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 Essential Human Cytomegalovirus Gene UL52 Is Required for Cleavage-Packaging of the Viral Genome

2007 ◽  
Vol 82 (5) ◽  
pp. 2065-2078 ◽  
Author(s):  
Eva Maria Borst ◽  
Karen Wagner ◽  
Anne Binz ◽  
Beate Sodeik ◽  
Martin Messerle
Keyword(s):  
Human Cytomegalovirus ◽  
Viral Genome ◽  
Essential Role ◽  
Unit Length ◽  
Viral Dna ◽  
Subnuclear Localization ◽  
Viral Genomes ◽  
Nuclear Egress ◽  
C Terminus ◽  
Infected Cells

ABSTRACT Replication of human cytomegalovirus (HCMV) produces large DNA concatemers of head-to-tail-linked viral genomes that upon packaging into capsids are cut into unit-length genomes. The mechanisms underlying cleavage-packaging and the subsequent steps prior to nuclear egress of DNA-filled capsids are incompletely understood. The hitherto uncharacterized product of the essential HCMV UL52 gene was proposed to participate in these processes. To investigate the function of pUL52, we constructed a ΔUL52 mutant as well as a complementing cell line. We found that replication of viral DNA was not impaired in noncomplementing cells infected with the ΔUL52 virus, but viral concatemers remained uncleaved. Since the subnuclear localization of the known cleavage-packaging proteins pUL56, pUL89, and pUL104 was unchanged in ΔUL52-infected fibroblasts, pUL52 does not seem to act via these proteins. Electron microscopy studies revealed only B capsids in the nuclei of ΔUL52-infected cells, indicating that the mutant virus has a defect in encapsidation of viral DNA. Generation of recombinant HCMV genomes encoding epitope-tagged pUL52 versions showed that only the N-terminally tagged pUL52 supported viral growth, suggesting that the C terminus is crucial for its function. pUL52 was expressed as a 75-kDa protein with true late kinetics. It localized preferentially to the nuclei of infected cells and was found to enclose the replication compartments. Taken together, our results demonstrate an essential role for pUL52 in cleavage-packaging of HCMV DNA. Given its unique subnuclear localization, the function of pUL52 might be distinct from that of other cleavage-packaging proteins.

scholarly journals Assembly of monomeric human cytomegalovirus pUL104 into portal structures

2009 ◽  
Vol 90 (10) ◽  
pp. 2381-2385 ◽  
Author(s):  
Andreas Holzenburg ◽  
Alexandra Dittmer ◽  
Elke Bogner
Keyword(s):  
Human Cytomegalovirus ◽  
Gel Electrophoresis ◽  
Unit Length ◽  
Gel Permeation Chromatography ◽  
Viral Proteins ◽  
Cross Linking ◽  
Gel Permeation ◽  
Infected Cells ◽  
Candidate Protein ◽  
Native Gel

In order for human cytomegalovirus (HCMV) to replicate, concatemeric DNA has to be cleaved into unit-length genomes and packaged into preformed capsids. For packaging to take place and DNA to be translocated, a channel is required in the capsid. Viral capsid channels are generally formed by portal proteins. Here, we show by cross-linking, native gel electrophoresis of infected cells and gel permeation chromatography that the HCMV portal candidate protein pUL104 can form dimers and higher order multimers. Electron microscopy of purified monomeric pUL104 after 5 min incubation revealed that the protein had assembled into a multimeric form and that this form closely resembles complete portal assembly. This is the first study to show that pUL104 monomers have the ability to form portal complexes without additional viral proteins.

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 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 Interaction between the Human Cytomegalovirus Tegument Proteins UL94 and UL99 Is Essential for Virus Replication

2012 ◽  
Vol 86 (18) ◽  
pp. 9995-10005 ◽  
Author(s):  
Stacia L. Phillips ◽  
Daniel Cygnar ◽  
Alexandra Thomas ◽  
Wade A. Bresnahan
Keyword(s):  
Amino Acids ◽  
Human Cytomegalovirus ◽  
Infectious Virus ◽  
Viral Proteins ◽  
Tegument Protein ◽  
Dependent Manner ◽  
Tegument Proteins ◽  
Cytoplasmic Structure ◽  
Infected Cells ◽  
Secretory Apparatus

