Knockdown of hDaxx in normally non-permissive undifferentiated cells does not permit human cytomegalovirus immediate-early gene expression

The cellular protein human Daxx (hDaxx), a component of nuclear domain 10 structures, is known to mediate transcriptional repression of human cytomegalovirus immediate-early (IE) gene expression upon infection of permissive cell types, at least in part, by regulation of chromatin structure around the major IE promoter (MIEP). As it is now clear that differentiation-dependent regulation of the MIEP also plays a pivotal role in the control of latency and reactivation, we asked whether hDaxx-mediated repression is involved in differentiation-dependent MIEP regulation. We show that downregulation of hDaxx by using small interfering RNA technology in undifferentiated NT2D1 cells does not permit expression of viral IE genes, nor does it result in changes in chromatin structure around the MIEP. Viral IE gene expression is only observed upon cellular differentiation, suggesting little involvement of hDaxx in the regulation of the viral MIEP in undifferentiated cells.

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Role of the cellular protein hDaxx in human cytomegalovirus immediate-early gene expression

Journal of General Virology ◽

10.1099/vir.0.81566-0 ◽

2006 ◽

Vol 87 (5) ◽

pp. 1113-1121 ◽

Cited By ~ 80

Author(s):

Chris M. Preston ◽

Mary Jane Nicholl

Keyword(s):

Gene Expression ◽

Human Cytomegalovirus ◽

Transcriptional Response ◽

Cellular Protein ◽

Early Gene ◽

Immediate Early ◽

Simplex Virus ◽

Interfering Rna ◽

Stimulation Of

Human cytomegalovirus (HCMV) immediate-early (IE) transcription is stimulated by virion phosphoprotein pp71, the product of gene UL82. It has previously been shown that pp71 interacts with the cellular protein hDaxx and, in the studies presented here, the significance of this interaction was investigated for HCMV IE gene expression. In co-transfection experiments, the presence of hDaxx increased the transcriptional response of the HCMV major IE promoter (MIEP) to pp71, but it was not possible to determine whether the effect was due to an interaction between the two proteins or to stimulation of hDaxx synthesis by pp71. The use of small interfering RNA (siRNA) in long- and short-term transfection approaches reduced intracellular hDaxx levels to no more than 3 % of normal. Infection of hDaxx-depleted cells with herpes simplex virus recombinants containing the HCMV MIEP revealed significantly greater promoter activity when hDaxx levels were minimal. Similarly, reducing intracellular hDaxx amounts resulted in greater IE gene expression during infection with an HCMV mutant lacking pp71, but had no effect on IE transcription during infection with wild-type HCMV. The results suggest that hDaxx is not important as a positive-acting factor for the stimulation of HCMV IE transcription by pp71. Instead, it appears that hDaxx acts as a repressor of IE gene expression, and it is proposed here that the interaction of pp71 with hDaxx is important to relieve repression and permit efficient initiation of productive replication.

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Infection of Cells with Human Cytomegalovirus during S Phase Results in a Blockade to Immediate-Early Gene Expression That Can Be Overcome by Inhibition of the Proteasome

Journal of Virology ◽

10.1128/jvi.76.11.5369-5379.2002 ◽

2002 ◽

Vol 76 (11) ◽

pp. 5369-5379 ◽

Cited By ~ 54

Author(s):

Elizabeth A. Fortunato ◽

Veronica Sanchez ◽

Judy Y. Yen ◽

Deborah H. Spector

Keyword(s):

Gene Expression ◽

Dna Replication ◽

Human Cytomegalovirus ◽

Dna Content ◽

Laser Scanning ◽

Small Population ◽

S Phase ◽

Early Gene ◽

Immediate Early ◽

Laser Scanning Cytometry

ABSTRACT Cells infected with human cytomegalovirus (HCMV) after commencing DNA replication do not initiate viral immediate-early (IE) gene expression and divide before arresting. To determine the nature of this blockade, we examined cells that were infected 24 h after release from G0 using immunofluorescence, laser scanning cytometry, and fluorescence-activated cell sorting (FACS) analysis. Approximately 40 to 50% of the cells had 2N DNA content, became IE+ in the first 12 h, and arrested. Most but not all of the cells with >2N DNA content did not express IE antigens until after mitosis. To define the small population of IE+ cells that gradually accumulated within the S and G2/M compartments, cells were pulsed with bromodeoxyuridine (BrdU) just prior to S-phase infection and analyzed at 12 h postinfection for IE gene expression, BrdU positivity, and cell cycle position. Most of the BrdU+ cells were IE− and had progressed into G2/M or back to G1. The majority of the IE+ cells in S and G2/M were BrdU−. Only a few cells were IE+ BrdU+, and they resided in G2/M. Multipoint BrdU pulse-labeling revealed that, compared to cells actively synthesizing DNA at the beginning of the infection, a greater percentage of the cells that initiated DNA replication 4 h later could express IE antigens and proceed into S. Synchronization of the cells with aphidicolin also indicated that the blockade to the activation of IE gene expression was established in cells soon after initiation of DNA replication. It appears that a short-lived protein in S-phase cells may be required for IE gene expression, as it is partially restored by treatment with the proteasome inhibitor MG132.

