scholarly journals Evidence for a Role of the Cellular ND10 Protein PML in Mediating Intrinsic Immunity against Human Cytomegalovirus Infections

2006 ◽  
Vol 80 (16) ◽  
pp. 8006-8018 ◽  
Author(s):  
Nina Tavalai ◽  
Peer Papior ◽  
Sabine Rechter ◽  
Martina Leis ◽  
Thomas Stamminger
Keyword(s):  
Human Cytomegalovirus ◽  
Defense Mechanism ◽  
De Novo ◽  
Human Fibroblasts ◽  
Dependent Manner ◽  
Dna Uptake ◽  
Viral Dna ◽  
Antiviral Function ◽  
Associated Proteins ◽  
Cytomegalovirus Infections

ABSTRACT Several viruses, including human cytomegalovirus (HCMV), encode proteins that colocalize with a cellular subnuclear structure known as ND10. Since only viral DNA deposited at ND10 initiates transcription, ND10 structures were hypothesized to be essential for viral replication. On the other hand, interferon treatment induces an up-regulation of ND10 structures and viruses have evolved polypeptides that disperse the dot-like accumulation of ND10 proteins, suggesting that ND10 could also be part of an intrinsic defense mechanism. In order to obtain evidence for either a proviral or an antiviral function of ND10, we generated primary human fibroblasts with a stable, short interfering RNA-mediated knockdown (kd) of PML. In these cells, other ND10-associated proteins like hDaxx showed a diffuse nuclear distribution. Interestingly, we observed that HCMV infection induced the de novo formation of ND10-like hDaxx and Sp100 accumulations that colocalized with IE2 and were disrupted, in the apparent absence of PML, in an IE1-dependent manner during the first hours after infection. Furthermore, infection of PML-kd cells with wild-type HCMV at a low multiplicity of infection resulted in enhanced replication. In particular, a significantly increased plaque formation was detected, suggesting that more cells are able to support initiation of replication in the absence of PML. While there was no difference in viral DNA uptake between PML-kd and control cells, we observed a considerable increase in the number of immediate-early (IE) protein-positive cells, indicating that the depletion of PML augments the initiation of viral IE gene expression. These results strongly suggest that PML functions as part of an intrinsic immune mechanism against cytomegalovirus infections.

scholarly journals Nonnucleoside Pyrrolopyrimidines with a Unique Mechanism of Action against Human Cytomegalovirus

1999 ◽  
Vol 43 (8) ◽  
pp. 1888-1894 ◽  
Author(s):  
Jennie G. Jacobson ◽  
Thomas E. Renau ◽  
M. Reza Nassiri ◽  
Dominica G. Sweier ◽  
Julie M. Breitenbach ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Virus Titer ◽  
Enzyme Linked Immunosorbent Assay ◽  
Yield Reduction ◽  
Dependent Manner ◽  
Viral Dna ◽  
Human Foreskin Fibroblasts ◽  
Combination Studies ◽  
Hcmv Replication ◽  
Viral Titers

ABSTRACT Based upon a prior study which evaluated a series of nonnucleoside pyrrolo[2,3-d]pyrimidines as inhibitors of human cytomegalovirus (HCMV), we have selected three active analogs for detailed study. In an HCMV plaque-reduction assay, compounds 828, 951, and 1028 had 50% inhibitory concentrations (IC50s) of 0.4 to 1.0 μM. Similar results were obtained when 828 and 951 were examined by HCMV enzyme-linked immunosorbent assay (IC50s = 1.9 and 0.4 μM, respectively) and when 828 was tested in a viral DNA-DNA hybridization assay (IC50 = 1.3 μM). In yield-reduction assays with a low multiplicity of infection (MOI), all three compounds caused multiple log10 reductions in virus titer, and the activities of these compounds were comparable to the activity of ganciclovir (GCV; IC90 = 0.2 μM). In contrast to the reduction of viral titers by GCV, the reduction of viral titers by 828, 951, and 1028 decreased with increasing MOI. Cytotoxicity in human foreskin fibroblasts and KB cells ranged from 32 to >100 μM. In addition, 828 (the only compound tested) was less toxic against human bone marrow progenitor cells than GCV. Time-of-addition and time-of-removal studies established that the three pyrrolopyrimidines inhibited HCMV replication before GCV had an effect on viral DNA synthesis but after viral adsorption. Compound 828 was equally effective against GCV-sensitive and GCV-resistant HCMV clinical isolates. Combination studies with 828 and GCV showed that the effects of the two compounds on HCMV were additive but not synergistic. Taken together, the data indicate that these pyrrolopyrimidines target a viral protein that is required in an MOI-dependent manner and that is expressed early in the HCMV replication cycle.

