Department of Pediatrics, Laboratory for Molecular Biology, Charité, CCM-Ziegelstr. 59, Humboldt-University, Berlin, Germany
Correspondence
Christian Hagemeier
christian.hagemeier{at}charite.de
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ABSTRACT |
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Introduction |
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The HCMV 86 kDa immediate-early protein 2 (IE2) is not only an essential transcriptional activator of early viral gene expression (Gebert et al., 1997; Heider et al., 2002
; Marchini et al., 2001
) but also a cell-cycle regulator that can reproduce some important features of the deregulated cell-cycle phenotype in HCMV infection. It blocks cell-cycle progression at the beginning of S phase in various cell types (Murphy et al., 2000
; Wiebusch & Hagemeier, 1999
, 2001
), including primary fibroblasts (Kronschnabl et al., 2002
). This arrest resembles the specific inhibition of DNA replication by chemical inhibitors such as hydroxyurea (HU) or aphidicoline since it is dominant over cyclincdk activities and leaves the RbE2F pathway untouched (Wiebusch & Hagemeier, 2001
). Moreover, cyclin E expression and activity are up-regulated in IE2-arrested cells (Wiebusch & Hagemeier, 2001
) and the mRNA levels of numerous genes involved in nucleotide synthesis and DNA replication are elevated (Song & Stinski, 2002
). Other findings also point towards IE2 as a factor with an ambivalent role in regulating cell proliferation: IE2 relieves the flat-cell phenotype of pRb-transfected Saos cells (Fortunato et al., 1997
) and rescues the defect of ts13 cells in cyclin transcription (Lukac et al., 1997
) in both cases the cell cycle is still blocked. IE2 even forces quiescent REF-52 cells to re-enter the cell cycle and synthesize at least limited amounts of DNA (Castillo et al., 2000
). Thus, there is considerable evidence that IE2 triggers both proliferative and anti-proliferative cellular activities but it is unknown how these functions integrate mechanistically on IE2.
Activation of cyclin Ecdk2 in HCMV-infected cells is mainly achieved by a strong increase in cyclin E transcription (Bresnahan et al., 1998; Salvant et al., 1998
). Other HCMV-induced alterations that are known to contribute to the cyclin Ecdk2 activation are the translocation of cdk2 from the cytoplasm into the nucleus (Bresnahan et al., 1997b
) and the enhanced degradation of the cdk inhibitor p21WAF1,CIP1 (Chen et al., 2001
). How IE2 could contribute towards cyclin Ecdk2 regulation is not fully understood. One report investigating the requirements for cyclin E activation in infected cells questioned the importance of IE2. It demonstrated that cyclin E induction depended on early viral gene expression and was mainly associated with a change in cyclin E promoter occupancy concerning the composition of E2Fpocket protein complexes (McElroy et al., 2000
). In contrast, in vitro experiments showing binding of IE2 to the cyclin E promoter (Bresnahan et al., 1998
) and a functional IE2p21WAF1,CIP1 interaction (Sinclair et al., 2000
) have suggested a direct involvement of IE2 in cyclin Ecdk2 activation on the transcriptional as well as the post-translational level. However, even the fact that IE2 transfection leads to an up-regulation of cyclin E promoter activity (Bresnahan et al., 1998
), cyclin E mRNA level (Song & Stinski, 2002
; Wiebusch & Hagemeier, 2001
), cyclin E protein level and cyclin E-associated kinase activity (Wiebusch & Hagemeier, 2001
) cannot unequivocally qualify cyclin E as a primary IE2 target. Since the beginning of S phase where IE2-expressing cells accumulate coincides with maximum cyclin E expression and maximum cyclin Ecdk2 activity (Dulic et al., 1992
; Koff et al., 1992
), it is also conceivable that cyclin E only serves as a cell-cycle marker for IE2-arrested cells.
Therefore, the work presented here is aimed at examining whether IE2 can serve as a genuine activator of cyclin E gene expression and looking at the possible functional implications of such an activation in IE2-expressing cells.
