scholarly journals Human cytomegalovirus interferes with signal transducer and activator of transcription (STAT) 2 protein stability and tyrosine phosphorylation

2008 ◽  
Vol 89 (10) ◽  
pp. 2416-2426 ◽  
Author(s):  
Vu Thuy Khanh Le ◽  
Mirko Trilling ◽  
Manuel Wilborn ◽  
Hartmut Hengel ◽  
Albert Zimmermann
Keyword(s):  
Tyrosine Phosphorylation ◽  
Human Cytomegalovirus ◽  
Proteasome Inhibition ◽  
Early Gene ◽  
Similar Degree ◽  
Signal Transducer ◽  
Protein Levels ◽  
Ubiquitin Proteasome ◽  
Infected Cells ◽  
Hcmv Replication

We have investigated the role of signal transducer and activator of transcription (STAT) 2 during human cytomegalovirus (HCMV) replication and found that protein levels of STAT2 are downregulated. STAT2 downregulation was observed in HCMV clinical isolates and laboratory strains with the exception of strain Towne. The HCMV-induced loss of STAT2 protein occurred despite an increased accumulation of STAT2 mRNA; it required HCMV early gene expression. The decrease in STAT2 was sensitive to proteasome inhibition, suggesting degradation of STAT2 via the ubiquitin proteasome pathway. Notably, pUL27, the HCMV homologue of the mouse CMV pM27 protein, which mediates the selective proteolysis of STAT2, did not induce STAT2 downregulation. Moreover, preceding STAT2 degradation, alpha/beta interferon (IFN)-receptor-mediated tyrosine phosphorylation of STAT2 was inhibited in HCMV-infected cells. This effect was paralleled by impaired tyrosine activation of STAT1 and STAT3. Accordingly, IFNs affected the replication efficiency of STAT2 degrading and non-degrading HCMV strains to a similar degree. In summary, HCMV abrogates IFN receptor signalling at multiple checkpoints by independent mechanisms including UL27-independent degradation of STAT2 and a preceding blockade of STAT2 phosphorylation.

scholarly journals Targeting the latent human cytomegalovirus reservoir for T-cell-mediated killing with virus-specific nanobodies

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Timo W. M. De Groof ◽  
Elizabeth G. Elder ◽  
Eleanor Y. Lim ◽  
Raimond Heukers ◽  
Nick D. Bergkamp ◽  
...  
Keyword(s):  
Gene Expression ◽  
Human Cytomegalovirus ◽  
Early Gene ◽  
Latent Reservoir ◽  
Solid Organ ◽  
Cell Transplant ◽  
Viral Receptor ◽  
Transplant Patients ◽  
Latently Infected ◽  
Infected Cells

AbstractLatent human cytomegalovirus (HCMV) infection is characterized by limited gene expression, making latent HCMV infections refractory to current treatments targeting viral replication. However, reactivation of latent HCMV in immunosuppressed solid organ and stem cell transplant patients often results in morbidity. Here, we report the killing of latently infected cells via a virus-specific nanobody (VUN100bv) that partially inhibits signaling of the viral receptor US28. VUN100bv reactivates immediate early gene expression in latently infected cells without inducing virus production. This allows recognition and killing of latently infected monocytes by autologous cytotoxic T lymphocytes from HCMV-seropositive individuals, which could serve as a therapy to reduce the HCMV latent reservoir of transplant patients.

scholarly journals Cyclin-Dependent Kinase Activity Is Required for Efficient Expression and Posttranslational Modification of Human Cytomegalovirus Proteins and for Production of Extracellular Particles

2006 ◽  
Vol 80 (12) ◽  
pp. 5886-5896 ◽  
Author(s):  
Veronica Sanchez ◽  
Deborah H. Spector
Keyword(s):  
Steady State ◽  
Human Cytomegalovirus ◽  
Virus Titer ◽  
State Level ◽  
Early Gene ◽  
Tegument Protein ◽  
Cyclin Dependent Kinase ◽  
Viral Dna ◽  
Infected Cells ◽  
Steady State Levels