scholarly journals DNA Microarrays of the Complex Human Cytomegalovirus Genome: Profiling Kinetic Class with Drug Sensitivity of Viral Gene Expression

1999 ◽  
Vol 73 (7) ◽  
pp. 5757-5766 ◽  
Author(s):  
James Chambers ◽  
Ana Angulo ◽  
Dhammika Amaratunga ◽  
Hongqing Guo ◽  
Ying Jiang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Human Cytomegalovirus ◽  
Dna Sequences ◽  
De Novo ◽  
Expression Profiles ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Viral Dna ◽  
Viral Protein Synthesis ◽  
Entire Genome

ABSTRACT We describe, for the first time, the generation of a viral DNA chip for simultaneous expression measurements of nearly all known open reading frames (ORFs) in the largest member of the herpesvirus family, human cytomegalovirus (HCMV). In this study, an HCMV chip was fabricated and used to characterize the temporal class of viral gene expression. The viral chip is composed of microarrays of viral DNA prepared by robotic deposition of oligonucleotides on glass for ORFs in the HCMV genome. Viral gene expression was monitored by hybridization to the oligonucleotide microarrays with fluorescently labelled cDNAs prepared from mock-infected or infected human foreskin fibroblast cells. By using cycloheximide and ganciclovir to block de novo viral protein synthesis and viral DNA replication, respectively, the kinetic classes of array elements were classified. The expression profiles of known ORFs and many previously uncharacterized ORFs provided a temporal map of immediate-early (α), early (β), early-late (γ1), and late (γ2) genes in the entire genome of HCMV. Sequence compositional analysis of the 5′ noncoding DNA sequences of the temporal classes, performed by using algorithms that automatically search for defined and recurring motifs in unaligned sequences, indicated the presence of potential regulatory motifs for β, γ1, and γ2 genes. In summary, these fabricated microarrays of viral DNA allow rapid and parallel analysis of gene expression at the whole viral genome level. The viral chip approach coupled with global biochemical and genetic strategies should greatly speed the functional analysis of established as well as newly discovered large viral genomes.

scholarly journals A Strong Negative Transcriptional Regulatory Region between the Human Cytomegalovirus UL127 Gene and the Major Immediate-Early Enhancer

1999 ◽  
Vol 73 (11) ◽  
pp. 9039-9052 ◽  
Author(s):  
Christopher A. Lundquist ◽  
Jeffrey L. Meier ◽  
Mark F. Stinski
Keyword(s):  
Human Cytomegalovirus ◽  
Viral Genome ◽  
De Novo ◽  
Human Fibroblasts ◽  
Regulatory Region ◽  
Fold Increase ◽  
Immediate Early ◽  
Wild Type ◽  
Viral Protein Synthesis ◽  
Unique Region

