Resistance to Methylation de novo of the Human Cytomegalovirus Immediate Early Enhancer in a Model for Virus Latency and Reactivation in vitro

To define mechanistically how the human cytomegalovirus (HCMV) major immediate-early (IE) proteins induce early-gene transcription, the IE1 72-kDa protein, the IE2 55-kDa protein, and the IE2 86-kDa protein were analyzed for their ability to activate transcription from an HCMV early promoter in vivo and in vitro. In transient-expression assays in U373MG astrocytoma/glioblastoma and HeLa cells, only the IE2 86-kDa protein was able to activate the HCMV early promoter to high levels. In HeLa cells, the IE1 72-kDa protein was able to activate the promoter to a low but detectable level, and the level of promoter activity observed in response to the IE2 86-kDa protein was increased synergistically following cotransfection of the constructs expressing both IE proteins. To examine the interaction of the HCMV IE proteins with the RNA polymerase II transcription machinery, we assayed the ability of Escherichia coli-synthesized proteins to activate the HCMV early promoter in nuclear extracts prepared from U373MG cells, HeLa cells, and Drosophila embryos. The results of the in vitro experiments correlated well with those obtained in vivo. The basal activity of the promoter was minimal in both the HeLa and U373MG extracts but was stimulated 6- to 10-fold by the IE2 86-kDa protein. With a histone H1-deficient extract from Drosophila embryos, the HCMV early promoter was quite active and was stimulated two- to fourfold by the IE2 86-kDa protein. Addition of histone H1 at 1 molecule per 40 to 50 bp of DNA template significantly repressed basal transcription from this promoter. However, the IE2 86-kDa protein, but none of the other IE proteins, was able to counteract the H1-mediated repression and stimulate transcription at least 10- to 20-fold. The promoter specificity of the activation was demonstrated by the inability of the IE2 86-kDa protein to activate the Drosophila Krüppel promoter in either the presence or absence of histone H1. These results suggest that one mechanism of transcription activation by the IE2 86-kDa protein involves antirepression.

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Inactivation and Disassembly of the Anaphase-Promoting Complex during Human Cytomegalovirus Infection Is Associated with Degradation of the APC5 and APC4 Subunits and Does Not Require UL97-Mediated Phosphorylation of Cdh1

Journal of Virology ◽

10.1128/jvi.01260-10 ◽

2010 ◽

Vol 84 (20) ◽

pp. 10832-10843 ◽

Cited By ~ 27

Author(s):

Karen Tran ◽

Jeremy P. Kamil ◽

Donald M. Coen ◽

Deborah H. Spector

Keyword(s):

Human Cytomegalovirus ◽

De Novo ◽

Cellular Protein ◽

Early Gene ◽

Mass Spectrometry Analysis ◽

Wild Type Virus ◽

Viral Gene ◽

Anaphase Promoting Complex ◽

Core Complex

ABSTRACT Infection of quiescent cells by human cytomegalovirus (HCMV) elicits severe cell cycle deregulation, resulting in a G1/S arrest, which can be partly attributed to the inactivation of the anaphase-promoting complex (APC). As we previously reported, the premature phosphorylation of its coactivator Cdh1 and/or the dissociation of the core complex can account for the inactivation. We have expanded on these results and further delineated the key components required for disabling the APC during HCMV infection. The viral protein kinase UL97 was hypothesized to phosphorylate Cdh1, and consistent with this, phosphatase assays utilizing a virus with a UL97 deletion mutation (ΔUL97 virus) indicated that Cdh1 is hypophosphorylated at early times in the infection. Mass spectrometry analysis demonstrated that UL97 can phosphorylate Cdh1 in vitro, and the majority of the sites identified correlated with previously characterized cyclin-dependent kinase (Cdk) consensus sites. Analysis of the APC core complex during ΔUL97 virus infection showed APC dissociation occurring at the same time as during infection with wild-type virus, suggesting that the UL97-mediated phosphorylation of Cdh1 is not required for this to occur. Further investigation of the APC subunits showed a proteasome-dependent loss of the APC5 and APC4 subunits that was temporally associated with the disassembly of the APC. Immediate early viral gene expression was not sufficient for the degradation of APC4 and APC5, indicating that a viral early gene product(s), possibly in association with a de novo-synthesized cellular protein(s), is involved.

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Activation of Peroxisome Proliferator-Activated Receptor Gamma by Human Cytomegalovirus for De Novo Replication Impairs Migration and Invasiveness of Cytotrophoblasts from Early Placentas

Journal of Virology ◽

10.1128/jvi.01779-09 ◽

2009 ◽

Vol 84 (6) ◽

pp. 2946-2954 ◽

Cited By ~ 37

Author(s):

Benjamin Rauwel ◽

Bernard Mariamé ◽

Hélène Martin ◽

Ronni Nielsen ◽

Sophie Allart ◽

...