ABSTRACT We have previously shown that the addition of the cyclin-dependent kinase (cdk) inhibitor Roscovitine at the beginning of infection of cells with human cytomegalovirus (HCMV) significantly disrupts immediate-early gene expression and the progression of the infection. In the present study, we have examined the effects of cdk inhibition on late viral events by delaying addition of Roscovitine until 24 h postinfection. Although viral DNA replication was inhibited two- to threefold by treatment of infected cells with Roscovitine, the drop did not correspond to the 1- to 2-log-unit decrease in virus titer. Quantification of viral DNA in the supernatant from cells revealed that there was a significant reduction in the production or release of extracellular particles. We observed a lag in the expression of several viral proteins but there was a significant decrease in the steady-state levels of IE2-86. Likewise, the steady-state level of the essential tegument protein UL32 (pp150) was reduced. The levels of pp150 and IE2-86 mRNA were not greatly affected by treatment with Roscovitine and thus did not correlate with the reduced levels of protein. In contrast, the expression of the tegument protein ppUL69 was higher in drug-treated samples, and the protein accumulated in a hyperphosphorylated form. ppUL69 localized to intranuclear aggregates that did not overlap with viral replication centers in cells treated with Roscovitine. Taken together, these data indicate that cdk activity is required at multiple steps during HCMV infection, including the expression, modification, and localization of virus-encoded proteins.

scholarly journals The Human Cytomegalovirus UL51 Protein Is Essential for Viral Genome Cleavage-Packaging and Interacts with the Terminase Subunits pUL56 and pUL89

2012 ◽  
Vol 87 (3) ◽  
pp. 1720-1732 ◽  
Author(s):  
Eva Maria Borst ◽  
Jennifer Kleine-Albers ◽  
Ildar Gabaev ◽  
Marina Babić ◽  
Karen Wagner ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Unit Length ◽  
Viral Proteins ◽  
Enzymatic Process ◽  
Genome Packaging ◽  
Subnuclear Localization ◽  
Promising Target ◽  
Synthetic Ligand ◽  
Infected Cells ◽  
Hcmv Replication

ABSTRACTCleavage of human cytomegalovirus (HCMV) genomes as well as their packaging into capsids is an enzymatic process mediated by viral proteins and therefore a promising target for antiviral therapy. The HCMV proteins pUL56 and pUL89 form the terminase and play a central role in cleavage-packaging, but several additional viral proteins, including pUL51, had been suggested to contribute to this process, although they remain largely uncharacterized. To study the function of pUL51 in infected cells, we constructed HCMV mutants encoding epitope-tagged versions of pUL51 and used a conditionally replicating virus (HCMV-UL51-ddFKBP), in which pUL51 levels could be regulated by a synthetic ligand. In cells infected with HCMV-UL51-ddFKBP, viral DNA replication was not affected when pUL51 was knocked down. However, no unit-length genomes and no DNA-filled C capsids were found, indicating that cleavage of concatemeric HCMV DNA and genome packaging into capsids did not occur in the absence of pUL51. pUL51 was expressed mainly with late kinetics and was targeted to nuclear replication compartments, where it colocalized with pUL56 and pUL89. Upon pUL51 knockdown, pUL56 and pUL89 were no longer detectable in replication compartments, suggesting that pUL51 is needed for their correct subnuclear localization. Moreover, pUL51 was found in a complex with the terminase subunits pUL56 and pUL89. Our data provide evidence that pUL51 is crucial for HCMV genome cleavage-packaging and may represent a third component of the viral terminase complex. Interference with the interactions between the terminase subunits by antiviral drugs could be a strategy to disrupt the HCMV replication cycle.

scholarly journals The Clinically Approved Antifungal Drug Posaconazole Inhibits Human Cytomegalovirus Replication