Human cytomegalovirus (HCMV) virions are structurally complex, and the mechanisms by which they are assembled are poorly understood, especially with respect to the cytoplasmic phase of assembly, during which the majority of the tegument is acquired and final envelopment occurs. These processes occur at a unique cytoplasmic structure called the assembly complex, which is formed through a reorganization of the cellular secretory apparatus. The HCMV tegument protein UL99 (pp28) is essential for viral replication at the stage of secondary envelopment. We previously demonstrated that UL99 interacts with the essential tegument protein UL94 in infected cells as well as in the absence of other viral proteins. Here we show that UL94 and UL99 alter each other's localization and that UL99 stabilizes UL94 in a binding-dependent manner. We have mapped the interaction between UL94 and UL99 to identify the amino acids of each protein that are required for their interaction. Mutation of these amino acids in the context of the viral genome demonstrates that HCMV is completely defective for replication in the absence of the interaction between UL94 and UL99. Further, we demonstrate that in the absence of their interaction, both UL94 and UL99 exhibit aberrant localization and do not accumulate at the assembly complex during infection. Taken together, our data suggest that the interaction between UL94 and UL99 is essential for the proper localization of each protein to the assembly complex and thus for the production of infectious virus.

scholarly journals Recruitment of Human Cytomegalovirus Immediate-Early 2 Protein onto Parental Viral Genomes in Association with ND10 in Live-Infected Cells

2007 ◽  
Vol 81 (18) ◽  
pp. 10123-10136 ◽  
Author(s):  
George Sourvinos ◽  
Nina Tavalai ◽  
Anja Berndt ◽  
Demetrios A. Spandidos ◽  
Thomas Stamminger
Keyword(s):  
Viral Replication ◽  
Human Cytomegalovirus ◽  
Spatial Organization ◽  
Human Fibroblasts ◽  
Nuclear Bodies ◽  
Immediate Early ◽  
Viral Dna ◽  
Subnuclear Localization ◽  
Pml Nuclear Bodies ◽  
Infected Cells

ABSTRACT The human cytomegalovirus (HCMV) immediate-early 2 (IE2) transactivator has previously been shown to form intranuclear, dot-like accumulations in association with subnuclear structures known as promyelocytic leukemia protein (PML) nuclear bodies or ND10. We recently observed that IE2 can form dot-like structures even after infection of PML knockdown cells, which lack genuine ND10. To further analyze the determinants of IE2 subnuclear localization, a recombinant HCMV expressing IE2 fused to the enhanced green fluorescent protein was constructed. We infected primary human fibroblasts expressing Sp100 fused to the autofluorescent protein mCherry while performing live-cell imaging experiments. These experiments revealed a very dynamic association of IE2 dots with ND10 structures during the first hours postinfection: juxtaposed structures rapidly fused to precise colocalizations, followed by segregation, and finally, the dispersal of ND10 accumulations. Furthermore, by infecting PML knockdown cells we determined that the number of IE2 accumulations was dependent on the multiplicity of infection. Since time-lapse microscopy in live-infected cells revealed that IE2 foci developed into viral replication compartments, we hypothesized that viral DNA could act as a determinant of IE2 accumulations. Direct evidence that IE2 molecules are associated with viral DNA early after HCMV infection was obtained using fluorescence in situ hybridization. Finally, a DNA-binding-deficient IE2 mutant could no longer be recruited into viral replication centers, suggesting that the association of IE2 with viral DNA is mediated by a direct DNA contact. Thus, we identified viral DNA as an important determinant of IE2 subnuclear localization, which suggests that the formation of a virus-induced nucleoprotein complex and its spatial organization is likely to be critical at the early stages of a lytic infection.

scholarly journals Hypoxia-Inducible Factor 1α (HIF1α) Suppresses Virus Replication in Human Cytomegalovirus Infection by Limiting Kynurenine Synthesis

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Lisa M. Wise ◽  
Yuecheng Xi ◽  
John G. Purdy
Keyword(s):  
Aryl Hydrocarbon Receptor ◽  
Virus Replication ◽  
Human Cytomegalovirus ◽  
Hypoxia Inducible Factor ◽  
Hypoxia Inducible Factor 1Α ◽  
Indoleamine 2,3 Dioxygenase ◽  
Aryl Hydrocarbon ◽  
Infected Cells ◽  
Hcmv Replication ◽  
Rate Limiting