Keyword(s):

Human Cytomegalovirus ◽

De Novo ◽

Present Report ◽

Primary Cultures ◽

First Trimester ◽

Peroxisome Proliferator ◽

Mobility Shift ◽

Peroxisome Proliferator Activated Receptor ◽

Human Trophoblast

ABSTRACT Human cytomegalovirus (HCMV) contributes to pathogenic processes in immunosuppressed individuals, in fetuses, and in neonates. In the present report, by using reporter gene activation assays and confocal microscopy in the presence of a specific antagonist, we show for the first time that HCMV infection induces peroxisome proliferator-activated receptor gamma (PPARγ) transcriptional activity in infected cells. We demonstrate that the PPARγ antagonist dramatically impairs virus production and that the major immediate-early promoter contains PPAR response elements able to bind PPARγ, as assessed by electrophoretic mobility shift and chromatin immunoprecipitation assays. Due to the key role of PPARγ in placentation and its specific trophoblast expression within the human placenta, we then provided evidence that by activating PPARγ human cytomegalovirus dramatically impaired early human trophoblast migration and invasiveness, as assessed by using well-established in vitro models of invasive trophoblast, i.e., primary cultures of extravillous cytotrophoblasts (EVCT) isolated from first-trimester placentas and the EVCT-derived cell line HIPEC. Our data provide new clues to explain how early infection during pregnancy could impair implantation and placentation and therefore embryonic development.

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Integrin alpha 5 beta 1 expression negatively regulates cell growth: reversal by attachment to fibronectin.

Molecular Biology of the Cell ◽

10.1091/mbc.6.6.725 ◽

1995 ◽

Vol 6 (6) ◽

pp. 725-740 ◽

Cited By ~ 175

Author(s):

J A Varner ◽

D A Emerson ◽

R L Juliano

Keyword(s):

Cellular Proliferation ◽

De Novo ◽

Cell Attachment ◽

Growth Arrest ◽

Early Gene ◽

Immediate Early ◽

Specific Gene ◽

Cellular Quiescence

Cells selected for overexpression of the integrin alpha 5 beta 1 show decreased proliferation and loss of the transformed phenotype. We provide evidence that de novo expression of the integrin alpha 5 beta 1 in HT29 colon carcinoma cells results in the growth arrest of these cells as characterized by reduced DNA synthesis and cellular proliferation in vitro. In fact, expression of integrin alpha 5 beta 1 on these cells induces the transcription of growth arrest specific gene 1 (gas-1), a gene product known to induce cellular quiescence, but blocks transcription of the immediate early genes c-fos, c-jun, and jun B. In vivo, the alpha 5 beta 1 transfectants display dramatically reduced tumorigenicity as well as a highly differentiated phenotype when compared with their pSVneo-transfected counterparts. Surprisingly, ligation of alpha 5 beta 1 on these cells by cell attachment to a fibronectin substrate not only reverses the growth inhibition and gas-1 gene induction but activates immediate early gene transcription. These findings demonstrate that integrin alpha 5 beta 1 expression in the absence of attachment to fibronectin activates a signaling pathway leading to decreased cellular proliferation and that ligation of this receptor with fibronectin reverses this signal, thereby contributing to the proliferation of transformed cells.

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Recruitment of cdk9 to the Immediate-Early Viral Transcriptosomes during Human Cytomegalovirus Infection Requires Efficient Binding to Cyclin T1, a Threshold Level of IE2 86, and Active Transcription

Journal of Virology ◽

10.1128/jvi.02651-08 ◽

2009 ◽

Vol 83 (11) ◽

pp. 5904-5917 ◽

Cited By ~ 15

Author(s):

Anokhi J. Kapasi ◽

Charles L. Clark ◽

Karen Tran ◽

Deborah H. Spector

Keyword(s):

