrest for 3 days before replating at a lower density in the absence of serum. Two days later, fibroblasts were infected at an MOI of 5 with the Towne strain of HCMV prepared in serum-free medium. As shown in Fig. 1, cyclin B1 accumulated in HCMV-infected fibroblasts in the absence of serum stimulation as early as 24 h p.i. Minimal cyclin B1 expression was detected in mock-infected cells. These data indicated that cyclin B1 accumulation was not an artifact of the experimental design but was induced by viral infection. All subsequent experiments were performed in medium containing 10% fetal bovine serum with confluence-synchronized cells.



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  		FIG. 1. Accumulation of cyclin B1 in HCMV-infected cells. Confluence-synchronized HFF cells were trypsinized and replated at a lower density in serum-free medium. Two days later, cells were infected with serum-free HCMV Towne at an MOI of 5 in the presence or absence of serum (V+ and V-, respectively) or were mock infected with conditioned medium with or without serum (M+ or M-, respectively). At the time points indicated, samples were harvested and processed for Western blotting with antibodies against cyclin B1. Equivalent cell numbers were loaded in all lanes.

Accumulation of cyclin B1 is controlled at the levels of both synthesis and degradation. In earlier studies, we had obtained evidence that suggested that cyclin B1 accumulated earlier in infected cells than in uninfected cells. The kinetics of cyclin B1 expression were determined by Western blotting. Figure 2A shows the fluctuation of cyclin B1 expression over time in mock- and virus-infected cells. In mock-infected cells, a low level of cyclin B1 was detectable in G1 through 10 h p.i. The level then dropped slightly at 14 h p.i., consistent with degradation of cyclin B1 mediated by the E3 ubiquitin ligase APC. As the cells leave G1 phase, the levels of cyclin B1 begin to rise again (Fig. 2A, 18 h p.i.). The expression of cyclin B1 was highest at 24 h p.i. in mock-infected samples, consistent with S phase. The levels dropped slightly by 48 h p.i. and disappeared by 96 h p.i., when mock-infected cells reached confluence (Fig. 2A and data not shown). In contrast, high levels of cyclin B1 were detected as early as 10 h p.i. in virus-infected cells (Fig. 2A). In addition, the steady-state level of cyclin B1 in HCMV-infected cells did not fluctuate significantly at late times during the infection, and cyclin B1 was detected as late as 96 h p.i. As a loading control, lanes containing equivalent cell numbers were probed with an antibody against ß-actin, which does not fluctuate during the cell cycle.



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  		FIG. 2. Accumulation of cyclin B1 in HCMV-infected cells is controlled at the levels of synthesis and degradation. (A) G0-arrested HFF cells were released from confluence by replating at a lower density. Cells were infected with HCMV Towne at an MOI of 5 (V) or were mock infected with conditioned medium (M) at the time of replating. Cells were harvested at the time points indicated and samples were processed for Western blotting for cyclin B1. Equivalent cell numbers were loaded in all lanes. Panels shown represent two independent experiments. As a loading control, lanes containing equivalent cell numbers were probed with an antibody against ß-actin. (B) G0-synchronized HFF cells were infected as described for panel A. Three hours prior to harvesting, cells were treated with the proteasome inhibitor MG132 at the concentrations indicated. Cells were harvested at 18 and 21 h p.i., and the samples were processed for Western blotting with antibodies against cyclin B1. Lanes contain equivalent cell numbers.

Previous work from our laboratory demonstrated that expression of cyclin B1 mRNA was slightly increased in HCMV-infected cells at 12 h p.i.; however, this slight increase was not sufficient to account for the early appearance and accumulation of cyclin B1 protein (49). We inferred that another mechanism might also contribute to the accumulation of cyclin B1 protein at early times during infection. Because the level of cyclin B1 during G1 phase of uninfected cells is regulated by ubiquitination catalyzed by the APC E3 ubiquitin ligase and proteasome-mediated degradation, we examined whether the accumulation of cyclin B1 in HCMV-infected cells early in the infection might be the result of decreased degradation. G0-synchronized human fibroblasts were infected with HCMV Towne at an MOI of 5 as for the previous experiments. Three hours before harvesting at 18 and 21 h p.i., cultures were treated with the proteasome inhibitor MG132 dissolved in DMSO or with DMSO alone as a control. As shown in Fig. 2B, the addition of proteasome inhibitor to mock-infected cultures had a marked effect on the expression of cyclin B1 at 18 and 21 h p.i. In contrast, the addition of MG132 had a less significant effect on the expression of cyclin B1 in virus-infected cells (Fig. 2B). At 18 h p.i., the inhibition of the proteasome resulted in a moderate increase in the level of cyclin B1 in the infected cells, but by 21 h p.i., MG132 had no effect on cyclin B1 accumulation. These data suggest that the increase in cyclin B1 observed for HCMV-infected cells is the result of both increased transcription and decreased proteasome-mediated degradation.

