Cell Cycle Arrest by Human Cytomegalovirus 86-kDa IE2 Protein Resembles Premature Senescence

ABSTRACT Primary human embryo lung fibroblasts and adult diploid fibroblasts infected by the human cytomegalovirus (HCMV) display β-galactosidase (β-Gal) activity at neutral pH (senescence-associated β-Gal [SA-β-Gal] activity) and overexpression of the plasminogen activator inhibitor type 1 (PAI-1) gene, two widely recognized markers of the process designated premature cell senescence. This activity is higher when cells are serum starved for 48 h before infection, a process that speeds and facilitates HCMV infection but that is insufficient by itself to induce senescence. Fibroblasts infected by HCMV do not incorporate bromodeoxyuridine, a prerequisite for the formal definition of senescence. At the molecular level, cells infected by HCMV, beside the accumulation of large amounts of the cell cycle regulators p53 and pRb, the latter in its hyperphosphorylated form, display a strong induction of the cyclin-dependent kinase inhibitor (cdki) p16INK4a, a direct effector of the senescence phenotype in fibroblasts, and a decrease of the cdki p21CIP1/WAF. Finally, a replicative senescence state in the early phases of infection significantly increased the number of cells permissive to virus infection and enhanced HCMV replication. HCMV infection assays carried out in the presence of phosphonoformic acid, which inhibits the virus DNA polymerase and the expression of downstream genes, indicated that immediate-early and/or early (α) genes are sufficient for the induction of SA-β-Gal activity. When baculovirus vectors expressing HCMV IE1-72 or IE2-86 proteins were inoculated into fibroblasts, the increase of p16INK4a (observed predominantly with IE2-86) was similar to that observed with the whole virus, as was the induction of SA-β-Gal activity, suggesting that the viral IE2 gene leads infected cells into senescence. Altogether our results demonstrate for the first time that HCMV, after arresting the cell cycle and inhibiting apoptosis, triggers the cellular senescence program, probably through the p16INK4a and p53 pathways.

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Aurora kinases A and B are up-regulated by Myc and are essential for maintenance of the malignant state

Blood ◽

10.1182/blood-2009-11-251074 ◽

2010 ◽

Vol 116 (9) ◽

pp. 1498-1505 ◽

Cited By ~ 140

Author(s):

Jürgen den Hollander ◽

Sara Rimpi ◽

Joanne R. Doherty ◽

Martina Rudelius ◽

Andreas Buck ◽

...

Keyword(s):

Cell Cycle ◽

Kinase Activity ◽

Kinase Inhibitor ◽

Aurora Kinase ◽

Cell Cycle Regulators ◽

Aurora Kinase Inhibitor ◽

Aurora Kinases ◽

Protein Levels ◽

Malignant State ◽

E Boxes

Myc oncoproteins promote continuous cell growth, in part by controlling the transcription of key cell cycle regulators. Here, we report that c-Myc regulates the expression of Aurora A and B kinases (Aurka and Aurkb), and that Aurka and Aurkb transcripts and protein levels are highly elevated in Myc-driven B-cell lymphomas in both mice and humans. The induction of Aurka by Myc is transcriptional and is directly mediated via E-boxes, whereas Aurkb is regulated indirectly. Blocking Aurka/b kinase activity with a selective Aurora kinase inhibitor triggers transient mitotic arrest, polyploidization, and apoptosis of Myc-induced lymphomas. These phenotypes are selectively bypassed by a kinase inhibitor-resistant Aurkb mutant, demonstrating that Aurkb is the primary therapeutic target in the context of Myc. Importantly, apoptosis provoked by Aurk inhibition was p53 independent, suggesting that Aurka/Aurkb inhibitors will show efficacy in treating primary or relapsed malignancies having Myc involvement and/or loss of p53 function.

