UL82 virion protein activates expression of immediate early viral genes in human cytomegalovirus-infected cells

ABSTRACT We have characterized the function of the human cytomegalovirus US24 gene, a US22 gene family member. Two US24-deficient mutants (BADinUS24 and BADsubUS24) exhibited a 20- to 30-fold growth defect, compared to their wild-type parent (BADwt), after infection at a relatively low (0.01 PFU/cell) or high (1 PFU/cell) input multiplicity. Representative virus-encoded proteins and viral DNA accumulated with normal kinetics to wild-type levels after infection with mutant virus when cells received equal numbers of mutant and wild-type infectious units. Further, the proteins were properly localized and no ultrastructural differences were found by electron microscopy in mutant-virus-infected cells compared to wild-type-virus-infected cells. However, virions produced by US24-deficient mutants had a 10-fold-higher genome-to-PFU ratio than wild-type virus. When infections were performed using equal numbers of input virus particles, the expression of immediate-early, early, and late viral proteins was substantially delayed and decreased in the absence of US24 protein. This delay is not due to inefficient virus entry, since two tegument proteins and viral DNA moved to the nucleus equally well in mutant- and wild-type-virus-infected cells. In summary, US24 is a virion protein and virions produced by US24-deficient viruses exhibit a block to the human cytomegalovirus replication cycle after viral DNA reaches the nucleus and before immediate-early mRNAs are transcribed.

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Inhibitory Effects of Statins on Expression of Immediate–Early 1 Protein of Human Cytomegalovirus in Virus-infected Cells

Journal of Experimental & Clinical Medicine ◽

10.1016/j.jecm.2013.08.001 ◽

2013 ◽

Vol 5 (5) ◽

pp. 187-193 ◽

Cited By ~ 3

Author(s):

Hidetaka Sadanari ◽

Tsugiya Murayama ◽

Xin Zheng ◽

Rie Yamada ◽

Keiko Matsubara ◽

...

Keyword(s):

Human Cytomegalovirus ◽

Immediate Early ◽

Inhibitory Effects ◽

Infected Cells

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Activation of the NF-κB Pathway in Human Cytomegalovirus-Infected Cells Is Necessary for Efficient Transactivation of the Major Immediate-Early Promoter

Journal of Virology ◽

10.1128/jvi.78.9.4498-4507.2004 ◽

2004 ◽

Vol 78 (9) ◽

pp. 4498-4507 ◽

Cited By ~ 98

Author(s):

Ian B. DeMeritt ◽

Liesl E. Milford ◽

Andrew D. Yurochko

Keyword(s):

Human Cytomegalovirus ◽

Time Course ◽

Hcmv Infection ◽

Dominant Negative ◽

Immediate Early ◽

Primary Role ◽

Early Promoter ◽

Ikk Complex ◽

Infected Cells ◽

Major Immediate Early Promoter

ABSTRACT We previously demonstrated that human cytomegalovirus (HCMV) infection induced the activation of the cellular transcription factor NF-κB. Here, we investigate the mechanism for the HCMV-induced NF-κB activation and the role that the induced NF-κB plays in transactivation of the major immediate-early promoter (MIEP) and production of immediate-early (IE) proteins. Using a dominant-negative inhibitor of NF-κB, the IκB-superrepressor, we demonstrated that active NF-κB is critical for transactivation of the HCMV MIEP. Investigation of the mechanisms of NF-κB activation following HCMV infection showed a rapid and sustained decrease in the inhibitors of NF-κB, IκBα and IκBβ. Because the IκB kinases (IKKs) regulate the degradation of the IκBs, virus-mediated changes in the IKKs were examined next. Using dominant-negative forms of the IKKs, we showed significant decreases in transactivation of the MIEP in the presence of these mutants. In addition, protein levels of members of the IKK complex and IKK kinase activity were upregulated throughout the time course of infection. Lastly, the role NF-κB plays in HCMV IE mRNA and protein production during infection was examined. Using aspirin and MG-132, we demonstrated that production of IE protein and mRNA was significantly decreased and delayed in infected cells treated with these drugs. Together, the results of these studies suggest that virus-mediated NF-κB activation, through the dysregulation of the IKK complex, plays a primary role in the initiation of the HCMV gene cascade in fibroblasts and may provide new targets for therapeutic intervention.