Viral early gene expression is required for the accumulation of cyclin B1. We next sought to determine what stage of viral gene expression was necessary for the induction of cyclin B1 expression. To do this, we performed cycloheximide block and release experiments as previously described (38). Briefly, confluence-synchronized cells were infected in the presence of cycloheximide. At 3 h p.i., cells were released from the cycloheximide block, and at various times postrelease, actinomycin D was added to prevent further transcription. All cells were harvested at 18 h p.i. As shown in Fig. 3, the earliest induction of cyclin B1 in the HCMV-infected cells was observed when a 4-h window was allowed between cycloheximide release and the addition of actinomycin D (ActD lane 7-18). At this same time point, we can detect immediate-early (IE) gene expression as well as a very small amount of the HCMV DNA polymerase accessory protein UL44. Cyclin B1 expression was not detected at earlier time points despite the presence of IE proteins, indicating that cyclin B1 is not induced with the kinetics of an IE protein. When a 10-h time interval was allowed between the removal of cycloheximide and the addition of actinomycin D (Fig. 3, ActD lane 13-18), cyclin B1 levels declined in the mock-infected cells but continued to increase in the HCMV-infected cells. Taken together, these data suggest that the induction of cyclin B1 in virus-infected cells occurs at early times during infection and that sustained accumulation likely requires some viral early gene expression.



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  		FIG. 3. Sustained accumulation of cyclin B1 requires some viral early gene expression. G0-synchronized cells were released from confluence and infected at an MOI of 5 with HCMV Towne (V) or were mock infected (M) in the presence or absence of cycloheximide (CHX). At 3 h p.i., the drug was removed. At the time points indicated, cells were treated with actinomycin D (ActD), which was incubated with the cells for the time intervals specified. All samples were harvested at 18 h p.i. and were processed for Western blotting with antibodies against IE1/2 and early (UL44) viral proteins as well as cyclin B1. Lanes contain equivalent protein contents.

Cdk1 accumulates in HCMV-infected cells in its active form. We also examined the expression of Cdk1 in HCMV-infected cells. We found that expression of Cdk1 fluctuated throughout the cell cycle in mock-infected cells (Fig. 4A). At 4 h p.i., there was no detectable Cdk1 in mock or viral samples. By 24 h p.i., Cdk1 was present at high levels in the mock samples. The expression of Cdk1 declined by 96 h p.i. in mock-infected cells as they became confluent, comparable to what was observed for cyclin B1 (Fig. 4A and data not shown). In contrast, the steady-state level of Cdk1 appeared to increase over time in HCMV-infected cell samples. In addition, we detected predominantly the faster migrating form of Cdk1, which represents the active form of Cdk1 that has not been modified by Myt1 or Wee1 (Fig. 4A, compare lane 24 M to lane 24 V). The decrease in Cdk1 Tyr15 phosphorylation in viral samples was confirmed by probing Western blots with an antibody against the phosphorylated Tyr15 epitope on Cdk1 (Fig. 4B). Note that this antibody cross-reacts with a similar epitope on Cdk2, which runs slightly faster than Cdk1 on these gels. We observed that overall the levels of the phosphorylated proteins were low in viral samples at all time points; however, there was some enrichment of the pTyr15 form of Cdk1 in immunoprecipitates of cyclin B1 from virus-infected cells (Fig. 4B and C). These results were consistent with our earlier findings showing that Cdk1/cyclin B1 complexes were active in HCMV-infected cells throughout the infection (24).