Human Cytomegalovirus ◽

De Novo ◽

Threshold Level ◽

Immediate Early ◽

Cyclin T1 ◽

Cellular Factors ◽

Active Transcription ◽

Exogenous Expression ◽

Kinetics Of ◽

De Novo Protein Synthesis

ABSTRACT Human cytomegalovirus (HCMV) infection results in the formation of nuclear viral transcriptosomes, which are sites dedicated to viral immediate-early (IE) transcription. At IE times of the infection, viral and cellular factors, including several components of transcription such as cyclin-dependent kinase 9 (cdk9), localize at these sites. To determine the mechanism and requirements of specific recruitment of cdk9 to the viral transcriptosomes, infection in the presence of inhibitor drugs and infection of cell lines expressing exogenous mutant cdk9 were performed. We found that cdk9 localization to the viral transcriptosomes requires de novo protein synthesis. In addition, active transcription is required for recruitment and maintenance of cdk9 at the viral transcriptosomes. In cells infected with a recombinant IE2 HCMV (IE2 86 ΔSX virus) in which IE2 gene expression is greatly reduced, cdk9 localization at the transcriptosome is delayed and corresponds to the kinetics of accumulation of the IE2 protein at these sites. Infection in the presence of the cdk9 inhibitors Flavopiridol and DRB (5,6-dichloro-1-β-d-ribofuranosylbenzimidazole) allowed cdk9 localization to the viral transcriptosomes. A kinase-inactive cdk9 (D167N) expressed during the infection also localizes to the viral transcriptosomes, indicating that kinase activity of cdk9 is not a requirement for its localization to the sites of IE transcription. Exogenous expression of additional cdk9 mutants indicates that binding of Brd4 to the cdk9 complex is not required but that efficient binding to cyclin T1 is essential.

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A Strong Negative Transcriptional Regulatory Region between the Human Cytomegalovirus UL127 Gene and the Major Immediate-Early Enhancer

Journal of Virology ◽

10.1128/jvi.73.11.9039-9052.1999 ◽

1999 ◽

Vol 73 (11) ◽

pp. 9039-9052 ◽

Cited By ~ 28

Author(s):

Christopher A. Lundquist ◽

Jeffrey L. Meier ◽

Mark F. Stinski

Keyword(s):

Human Cytomegalovirus ◽

Viral Genome ◽

De Novo ◽

Human Fibroblasts ◽

Regulatory Region ◽

Fold Increase ◽

Immediate Early ◽

Wild Type ◽

Viral Protein Synthesis ◽

Unique Region

ABSTRACT The region of the human cytomegalovirus (CMV) genome between the UL127 open reading frame and the major immediate-early (MIE) enhancer is referred to as the unique region. DNase I protection analysis with human cell nuclear extracts demonstrated multiple protein binding sites in this region of the viral genome (P. Ghazal, H. Lubon, C. Reynolds-Kohler, L. Hennighausen, and J. A. Nelson, Virology 174:18–25, 1990). However, the function of this region in the context of the viral genome is not known. In wild-type human CMV-infected human fibroblasts, cells permissive for viral replication, there is little to no transcription from UL127. We determined that the unique region prevented transcription from the UL127 promoter but had no effect on the divergent MIE promoter. In transient-transfection assays, the basal level of expression from the UL127 promoter increased significantly when the wild-type unique sequences were mutated. In recombinant viruses with similar mutations in the unique region, expression from the UL127 promoter occurred only after de novo viral protein synthesis, typical of an early viral promoter. A 111-bp deletion-substitution of the unique sequence caused approximately a 20-fold increase in the steady-state level of RNA from the UL127 promoter and a 245-fold increase in the expression of a downstream indicator gene. This viral negative regulatory region was also mutated at approximately 50-bp regions proximal and distal to the UL127 promoter. Although some repressive effects were detected in the distal region, mutations of the region proximal to the UL127 promoter had the most significant effects on transcription. Within the proximal and distal regions, there are potential cis sites for known eucaryotic transcriptional repressor proteins. This region may also bind unknown viral proteins. We propose that the unique region upstream of the UL127 promoter and the MIE enhancer negatively regulates the expression from the UL127 promoter in permissive human fibroblast cells. This region may be a regulatory boundary preventing the effects of the very strong MIE enhancer on this promoter.

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Effects of allitridin on transcription of immediate-early,early and late genes of human cytomegalovirus in vitro

China Journal of Chinese Materia Medica ◽

10.4268/cjcmm20111431 ◽

2011 ◽

Author(s):

ZHANG Ju

Keyword(s):

Human Cytomegalovirus ◽

Immediate Early ◽

Late Genes

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Transcriptional regulation of the human cytomegalovirus major immediate-early gene is associated with induction of DNase I-hypersensitive sites

Molecular and Cellular Biology ◽

10.1128/mcb.6.2.452-461.1986 ◽

1986 ◽

Vol 6 (2) ◽

pp. 452-461 ◽

Cited By ~ 2

Author(s):

J A Nelson ◽

M Groudine

Keyword(s):

Human Cytomegalovirus ◽

Structural Features ◽

In Vitro Transcription ◽

Dnase I ◽

Early Gene ◽

Immediate Early ◽

Dnase I Hypersensitive Sites ◽

Teratocarcinoma Cells ◽

Hypersensitive Sites

Human teratocarcinoma cells were used to examine structural features associated with expression of the major immediate-early (IE) gene of human cytomegalovirus. By immunofluorescence, comparison of RNA levels, and in vitro transcription of nuclei, we showed that the major IE gene is inactive in undifferentiated but active in differentiated cells. Therefore, the block in human cytomegalovirus replication in teratocarcinoma cells appears to be at the transcriptional level, in one of the initial genes transcribed. In addition, the in vitro transcription experiments demonstrated that in permissive infections the gene was transcriptionally inactive late in infection. A comparison of the structural features of the promoter region with the active and inactive IE genes showed the presence of constitutive and inducible DNase I-hypersensitive sites. The majority of the constitutive sites existed at -175, -275, -375, -425, and -525 relative to the cap site in an area which has been shown to be capable of simian virus 40 enhancer function. In contrast, the inducible DNase I sites were located outside this region at -650, -775, -875, and -975.