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MicroRNA miR-21 Attenuates Human Cytomegalovirus Replication in Neural Cells by Targeting Cdc25a

Journal of Virology ◽

10.1128/jvi.01740-14 ◽

2014 ◽

Vol 89 (2) ◽

pp. 1070-1082 ◽

Cited By ~ 43

Author(s):

Ya-Ru Fu ◽

Xi-Juan Liu ◽

Xiao-Jun Li ◽

Zhang-zhou Shen ◽

Bo Yang ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Stem Cells ◽

Birth Defects ◽

Human Cytomegalovirus ◽

Hcmv Infection ◽

Viral Gene Expression ◽

Viral Gene ◽

Gene Products ◽

Hcmv Replication

ABSTRACTCongenital human cytomegalovirus (HCMV) infection is a leading cause of birth defects, primarily manifesting as neurological disorders. HCMV infection alters expression of cellular microRNAs (miRs) and induces cell cycle arrest, which in turn modifies the cellular environment to favor virus replication. Previous observations found that HCMV infection reduces miR-21 expression in neural progenitor/stem cells (NPCs). Here, we show that infection of NPCs and U-251MG cells represses miR-21 while increasing the levels of Cdc25a, a cell cycle regulator and known target of miR-21. These opposing responses to infection prompted an investigation of the relationship between miR-21, Cdc25a, and viral replication. Overexpression of miR-21 in NPCs and U-251MG cells inhibited viral gene expression, genome replication, and production of infectious progeny, while shRNA-knockdown of miR-21 in U-251MG cells increased viral gene expression. In contrast, overexpression of Cdc25a in U-251MG cells increased viral gene expression and production of infectious progeny and overcame the inhibitory effects of miR-21 overexpression. Three viral gene products—IE1, pp71, and UL26—were shown to inhibit miR-21 expression at the transcriptional level. These results suggest that Cdc25a promotes HCMV replication and elevation of Cdc25a levels after HCMV infection are due in part to HCMV-mediated repression of miR-21. Thus, miR-21 is an intrinsic antiviral factor that is modulated by HCMV infection. This suggests a role for miR-21 downregulation in the neuropathogenesis of HCMV infection of the developing CNS.IMPORTANCEHuman cytomegalovirus (HCMV) is a ubiquitous pathogen and has very high prevalence among population, especially in China, and congenital HCMV infection is a major cause for birth defects. Elucidating virus-host interactions that govern HCMV replication in neuronal cells is critical to understanding the neuropathogenesis of birth defects resulting from congenital infection. In this study, we confirm that HCMV infection downregulates miR-21 but upregulates Cdc25a. Further determined the negative effects of cellular miRNA miR-21 on HCMV replication in neural progenitor/stem cells and U-251MG glioblastoma/astrocytoma cells. More importantly, our results provide the first evidence that miR-21 negatively regulates HCMV replication by targeting Cdc25a, a vital cell cycle regulator. We further found that viral gene products of IE1, pp71, and UL26 play roles in inhibiting miR-21 expression, which in turn causes increases in Cdc25a and benefits HCMV replication. Thus, miR-21 appears to be an intrinsic antiviral factor that represents a potential target for therapeutic intervention.

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CCNE1 and E2F1 Partially Suppress G1 Phase Arrest Caused by Spliceostatin A Treatment

International Journal of Molecular Sciences ◽

10.3390/ijms222111623 ◽

2021 ◽

Vol 22 (21) ◽

pp. 11623

Author(s):

Kei Kikuchi ◽

Daisuke Kaida

Keyword(s):

Cell Cycle ◽

Cell Cycle Progression ◽

Kinase Inhibitor ◽

G1 Phase ◽

Cell Cycle Regulators ◽

Protein Levels ◽

Cyclin Dependent Kinase Inhibitor ◽

Phase Arrest ◽

G1 Phase Arrest ◽

Spliceostatin A

The potent splicing inhibitor spliceostatin A (SSA) inhibits cell cycle progression at the G1 and G2/M phases. We previously reported that upregulation of the p27 cyclin-dependent kinase inhibitor encoded by CDKN1B and its C-terminal truncated form, namely p27*, which is translated from CDKN1B pre-mRNA, is one of the causes of G1 phase arrest caused by SSA treatment. However, the detailed molecular mechanism underlying G1 phase arrest caused by SSA treatment remains to be elucidated. In this study, we found that SSA treatment caused the downregulation of cell cycle regulators, including CCNE1, CCNE2, and E2F1, at both the mRNA and protein levels. We also found that transcription elongation of the genes was deficient in SSA-treated cells. The overexpression of CCNE1 and E2F1 in combination with CDKN1B knockout partially suppressed G1 phase arrest caused by SSA treatment. These results suggest that the downregulation of CCNE1 and E2F1 contribute to the G1 phase arrest induced by SSA treatment, although they do not exclude the involvement of other factors in SSA-induced G1 phase arrest.