ABSTRACT Human cytomegalovirus (HCMV) replication depends on the activities of several host regulators of metabolism. Hypoxia-inducible factor 1α (HIF1α) was previously proposed to support virus replication through its metabolic regulatory function. HIF1α protein levels rise in response to HCMV infection in nonhypoxic conditions, but its effect on HCMV replication was not investigated. We addressed the role of HIF1α in HCMV replication by generating primary human cells with HIF1α knocked out using CRISPR/Cas9. When HIF1α was absent, we found that HCMV replication was enhanced, showing that HIF1α suppresses viral replication. We used untargeted metabolomics to determine if HIF1α regulates metabolite concentrations in HCMV-infected cells. We discovered that in HCMV-infected cells, HIF1α suppresses intracellular and extracellular concentrations of kynurenine. HIF1α also suppressed the expression of indoleamine 2,3-dioxygenase 1 (IDO1), the rate-limiting enzyme in kynurenine synthesis. In addition to its role in tryptophan metabolism, kynurenine acts as a signaling messenger by activating aryl hydrocarbon receptor (AhR). Inhibiting AhR reduces HCMV replication, while activating AhR with an exogenous ligand increases virus replication. Moreover, we found that feeding kynurenine to cells promotes HCMV replication. Overall, our findings indicate that HIF1α reduces HCMV replication by regulating metabolism and metabolite signaling. IMPORTANCE Viruses, including human cytomegalovirus (HCMV), reprogram cellular metabolism using host metabolic regulators to support virus replication. Alternatively, in response to infection, the host can use metabolism to limit virus replication. Here, our findings show that the host uses hypoxia-inducible factor 1α (HIF1α) as a metabolic regulator to reduce HCMV replication. Further, we found that HIF1α suppresses kynurenine synthesis, a metabolite that can promote HCMV replication by signaling through the aryl hydrocarbon receptor (AhR). In infected cells, the rate-limiting enzyme in kynurenine synthesis, indoleamine 2,3-dioxygenase 1 (IDO1), is suppressed by a HIF1α-dependent mechanism. Our findings describe a functional connection between HIF1α, IDO1, and AhR that allows HIF1α to limit HCMV replication through metabolic regulation, advancing our understanding of virus-host interactions.

scholarly journals Nitric Oxide Circumvents Virus-Mediated Metabolic Regulation during Human Cytomegalovirus Infection

mBio ◽  
2020 ◽  
Vol 11 (6) ◽  
pp. e02630-20
Author(s):  
Rebekah L. Mokry ◽  
Megan L. Schumacher ◽  
Neil Hogg ◽  
Scott S. Terhune
Keyword(s):  
Nitric Oxide ◽  
Human Cytomegalovirus ◽  
Mitochondrial Respiration ◽  
Hcmv Infection ◽  
Cellular Metabolism ◽  
Glutamine Metabolism ◽  
Cell Type ◽  
Cellular Processes ◽  
Infected Cells ◽  
Hcmv Replication

ABSTRACTNitric oxide is a versatile and critical effector molecule that can modulate many cellular functions. Although recognized as a regulator of infections, the inhibitory mechanism of nitric oxide against human cytomegalovirus (HCMV) replication remains elusive. We demonstrate that nitric oxide attenuates viral replication by interfering with HCMV-mediated modulation of several cellular processes. Nitric oxide exposure reduced HCMV genome synthesis and infectious viral progeny with cell-type-dependent differences observed. Mitochondrial respiration was severely reduced in both uninfected and HCMV-infected cells during exposure with little impact on ATP levels indicating changes in cellular metabolism. Metabolomics identified significantly altered small molecules in multiple pathways during nitric oxide exposure including nucleotide biosynthesis, tricarboxylic acid (TCA) cycle, and glutamine metabolism. Glutathione metabolites were increased coinciding with a reduction in the glutathione precursor glutamine. This shift was accompanied by increased antioxidant enzymes. Glutamine deprivation mimicked defects in HCMV replication and mitochondrial respiration observed during nitric oxide exposure. These data suggest that nitric oxide limits glutaminolysis by shuttling glutamine to glutathione synthesis. In addition, lipid intermediates were severely altered, which likely contributes to the observed increase in defective viral particles. Nitric oxide disrupts multiple cellular processes, and we had limited success in rescuing replication defects by supplementing with metabolic intermediates. Our studies indicate that nitric oxide attenuation of HCMV is multifactorial with interference in viral manipulation of cellular metabolism playing a central role.IMPORTANCE Human cytomegalovirus is a prevalent pathogen that can cause serious disease in patients with compromised immune systems, including transplant patients and during congenital infection. HCMV lytic replication likely occurs in localized sites of infection with immune cells infiltrating and releasing nitric oxide with other effector molecules. This nonspecific immune response results in both uninfected and infected cells exposed to high levels of nitric oxide. The absence of nitric oxide synthase has been associated with lethal HCMV infection. We demonstrate that nitric oxide inhibition of HCMV replication is multifactorial and cell type dependent. Our results indicate that nitric oxide controls replication by interfering with viral modulation of cellular metabolism while also affecting proliferation and mitochondrial respiration of neighboring uninfected cells. These studies identify the mechanism and contribution of nitric oxide during immune control of HCMV infection and provide insight into its role in other viral infections.

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