2020 ◽  
Vol 64 (10) ◽  
Author(s):  
Beatrice Mercorelli ◽  
Anna Luganini ◽  
Marta Celegato ◽  
Giorgio Palù ◽  
Giorgio Gribaudo ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Fungal Infections ◽  
Antimicrobial Effect ◽  
Virus Particles ◽  
Human Cytochrome ◽  
Passage Number ◽  
Infected Cells ◽  
Hcmv Replication ◽  
Different Strains ◽  
Low Passage

ABSTRACT Posaconazole (PCZ) is a clinically approved drug used predominantly for prophylaxis and salvage therapy of fungal infections. Here, we report its previously undescribed anti-human cytomegalovirus (HCMV) activity. By using antiviral assays, we demonstrated that PCZ, along with other azolic antifungals, has a broad anti-HCMV activity, being active against different strains, including low-passage-number clinical isolates and strains resistant to viral DNA polymerase inhibitors. Using a pharmacological approach, we identified the inhibition of human cytochrome P450 51 (hCYP51), or lanosterol 14α demethylase, a cellular target of posaconazole in infected cells, as a mechanism of anti-HCMV activity of the drug. Indeed, hCYP51 expression was stimulated upon HCMV infection, and the inhibition of its enzymatic activity by either the lanosterol analog VFV {(R)-N-(1-(3,4′-difluoro-[1,1′-biphenyl]-4-yl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadiazol-2-yl)benzamide} or PCZ decreased HCMV yield and infectivity of released virus particles. Importantly, we observed that the activity of the first-line anti-HCMV drug ganciclovir was boosted tenfold by PCZ and that ganciclovir (GCV) and PCZ act synergistically in inhibiting HCMV replication. Taken together, these findings suggest that this clinically approved drug deserves further investigation in the development of host-directed antiviral strategies as a candidate anti-HCMV drug with a dual antimicrobial effect.

scholarly journals Wnt Modulating Agents Inhibit Human Cytomegalovirus Replication

2013 ◽  
Vol 57 (6) ◽  
pp. 2761-2767 ◽  
Author(s):  
Arun Kapoor ◽  
Ran He ◽  
Rajkumar Venkatadri ◽  
Michael Forman ◽  
Ravit Arav-Boger
Keyword(s):  
Stem Cell ◽  
Cancer Stem Cell ◽  
Human Cytomegalovirus ◽  
Wnt Pathway ◽  
Critical Role ◽  
Wnt Signaling Pathway ◽  
Negative Regulator ◽  
Infected Cells ◽  
Hcmv Replication

ABSTRACTInfection with human cytomegalovirus (HCMV) continues to be a threat for pregnant women and immunocompromised hosts. Although limited anti-HCMV therapies are available, development of new agents is desired. The Wnt signaling pathway plays a critical role in embryonic and cancer stem cell development and is targeted by gammaherpesviruses, Epstein-Barr virus (EBV), and Kaposi's sarcoma-associated herpesvirus (KSHV). HCMV infects stem cells, including neural progenitor cells, during embryogenesis. To investigate the role of Wnt in HCMV replicationin vitro, we tested monensin, nigericin, and salinomycin, compounds that inhibit cancer stem cell growth by modulating the Wnt pathway. These compounds inhibited the replication of HCMV Towne and a clinical isolate. Inhibition occurred prior to DNA replication but persisted throughout the full replication cycle. There was a significant decrease in expression of IE2, UL44, and pp65 proteins. HCMV infection resulted in a significant and sustained decrease in expression of phosphorylated and total lipoprotein receptor-related protein 6 (pLRP6 and LRP6, respectively), Wnt 5a/b, and β-catenin and a modest decrease in Dvl2/3, while levels of the negative regulator axin 1 were increased. Nigericin decreased the expression of pLRP6, LRP6, axin 1, and Wnt 5a/b in noninfected and HCMV-infected cells. For all three compounds, a correlation was found between expression levels of Wnt 5a/b and axin 1 and HCMV inhibition. The decrease in Wnt 5a/b and axin 1 expression was more significant in HCMV-infected cells than noninfected cells. These data illustrate the complex effects of HCMV on the Wnt pathway and the fine balance between Wnt and HCMV, resulting in abrogation of HCMV replication. Additional studies are required to elucidate how HCMV targets Wnt for its benefit.

