Human Cytomegalovirus Inhibits Cellular DNA Synthesis and Arrests Productively Infected Cells in Late G1

Bresnahan, Wade A.; Boldogh, István; Thompson, E. Aubrey; Albrecht, Thomas

doi:10.1006/viro.1996.0516

Cited by 209 publications

(258 citation statements)

References 4 publications

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“…Nevertheless, the crystal structure study suggests that cyclin A-binding exposes the T-loop of CDK2 making it a better substrate for phosphorylation by CAK (52), in agreement with a more recent study clearly showing that mammalian CAK preferentially phosphorylates cyclin⅐CDK2 complexes (21), as previously demonstrated for cdc2 (14). The nuclear translocation of CDK2 (40,53) associated with cyclin⅐CDK2 complex formation should also favor CDK2 phosphorylation by permitting the colocalization with nuclear CAK (54). Moreover, cyclin binding protects Thr-160-phosphorylated CDK2 from dephosphorylation by the CDK-associated phosphatase KAP (55).…”

Section: Discussionsupporting

confidence: 86%

“…An even tinier fraction of CDK2, consisting only of the Thr-160ϩTyr-15-phosphorylated forms, was complexed with cyclin E and possibly p21. It is likely to be totally inactive (5,40). At variance with cyclin E-bound CDK2, CDK2 associated with cyclin D1 and p21 was only faintly phosphorylated at Thr-160, even in serum-stimulated cells (not shown), in agreement with previous reports indicating that cyclin D1 is an inhibitor for CDK2 that prevents its phosphorylation by CAK (45,61,62).…”

Section: Table I Summary Of the Main Observationssupporting

confidence: 92%

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Phosphorylations of Cyclin-dependent Kinase 2 Revisited Using Two-dimensional Gel Electrophoresis

Coulonval

Bockstaele²,

Paternot³

et al. 2003

Journal of Biological Chemistry

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To control the G 1 /S transition and the progression through the S phase, the activation of the cyclin-dependent kinase (CDK) 2 involves the binding of cyclin E then cyclin A, the activating Thr-160 phosphorylation within the T-loop by CDK-activating kinase (CAK), inhibitory phosphorylations within the ATP binding region at Tyr-15 and Thr-14, dephosphorylation of these sites by cdc25A, and release from Cip/Kip family (p27 kip1 and p21 cip1 ) CDK inhibitors. To re-assess the precise relationship between the different phosphorylations of CDK2, and the influence of cyclins and CDK inhibitors upon them, we introduce here the use of the high resolution power of two-dimensional gel electrophoresis, combined to Tyr-15-or Thr-160-phosphospecific antibodies. The relative proportions of the potentially active forms of CDK2 (phosphorylated at Thr-160 but not Tyr-15) and inactive forms (non-phosphorylated, phosphorylated only at Tyr-15, or at both Tyr-15 and Thr-160), and their respective association with cyclin E, cyclin A, p21, and p27, were demonstrated during the mitogenic stimulation of normal human fibroblasts. Novel observations modify the current model of the sequential CDK2 activation process: (i) Tyr-15 phosphorylation induced by serum was not restricted to cyclin-bound CDK2; (ii) Thr-160 phosphorylation engaged the entirety of Tyr-15-phosphorylated CDK2 associated not only with a cyclin but also with p27 and p21, suggesting that Cip/ Kip proteins do not prevent CDK2 activity by impairing its phosphorylation by CAK; (iii) the potentially active CDK2 phosphorylated at Thr-160 but not Tyr-15 represented a tiny fraction of total CDK2 and a minor fraction of cyclin A-bound CDK2, underscoring the rate-limiting role of Tyr-15 dephosphorylation by cdc25A.The major events of the eukaryotic cell cycle depend on the sequential and ordered formation, activation (by phosphorylation and/or dephosphorylation) and then inactivation of different complexes of cyclin-dependent kinases (CDKs). 1 CDK2 plays an essential role in controlling the G 1 /S transition and the progression through the S phase: it participates in the inactivating phosphorylations of pRb and related p130 (1-4), and it phosphorylates other key substrates whose activities are necessary to trigger and organize the DNA synthesis phase while preventing DNA re-replication (5-9). Because the critical roles of CDK2, both in positive and negative mitogenic controls of cell cycle and as the end point target of DNA damage checkpoint mechanisms, have been well established (10 -13), understanding the details of CDK2 regulation is of fundamental importance.Based on the cdc2/CDK1 activation model (14 -16) and on structural (17) and enzymatic studies (18 -24), the consensual framework of CDK2 activation is the following. The binding of a cyclin partner (cyclin E at the G 1 /S transition, cyclin A during the S phase) confers a low basal activity to the cyclin⅐CDK2 complex and enables subsequent phosphorylation of CDK2 by the CDK-activating kinase (CAK/cyclin H-CDK7) on a conser...