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Functional Interaction between the pp71 Protein of Human Cytomegalovirus and the PML-Interacting Protein Human Daxx

Journal of Virology ◽

10.1128/jvi.76.11.5769-5783.2002 ◽

2002 ◽

Vol 76 (11) ◽

pp. 5769-5783 ◽

Cited By ~ 120

Author(s):

Heike Hofmann ◽

Hilde Sindre ◽

Thomas Stamminger

Keyword(s):

Human Cytomegalovirus ◽

Protein Function ◽

Fluorescent Protein ◽

De Novo ◽

Human Fibroblasts ◽

Specific Interaction ◽

Immediate Early ◽

Regulatory Function ◽

Viral Protein Synthesis ◽

Interacting Protein

ABSTRACT The tegument protein pp71 (UL82) of human cytomegalovirus (HCMV) has previously been shown to transactivate the major immediate-early enhancer-promoter of HCMV. Furthermore, this protein is able to enhance the infectivity of viral DNA and to accelerate the infection cycle, suggesting an important regulatory function during viral replication. To gain insight into the underlying mechanisms that are used by pp71 to exert these pleiotropic effects, we sought for cellular factors interacting with pp71 in a yeast two-hybrid screen. Here, we report the isolation of the human Daxx (hDaxx) protein as a specific interaction partner of HCMV pp71. hDaxx, which was initially described as an adapter protein involved in apoptosis regulation, has recently been identified as a nuclear protein that interacts and colocalizes with PML in the nuclear domain ND10. In order to assess whether pp71 can also be detected in ND10 structures, a vector expressing pp71 in fusion with the green fluorescent protein was used for transfection of human fibroblasts. This revealed a colocalization of pp71 with the ND10 proteins PML and Sp100. In addition, cotransfection of a hDaxx expression vector resulted in an enhanced recruitment of pp71 to ND10. Targeting of pp71 to nuclear dots could also be observed in infected human fibroblasts in the absence of de novo viral protein synthesis. Moreover, cotransfection experiments revealed that pp71-mediated transactivation of the major immediate-early enhancer-promoter was synergistically enhanced in the presence of hDaxx. These results suggest an important role of hDaxx for pp71 protein function.

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Pentoxifylline Promotes Replication of Human Cytomegalovirus In Vivo and In Vitro

Blood ◽

10.1182/blood.v89.10.3682 ◽

1997 ◽

Vol 89 (10) ◽

pp. 3682-3690 ◽

Cited By ~ 1

Author(s):

Kerstin Staak ◽

Susanna Prösch ◽

Joachim Stein ◽

Christina Priemer ◽

Ralf Ewert ◽

...

Keyword(s):

Transcription Factor ◽

Human Cytomegalovirus ◽

Peak Plasma ◽

Immediate Early ◽

Lung Fibroblasts ◽

Sequence Motif ◽

Peak Plasma Level ◽

Hcmv Disease

Abstract OKT3 monoclonal antibody (MoAb) therapy is well established in the prevention and therapy of acute rejection in transplant patients. Unfortunately, this therapy is associated with several short-term (cytokine release syndrome) and long-term (infections, EBV-related lymphoma) side effects. Recently, we were able to demonstrate an association between the TNFα release following the first OKT3 MoAb infusions and the appearance of human cytomegalovirus (HCMV) reactivation several days later. In order to prevent this TNFα associated HCMV reactivation patients were additionally treated with pentoxifylline (PTX), a methylxanthine derivative that has been shown to suppress TNFα induction. Although the TNFα peak plasma level following OKT3 MoAb treatment was markedly reduced, the incidence of HCMV reactivation and HCMV disease was not influenced. In transient transfection experiments using HCMV immediate early enhancer/promoter CAT reporter gene constructs PTX enhanced the promoter activity independently from TNFα in premonocytic cells. Furthermore, PTX acted synergistically with TNFα. In virus-infected human embryonal lung fibroblasts HCMV replication was triggered in the presence of both PTX and TNFα, while either substance alone had only marginal effects. The stimulatory effect of PTX on the immediate early (IE) enhancer/promoter was mediated via CREB/ATF, a eukaryotic transcription factor that binds to the 19 bp sequence motif in the enhancer region, while TNFα stimulation was mediated by activation of the transcription factor NF-kB and its binding to the 18 bp sequence motif in the enhancer. These data suggest a potential side effect of cAMP-elevating drugs such as PTX.

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