ABSTRACT The region of the human cytomegalovirus (CMV) genome between the UL127 open reading frame and the major immediate-early (MIE) enhancer is referred to as the unique region. DNase I protection analysis with human cell nuclear extracts demonstrated multiple protein binding sites in this region of the viral genome (P. Ghazal, H. Lubon, C. Reynolds-Kohler, L. Hennighausen, and J. A. Nelson, Virology 174:18–25, 1990). However, the function of this region in the context of the viral genome is not known. In wild-type human CMV-infected human fibroblasts, cells permissive for viral replication, there is little to no transcription from UL127. We determined that the unique region prevented transcription from the UL127 promoter but had no effect on the divergent MIE promoter. In transient-transfection assays, the basal level of expression from the UL127 promoter increased significantly when the wild-type unique sequences were mutated. In recombinant viruses with similar mutations in the unique region, expression from the UL127 promoter occurred only after de novo viral protein synthesis, typical of an early viral promoter. A 111-bp deletion-substitution of the unique sequence caused approximately a 20-fold increase in the steady-state level of RNA from the UL127 promoter and a 245-fold increase in the expression of a downstream indicator gene. This viral negative regulatory region was also mutated at approximately 50-bp regions proximal and distal to the UL127 promoter. Although some repressive effects were detected in the distal region, mutations of the region proximal to the UL127 promoter had the most significant effects on transcription. Within the proximal and distal regions, there are potential cis sites for known eucaryotic transcriptional repressor proteins. This region may also bind unknown viral proteins. We propose that the unique region upstream of the UL127 promoter and the MIE enhancer negatively regulates the expression from the UL127 promoter in permissive human fibroblast cells. This region may be a regulatory boundary preventing the effects of the very strong MIE enhancer on this promoter.

scholarly journals Functional Interaction between the pp71 Protein of Human Cytomegalovirus and the PML-Interacting Protein Human Daxx

2002 ◽  
Vol 76 (11) ◽  
pp. 5769-5783 ◽  
Author(s):  
Heike Hofmann ◽  
Hilde Sindre ◽  
Thomas Stamminger
Keyword(s):  
Human Cytomegalovirus ◽  
Protein Function ◽  
Fluorescent Protein ◽  
De Novo ◽  
Human Fibroblasts ◽  
Specific Interaction ◽  
Immediate Early ◽  
Regulatory Function ◽  
Viral Protein Synthesis ◽  
Interacting Protein

ABSTRACT The tegument protein pp71 (UL82) of human cytomegalovirus (HCMV) has previously been shown to transactivate the major immediate-early enhancer-promoter of HCMV. Furthermore, this protein is able to enhance the infectivity of viral DNA and to accelerate the infection cycle, suggesting an important regulatory function during viral replication. To gain insight into the underlying mechanisms that are used by pp71 to exert these pleiotropic effects, we sought for cellular factors interacting with pp71 in a yeast two-hybrid screen. Here, we report the isolation of the human Daxx (hDaxx) protein as a specific interaction partner of HCMV pp71. hDaxx, which was initially described as an adapter protein involved in apoptosis regulation, has recently been identified as a nuclear protein that interacts and colocalizes with PML in the nuclear domain ND10. In order to assess whether pp71 can also be detected in ND10 structures, a vector expressing pp71 in fusion with the green fluorescent protein was used for transfection of human fibroblasts. This revealed a colocalization of pp71 with the ND10 proteins PML and Sp100. In addition, cotransfection of a hDaxx expression vector resulted in an enhanced recruitment of pp71 to ND10. Targeting of pp71 to nuclear dots could also be observed in infected human fibroblasts in the absence of de novo viral protein synthesis. Moreover, cotransfection experiments revealed that pp71-mediated transactivation of the major immediate-early enhancer-promoter was synergistically enhanced in the presence of hDaxx. These results suggest an important role of hDaxx for pp71 protein function.

scholarly journals Detection of human cytomegalovirus DNA replication in non-permissive Vero and 293 cells

2003 ◽  
Vol 84 (3) ◽  
pp. 639-645 ◽  
Author(s):  
Victoria Ellsmore ◽  
G. Gordon Reid ◽  
Nigel D. Stow
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Human Cytomegalovirus ◽  
Human Fibroblasts ◽  
Cell Types ◽  
Vero Cells ◽  
Progeny Virus ◽  
Viral Dna ◽  
Viral Origin ◽  
293 Cells