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Methods |
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Plasmids.
pSG5 (Green et al., 1988) and the expression plamids for CD20, IE2(195-579) and cyclin E [pSG5-CD20, pSG5-3HA-IE2(195-579) and pCMX-CycE] have been previously described (Wiebusch & Hagemeier, 2001
). For IE2 expression, the vector pSG5-IE2-His was used containing full-length IE2 cDNA of AD169 origin C-terminally fused to a tag of six histidine codons.
DNA transfections.
Cells were transfected using the calcium phosphate co-precipitation method, as previously described (Wiebusch & Hagemeier, 1999). The DNA precipitates were left on the cells for 1416 h. The transfection medium was replaced with fresh culture medium supplemented with 1 mM mimosine, 1 mM HU or 10 µM 5-bromo-2'-deoxyuridine (BrdU) as indicated. Nocodazole was added at a final concentration of 50 ng ml-1 24 h after removal of the DNA precipitates. The time-points of cell harvest are specified in the figure legends.
Cell sorting.
Transfected cells were labelled with a CD20 antibody (clone 2H7; Pharmingen) and separated from untransfected cells by magnetic affinity cell sorting (MACS), as previously described (Wiebusch & Hagemeier, 2001). To control the sorting efficiency, aliquots of sorted and unsorted cells were stained with an FITC-conjugated secondary antibody (F0313; DAKO) and analysed by flow cytometry for CD20 expression. To control for IE2 co-expression, cells were permeabilized by overnight incubation in 70 % ethanol in PBS, blocked in 0·5 % BSA in PBS and labelled with a primary IE1/2-specific antibody (clone E13; Argene) and a secondary FITC-conjugated mouse IgG1-specific antibody (clone A85-1; Pharmingen). Both antibody-binding reactions were carried out for 15 min at room temperature using 5 µg antibodies ml-1 in 0·5 % BSA in PBS, followed by a washing step with 0·5 % BSA in PBS. Finally, cells were suspended in PBS and analysed by flow cytometry.
Cell-cycle analysis.
The overall DNA content of transfected cells was determined by propidium iodide staining and flow cytometry, as previously described (Wiebusch & Hagemeier, 1999). The BrdU incorporation assay for detection of newly synthesized DNA was carried out as described by Wiebusch & Hagemeier (2001)
. Briefly, following CD20-directed cell sorting, BrdU-treated cells were permeabilized in 70 % ethanol and stained by indirect immunofluorescence using an anti-BrdU antibody (clone 3D4; Pharmingen) and an isotype-specific secondary antibody (clone A85-1; Pharmingen) to avoid cross-reaction with the CD20 antibody used for cell sorting. Cells were then co-stained with propidium iodide and analysed by flow cytometry.
Immunoblot analysis and kinase assay.
After sorting, cell extracts of CD20-positive cells were prepared as previously described (Wiebusch & Hagemeier, 2001). These were used for immunoblot analysis and cyclin E kinase assays. Both assays were performed exactly as previously described (Wiebusch & Hagemeier, 2001
) employing the cyclin E monoclonal antibodies HE12 (Santa Cruz) for immunoblotting and HE111 (Santa Cruz) for immunoprecipitation of cyclin E kinase activity. Blots were routinely controlled for equal loading and protein transfer by Ponceau S staining. To control the expression level of IE2 and its mutant IE2(195-579), blots were probed with a rabbit antiserum recognizing the C-terminal region of IE2 (a kind gift from Thomas Stamminger, Erlangen, Germany).
Ribonuclease protection assay (RPA).
Total RNAs from CD20-positive cells were isolated using Trizol reagent (Gibco) according to the manufacturer's instructions. Two µg of each preparation was taken as input in a multiprobe RPA (Riboquant; Pharmingen). A defined pool of radioactively labelled probes was generated by in vitro transcription from the template set hCyc-2 (Pharmingen) and hybridized with the input RNA. The samples were subsequently digested with an RNase A/T1 mixture, purified by ethanol precipitation, separated by denaturing PAGE and subjected to autoradiography.