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.

scholarly journals Checks and balances between human cytomegalovirus replication and indoleamine-2,3-dioxygenase

2014 ◽  
Vol 95 (3) ◽  
pp. 659-670 ◽  
Author(s):  
Albert Zimmermann ◽  
Sebastian Hauka ◽  
Marco Maywald ◽  
Vu Thuy Khanh Le ◽  
Silvia K. Schmidt ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Human Cytomegalovirus ◽  
Cell Types ◽  
Interferon Γ ◽  
Counter Measures ◽  
Indoleamine 2,3 Dioxygenase ◽  
Infected Cells ◽  
Hcmv Replication ◽  
Ifn Γ ◽  
The Impact

Despite a rigorous blockade of interferon-γ (IFN-γ) signalling in infected fibroblasts as a mechanism of immune evasion by human cytomegalovirus (HCMV), IFN-γ induced indoleamine-2,3-dioxygenase (IDO) has been proposed to represent the major antiviral restriction factor limiting HCMV replication in epithelial cells. Here we show that HCMV efficiently blocks transcription of IFN-γ-induced IDO mRNA both in infected fibroblasts and epithelial cells even in the presence of a preexisting IFN-induced antiviral state. This interference results in severe suppression of IDO bioactivity in HCMV-infected cells and restoration of vigorous HCMV replication. Depletion of IDO expression nonetheless substantially alleviated the antiviral impact of IFN-γ treatment in both cell types. These findings highlight the effectiveness of this IFN-γ induced effector gene in restricting HCMV productivity, but also the impact of viral counter-measures.

scholarly journals Human Cytomegalovirus miR-UL70-3p Downregulates the H2O2-Induced Apoptosis by Targeting the Modulator of Apoptosis-1 (MOAP1)

2021 ◽  
Vol 23 (1) ◽  
pp. 18
Author(s):  
Abhishek Pandeya ◽  
Raj Kumar Khalko ◽  
Anup Mishra ◽  
Nishant Singh ◽  
Sukhveer Singh ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Molecular Mechanisms ◽  
Ectopic Expression ◽  
Antiviral Responses ◽  
Persistent Infections ◽  
Innate Immune Mechanism ◽  
Infected Cells ◽  
Hcmv Replication ◽  
Induced Apoptosis

Human Cytomegalovirus (HCMV) is a prototypic beta herpesvirus, causing persistent infections in humans. There are medications that are used to treat the symptoms; however, there is no cure yet. Thus, understanding the molecular mechanisms of HCMV replication and its persistence may reveal new prevention strategies. HCMV evasive strategies on the antiviral responses of the human host largely rely on its significant portion of genome. Numerous studies have highlighted the importance of miRNA-mediated regulation of apoptosis, which is an innate immune mechanism that eradicates virus-infected cells. In this study, we explore the antiapoptotic role of hcmv-miR-UL70-3p in HEK293T cells. We establish that hcmv-miR-UL70-3p targets the proapoptotic gene Modulator of Apoptosis-1 (MOAP1) through interaction with its 3’UTR region of mRNA. The ectopic expression of hcmv-miR-UL70-3p mimic significantly downregulates the H2O2-induced apoptosis through the translational repression of MOAP1. Silencing of MOAP1 through siRNA also inhibits the H2O2-induced apoptosis, which further supports the hcmv-miR-UL70-3p mediated antiapoptotic effect by regulating MOAP1 expression. These results uncover a role for hcmv-miR-UL70-3p and its target MOAP1 in regulating apoptosis.

scholarly journals Establishment of a Cell-Based Assay for Screening of Compounds Inhibiting Very Early Events in the Cytomegalovirus Replication Cycle and Characterization of a Compound Identified Using the Assay