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Human Cytomegalovirus IE1-72 Activates Ataxia Telangiectasia Mutated Kinase and a p53/p21-Mediated Growth Arrest Response

Journal of Virology ◽

10.1128/jvi.79.17.11467-11475.2005 ◽

2005 ◽

Vol 79 (17) ◽

pp. 11467-11475 ◽

Cited By ~ 46

Author(s):

Jonathan P. Castillo ◽

Fiona M. Frame ◽

Harry A. Rogoff ◽

Mary T. Pickering ◽

Andrew D. Yurochko ◽

...

Keyword(s):

Cell Cycle ◽

Human Cytomegalovirus ◽

Ataxia Telangiectasia ◽

Cell Cycle Progression ◽

Hcmv Infection ◽

S Phase ◽

Nuclear Accumulation ◽

Ataxia Telangiectasia Mutated ◽

P53 Response ◽

Nuclear Shuttling

ABSTRACT Human cytomegalovirus (HCMV) encodes several proteins that can modulate components of the cell cycle machinery. The UL123 gene product, IE1-72, binds the Rb-related, p107 protein and relieves its repression of E2F-responsive promoters; however, it is unable to induce quiescent cells to enter S phase in wild-type (p53 +/+ ) cells. IE1-72 also induces p53 accumulation through an unknown mechanism. We present here evidence suggesting that IE1-72 may activate the p53 pathway by increasing the levels of p19Arf and by inducing the phosphorylation of p53 at Ser15. Phosphorylation of this residue by IE1-72 expression alone or HCMV infection is found to be dependent on the ataxia-telangiectasia mutated kinase. IE2-86 expression leads to p53 phosphorylation and may contribute to this phenotype in HCMV-infected cells. We also found that IE1-72 promotes p53 nuclear accumulation by abrogating p53 nuclear shuttling. These events result in the stimulation of p53 activity, leading to a p53- and p21-dependent inhibition of cell cycle progression from G1 to S phase in cells transiently expressing IE1-72. Thus, like many of the small DNA tumor viruses, the first protein expressed upon HCMV infection activates a p53 response by the host cell.

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Aurora Kinase Inhibitor ZM447439 Blocks Chromosome-induced Spindle Assembly, the Completion of Chromosome Condensation, and the Establishment of the Spindle Integrity Checkpoint inXenopusEgg Extracts

Molecular Biology of the Cell ◽

10.1091/mbc.e04-10-0891 ◽

2005 ◽

Vol 16 (3) ◽

pp. 1305-1318 ◽

Cited By ~ 91

Author(s):

Bedrick B. Gadea ◽

Joan V. Ruderman

Keyword(s):

Cell Cycle ◽

Mitotic Spindle ◽

Kinase Inhibitor ◽

Spindle Assembly ◽

Aurora Kinase ◽

Chromosome Morphology ◽

Chromosome Condensation ◽

Embryonic Cell ◽

Cell Cycle Regulators ◽

Cell Cycles

The Aurora family kinases contribute to accurate progression through several mitotic events. ZM447439 (“ZM”), the first Aurora family kinase inhibitor to be developed and characterized, was previously found to interfere with the mitotic spindle integrity checkpoint and chromosome segregation. Here, we have used extracts of Xenopus eggs, which normally proceed through the early embryonic cell cycles in the absence of functional checkpoints, to distinguish between ZM's effects on the basic cell cycle machinery and its effects on checkpoints. ZM clearly had no effect on either the kinetics or amplitude in the oscillations of activity of several key cell cycle regulators. It did, however, have striking effects on chromosome morphology. In the presence of ZM, chromosome condensation began on schedule but then failed to progress properly; instead, the chromosomes underwent premature decondensation during mid-mitosis. ZM strongly interfered with mitotic spindle assembly by inhibiting the formation of microtubules that are nucleated/stabilized by chromatin. By contrast, ZM had little effect on the assembly of microtubules by centrosomes at the spindle poles. Finally, under conditions where the spindle integrity checkpoint was experimentally induced, ZM blocked the establishment, but not the maintenance, of the checkpoint, at a point upstream of the checkpoint protein Mad2. These results show that Aurora kinase activity is required to ensure the maintenance of condensed chromosomes, the generation of chromosome-induced spindle microtubules, and activation of the spindle integrity checkpoint.