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Results |
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Thus, we applied an experimental system that we have successfully used before (Wiebusch & Hagemeier, 2001), which relies on the co-transfection of cDNAs for IE2 and the cell-surface marker CD20 into U373 cells, a cell line in which HCMV can replicate. This enabled us: (i) to determine the cell-cycle stage of IE2-expressing cells, and (ii) to physically separate IE2-expressing cells via MACS for biochemical analysis (see Methods; Wiebusch & Hagemeier, 2001
). In addition, we made use of two drugs that are known to arrest cell-cycle progression (Fig. 1
A): mimosine, which can block cells in a dose-dependent manner in G1, and hydroxyurea (HU), which inhibits ongoing DNA synthesis in early S phase (Krude, 1999
, Wiebusch & Hagemeier, 2001
). Since cells take up transfected DNA primarily during mitosis at times when the nuclear membrane breaks down, these cells become functionally synchronized by the transfection procedure itself (Adams et al., 1997
; Rodriguez et al., 1999
; unpublished observations). Therefore, in the presence of mimosine or HU, control-transfected, i.e. CD20-positive, cells synchronously ran into a cell-cycle block that appeared to be characterized by a G1-like DNA content (Fig. 1B
, top panel). However, HU-treated cells had elevated levels of cyclin E protein (Fig. 1C
, lane 2) reflecting the fact that these cells were arrested right at the beginning of S phase, which could be directly demonstrated by these cells having incorporated a small amount of BrdU (data not shown; Wiebusch & Hagemeier, 2001
). In contrast, mimosine-arrested cells were truly blocked in G1 (for BrdU incorporation analysis, see below) and importantly, these cells had non-elevated levels of cyclin E protein (Fig. 1C
, lane 3). Although mimosine has also been reported to block cells in early S phase (Hughes & Cook, 1996
), this was clearly not the case in the transfected, CD20-positive population, since after transfection these cells synchronously leave mitosis and pass into G1 before becoming arrested in this phase.
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Together these data showed that in our experimental system of functionally synchronized cells, mimosine not only arrested transfected cells in G1 but, furthermore, the time point of arrest within G1 was prior to the physiological boost of cyclin E induction late in G1. This assay system should therefore allow us to measure any genuine effect of IE2 on the expression level of cyclin E.
Cyclin E activation by IE2 is independent of the cell-cycle stage
We next transfected cells with either an IE2 expression plasmid or its parental empty plasmid, both in the presence and absence of mimosine, to test for IE2-mediated cell-cycle-independent activation of cyclin E. First, we asked whether IE2 expression would interfere with the mimosine-dependent cell-cycle block in G1. Whereas in the absence of mimosine control-transfected cells were evenly distributed over S phase, IE2-expressing cells were blocked at the beginning of S (Fig. 2A, plots 1 and 2; B) as shown by BrdU incorporation assays. These results are consistent with previous data (Wiebusch & Hagemeier, 2001
). In contrast, mimosine treatment, as expected from the data presented in Fig. 1
, led to a clear-cut block of cell-cycle progression in G1, irrespective of the presence or absence of IE2 (Fig. 2A
, plots 3 and 4; B). Thus, IE2 expression did not overcome the mimosine-induced G1 arrest.