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Section: Discussionsupporting

confidence: 86%

Section: Table I Summary Of the Main Observationssupporting

confidence: 92%

Phosphorylations of Cyclin-dependent Kinase 2 Revisited Using Two-dimensional Gel Electrophoresis

Coulonval

Bockstaele²,

Paternot³

et al. 2003

Journal of Biological Chemistry

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“…In other virus infections, including those by Human cytomegalovirus (HCMV), host DNA is not replicated (Morin et al, 1996). HCMV, a much larger and perhaps more sophisticated virus than HPV and TGMV, activates cell cycle progression and nucleotide synthesis but blocks cells at late G1, thereby inhibiting cellular DNA synthesis and allowing exclusive viral access to nucleotide pools (Bresnahan et al, 1996). Our data suggest that TGMV, like the similarly sized HPV, stimulates differentiated cells to enter S-phase but does not prevent cellular DNA synthesis.…”

Section: Host Dna Is Replicated In Tgmv-infected Cellsmentioning

confidence: 69%

Host DNA Replication Is Induced by Geminivirus Infection of Differentiated Plant Cells

2002

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The geminivirus Tomato golden mosaic virus (TGMV) replicates in differentiated plant cells using host DNA synthesis machinery. We used 5-bromo-2-deoxyuridine (BrdU) incorporation to examine DNA synthesis directly in infected Nicotiana benthamiana plants to determine if viral reprogramming of host replication controls had an impact on host DNA replication. Immunoblot analysis revealed that up to 17-fold more BrdU was incorporated into chromosomal DNA of TGMV-infected versus mock-infected, similarly treated healthy leaves. Colocalization studies of viral DNA and BrdU demonstrated that BrdU incorporation was specific to infected cells and was associated with both host and viral DNA. TGMV and host DNA synthesis were inhibited differentially by aphidicolin but were equally sensitive to hydroxyurea. Short BrdU labeling times resulted in some infected cells showing punctate foci associated with host DNA. Longer periods showed BrdU label uniformly throughout host DNA, some of which showed condensed chromatin, only in infected nuclei. By contrast, BrdU associated with viral DNA was centralized and showed uniform, compartmentalized labeling. Our results demonstrate that chromosomal DNA is replicated in TGMV-infected cells.

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“…Nevertheless, the incomplete restoration also suggests that other histone chaperons, such as ASF1a [43] and HIRA (unpublished data), might be involved through an IE2-independent manner. In addition, IE2-mediated CAF1 mislocation could contribute to the reported cell cycle arrest and block in host DNA synthesis during HCMV infection [21,22,[67][68][69]. CAF1 also functions in DNA repair and heterochromatin silencing [70,71], which can both be affected once CAF1 is mislocated by IE2.…”

Section: Host Effects Caused By Hcmv Hijacking Of Caf1mentioning

confidence: 99%

Host-viral effects of chromatin assembly factor 1 interaction with HCMV IE2

Lee

et al. 2011

Cell Res

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Chromatin assembly factor 1 (CAF1) consisting of p150, p60 and p48 is known to assemble histones onto newly synthesized DNA and thus maintain the chromatin structure. Here, we show that CAF1 expression was induced in human cytomegalovirus (HCMV)-infected cells, concomitantly with global chromatin decondensation. This apparent conflict was thought to result, in part, from CAF1 mislocalization to compartments of HCMV DNA synthesis through binding of its largest subunit p150 to viral immediate-early protein 2 (IE2). p150 interaction with p60 and IE2 facilitated HCMV DNA synthesis. The IE2Q548R mutation, previously reported to result in impaired HCMV growth with unknown mechanism, disrupted IE2/p150 and IE2/histones association in our study. Moreover, IE2 interaction with histones partly depends on p150, and the HCMV-induced chromatin decondensation was reduced in cells ectopically expressing the p150 mutant defective in IE2 binding. These results not only indicate that CAF1 was hijacked by IE2 to facilitate the replication of the HCMV genome, suggesting chromatin assembly plays an important role in herpesviral DNA synthesis, but also provide a model of the virus-induced chromatin instability through CAF1.

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Human Cytomegalovirus Inhibits Cellular DNA Synthesis and Arrests Productively Infected Cells in Late G1

Cited by 209 publications

References 4 publications

Phosphorylations of Cyclin-dependent Kinase 2 Revisited Using Two-dimensional Gel Electrophoresis

Phosphorylations of Cyclin-dependent Kinase 2 Revisited Using Two-dimensional Gel Electrophoresis

Host DNA Replication Is Induced by Geminivirus Infection of Differentiated Plant Cells

Host-viral effects of chromatin assembly factor 1 interaction with HCMV IE2

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