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Inhibition of human cytomegalovirus immediate-early gene expression by an antisense oligonucleotide complementary to immediate-early RNA.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.9.2004 ◽

1996 ◽

Vol 40 (9) ◽

pp. 2004-2011 ◽

Cited By ~ 97

Author(s):

K P Anderson ◽

M C Fox ◽

V Brown-Driver ◽

M J Martin ◽

R F Azad

Keyword(s):

Gene Expression ◽

Cell Culture ◽

Antiviral Activity ◽

Protein Expression ◽

Human Cytomegalovirus ◽

Host Cells ◽

Early Gene ◽

Immediate Early ◽

Mrna Levels ◽

Virus Adsorption

ISIS 2922 is a phosphorothioate oligonucleotide that is complementary to human cytomegalovirus (CMV) immediate-early (IE) RNA and that exhibits potent and specific antiviral activity against CMV in cell culture assays. Specific assay systems were developed to separately characterize the antisense and nonantisense components of the antiviral activity mediated by ISIS 2922. In U373 cells transformed with cDNA encoding the CMV IE 55-kDa (IE55) protein, expression was inhibited at nanomolar concentrations comparable to effective concentrations in antiviral assays. The specificity of inhibition was demonstrated by using control oligonucleotides incorporating progressive base changes to destabilize oligonucleotide-RNA base pairing and by showing a lack of inhibition of the CMV IE72 product expressed from the same promoter. Inhibition of IE55 protein expression correlated with a reduction in mRNA levels consistent with an RNase H-mediated termination event. Studies with virus-infected cells demonstrated that antisense and nonantisense mechanisms contribute to the antiviral activity of ISIS 2922. Base complementarity to target RNA was important for optimal activity in antiviral assays, but base changes affecting parameters other than hybridization affinity also influenced antiviral activity. Sequence-independent inhibition of virus adsorption to host cells by phosphorothioate oligonucleotides was also observed at high concentrations. Therefore, at least three different mechanisms may contribute to the antiviral activity of ISIS 2922 in cell culture: antisense-mediated inhibition of target gene expression; nonantisense, sequence-dependent inhibition of virus replication; and sequence-independent inhibition of virus adsorption to host cells.

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Repression of human cytomegalovirus major immediate early gene expression in a monocytic cell line

Journal of General Virology ◽

10.1099/0022-1317-73-2-433 ◽

1992 ◽

Vol 73 (2) ◽

pp. 433-435 ◽

Cited By ~ 60

Author(s):

J. H. Sinclair ◽

J. Baillie ◽

L. A. Bryant ◽

J. A. Taylor-Wiedeman ◽

J. G. P. Sissons

Keyword(s):

Gene Expression ◽

Cell Line ◽

Human Cytomegalovirus ◽

Immediate Early Gene ◽

Early Gene ◽

Immediate Early ◽

Monocytic Cell ◽

Monocytic Cell Line ◽

Early Gene Expression

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Lytic infection of permissive cells with human cytomegalovirus is regulated by an intrinsic ‘pre-immediate-early’ repression of viral gene expression mediated by histone post-translational modification

Journal of General Virology ◽

10.1099/vir.0.012526-0 ◽

2009 ◽

Vol 90 (10) ◽

pp. 2364-2374 ◽

Cited By ~ 56

Author(s):

Ian J. Groves ◽

Matthew B. Reeves ◽

John H. Sinclair

Keyword(s):