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Given that the IE2 mutant protein failed to activate cyclin E (see above), this suggested to us that IE2-mediated cyclin E induction is a prerequisite for S-phase entry. Conversely, lack of cyclin E expression in IE2(195-579)-transfected cells appeared to go hand in hand with a lack of S-phase entry. To test this hypothesis directly, we co-expressed cyclin E and IE2(195-579) and asked whether, under these conditions, cells would now enter S phase as measured by BrdU incorporation. As predicted, cyclin E was found to completely rescue the S-phase entry of IE2(195-579)-expressing cells (Fig. 5A, plot 4). At the same time, full-length IE2 was also able to rescue S-phase entry when co-expressed with the IE2 mutant form (Fig. 5A
, plot 6), also implying that IE2(195-579) does not function as a dominant negative form of IE2. Quantification of the rescue experiments showed that co-expression of cyclin E induced a more pronounced S-phase entry in IE2(195-579)-transfected cells than co-expression of IE2 (49 % versus 38 %, Fig. 5B
). This was most likely due to the significantly higher levels of cyclin E protein in these cells (data not shown). In accordance with this notion, a significantly higher proportion of S phase cells was also seen after the sole transfection of cyclin E (59 %) versus IE2 (40 %). These findings further support the view that IE2-mediated cyclin E induction drives IE2(195-579)-arrested cells across the G1/S border.
Although S-phase entry in IE2(195-579)-expressing cells could be rescued by raising cyclin E protein levels as demonstrated, these cells did not progress through S phase and into G2 (Fig. 5A, B). Instead, they remained in an early S phase compartment, like cells arrested by IE2 [Fig. 5B
, compare the G0/G1 : early S ratios between cells expressing IE2 (1 : 1·2), IE2(195-579) plus cyclin E (1 : 2·3) and IE2(195-579) plus IE2 (1 : 1·1)]. Again, the direct overexpression of cyclin E appeared to empty the G1 compartment more rapidly than IE2. As demonstrated by immunoblot analysis, we could exclude cross-regulation of the IE2 mutant by cyclin E (Fig. 5C
, left-hand panel, lanes 3 and 4) and of cyclin E by the IE2 mutant (Fig. 5C
, right-hand panel, lanes 4 and 5) to account for the differences in BrdU incorporation.
Together these data suggest that IE2, by transcriptionally up-regulating cyclin E, promotes G1/S transition before cells finally arrest in early S phase. Thus, in addition to the demonstrated cell-cycle-arrest function of IE2, we can also assign an independent and genuine proliferative capacity to IE2. This view is consistent with the observation of the untimely induction of cyclin E in mimosine-arrested IE2-expressing cells shown in Fig. 3.
IE2(195-579) retains the ability to arrest cells in early S phase
The rescue experiments shown in Fig. 5 suggested that cells finally arrest in early S phase after traversing the G1/S border. In order to test further whether the cell-cycle arrest imposed by the mutant form of IE2 is dominant over cdk activity, as for IE2, we used nocodazole treatment in the experimental set-up described in Fig. 5
. Nocodazole is a spindle poison and blocks cells at the beginning of mitosis. Under this treatment, cycling control-transfected cells primarily accumulated in G2/M as expected (Fig. 6
, plot 1). In contrast, a large proportion of IE2- and IE2(195-579)-expressing cells remained arrested in S and G1, respectively (Fig. 6
, plots 2 and 3). Importantly, the same was true for cells overexpressing cyclin E in addition to the mutant form of IE2 (Fig. 6
, plot 4). This demonstrated that, although cyclin E expression drives cells arrested by IE2(195-579) in G1 into S phase (see Fig. 5A
, plot 4), the majority of these cells do not progress further through S phase and do not enter G2. Cyclin E overexpression alone first accelerated G1/S transition and then slowed down S-phase progression (Fig. 6
, plot 5). This resulted in a high proportion of a pan-S cell population that slowly emptied into G2, which is consistent with previously published observations (Resnitzky et al., 1994
; Spruck et al., 1999
). As expected, cells expressing both IE2 and the IE2 mutant remained to a great extent in the G1/S compartment, demonstrating the cell-cycle arrest suggested from the findings shown in Fig. 5
.