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Human cytomegalovirus infection stimulates Cl−/ HCO 3 − exchanger activity in human fibroblasts

AJP Cell Physiology ◽

10.1152/ajpcell.1998.275.2.c515 ◽

1998 ◽

Vol 275 (2) ◽

pp. C515-C526 ◽

Cited By ~ 25

Author(s):

Lilia M. Maglova ◽

William E. Crowe ◽

Aníbal A. Altamirano ◽

John M. Russell

Keyword(s):

Human Cytomegalovirus ◽

Hcmv Infection ◽

Human Lung ◽

Human Fibroblasts ◽

Cell Types ◽

Recovery Phase ◽

Lung Fibroblasts ◽

Continuous Exposure ◽

Infected Cells ◽

Human Cytomegalovirus Infection

The effects of human cytomegalovirus (HCMV) infection on Cl−/[Formula: see text]exchanger activity in human lung fibroblasts (MRC-5 cells) were studied using fluorescent, ion-sensitive dyes. The intracellular pH (pHi) of mock- and HCMV-infected cells bathed in a solution containing 5% CO2-25 mM[Formula: see text] were nearly the same. However, replacement of external Cl−with gluconate caused an H2DIDS-inhibitable (100 μM) increase in the pHi of HCMV-infected cells but not in mock-infected cells. Continuous exposure to hyperosmotic external media containing CO2/[Formula: see text]caused the pHi of both cell types to increase. The pHi remained elevated in mock-infected cells. However, in HCMV-infected cells, the pHi peaked and then recovered toward control values. This pHirecovery phase was completely blocked by 100 μM H2DIDS. In the presence of CO2/[Formula: see text], there was an H2DIDS-sensitive component of net Cl− efflux (external Cl− was substituted with gluconate) that was less in mock- than in HCMV-infected cells. When nitrate was substituted for external Cl− (in the nominal absence of CO2/[Formula: see text]), the H2DIDS-sensitive net Cl− efflux was much greater from HCMV- than from mock-infected cells. In mock-infected cells, H2DIDS-sensitive, net Cl− efflux decreased as pHi increased, whereas for HCMV-infected cells, efflux increased as pHi increased. All these results are consistent with an HCMV-induced enhancement of Cl−/[Formula: see text]exchanger activity.

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The aryl hydrocarbon receptor facilitates the human cytomegalovirus-mediated G1/S block to cell cycle progression

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2026336118 ◽

2021 ◽

Vol 118 (12) ◽

pp. e2026336118

Author(s):

Pooya Naseri-Nosar ◽

Maciej T. Nogalski ◽

Thomas Shenk

Keyword(s):

Cell Cycle ◽

Aryl Hydrocarbon Receptor ◽

Human Cytomegalovirus ◽

Cell Cycle Progression ◽

Hcmv Infection ◽

Cell Interaction ◽

Cycle Progression ◽

Aryl Hydrocarbon ◽

Cell Transcriptome ◽

Cell Cycle Block

The tryptophan metabolite, kynurenine, is known to be produced at elevated levels within human cytomegalovirus (HCMV)-infected fibroblasts. Kynurenine is an endogenous aryl hydrocarbon receptor (AhR) ligand. Here we show that the AhR is activated following HCMV infection, and pharmacological inhibition of AhR or knockdown of AhR RNA reduced the accumulation of viral RNAs and infectious progeny. RNA-seq analysis of infected cells following AhR knockdown showed that the receptor alters the levels of numerous RNAs, including RNAs related to cell cycle progression. AhR knockdown alleviated the G1/S cell cycle block that is normally instituted in HCMV-infected fibroblasts, consistent with its known ability to regulate cell cycle progression and cell proliferation. In sum, AhR is activated by kynurenine and perhaps other ligands produced during HCMV infection, it profoundly alters the infected-cell transcriptome, and one outcome of its activity is a block to cell cycle progression, providing mechanistic insight to a long-known element of the virus–host cell interaction.