Gene Expression ◽

Human Cytomegalovirus ◽

Chromatin Structure ◽

Viral Gene Expression ◽

Lytic Infection ◽

Immediate Early ◽

Late Gene ◽

Viral Gene ◽

Gene Promoters ◽

Late Gene Expression

Human cytomegalovirus (HCMV) lytic gene expression occurs in a regulated cascade, initiated by expression of the viral major immediate-early (IE) proteins. Transcribed from the major IE promoter (MIEP), the major IE genes regulate viral early and late gene expression. This study found that a substantial proportion of infecting viral genomes became associated with histones immediately upon infection of permissive fibroblasts at low m.o.i. and these histones bore markers of repressed chromatin. As infection progressed, however, the viral MIEP became associated with histone marks, which correlate with the known transcriptional activity of the MIEP at IE time points. Interestingly, this chromatin-mediated repression of the MIEP at ‘pre-IE’ times of infection could be overcome by inhibition of histone deacetylases, as well as by infection at high m.o.i., and resulted in a temporal advance of the infection cycle by inducing premature viral early and late gene expression and DNA replication. As well as the MIEP, and consistent with previous observations, the viral early and late promoters were also initially associated with repressive chromatin. However, changes in histone modifications around these promoters also occurred as infection progressed, and this correlated with the known temporal regulation of the viral early and late gene expression cascade. These data argue that the chromatin structure of all classes of viral genes are initially repressed on infection of permissive cells and that the chromatin structure of HCMV gene promoters plays an important role in regulating the time course of viral gene expression during lytic infection.

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The Immediate Early Gene 1 Product of Human Cytomegalovirus Is Sufficient for Up-Regulation of Interleukin-8 Gene Expression

Biochemical and Biophysical Research Communications ◽

10.1006/bbrc.2000.3923 ◽

2000 ◽

Vol 279 (1) ◽

pp. 298-304 ◽

Cited By ~ 37

Author(s):

Tsugiya Murayama ◽

Naofumi Mukaida ◽

Hidetaka Sadanari ◽

Nobuo Yamaguchi ◽

Khalid S.A. Khabar ◽

...

Keyword(s):

Gene Expression ◽

Human Cytomegalovirus ◽

Immediate Early Gene ◽

Interleukin 8 ◽

Early Gene ◽

Immediate Early

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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

Journal of Virology ◽

10.1128/jvi.74.9.4192-4206.2000 ◽

2000 ◽

Vol 74 (9) ◽

pp. 4192-4206 ◽

Cited By ~ 33

Author(s):

Anita K. McElroy ◽

Roopashree S. Dwarakanath ◽

Deborah H. Spector

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Human Cytomegalovirus ◽

Cyclin E ◽

Early Gene ◽

Immediate Early ◽

Mrna Levels ◽

Early Gene Expression ◽

Cell Extracts ◽

Infected Cells

ABSTRACT We have previously shown that many cell cycle regulatory gene products are markedly affected by infection of primary fibroblasts with human cytomegalovirus (HCMV) (F. M. Jault, J. M. Jault, F. Ruchti, E. A. Fortunato, C. Clark, J. Corbeil, D. D. Richman, and D. H. Spector, J. Virol. 69:6697–6704, 1995). One of these proteins, cyclin E, is a key determinant of cell cycle progression during G1, and its mRNA levels are significantly increased in HCMV-infected fibroblasts (B. S. Salvant, E. A. Fortunato, and D. H. Spector, J. Virol. 72:3729–3741, 1998). To determine the molecular basis of this effect, we have examined the events that occur at the endogenous cyclin E promoter during the course of infection. In vivo dimethyl sulfate footprinting of the cyclin E promoter revealed several regions of protection and hypersensitivity that were unique to infected cells. In accord with this observation, we find that the virus-induced cyclin E transcripts initiate downstream of the start site identified in mock-infected cells, in regions where these newly appearing protected and hypersensitive sites occur. Viral gene expression is required for this induction. However, the viral immediate-early proteins IE1-72 and IE2-86, either alone or in combination, cannot induce expression of the endogenous cyclin E. The virus must progress past the immediate-early phase and express an early gene product(s) for activation of cyclin E expression. Moreover, IE1-72 does not appear to be required, as infection of cells with an HCMV mutant containing a deletion in the IE1-72 gene leads to full upregulation of cyclin E expression. Using electrophoretic mobility shift assays with infected cell extracts and a region of the cyclin E promoter that includes two previously defined E2F sites as the probe, we detected the appearance of an infection-specific banding pattern. One of the infection-specific bands contained the proteins E2F-4, DP-1, and p130, which were maintained in the infected cells as uniquely phosphorylated species. These results suggest that an altered E2F-4–DP-1–p130 complex along with viral early gene expression may play a role in the transcriptional regulation of cyclin E mRNA during HCMV infection.

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