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Discussion |
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The initial question of this study, whether IE2 is a genuine activator of cyclin E or not, was answered by experiments employing the plant amino acid mimosine to arrest IE2-expressing cells in G1 before they entered into the IE2 block and the time window of periodic cyclin E induction around the G1/S transition phase (Figs 13). Mimosine is known to arrest cells 24 h before the onset of S phase by as yet undefined mechanisms (Krude, 1999
). Since cyclin E does not start to accumulate until 2 h before S-phase entry (Ekholm et al., 2001
), our finding of low cyclin E expression in mimosine-arrested cells is fully consistent with what one would expect from published evidence. Therefore, the fact that IE2 promotes up-regulation of cyclin E in these cells without having any obvious effect on the mimosine arrest itself demonstrates that IE2 can act as a cell-cycle-independent activator of cyclin E.
How does IE2 activate cyclin E? An earlier study (Bresnahan et al., 1998) suggested a model based on transient transfection assays employing cyclin E promoter constructs whereby IE2 directly transactivates cyclin E transcription. Two of our experimental results supported such a view. Firstly, IE2 expression led to an increase in endogenous cyclin E mRNA abundance (Fig. 3A, F
ig. 4A), and secondly, the IE2 mutant IE2(195-579), which lacks the transcriptional activator function of IE2, was deficient in cyclin E activation (Fig. 4
; Wiebusch & Hagemeier, 2001
). Since the same mutant still contains the major binding regions for pRb and p21WAF1,CIP1 (Fortunato et al., 1997
, 2000
; Sinclair et al., 2000
), it seems unlikely that an IE2 interaction with one of these proteins causes the untimely up-regulation of cyclin E that we observed in U373 cells. Furthermore, p21WAF1,CIP1 expression levels are very low in this p53-negative cell line, arguing against the functional relevance of IE2 targeting p21WAF1,CIP1 in these cells. However, considering the fact that cyclin Ecdk2 activity is a convergence point for multiple negative control mechanisms in G1/S (Bartek & Lukas, 2001
), it cannot be excluded that IE2 generally exerts additional functions at a post-transcriptional level contributing to the constitutive activation of cyclin E. For instance, IE2 activates cyclin Ecdk2, even in p16INK4a-arrested cells (unpublished observation), where cyclin Ecdk2 is normally held inactive by p21WAF1,CIP1 that has been set free from cyclin-Dcdk4 complexes (Jiang et al., 1998
). This supports the view that under conditions of high p21WAF1,CIP1 availability, as in differentiated cells, an IE2-mediated inactivation of this cdk inhibitor may become important for cyclin E activation (Sinclair et al., 2000
). Similarly, it should be interesting to investigate whether IE2 has an influence on SCFCDC4-mediated proteolysis of cyclin E, which is normally initiated shortly after S-phase entry (Strohmaier et al., 2001
).
A number of reports have shown that premature activation of cyclin Ecdk2 by constitutive overexpression of cyclin E induces S phase, even in growth-arrested cells (Leone et al., 1999; Lukas et al., 1997
). Accordingly, the ability of IE2 to up-regulate cyclin E expression independent of the cell-cycle state may be one explanation of how IE2 expression leads to S-phase entry in quiescent cells (Castillo et al., 2000
). Since cyclin Ecdk2 is a key regulator of the pocket protein/E2F pathway, which controls the transcription of numerous genes coupled to proliferation and DNA synthesis (DeGregori et al., 1995
), it is feasible that the activation of E2F-responsive gene expression observed in IE2-transfected fibroblasts (Song & Stinski, 2002
) is, in addition to the direct transactivation of certain genes by IE2, the consequence of more general IE2 functions, such as cyclin Ecdk2 activation or pRb inactivation (Hagemeier et al., 1994
).
There is considerable evidence that cyclin E overexpression contributes to the development of many types of human cancer (Donnellan & Chetty, 1999). Therefore, the finding that IE2 activates cyclin E constitutively also suggests a latent oncogenic activity for this viral regulator. Given the functional relationship between IE2 and viral oncogenes such as E1A and SV40 large T antigen [namely the interactions with pRb (Hagemeier et al., 1994
) and p53 (Speir et al., 1994
)] and regarding the mutagenic (Shen et al., 1997
) and anti-apoptotic (Yu & Alwine, 2002
; Zhu et al., 1995
) capabilities of IE2, one could predict that a still undiscovered IE2 species specifically lacking the cell-cycle-arrest function could have considerable oncogenic potential.