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  		FIG. 4. Time course of Cdk1 expression and cyclin B1-associated kinase activity in HCMV-infected cells. G0-synchronized cells were released from confluence and infected at an MOI of 5 with HCMV Towne (V) or were mock infected with conditioned medium (M). Cells were harvested at the time points indicated. Samples were processed for Western blotting with antibodies against Cdk1 (A) and the phosphorylated Tyr15 epitope of Cdk1 (B). Equivalent cell numbers were loaded in all lanes. (C) G0-synchronized cells were infected as described above. Pellets containing an equivalent number of cells were solubilized and immunodepleted of cyclin B1. Immunoprecipitates were washed and divided into samples for Western blots and in vitro kinase assays as described in Materials and Methods. Western blots were probed with cyclin B1-, Cdk1-, and pTyr15-specific antibodies. *, for visual purposes, lanes 72 M and 72 V in the panel showing reactivity with pTyr15 antibody are from a longer exposure of the Western blot. =, inactive forms of Cdk1; dots, faster migrating, active form of Cdk1.

To determine the relative activity of the Cdk1/cyclin B1 complexes in the mock- and HCMV-infected cells, we subjected whole-cell lysates to immunoprecipitation with an antibody against cyclin B1 under conditions in which cyclin B1 was completely depleted from the lysate (data not shown). The precipitates were then assessed for the ability to phosphorylate histone H1 in an in vitro kinase assay. Surprisingly, histone H1 kinase assays revealed that the highest levels of cyclin B1-associated kinase activity were not observed for the HCMV-infected cells until the late phases of the infection (Fig. 4C). The active form of Cdk1 was coprecipitated with cyclin B1 in the viral samples at each of the time points tested (denoted by dots in Fig. 4C), and there was an increase in histone H1 kinase activity over time in the viral samples. In mock-infected cells, the levels of cyclin B1-associated kinase activity fluctuated over the cell cycle and peaked at 48 h p.i. At this time point, there was a detectable level of the active form of Cdk1 in immunoprecipitates from mock-infected cells; however, because the relative abundance of the active form of Cdk1 does not correlate with the levels of kinase activity in the mock and viral samples at 48 h p.i., these data suggest that there may be an additional level of regulation that controls cyclin B1-associated kinase activity in HCMV-infected cells.

Down-regulation of the Myt1 kinase pathway. The detection of the active form of Cdk1 in virus-infected cells prompted us to investigate if the alteration of the Myt1 kinase inhibitory pathway might be one mechanism underlying the activation of Cdk1. Previous work from our laboratory showed that the ERK kinases were up-regulated shortly after infection of cells with HCMV (47). The activity of one of the substrates that are positively regulated by the ERKs, p90Rsk1, was also increased. This was of particular interest because p90Rsk1 phosphorylates Myt1 and inhibits its activity (45), consistent with the data shown in Fig. 4. In mock-infected cells, the steady-state level of p90Rsk1 did not fluctuate significantly throughout the cell cycle (Fig. 5A). In contrast, there was a reproducible decrease in the level of p90Rsk1 in the viral samples at 8 h p.i. relative to the control samples. The levels of total p90Rsk1 then increased by 24 h p.i., and the protein accumulated to high levels late in infection. A very different pattern of expression was observed when the Western blots were probed with an antibody specific for p90Rsk1 phosphorylated at Ser381, which is the active form of the kinase (Fig. 5B) (10). Although the steady-state level of total p90Rsk1 was lower at 8 h p.i. in the viral sample, the level of active, phospho-Ser381-p90Rsk1 was higher in the viral lysate than in the mock sample at the same time point. Interestingly, later in the infection, the levels of phospho-Ser381-p90Rsk1 dropped in the viral samples while the total amount of p90Rsk1 greatly increased (compare Fig. 5A and B). These results paralleled observations by Rodems and Spector that high levels of p90Rsk1 activity appeared at early times but were not maintained at late stages of the infection (47).