Human cytomegalovirus (HCMV) displays an exceptionally restricted host range in tissue culture with human fibroblasts being the principal fully permissive system. Nevertheless, immediate early (IE) proteins are expressed following infection of many non-permissive cell types of human, simian and murine origin, and viral origin-dependent DNA synthesis has been reconstituted by transfection of plasmids into Vero cells, a non-permissive line from African green monkey. We have examined the accumulation of HCMV strain AD169 DNA, and the replication of transfected HCMV origin-containing plasmids, in infected Vero and human embryonic kidney 293 cells, which were previously reported to express the major IE protein in a small proportion of infected cells but to be non-permissive for viral DNA synthesis. In Vero cells accumulation of origin-containing plasmid but not viral DNA occurred, whilst in 293 cells both DNAs accumulated. Immunofluorescence experiments indicated that following infection with 3 p.f.u. per cell, a small fraction of both cell types expressed the UL44 DNA replication protein. Neither cell line, however, supported the generation of infectious progeny virus. These results suggest that IE proteins expressed in Vero and 293 cells can induce the synthesis of early proteins capable of functioning in viral DNA replication, but there is a failure in later events on the pathway to infectious virus production. This provides further support for transfected Vero cells being a valid system in which to study HCMV DNA synthesis, and suggests that 293 cells may also prove useful in similar experiments.

scholarly journals Novel virus-associated proteins encoded by UL112–113 of human cytomegalovirus

2009 ◽  
Vol 90 (12) ◽  
pp. 2840-2848 ◽  
Author(s):  
Shang-Kwei Wang ◽  
Cheng-Hui Hu ◽  
Miao-Chan Lu ◽  
Chang-Yih Duh ◽  
Pao-Chi Liao ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Lytic Infection ◽  
Tandem Mass ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Reading Frame ◽  
Dense Bodies ◽  
Associated Proteins ◽  
Novel Virus ◽  
Infectious Cycle

Evidence suggests that the products of the human cytomegalovirus (HCMV) UL112–113 genes are involved in viral DNA replication during lytic infection. A polyclonal antibody was raised against the UL112 open reading frame (ORF) to characterize its function in detail. Immunoblots utilizing the UL112 antibody identified seven distinct protein bands (p20, p26, p28, p34, p43, p50 and p84) expressed during the HCMV infectious cycle. After screening a cDNA library constructed from cells 72 h after infection with HCMV, only four different cDNA protein-producing constructs were obtained, and their ORFs corresponded to p34, p43, p50 and p84. The proteins p20, p26 and p28 were further shown to be selectively included within mature HCMV particles, virions, non-infectious enveloped particles and dense bodies. Immunoaffinity protein purification was used to prepare the samples for liquid chromatography coupled to tandem mass spectrometry. This analysis revealed that p20, p26 and p28 were derived from the UL112 ORF, most likely through post-translational proteolytic cleavage.

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 ATRX limits the accessibility of histone H3-occupied HSV genomes during lytic infection

2021 ◽  
Vol 17 (4) ◽  
pp. e1009567
Author(s):  
Joseph M. Cabral ◽  
Camille H. Cushman ◽  
Catherine N. Sodroski ◽  
David M. Knipe
Keyword(s):  
Viral Replication ◽  
De Novo ◽  
Human Fibroblasts ◽  
Histone H3 ◽  
Gc Content ◽  
Lytic Infection ◽  
Nuclear Entry ◽  
Viral Dna ◽  
Total Histone ◽  
Viral Genomes

Histones are rapidly loaded on the HSV genome upon entry into the nucleus of human fibroblasts, but the effects of histone loading on viral replication have not been fully defined. We showed recently that ATRX is dispensable for de novo deposition of H3 to HSV genomes after nuclear entry but restricted infection through maintenance of viral heterochromatin. To further investigate the roles that ATRX and other histone H3 chaperones play in restriction of HSV, we infected human fibroblasts that were systematically depleted of nuclear H3 chaperones. We found that the ATRX/DAXX complex is unique among nuclear H3 chaperones in its capacity to restrict ICP0-null HSV infection. Only depletion of ATRX significantly alleviated restriction of viral replication. Interestingly, no individual nuclear H3 chaperone was required for deposition of H3 onto input viral genomes, suggesting that during lytic infection, H3 deposition may occur through multiple pathways. ChIP-seq for total histone H3 in control and ATRX-KO cells infected with ICP0-null HSV showed that HSV DNA is loaded with high levels of histones across the entire viral genome. Despite high levels of H3, ATAC-seq analysis revealed that HSV DNA is highly accessible, especially in regions of high GC content, and is not organized largely into ordered nucleosomes during lytic infection. ATRX reduced accessibility of viral DNA to the activity of a TN5 transposase and enhanced accumulation of viral DNA fragment sizes associated with nucleosome-like structures. Together, these findings support a model in which ATRX restricts viral infection by altering the structure of histone H3-loaded viral chromatin that reduces viral DNA accessibility for transcription. High GC rich regions of the HSV genome, especially the S component inverted repeats of the HSV-1 genome, show increased accessibility, which may lead to increased ability to transcribe the IE genes encoded in these regions during initiation of infection.