Intriguingly, the lack of cyclin E activating function in IE2(195-579) revealed a true G1 arrest activity that is inherent to IE2. This activity normally appears to be overcome by the full-length IE2-mediated induction of cyclin E, which pushes these cells into early S phase where they finally arrest (Fig. 7). This underlines the importance of the IE2-mediated cyclin E up-regulation.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bartek, J. & Lukas, J. (2001). Pathways governing G1/S transition and their response to DNA damage. FEBS Lett 490, 117122.[CrossRef][Medline]
Biron, K. K., Fyfe, J. A., Stanat, S. C., Leslie, L. K., Sorrell, J. B., Lambe, C. U. & Coen, D. M. (1986). A human cytomegalovirus mutant resistant to the nucleoside analog 9-([2-hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine (BW B759U) induces reduced levels of BW B759U triphosphate. Proc Natl Acad Sci U S A 83, 87698773.[Abstract]
Bresnahan, W. A., Boldogh, I., Thompson, E. A. & Albrecht, T. (1996). Human cytomegalovirus inhibits cellular DNA synthesis and arrests productively infected cells in late G1. Virology 224, 150160.[CrossRef][Medline]
Bresnahan, W. A., Boldogh, I., Chi, P., Thompson, E. A. & Albrecht, T. (1997a). Inhibition of cellular Cdk2 activity blocks human cytomegalovirus replication. Virology 231, 239247.[CrossRef][Medline]
Bresnahan, W. A., Thompson, E. A. & Albrecht, T. (1997b). Human cytomegalovirus infection results in altered Cdk2 subcellular localization. J Gen Virol 78, 19931997.[Abstract]
Bresnahan, W. A., Albrecht, T. & Thompson, E. A. (1998). The cyclin E promoter is activated by human cytomegalovirus 86-kDa immediate early protein. J Biol Chem 273, 2207522082.
Castillo, J. P., Yurochko, A. D. & Kowalik, T. F. (2000). Role of human cytomegalovirus immediate-early proteins in cell growth control. J Virol 74, 80288037.
Chen, Z., Knutson, E., Kurosky, A. & Albrecht, T. (2001). Degradation of p21cip1 in cells productively infected with human cytomegalovirus. J Virol 75, 36133625.
DeGregori, J., Kowalik, T. & Nevins, J. R. (1995). Cellular targets for activation by the E2F1 transcription factor include DNA synthesis- and G1/S-regulatory genes. Mol Cell Biol 15, 42154224.[Abstract]
Dittmer, D. & Mocarski, E. S. (1997). Human cytomegalovirus infection inhibits G1/S transition. J Virol 71, 16291634.[Abstract]
Donnellan, R. & Chetty, R. (1999). Cyclin E in human cancers. FASEB J 13, 773780.
Dulic, V., Lees, E. & Reed, S. I. (1992). Association of human cyclin E with a periodic G1S phase protein kinase. Science 257, 19581961.[Medline]
Ekholm, S. V., Zickert, P., Reed, S. I. & Zetterberg, A. (2001). Accumulation of cyclin E is not a prerequisite for passage through the restriction point. Mol Cell Biol 21, 32563265.
Fortunato, E. A., Sommer, M. H., Yoder, K. & Spector, D. H. (1997). Identification of domains within the human cytomegalovirus major immediate-early 86-kilodalton protein and the retinoblastoma protein required for physical and functional interaction with each other. J Virol 71, 81768185.[Abstract]
Fortunato, E. A., McElroy, A. K., Sanchez, I. & Spector, D. H. (2000). Exploitation of cellular signaling and regulatory pathways by human cytomegalovirus. Trends Microbiol 8, 111119.[CrossRef][Medline]
Gebert, S., Schmolke, S., Sorg, G., Floss, S., Plachter, B. & Stamminger, T. (1997). The UL84 protein of human cytomegalovirus acts as a transdominant inhibitor of immediate-early-mediated transactivation that is able to prevent viral replication. J Virol 71, 70487060.[Abstract]
Green, S., Issemann, I. & Sheer, E. (1988). A versatile in vivo and in vitro eukaryotic expression vector for protein engineering. Nucleic Acids Res 16, 369.[Medline]
Gribaudo, G., Riera, L., Lembo, D. & 8 other authors (2000). Murine cytomegalovirus stimulates cellular thymidylate synthase gene expression in quiescent cells and requires the enzyme for replication. J Virol 74, 49794987.