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  		FIG. 5. Down-regulation of factors that mediate Cdk1 phosphorylation in HCMV-infected cells. G0-synchronized cells were released from confluence and infected at an MOI of 5 with HCMV Towne (V) or mock infected with conditioned medium (M). Cells were harvested at the time points indicated. Samples were processed for Western blotting with antibodies against total p90Rsk1 (A), Ser381-phosphorylated p90Rsk1 (B), Myt1 (C), and Wee1 (D). Equivalent cell numbers were loaded in all lanes. (E) Lower levels of Wee1 result from enhanced degradation. G0-synchronized HFF cells were infected as described above. Three hours prior to harvesting, cells were treated with the proteasome inhibitor MG132 at the concentration indicated. Cells were harvested at 22 h p.i., and the samples were processed for Western blotting with antibodies against Wee1.

Based on the above observations, phosphorylation of the Myt1 kinase and its subsequent inactivation should be more efficient in HCMV-infected cells at early times in the infection; however, since the levels of active p90Rsk1 were similar in the mock and viral samples at 24 h p.i., the inactivation of the Myt1 kinase might be comparable at this time point. To determine if there were any differences in the total amount of Myt1 present, we performed Western blot analyses of the steady-state levels of Myt1. As shown in Fig. 5C, the levels of Myt1 in both the mock- and HCMV-infected cells were very low at 8 h p.i. The levels then increased in the mock and viral samples, but the levels in the infected cells were significantly lower than that in mock-infected controls at 24 h p.i. and later time points. These results suggested that the Myt1 pathway for inhibition of Cdk1 activity was compromised in virus-infected cells as a result of high levels of p90Rsk1 at early times during the infection and reduced expression of Myt1 throughout the infection.

Alteration of Wee1 expression. Since inhibitory phosphates can be added to Cdk1 by both Myt1 and Wee1, we also proceeded to determine whether viral down-regulation of Wee1 might be another mechanism contributing to the activation of Cdk1. The expression of this protein was reported to be cell cycle regulated (39, 56), consistent with the results shown in Fig. 5D. The level of Wee1 was below the limit of detection at 6 h p.i. in both mock- and virus-infected cells. In mock samples, the highest levels of Wee1 were detected at 24 h p.i., which coincides with S phase (49). The levels of Wee1 then dropped as the cells became asynchronous at 48 h p.i. and at later time points. These data were consistent with the regulated synthesis and degradation patterns of Wee1 that have previously been reported. Wee1 expression was delayed in HCMV-infected cells, and the protein was not detected in viral samples at 24 h p.i. Thus, the absence of Wee1 coupled with the low levels of Myt1 is consistent with the reduced level of the inactive phosphorylated forms of Cdk1 detected in infected cells at 24 h p.i. After 24 h p.i., we detected forms of Wee1 with altered mobilities in virus-infected cells (Fig. 5D). Two of the slower migrating forms are likely the hyperphosphorylated inactive Wee1; however, because we detected three forms instead of the previously described doublet of Wee1 (56), we do not know whether these forms are inactive. In addition, the small amount of Wee1 coupled with the lack of a high-affinity antibody precluded direct measurement of activity. Nevertheless, these results suggest that the Wee1 inhibitory pathway is down-regulated in HCMV-infected cells within the first 24 h of infection and that this is one mechanism that contributes to the activity of Cdk1/cyclin B1.

The expression of Wee1 is regulated at both the transcriptional and posttranslational levels during the cell cycle. Recent evidence suggests that Wee1 is targeted for degradation by Tome-1, which acts as part of the Skp1-Cullin-F-box protein (SCF)ubiquitin ligase (reviewed in references 30 and 32; also see reference 3). Tome-1 itself is subject to proteasome-mediated degradation and is a substrate for the APC during early G1 of the cell cycle. The destruction of Tome-1 allows the accumulation of Wee1 in late G1 phase to ensure that Cdk1 is not activated prematurely. As shown in Fig. 2, cyclin B1 accumulated in infected cells at early times. Since cyclin B1 and Tome-1 are targets of the same E3 ubiquitin ligase, we reasoned that if Tome-1 accumulated, then the SCF E3 ubiquitin ligase might be able to target Wee1 for degradation more efficiently in the virus-infected cells. To address whether the lower level of Wee1 during early phases of the infection was due to decreased gene expression or to enhanced degradation of Wee1, we treated mock- or HCMV-infected cells with the proteasome inhibitor MG132 for 3 h before samples were harvested at 22 h p.i. Cell pellets were processed for Western blotting with an antibody directed against Wee1. Accumulation of Wee1 was observed in the virus-infected cells upon treatment with the proteasome inhibitor, suggesting that early in the infection Wee1 is targeted for degradation. This result indicates that the proteasome is active at early phases of HCMV infection and provides indirect evidence that the APC is less active in infected cells.

