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

1996 ◽  
Vol 40 (9) ◽  
pp. 2004-2011 ◽  
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.

scholarly journals 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

2000 ◽  
Vol 74 (9) ◽  
pp. 4192-4206 ◽  
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.

Shear stress induces hepatocyte PAI-1 gene expression through cooperative Sp1/Ets-1 activation of transcription

2006 ◽  
Vol 291 (1) ◽  
pp. G26-G34 ◽  
Author(s):  
Hideki Nakatsuka ◽  
Takaaki Sokabe ◽  
Kimiko Yamamoto ◽  
Yoshinobu Sato ◽  
Katsuyoshi Hatakeyama ◽  
...  
Keyword(s):  
Gene Expression ◽  
Shear Stress ◽  
Consensus Sequence ◽  
Early Gene ◽  
Immediate Early ◽  
Mrna Levels ◽  
Consensus Sequences ◽  
Static Conditions ◽  
Stress Dependent ◽  
Pai 1

Partial hepatectomy causes hemodynamic changes that increase portal blood flow in the remaining lobe, where the expression of immediate-early genes, including plasminogen activator inhibitor-1 (PAI-1), is induced. We hypothesized that a hyperdynamic circulatory state occurring in the remaining lobe induces immediate-early gene expression. In this study, we investigated whether the mechanical force generated by flowing blood, shear stress, induces PAI-1 expression in hepatocytes. When cultured rat hepatocytes were exposed to flow, PAI-1 mRNA levels began to increase within 3 h, peaked at levels significantly higher than the static control levels, and then gradually decreased. The flow-induced PAI-1 expression was shear stress dependent rather than shear rate dependent and accompanied by increased hepatocyte production of PAI-1 protein. Shear stress increased PAI-1 transcription but did not affect PAI-1 mRNA stability. Functional analysis of the 2.1-kb PAI-1 5′-promoter indicated that a 278-bp segment containing transcription factor Sp1 and Ets-1 consensus sequences was critical to the shear stress-dependent increase of PAI-1 transcription. Mutations of both the Sp1 and Ets-1 consensus sequences, but not of either one alone, markedly prevented basal PAI-1 transcription and abolished the response of the PAI-1 promoter to shear stress. EMSA and chromatin immunoprecipitation assays showed binding of Sp1 and Ets-1 to each consensus sequence under static conditions, which increased in response to shear stress. In conclusion, hepatocyte PAI-1 expression is flow sensitive and transcriptionally regulated by shear stress via cooperative interactions between Sp1 and Ets-1.

scholarly journals 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

2002 ◽  
Vol 76 (11) ◽  
pp. 5369-5379 ◽  
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.

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

1992 ◽  
Vol 73 (2) ◽  
pp. 433-435 ◽  
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

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

2000 ◽  
Vol 279 (1) ◽  
pp. 298-304 ◽  
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

scholarly journals Inhibition of human cytomegalovirus immediate early gene expression and growth by a novel RNase P ribozyme variant

PLoS ONE ◽  
2017 ◽  
Vol 12 (10) ◽  
pp. e0186791 ◽  
Author(s):  
Xu Sun ◽  
Weijie Chen ◽  
Lingling He ◽  
Jingxue Sheng ◽  
Yujun Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Human Cytomegalovirus ◽  
Rnase P ◽  
Immediate Early Gene ◽  
Early Gene ◽  
Immediate Early ◽  
Early Gene Expression

Human cytomegalovirus immediate-early-gene expression disrupts embryogenesis in transgenic Drosophila

2007 ◽  
Vol 17 (1) ◽  
pp. 105-119 ◽  
Author(s):  
Racheli Steinberg ◽  
Yonat Shemer-Avni ◽  
Noa Adler ◽  
Shira Neuman-Silberberg
Keyword(s):  
Gene Expression ◽  
Human Cytomegalovirus ◽  
Immediate Early Gene ◽  
Early Gene ◽  
Immediate Early ◽  
Early Gene Expression ◽  
Transgenic Drosophila

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

2007 ◽  
Vol 88 (11) ◽  
pp. 2935-2940 ◽  
Author(s):  
Ian J. Groves ◽  
John H. Sinclair
Keyword(s):  
Gene Expression ◽  
Human Cytomegalovirus ◽  
Chromatin Structure ◽  
Transcriptional Repression ◽  
Cell Types ◽  
Cellular Protein ◽  
Early Gene ◽  
Immediate Early ◽  
Undifferentiated Cells ◽  
Interfering Rna

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.

scholarly journals The Human Cytomegalovirus Major Immediate-Early Distal Enhancer Region Is Required for Efficient Viral Replication and Immediate-Early Gene Expression

2000 ◽  
Vol 74 (4) ◽  
pp. 1602-1613 ◽  
Author(s):  
Jeffery L. Meier ◽  
Jonathan A. Pruessner
Keyword(s):  
Gene Expression ◽  
Human Cytomegalovirus ◽  
Early Gene ◽  
Immediate Early ◽  
Regulatory Function ◽  
Wild Type Virus ◽  
Cell Type ◽  
Distal Enhancer ◽  
Unique Region ◽  
Enhancer Region

ABSTRACT The human cytomegalovirus (HCMV) major immediate-early (MIE) genes, encoding IE1 p72 and IE2 p86, are activated by a complex enhancer region (base positions -65 to -550) that operates in a cell type- and differentiation-dependent manner. The expression of MIE genes is required for HCMV replication. Previous studies analyzing functions of MIE promoter-enhancer segments suggest that the distal enhancer region variably modifies MIE promoter activity, depending on cell type, stimuli, or state of differentiation. To further understand the mechanism by which the MIE promoter is regulated, we constructed and analyzed several different recombinant HCMVs that lack the distal enhancer region (-300 to -582, -640, or -1108). In human fibroblasts, the HCMVs without the distal enhancer replicate normally at high multiplicity of infection (MOI) but replicate poorly at low MOI in comparison to wild-type virus (WT) or HCMVs that lack the neighboring upstream unique region and modulator (-582 or -640 to -1108). The growth aberrancy was normalized after restoring the distal enhancer in a virus lacking this region. For HCMVs without a distal enhancer, the impairment in replication at low MOI corresponds to a deficiency in production of MIE RNAs compared to WT or virus lacking the unique region and modulator. An underproduction of viral US3 RNA was also evident at low MOI. Whether lower production of IE1 p72 and IE2 p86 causes a reduction in expression of the immediate-early (IE) class US3 gene remains to be determined. We conclude that the MIE distal enhancer region possesses a mechanism for augmenting viral IE gene expression and genome replication at low MOI, but this regulatory function is unnecessary at high MOI.

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