Hagemeier, C., Caswell, R., Hayhurst, G., Sinclair, J. & Kouzarides, T. (1994). Functional interaction between the HCMV IE2 transactivator and the retinoblastoma protein. EMBO J 13, 28972903.[Abstract]
Heider, J. A., Bresnahan, W. A. & Shenk, T. E. (2002). Construction of a rationally designed human cytomegalovirus variant encoding a temperature-sensitive immediate-early 2 protein. Proc Natl Acad Sci U S A 99, 31413146.
Hughes, T. A. & Cook, P. R. (1996). Mimosine arrests the cell cycle after cells enter S-phase. Exp Cell Res 222, 275280.[CrossRef][Medline]
Jault, F. M., Jault, J.-M., Ruchti, F., Fortunato, E. A., Clark, C., Corbeil, J., Richman, D. D. & Spector, D. H. (1995). Cytomegalovirus infection induces high levels of cyclins, phosphorylated Rb and p53, leading to cell cycle arrest. J Virol 69, 66976704.[Abstract]
Jiang, H., Chou, H. S. & Zhu, L. (1998). Requirement of cyclin ECdk2 inhibition in p16(INK4a)-mediated growth suppression. Mol Cell Biol 18, 52845290.
Kalejta, R. F. & Shenk, T. (2002). Manipulation of the cell cycle by human cytomegalovirus. Front Biosci 7, D295306.[Medline]
Koff, A., Giordano, A., Desai, D. & 7 other authors (1992). Formation and activation of a cyclin Ecdk2 complex during the G1 phase of the human cell cycle. Science 257, 16891694.[Medline]
Kronschnabl, M., Marschall, M. & Stamminger, T. (2002). Efficient and tightly regulated expression systems for the human cytomegalovirus major transactivator protein IE2p86 in permissive cells. Virus Res 83, 89102.[CrossRef][Medline]
Krude, T. (1999). Mimosine arrests proliferating human cells before onset of DNA replication in a dose-dependent manner. Exp Cell Res 247, 148159.[CrossRef][Medline]
Leone, G., DeGregori, J., Jakoi, L., Cook, J. G. & Nevins, J. R. (1999). Collaborative role of E2F transcriptional activity and G1 cyclindependent kinase activity in the induction of S phase. Proc Natl Acad Sci U S A 96, 66266631.
Lu, M. & Shenk, T. (1996). Human cytomegalovirus infection inhibits cell cycle progression at multiple points, including the transition from G1 to S. J Virol 70, 88508857.[Abstract]
Lukac, D. M., Harel, N. Y., Tanese, N. & Alwine, J. C. (1997). TAF-like functions of human cytomegalovirus immediate-early proteins. J Virol 71, 72277239.[Abstract]
Lukas, J., Herzinger, T., Hansen, K., Moroni, M. C., Resnitzky, D., Helin, K., Reed, S. I. & Bartek, J. (1997). Cyclin E-induced S phase without activation of the pRb/E2F pathway. Genes & Dev 11, 14791492.[Abstract]
McElroy, A. K., Dwarakanath, R. S. & Spector, D. H. (2000). Dysregulation of cyclin E gene expression in human cytomegalovirus-infected cells requires viral early gene expression and is associated with changes in the Rb-related protein p130. J Virol 74, 41924206.