HCMV infection leads to accumulation of Cdc25 phosphatases. The activity of Cdk1/cyclin B complexes is positively regulated by the Cdc25 phosphatases, which remove the inhibitory phosphates from Cdk1 (14, 17, 29, 31). Thus, it was possible that a positive effect of the virus on these phosphatases was also involved in maintaining high levels of active Cdk1 in infected cells. Therefore, we determined the expression of Cdc25B and Cdc25C in mock- and HCMV-infected, confluence-synchronized HFF cells by Western blotting. As shown in Fig. 6, the levels of Cdc25B and Cdc25C fluctuated during the cell cycle in mock-infected cells. Cdc25B expression was barely detectable at 24 h p.i., peaked at 48 h p.i., and then dropped at 72 h p.i. as the mock-infected cells approached confluence (Fig. 6A and data not shown). In contrast, we detected Cdc25B at 24 h p.i. in the virus-infected samples, and the protein accumulated throughout the duration of the experiment. In addition, we observed a slight mobility difference in the HCMV-infected samples.



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  		FIG. 6. Up-regulation of Cdc25 phosphatase expression in HCMV-infected cells. G0-synchronized cells were released from confluence and infected at an MOI of 5 with HCMV Towne (V) or mock infected with conditioned medium (M). Cells were harvested at the time points indicated. Samples were processed for Western blotting with antibodies against Cdc25B (A) and Cdc25C (B). Equivalent cell numbers were loaded in all lanes.

The pattern of Cdc25C accumulation in mock-infected cells was similar to that observed for Cdc25B in that expression was cell cycle regulated; however, the level of Cdc25C in the viral samples increased over the duration of the time course (Fig. 6B). At 24 and 48 h p.i., the level of Cdc25C in the mock samples was slightly higher than in the viral lysates. At 72 h p.i., we detected more of the phosphatase in the viral sample than in the control sample. These results suggested that at late times postinfection, the maintenance of Cdk1/cyclin B1 complexes could also be attributed to accumulation of the Cdc25 phosphatases that oppose the activity of Myt1 and Wee1.

Subcellular localization of Cdk1 and cyclin B1. Using immunofluorescent staining, we examined the subcellular localization of both Cdk1 and cyclin B1. The localization of Cdk1 in mock-infected cells is cell cycle regulated. At 48 h p.i., the mock-infected cultures contained cells at various stages of cell division (11, 54). Cells in G1 and S phase showed diffuse cytoplasmic staining for Cdk1 as well as punctate staining at the centrioles (Fig. 7). We also observed cells in S/G2 that contained Cdk1 in the nucleus and cytoplasm. In contrast, all cells in HCMV-infected cultures at 48 h p.i. displayed diffuse staining for Cdk1 in the nucleus and cytoplasm and at the centrioles (Fig. 7).



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  		FIG. 7. Localization of Cdk1 and cyclin B1 in HCMV-infected cells. G0-synchronized cells were released from confluence, infected with HCMV Towne at an MOI of 5 or mock infected, and seeded onto glass coverslips. At 48 h p.i., cells were washed twice with PBS prior to fixation with ice-cold methanol for 10 min. Cells were washed with PBS and subjected to immunostaining with monoclonal antibodies against Cdk1 (tetramethyl rhodamine isocyanate) and cyclin B1 (fluorescein isothiocyanate) as described in Materials and Methods. Hoechst staining indicates nuclei. Magnification, x1,000.

As expected, staining for cyclin B1 in mock-infected cells also showed that localization of this protein was regulated throughout the cell cycle (Fig. 7) (54). At 48 h p.i., cultures contained cells in early G1, with little or no observable cyclin B1, and cells in late G1 and S phases, during which cyclin B1 was detected primarily in the cytoplasm. In addition, cells in late G2 were detected by the accumulation of cyclin B1 in the nucleus. Interestingly, the immunofluorescence data suggest that the cyclin B1 level within individual HCMV-infected cells was lower than that in mock-infected controls in which cyclin B1 was detectable; however, all cells in the infected culture expressed cyclin B1, whereas the protein was detected in only a percentage of the mock-infected cells. The finding that cyclin B1 was uniformly distributed in the cytoplasm and nucleus of virus-infected cells (Fig. 7) was particularly interesting. In addition, intranuclear accumulation of cyclin B1, consistent with viral replication centers, was noted occasionally.