2008 ◽  
Vol 52 (7) ◽  
pp. 2420-2427 ◽  
Author(s):  
Yoshiko Fukui ◽  
Keiko Shindoh ◽  
Yumiko Yamamoto ◽  
Shin Koyano ◽  
Isao Kosugi ◽  
...  
Keyword(s):  
Cell Line ◽  
Viral Entry ◽  
Hcmv Infection ◽  
Early Gene ◽  
Dependent Manner ◽  
Human Lung Fibroblast ◽  
Protein Levels ◽  
A Cell ◽  
Infected Cells ◽  
Time Frames

ABSTRACT To simplify the detection of infectious human cytomegalovirus (HCMV), we generated a cell line that produced luciferase in a dose-dependent manner upon HCMV infection. Using this cell line, we identified anti-HCMV compounds from a diverse library of 9,600 compounds. One of them, 1-(3,5-dichloro-4-pyridyl)piperidine-4-carboxamide (DPPC), was effective against HCMV (Towne strain) infection of human lung fibroblast cells at a 50% effective concentration of 2.5 μM. DPPC also inhibited the growth of clinical HCMV isolates and guinea pig and mouse cytomegaloviruses. Experiments using various time frames for treatment of the cells with DPPC demonstrated that DPPC was effective during the first 24 h after HCMV infection. DPPC treatment decreased not only viral DNA replication but also IE1 and IE2 expression at mRNA and protein levels in the HCMV-infected cells. However, DPPC did not inhibit the attachment of HCMV particles to the cell surface. DPPC is a unique compound that targets the very early phase of cytomegalovirus infection, probably by disrupting a pathway that is important after viral entry but before immediate-early gene expression.

scholarly journals Human Cytomegalovirus IE72 Protein Interacts with the Transcriptional Repressor hDaxx To Regulate LUNA Gene Expression during Lytic Infection

2010 ◽  
Vol 84 (14) ◽  
pp. 7185-7194 ◽  
Author(s):  
Matthew Reeves ◽  
David Woodhall ◽  
Teresa Compton ◽  
John Sinclair
Keyword(s):  
Gene Expression ◽  
Human Cytomegalovirus ◽  
Direct Interaction ◽  
Lytic Infection ◽  
Early Gene ◽  
Productive Infection ◽  
Viral Promoters ◽  
Infected Cells ◽  
Sirna Knockdown ◽  
Interfering Rna

ABSTRACT A putative latency-associated transcript (LUNA) complementary to the human cytomegalovirus (HCMV) UL81-82 region previously identified in seropositive donors' monocytes is also expressed during lytic infection. Thus, the LUNA promoter is active during both lytic and latent infection. Consequently, the mechanisms regulating this promoter may provide further insight into factors that determine whether the outcome of HCMV infection is latent or lytic. By transfection, the LUNA promoter exhibited low but reproducible activity. Substantial activation by virus infection suggested that a viral factor was important for LUNA expression during lytic infection. IE72, a known transactivator of viral promoters, activated the LUNA promoter in cotransfection assays. Furthermore, coinfection with wild-type HCMV but not an IE72 deletion virus (CR208) also activated the LUNA promoter. Finally, diminished LUNA gene expression in CR208 virus-infected cells supported a role for IE72 in LUNA gene expression. The initial regulation of herpesvirus immediate-early gene expression is associated with proteins found at cellular nuclear domain 10 (ND10) bodies, such as PML, hDaxx, and ATRX. hDaxx transfection repressed LUNA promoter activity. Furthermore, we observed binding of hDaxx to the LUNA promoter, which was abrogated by IE72 gene expression via direct interaction. Finally, we show that small interfering RNA (siRNA) knockdown of the hDaxx interaction partner ATRX rescued LUNA gene expression in CR208-infected cells. Overall, these data show that hDaxx/ATRX-mediated repression of LUNA during lytic infection absolutely requires IE72 gene expression. It also suggests that the targeting of cellular factors by IE72 is important throughout the different phases of HCMV gene expression during productive infection.

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