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Human Cytomegalovirus Virions Differentially Incorporate Viral and Host Cell RNA during the Assembly Process

Journal of Virology ◽

10.1128/jvi.74.19.9078-9082.2000 ◽

2000 ◽

Vol 74 (19) ◽

pp. 9078-9082 ◽

Cited By ~ 58

Author(s):

Astrid E. Greijer ◽

Chantal A. J. Dekkers ◽

Jaap M. Middeldorp

Keyword(s):

Human Cytomegalovirus ◽

Viral Rna ◽

Gene Products ◽

Active Infection ◽

Virion Assembly ◽

Viral Genes ◽

Infected Cells ◽

Low Levels ◽

Cellular Dna ◽

Positive Results

ABSTRACT While analyzing human cytomegalovirus (HCMV) gene expression in infected cells by RNA-specific nucleic acid sequence-based amplification (NASBA), positive results were observed for HCMV RNA encoded by several viral genes immediately after the addition of the virus. UV-inactivated virus also gave a positive NASBA result without establishing active infection, suggesting that RNA was associated with the inoculum. Highly purified virions devoid of cellular contamination proved to be positive for viral RNA encoding both immediate-early (UL123) and late (UL65) gene products. Virion-associated RNA might be incorporated specifically or without selection during the virion assembly. In the latter case, cellular RNA would also be present in the virion. A high-abundant cellular RNA encoded by GAPDH and even U1A RNA, which is expressed at low levels, were detected in the virion fraction, whereas cellular DNA was absent. Virion fractionation revealed that cellular RNA was absent in purified de-enveloped capsids. In conclusion, cellular and viral RNA was present between the capsid and envelope of the virion, whereas in the capsid only viral RNA could be detected. The results suggest that virion-associated viral and cellular RNA is incorporated nonspecifically during virion assembly.

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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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Reversal of Human Cytomegalovirus Major Immediate-Early Enhancer/Promoter Silencing in Quiescently Infected Cells via the Cyclic AMP Signaling Pathway

Journal of Virology ◽

10.1128/jvi.01524-06 ◽

2007 ◽

Vol 81 (12) ◽

pp. 6669-6681 ◽

Cited By ~ 46

Author(s):

Michael J. Keller ◽

Allen W. Wu ◽

Janet I. Andrews ◽

Patrick W. McGonagill ◽

Eric E. Tibesar ◽

...

Keyword(s):

Protein Synthesis ◽

Cyclic Amp ◽

Signaling Pathway ◽

Human Cytomegalovirus ◽

Human Fibroblasts ◽

Lytic Cycle ◽

Immediate Early ◽

Infected Cells ◽

Ifn Γ ◽

Stimulation Of

ABSTRACT The human cytomegalovirus (HCMV) major immediate-early (MIE) enhancer contains five functional cyclic AMP (cAMP) response elements (CRE). Because the CRE in their native context do not contribute appreciably to MIE enhancer/promoter activity in lytically infected human fibroblasts and NTera2 (NT2)-derived neurons, we postulated that they might have a role in MIE enhancer/promoter reactivation in quiescently infected cells. Here, we show that stimulation of the cAMP signaling pathway by treatment with forskolin (FSK), an adenylyl cyclase activator, greatly alleviates MIE enhancer/promoter silencing in quiescently infected NT2 neuronal precursors. The effect is immediate, independent of de novo protein synthesis, associated with the phosphorylation of ATF-1 serine 63 and CREB serine 133, dependent on protein kinase A (PKA) and the enhancer's CRE, and linked to viral-lytic-cycle advancement. Coupling of FSK treatment with the inhibition of either histone deacetylases or protein synthesis synergistically activates MIE gene expression in a manner suggesting that MIE enhancer/promoter silencing is optimally relieved by an interplay of multiple regulatory mechanisms. In contrast, MIE enhancer/promoter silence is not overcome by stimulation of the gamma interferon (IFN-γ) signaling pathway, despite the enhancer having two IFN-γ-activated-site-like elements. We conclude that stimulation of the cAMP/PKA signaling pathway drives CRE-dependent MIE enhancer/promoter activation in quiescently infected cells, thus exposing a potential mode of regulation in HCMV reactivation.

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UL26-Deficient Human Cytomegalovirus Produces Virions with Hypophosphorylated pp28 Tegument Protein That Is Unstable within Newly Infected Cells

Journal of Virology ◽

10.1128/jvi.80.7.3541-3548.2006 ◽

2006 ◽

Vol 80 (7) ◽

pp. 3541-3548 ◽

Cited By ~ 38

Author(s):

Joshua Munger ◽

Dong Yu ◽

Thomas Shenk

Keyword(s):