To confirm the data obtained from immunofluorescent staining of HCMV-infected cultures, we also used biochemical fractionation and Western blotting to determine the localization of Cdk1 and cyclin B1 as well as proteins involved in the regulation of the kinase complex. Mock- and virus-infected cells were harvested at 24 and 48 h p.i. and separated into cytoplasmic and nuclear fractions by use of isotonic lysis buffer containing digitonin (55). Fractions corresponding to the soluble cytosolic compartment (C) (Fig. 8A) and to the insoluble and nuclear-associated pellet (N) (Fig. 8A) were collected. Lysates containing equivalent cell numbers were subjected to immunoblotting. As shown in Fig. 8A, more cyclin B1 was detected in the nuclear and insoluble fractions of both mock- and virus-infected cells at 24 and 48 h p.i. A change in the solubility of cyclin B1 was detected in mock samples between 24 and 48 h p.i. such that more cyclin B1 was detected in the soluble fraction at 48 h p.i. than at 24 h p.i. This shift could represent the cell cycle-regulated nuclear localization of cyclin B1 but more likely reflects a change in the association of cyclin B1 with a detergent-resistant compartment (4). A similar shift was observed in virus-infected cells between 24 and 48 h p.i.



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  		FIG. 8. Subcellular localization of Cdk1 and cyclin B1 in HCMV-infected cells. G0-synchronized cells were released from confluence and infected at an MOI of 5 with HCMV Towne (V) or mock infected with conditioned medium (M). Cells were harvested at the time points indicated and immediately subjected to fractionation procedures as described in Materials and Methods. Samples were resolved by SDS-PAGE and transferred to nitrocellulose filters for Western blotting with an antibody against cyclin B1 and control antibodies against a cytoplasmic marker (G6PD) and viral nuclear proteins (UL44 and IE1/2) (A), antibody against total Cdk1 and phospho-specific antibody against Tyr15 of Cdk1 (B), and antibodies against Cdc25B and Cdc25C (C). C, soluble, cytosolic fraction; N, insoluble proteins and nuclear pellet. Approximately equivalent cell numbers were loaded in all lanes. The dots in panel B denote the migration of Cdk2, which is also detected with the phospho-specific antibody.

As a control for the fractionation procedure, Western blots were probed with antibodies against several cellular and viral proteins whose subcellular localization is well established. As a control for the cytosolic fraction, we used glucose-6-phosphate dehydrogenase (G6PD). We detected approximately equal amounts of G6PD in the soluble cytosolic fractions of mock- and virus-infected cells at both time points (Fig. 8A). No G6PD was present in the insoluble and nuclear pellet fractions. To monitor contamination of cytosolic fractions with nuclear proteins, we probed for an early viral protein, UL44. UL44 was only detected in the insoluble and nuclear pellet fraction of viral samples (Fig. 8A). This was expected since UL44 is tightly associated with viral replication centers (16, 50). We also probed for another set of nuclear viral proteins, IE1 72 and IE2 86 (Fig. 8A). Both IE1 and IE2 were detected almost exclusively in the nuclear fractions, although some IE1 could be extracted with nonionic detergent, consistent with a previous report (50).

To determine the distribution of the active and inactive forms of Cdk1, we probed the fractions with antibodies against total Cdk1 and phosphorylated inactive Cdk1. These analyses revealed that different forms of Cdk1 were localized to specific biochemically defined compartments. At 24 and 48 h p.i., mock and viral samples contained the faster migrating active form of Cdk1 in the soluble cytosolic fraction (Fig. 8B, total Cdk1). To identify the inactive, phosphorylated forms of Cdk1, we probed Western blots with an antibody against the Cdk1 Tyr15-phosphorylated epitope described above. At 24 and 48 h p.i., we detected bands that comigrated with the slower migrating forms of Cdk1 in the soluble and insoluble fractions of the mock-infected samples (Fig. 8B, phospho-Tyr15). In addition, a faster migrating band was detected in the insoluble fraction of the mock sample, and further experiments showed that it corresponded to Cdk2 (Fig. 8B, phospho-Tyr15, and data not shown). In contrast, only a low level of Tyr15-phosphorylated Cdk1 appeared in the soluble and insoluble fractions of the viral samples at these times. Taken together, these data suggest that the majority of Cdk1 in HCMV-infected cells resides in the cytoplasm and is in its active form.