Marchini, A., Liu, H. & Zhu, H. (2001). Human cytomegalovirus with IE-2 (UL122) deleted fails to express early lytic genes. J Virol 75, 18701878.
Murphy, E. A., Streblow, D. N., Nelson, J. A. & Stinski, M. F. (2000). The human cytomegalovirus IE86 protein can block cell cycle progression after inducing transition into the S phase of permissive cells. J Virol 74, 71087118.
Resnitzky, D., Gossen, M., Bujard, H. & Reed, S. I. (1994). Acceleration of the G1/S phase transition by expression of cyclins D1 and E with an inducible system. Mol Cell Biol 14, 16691679.[Abstract]
Rodriguez, A., Armstrong, M., Dwyer, D. & Flemington, E. (1999). Genetic dissection of cell growth arrest functions mediated by the EpsteinBarr virus lytic gene product, Zta. J Virol 73, 90299038.
Salvant, B. S., Fortunato, E. A. & Spector, D. H. (1998). Cell cycle dysregulation by human cytomegalovirus: influence of the cell cycle phase at the time of infection and effects on cyclin transcription. J Virol 72, 37293741.
Sauer, K. & Lehner, C. F. (1995). The role of cyclin E in the regulation of entry into S phase. Prog Cell Cycle Res 1, 125139.[Medline]
Shen, Y., Zhu, H. & Shenk, T. (1997). Human cytomagalovirus IE1 and IE2 proteins are mutagenic and mediate "hit-and-run" oncogenic transformation in cooperation with the adenovirus E1A proteins. Proc Natl Acad Sci U S A 94, 33413345.
Sinclair, J., Baillie, J., Bryant, L. & Caswell, R. (2000). Human cytomegalovirus mediates cell cycle progression through G1 into early S phase in terminally differentiated cells. J Gen Virol 81, 15531565.
Song, Y. J. & Stinski, M. F. (2002). Effect of the human cytomegalovirus IE86 protein on expression of E2F-responsive genes: a DNA microarray analysis. Proc Natl Acad Sci U S A 99, 28362841.
Speir, E., Modali, R., Huang, E.-S., Leon, M. B., Shawl, F., Finkel, T. & Epstein, S. E. (1994). Potential role of human cytomegalovirus and p53 interaction in coronary restenosis. Science 265, 391394.[Medline]
Spruck, C. H., Won, K. A. & Reed, S. I. (1999). Deregulated cyclin E induces chromosome instability. Nature 401, 297300.[CrossRef][Medline]
Strohmaier, H., Spruck, C. H., Kaiser, P., Won, K. A., Sangfelt, O. & Reed, S. I. (2001). Human F-box protein hCdc4 targets cyclin E for proteolysis and is mutated in a breast cancer cell line. Nature 413, 316322.[CrossRef][Medline]
Wade, M., Kowalik, T. F., Mudryj, M., Huang, E. S. & Azizkhan, J. C. (1992). E2F mediates dihydrofolate reductase promoter activation and multiprotein complex formation in human cytomegalovirus infection. Mol Cell Biol 12, 43644374.[Abstract]
Wiebusch, L. & Hagemeier, C. (1999). Human cytomegalovirus 86-kilodalton IE2 protein blocks cell cycle progression in G1. J Virol 73, 92749283.
Wiebusch, L. & Hagemeier, C. (2001). The human cytomegalovirus immediate early 2 protein dissociates cellular DNA synthesis from cyclin-dependent kinase activation. EMBO J 20, 10861098.
Yu, Y. & Alwine, J. C. (2002). Human cytomegalovirus major immediate-early proteins and simian virus 40 large T antigen can inhibit apoptosis through activation of the phosphatidylinositide 3'-OH kinase pathway and the cellular kinase Akt. J Virol 76, 37313738.
Zhu, H., Shen, Y. & Shenk, T. (1995). Human cytomegalovirus IE1 and IE2 proteins block apoptosis. J Virol 69, 79607970.[Abstract]
Received 10 July 2002;
accepted 4 October 2002.