scholarly journals Human cytomegalovirus blocks canonical TGFβ signaling during lytic infection to limit induction of type I interferons

2021 ◽  
Author(s):  
Andrew H. Pham ◽  
Jennifer Mitchell ◽  
Sara Botto ◽  
Kara M. Pryke ◽  
Victor R. Defilippis ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Hcmv Infection ◽  
Human Fibroblasts ◽  
Lytic Replication ◽  
Mutant Virus ◽  
Type I Interferons ◽  
Type I ◽  
Dependent Manner ◽  
Tgfβ Receptor ◽  
Smad3 Expression

AbstractHuman cytomegalovirus (HCMV) microRNAs (miRNAs) significantly rewire host signaling pathways to support the viral lifecycle and regulate host cell responses. Here we show that SMAD3 expression is regulated by HCMV miR-UL22A and contributes to the IRF7-mediated induction of type I IFNs and IFN-stimulated genes (ISGs) in human fibroblasts. Addition of exogenous TGFβ interferes with the replication of a miR-UL22A mutant virus in a SMAD3-dependent manner in wild type fibroblasts, but not in cells lacking IRF7, indicating that downregulation of SMAD3 expression to limit IFN induction is important for efficient lytic replication. These findings uncover a novel interplay between SMAD3 and innate immunity during HCMV infection and highlight the role of viral miRNAs in modulating these responses.Author SummaryCells trigger the interferon (IFN) response to induce the expression of cellular genes that limit virus replication. In turn, viruses have evolved numerous countermeasures to avoid the effects of IFN signaling. Using a microRNA (miRNA) mutant virus we have uncovered a novel means of regulating the IFN response during human cytomegalovirus (HCMV) infection. Lytic HCMV infection induces the production of TGFβ, which binds to the TGFβ receptor and activates the receptor-associated SMAD SMAD3. SMAD3, together with IRF7, induces the expression of IFNβ and downstream IFN-stimulated genes in human fibroblasts. To counteract this, HCMV miR-UL22A, along with other HCMV gene products, directly targets SMAD3 for downregulation. Infection of fibroblasts with a miR-UL22A mutant virus results in enhanced type I IFN production in a SMAD3-dependent manner and the virus is impaired for growth in the presence of TGFβ, but only when both SMAD3 and IRF7 are present, highlighting the unique interaction between TGFβ and innate immune signaling.

scholarly journals Human cytomegalovirus blocks canonical TGFβ signaling during lytic infection to limit induction of type I interferons

2021 ◽  
Vol 17 (8) ◽  
pp. e1009380
Author(s):  
Andrew H. Pham ◽  
Jennifer Mitchell ◽  
Sara Botto ◽  
Kara M. Pryke ◽  
Victor R. DeFilippis ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Human Fibroblasts ◽  
Lytic Replication ◽  
Type I Interferons ◽  
Type I ◽  
Dependent Manner ◽  
Type I Ifns ◽  
Host Signaling ◽  
Smad3 Expression ◽  
Viral Lifecycle

Human cytomegalovirus (HCMV) microRNAs (miRNAs) significantly rewire host signaling pathways to support the viral lifecycle and regulate host cell responses. Here we show that SMAD3 expression is regulated by HCMV miR-UL22A and contributes to the IRF7-mediated induction of type I IFNs and IFN-stimulated genes (ISGs) in human fibroblasts. Addition of exogenous TGFβ interferes with the replication of a miR-UL22A mutant virus in a SMAD3-dependent manner in wild type fibroblasts, but not in cells lacking IRF7, indicating that downregulation of SMAD3 expression to limit IFN induction is important for efficient lytic replication. These findings uncover a novel interplay between SMAD3 and innate immunity during HCMV infection and highlight the role of viral miRNAs in modulating these responses.

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