We next examined the distribution of the Cdc25 phosphatases within these biochemically defined compartments. We detected most of the Cdc25B in the soluble cytosolic fractions of mock- and virus-infected cells at 24 and 48 h p.i. (Fig. 8C), although small quantities of Cdc25B did partition into the insoluble and nuclear pellet fractions of viral samples at both time points. Cdc25C was also detected in the soluble cytosolic fractions of both mock- and virus-infected cells at both time points (Fig. 8C). These data indicate that the active form of Cdk1 and the Cdc25 phosphatases reside in the same biochemically defined compartment, which corresponds to the cytosol.


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DISCUSSION
 
Previous studies from our laboratory demonstrated that the expression of cyclin B1 is maintained in HCMV-infected cells and that this accumulation is coincident with an increase in cyclin B1-associated kinase activity (24). In the present report, we describe the mechanisms governing the maintenance of cyclin B1 and regulation of Cdk1/cyclin B1 kinase activity. We show that these mechanisms involve both the inhibition of negative regulatory pathways for Cdk1/cyclin B1 and the activation of the positive regulators of these complexes (Fig. 9).

Our results showed that Cdk1 was below the level of detection until sometime prior to 24 h p.i., at which time Cdk1 was present predominantly in the faster migrating, active form in virus-infected cells. While the expression of Cdk1 appeared to be cell cycle regulated in mock-infected cells, as previously described (40, 57), active Cdk1 was maintained in the HCMV-infected samples throughout the time course. Cyclin B1 also accumulated in virus-infected cells. Cyclin B1 was detected earlier in the virus-infected samples, but at 24 h p.i., the level of cyclin B1 was higher in mock-infected controls than in the viral samples. The expression of cyclin B1 then declined in the mock samples over time. These observations are in contrast to what was reported for cells infected with herpes simplex virus type 1 (HSV-1), in which cyclin B1 expression was significantly decreased yet Cdk1 activity was high in infected cells (2). Later work showed that the HSV DNA polymerase accessory protein UL42 bound to Cdk1 in HSV-infected cells, resulting in an active Cdk1 kinase complex (1).

Through the use of cycloheximide block and release experiments coupled with actinomycin D inhibition of RNA synthesis, we determined that some early viral gene expression was required for the sustained accumulation of cyclin B1 (38). In addition, the time course showed that although the level of cyclin B1 was lower in viral samples at 24 h p.i., cyclin B1 expression was higher in viral lysates at the earlier time points. Previous Northern blot analyses showed that the early expression of cyclin B1 in HCMV-infected cells was due in part to accumulation of cyclin B1 transcripts (49). Turnover of cyclin B1 was also observed for the mock samples which likely corresponds to APC-mediated degradation during G1 (for review, see reference 18). The results from our experiments using the proteasome inhibitor MG132 suggest that this pathway leading to degradation of cyclin B1 is compromised in virus-infected cells. This notion is further supported by recent evidence from our laboratory that other substrates for the APC, such as Cdc6 and geminin, accumulate in HCMV-infected cells (5). In addition, the enhanced degradation of Wee1 that we observed for virus-infected cells provides indirect evidence that Tome-1, an APC substrate, also accumulates in HCMV-infected cells and that it directs turnover of Wee1 in conjunction with the SCF complex at early phases of the infection (Fig. 9).