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Human Cytomegalovirus Gene UL21a Encodes a Short-Lived Cytoplasmic Protein and Facilitates Virus Replication in Fibroblasts

Journal of Virology ◽

10.1128/jvi.01116-09 ◽

2009 ◽

Vol 84 (1) ◽

pp. 291-302 ◽

Cited By ~ 22

Author(s):

Anthony R. Fehr ◽

Dong Yu

Keyword(s):

Human Cytomegalovirus ◽

Large Scale ◽

Hcmv Infection ◽

Productive Infection ◽

Lung Fibroblasts ◽

Wild Type Virus ◽

Growth Defect ◽

Cytoplasmic Protein ◽

Wild Type ◽

Virus Growth

ABSTRACT The human cytomegalovirus (HCMV) gene UL21a was recently annotated by its conservation in chimpanzee cytomegalovirus. Two large-scale mutagenic analyses showed that mutations in overlapping UL21a/UL21 resulted in a severe defect of virus growth in fibroblasts. Here, we characterized UL21a and demonstrated its role in HCMV infection. We mapped a UL21a-specific transcript of ∼600 bp that was expressed with early kinetics. UL21a encoded pUL21a, a protein of ∼15 kDa, which was unstable and localized predominantly to the cytoplasm during HCMV infection or when expressed alone. Interestingly, pUL21a was drastically stabilized in the presence of proteasome inhibitor MG132, but its instability was independent of a functional ubiquitin-mediated pathway, suggesting that pUL21a underwent proteasome-dependent, ubiquitin-independent degradation. A UL21a deletion virus was attenuated in primary human newborn foreskin fibroblasts (HFFs) and embryonic lung fibroblasts (MRC-5), whereas a marker-rescued virus and mutant viruses lacking the neighboring or overlapping genes UL20, UL21, or UL21.5-UL23 replicated at wild-type levels. The growth defect of UL21a-deficient virus in MRC-5 cells was more pronounced than that in HFFs. At a high multiplicity of infection, the UL21a deletion virus synthesized viral proteins with wild-type kinetics but had a two- to threefold defect in viral DNA replication. More importantly, although pUL21a was not detected in the virion, progeny virions produced by the mutant virus were ∼10 times less infectious than wild-type virus, suggesting that UL21a is required for HCMV to establish efficient productive infection. We conclude that UL21a encodes a short-lived cytoplasmic protein and facilitates HCMV replication in fibroblasts.

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The Human Cytomegalovirus IE86 Protein Can Block Cell Cycle Progression after Inducing Transition into the S Phase of Permissive Cells

Journal of Virology ◽

10.1128/jvi.74.15.7108-7118.2000 ◽

2000 ◽

Vol 74 (15) ◽

pp. 7108-7118 ◽

Cited By ~ 103

Author(s):

Eain A. Murphy ◽

Daniel N. Streblow ◽

Jay A. Nelson ◽

Mark F. Stinski

Keyword(s):