Human Cytomegalovirus ◽

Deletion Mutant ◽

Open Reading Frame ◽

Early Gene ◽

Immediate Early ◽

Tegument Protein ◽

Protein Accumulation ◽

Reading Frame ◽

Tegument Proteins ◽

Infected Cells

ABSTRACT The human cytomegalovirus UL26 open reading frame encodes proteins of 21 and 27 kDa that result from the use of two different in-frame initiation codons. The UL26 protein is a constituent of the virion and thus is delivered to cells upon viral entry. We have characterized a mutant of human cytomegalovirus in which the UL26 open reading frame has been deleted. The UL26 deletion mutant has a profound growth defect, the magnitude of which is dependent on the multiplicity of infection. Two very early defects were discovered. First, even though they were present in normal amounts within mutant virions, the UL99-coded pp28 and UL83-coded pp65 tegument proteins were present in reduced amounts at the earliest times assayed within newly infected cells; second, there was a delay in immediate-early mRNA and protein accumulation. Further analysis revealed that although wild-type levels of the pp28 tegument protein were present in UL26 deletion mutant virions, the protein was hypophosphorylated. We conclude that the UL26 protein influences the normal phosphorylation of at least pp28 in virions and possibly additional tegument proteins. We propose that the hypophosphorylation of tegument proteins causes their destabilization within newly infected cells, perhaps disrupting the normal detegumentation process and leading to a delay in the onset of immediate-early gene expression.

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The Human Cytomegalovirus UL35 Gene Encodes Two Proteins with Different Functions

Journal of Virology ◽

10.1128/jvi.76.5.2460-2468.2002 ◽

2002 ◽

Vol 76 (5) ◽

pp. 2460-2468 ◽

Cited By ~ 29

Author(s):

Yingguang Liu ◽

Bonita J. Biegalke

Keyword(s):

Human Cytomegalovirus ◽

Complex Structure ◽

Protein A ◽

Immediate Early Gene ◽

Early Gene ◽

Immediate Early ◽

Terminal Portion ◽

Virion Protein ◽

Early Promoter ◽

Major Immediate Early Promoter

ABSTRACT The human cytomegalovirus (HCMV) virion is a complex structure that contains at least 30 proteins, many of which have been identified. We determined that the HCMV UL35 gene encodes two proteins, including a previously unidentified virion protein. A 22-kDa phosphoprotein (ppUL35A) was translated from a 1.2-kb UL35 transcript by 4 h postinfection; a second phosphoprotein of 75 kDa (ppUL35) was translated from a 2.2-kb transcript predominantly late in infection. The 22-kDa protein localized to the nucleus, while the 75-kDa protein localized to the juxtanuclear compartment and was packaged into virion particles. The 22-kDa protein was identical to the COOH-terminal end of the 75-kDa protein but was not found in virions, thus defining the NH2-terminal portion of the 75-kDa protein as essential for packaging. Expression of the 22-kDa protein inhibited activation of the major immediate-early promoter by ppUL82 (pp71), suggesting that the UL35 22-kDa protein may modulate expression of the major immediate-early gene.

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Characterization of phenyl pyrimidine derivatives that inhibit cytomegalovirus immediate-early gene expression

Antiviral Chemistry and Chemotherapy ◽

10.1177/2040206618763193 ◽

2018 ◽

Vol 26 ◽

pp. 204020661876319 ◽

Cited By ~ 1

Author(s):

Koh-Hei Yamada ◽

Ryuichi Majima ◽

Toyofumi Yamaguchi ◽

Naoki Inoue

Keyword(s):

Gene Expression ◽

Cell Line ◽

Human Cytomegalovirus ◽

Human Fibroblasts ◽

Early Gene ◽

Immediate Early ◽

Murine Cytomegalovirus ◽

Viral Attachment ◽

Varicella Zoster ◽

Infected Cells

Background Previously, we established a reporter cell line for human cytomegalovirus and screened anti-human cytomegalovirus compounds using the cell line. In this study, we characterized one of the identified compounds, 2,4-diamino-6–(4-methoxyphenyl)pyrimidine (coded as 35C10). Methods 50% Effective concentrations (EC50s) and 50% cytotoxic concentrations (CC50s) of 35C10 and its derivatives in human fibroblasts were determined by X-gal staining of the cells infected with human cytomegalovirus Towne strain expressing β-galactosidase. Results EC50 and CC50 of 35C10 were 4.3 µM and >200 µM, respectively. Among several 35C10 derivatives, only one lacking 4-amino group of pyrimidine showed a similar EC50. 35C10 weakly inhibited murine cytomegalovirus, herpes simplex virus type 1, and varicella-zoster virus. A “time of addition” experiment suggested that 35C10 inhibited an early phase of the infection. Although 35C10 did not inhibit viral attachment to the cells nor the delivery of viral DNA to the nuclei, it decreased the number of infected cells expressing immediate-early 1 and 2 (IE1/IE2) proteins. 35C10 also inhibited the activation of a promoter for TRL4 in the reporter cells upon human cytomegalovirus infection, but not in the same reporter cells transfected with a plasmid expressing IE2. Conclusion Our findings suggest that 35C10 is a novel compound that inhibits IE gene expression in human cytomegalovirus-infected cells.

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