Both Myt1 and Wee1 inhibit the activity of Cdk1. Myt1 is a dual-specificity protein kinase associated with the endoplasmic reticulum and the Golgi apparatus, and it can phosphorylate both Thr14 and Tyr15 of Cdk1 in vitro (7, 33, 34). In confluence-synchronized human fibroblasts, Myt1 expression is cell cycle regulated and is detectable during S phase. In contrast, Myt1 expression is reduced in HCMV-infected cells relative to mock-infected controls. Myt1 activity is itself inhibited by phosphorylation catalyzed by p90Rsk1 (45). This is significant because previous work by Rodems and Spector demonstrated an increase in p90Rsk1 kinase activity in HCMV-infected cells early in infection (47). Examination of phospho-Ser381-p90Rsk1, the active form of the protein, showed that this form of p90Rsk1 was present in infected cells, although the highest levels were detected at early times during infection (10). We also found that the expression of Wee1 was altered. We detected a delay in Wee1 expression in HCMV-infected cells early in infection and forms with altered mobilities late in infection. In addition, we found that the low level of Wee1 expression in virus-infected cells early in infection was due to enhanced degradation. Taken together, these data indicate that both Cdk1 inhibitory pathways are down-regulated in HCMV-infected cells (Fig. 9). Similarly, Wee1 expression was found to be down-regulated in HSV-1-infected cells, contributing to the activation of Cdk1 during infection (2).

HCMV infection had an opposite effect on the positive regulators of Cdk1/cyclin B1 activity (28, 29, 31). We found that both Cdc25B and Cdc25C accumulated in virus-infected cells, while the steady-state levels of these phosphatases fluctuated throughout the cell cycle in the mock-infected samples. These results were similar to the observed accumulation of hyperphosphorylated Cdc25C in HSV-1-infected cells (2). Whether the Cdc25 phosphatases are active within HCMV-infected cells has not been definitively determined. Due to the low specificities of the antibodies, phosphatase assays with immunoprecipitated Cdc25 proteins were inconclusive; however, additional information suggests that these proteins are active. First, we detected the faster-migrating form of Cdk1 in virus-infected cells, suggesting that inhibitory phosphates were removed. Second, we have also observed up-regulation of Polo-like kinase 1 (Plk1) levels in infected cells. Plk1 is a Ser/Thr kinase that phosphorylates and activates Cdc25C during mitosis (48). Finally, biochemical fractionation studies have shown that the active form of Cdk1, a fraction of cyclin B1, and the majority of Cdc25B and Cdc25C localize to the same soluble fraction in infected cells.

As one approach for identifying potential substrates of the Cdk1/cyclin B1 activity, we examined the subcellular localization of both proteins. By immunofluorescence, we found that cyclin B1 levels within individual infected cells were low compared to in mock-infected controls that had detectable cyclin B1; however, all cells in the infected cultures expressed cyclin B1, in contrast to only a percentage of mock-infected cells that expressed high levels of cyclin B1. The distribution of cyclin B1 in infected cells was also altered, with cyclin B1 present in both the nucleus and cytoplasm. In contrast, in mock-infected cells, the distribution of cyclin B1 is cell cycle regulated and cyclin B1 is predominantly cytoplasmic (54). In virus-infected cells, active Cdk1 localizes predominantly in the cytoplasm, as detected by immunofluorescence and biochemical fractionation; therefore, it seems likely that substrate specificity is controlled by cellular compartmentalization of the components of Cdk1/cyclin B1 complexes within infected cells. Taken together, these data suggest that Cdk1/cyclin B1 activity in HCMV-infected cells is regulated at multiple levels, including stabilization of the cyclin subunit, down-regulation of inhibitory pathways, accumulation of positive regulators of Cdk1/cyclin B1 activity, and spatial constraints.


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ACKNOWLEDGMENTS
 
We thank Robert Booher for his generous gift of antibodies against Myt1 kinase. We also thank R. Nakayama for his gift of an antibody against G6PD. We also acknowledge the members of the laboratory for helpful advice in preparation of the manuscript.

This work was supported by NIH grants CA34729 and CA73490. A.K.M. was supported by NIH training grant CA09345.


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FOOTNOTES
 
* Corresponding author. Mailing address: Molecular Biology Section, 9500 Gilman Dr., Pacific Hall Rm. 1224A, La Jolla, CA 92093-0366. Phone: (858) 534-4584. Fax: (858) 534-6083. E-mail: dspector{at}ucsd.edu. Back

{dagger} Present address: Department of Molecular Virology, USAMRIID, Ft. Detrick, MD 21702. Back


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Journal of Virology, December 2003, p. 13214-13224, Vol. 77, No. 24
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.24.13214-13224.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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