Cell Cycle ◽

Protein Expression ◽

Human Cytomegalovirus ◽

Cell Cycle Progression ◽

Hcmv Infection ◽

Fluorescent Protein ◽

Cell Types ◽

S Phase ◽

Cycle Progression ◽

Green Fluorescent

ABSTRACT Human cytomegalovirus (HCMV) infection of permissive cells has been reported to induce a cell cycle halt. One or more viral proteins may be involved in halting progression at different stages of the cell cycle. We investigated how HCMV infection, and specifically IE86 protein expression, affects the cell cycles of permissive and nonpermissive cells. We used a recombinant virus that expresses the green fluorescent protein (GFP) to determine the effects of HCMV on the cell cycle of permissive cells. Fluorescence by GFP allowed us to select for only productively infected cells. Replication-defective adenovirus vectors expressing the IE72 or IE86 protein were also used to efficiently transduce 95% or more of the cells. The adenovirus-expressed IE86 protein was determined to be functional by demonstrating negative autoregulation of the major immediate-early promoter and activation of an early viral promoter in the context of the viral genome. To eliminate adenovirus protein effects, plasmids expressing GFP for fluorescent selection of only transfected cells and wild-type IE86 protein or a mutant IE86 protein were tested in permissive and nonpermissive cells. HCMV infection induced the entry of U373 cells into the S phase. All permissive cells infected with HCMV were blocked in cell cycle progression and could not divide. After either transduction or transfection and IE86 protein expression, the number of all permissive or nonpermissive cell types in the S phase increased significantly, but the cells could no longer divide. The IE72 protein did not have a significant effect on the S phase. Since IE86 protein inhibits cell cycle progression, the IE2 gene in a human fibroblast IE86 protein-expressing cell line was sequenced. The IE86 protein in these retrovirus-transduced cells has mutations in a critical region of the viral protein. The locations of the mutations and the function of the IE86 protein in controlling cell cycle progression are discussed.

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The TGF-β1/p53/PAI-1 Signaling Axis in Vascular Senescence: Role of Caveolin-1

Biomolecules ◽

10.3390/biom9080341 ◽

2019 ◽

Vol 9 (8) ◽

pp. 341 ◽

Cited By ~ 7

Author(s):

Rohan Samarakoon ◽

Stephen P. Higgins ◽

Craig E. Higgins ◽

Paul J. Higgins

Keyword(s):

Cell Cycle ◽

Cell Cycle Arrest ◽

Replicative Senescence ◽

Plasminogen Activator Inhibitor ◽

Negative Regulator ◽

Plasminogen Activator Inhibitor 1 ◽

Caveolin 1 ◽

Cycle Arrest ◽

Tgf Β1 ◽

Pai 1

Stress-induced premature cellular senescence is a significant factor in the onset of age-dependent disease in the cardiovascular system. Plasminogen activator inhibitor-1 (PAI-1), a major TGF-β1/p53 target gene and negative regulator of the plasmin-based pericellular proteolytic cascade, is elevated in arterial plaques, vessel fibrosis, arteriosclerosis, and thrombosis, correlating with increased tissue TGF-β1 levels. Additionally, PAI-1 is necessary and sufficient for the induction of p53-dependent replicative senescence. The mechanism of PAI-1 transcription in senescent cells appears to be dependent on caveolin-1 signaling. Src kinases are upstream effectors of both FAK and caveolin-1 activation as FAKY577,Y861 and caveolin-1Y14 phosphorylation are not detected in TGF-β1-stimulated src family kinase (pp60c-src, Yes, Fyn) triple-deficient (SYF−/−/−) cells. However, restoration of pp60c-src expression in SYF-null cells rescued both caveolin-1Y14 phosphorylation and PAI-1 induction in response to TGF-β1. Furthermore, TGF-β1-initiated Src phosphorylation of caveolin-1Y14 is critical in Rho-ROCK-mediated suppression of the SMAD phosphatase PPM1A maintaining and, accordingly, SMAD2/3-dependent transcription of the PAI-1 gene. Importantly, TGF-β1 failed to induce PAI-1 expression in caveolin-1-null cells, correlating with reductions in both Rho-GTP loading and SMAD2/3 phosphorylation. These findings implicate caveolin-1 in expression controls on specific TGF-β1/p53 responsive growth arrest genes. Indeed, up-regulation of caveolin-1 appears to stall cells in G0/G1 via activation of the p53/p21 cell cycle arrest pathway and restoration of caveolin-1 in caveolin-1-deficient cells rescues TGF-β1 inducibility of the PAI-1 gene. Although the mechanism is unclear, caveolin-1 inhibits p53/MDM2 complex formation resulting in p53 stabilization, induction of p53-target cell cycle arrest genes (including PAI-1), and entrance into premature senescence while stimulating the ATM→p53→p21 pathway. Identification of molecular events underlying senescence-associated PAI-1 expression in response to TGF-β1/src kinase/p53 signaling may provide novel targets for the therapy of cardiovascular disease.

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