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Sample records for human cytomegalovirus ie1

  1. Human Cytomegalovirus IE1 Protein Disrupts Interleukin-6 Signaling by Sequestering STAT3 in the Nucleus

    PubMed Central

    Reitsma, Justin M.; Sato, Hiromi; Nevels, Michael

    2013-01-01

    In the canonical STAT3 signaling pathway, binding of agonist to receptors activates Janus kinases that phosphorylate cytoplasmic STAT3 at tyrosine 705 (Y705). Phosphorylated STAT3 dimers accumulate in the nucleus and drive the expression of genes involved in inflammation, angiogenesis, invasion, and proliferation. Here, we demonstrate that human cytomegalovirus (HCMV) infection rapidly promotes nuclear localization of STAT3 in the absence of robust phosphorylation at Y705. Furthermore, infection disrupts interleukin-6 (IL-6)-induced phosphorylation of STAT3 and expression of a subset of IL-6-induced STAT3-regulated genes, including SOCS3. We show that the HCMV 72-kDa immediate-early 1 (IE1) protein associates with STAT3 and is necessary to localize STAT3 to the nucleus during infection. Furthermore, expression of IE1 is sufficient to disrupt IL-6-induced phosphorylation of STAT3, binding of STAT3 to the SOCS3 promoter, and SOCS3 gene expression. Finally, inhibition of STAT3 nuclear localization or STAT3 expression during infection is linked to diminished HCMV genome replication. Viral gene expression is also disrupted, with the greatest impact seen following viral DNA synthesis. Our study identifies IE1 as a new regulator of STAT3 intracellular localization and IL-6 signaling and points to an unanticipated role of STAT3 in HCMV infection. PMID:23903834

  2. Human cytomegalovirus IE1 protein alters the higher-order chromatin structure by targeting the acidic patch of the nucleosome.

    PubMed

    Fang, Qianglin; Chen, Ping; Wang, Mingzhu; Fang, Junnan; Yang, Na; Li, Guohong; Xu, Rui-Ming

    2016-01-26

    Human cytomegalovirus (hCMV) immediate early 1 (IE1) protein associates with condensed chromatin of the host cell during mitosis. We have determined the structure of the chromatin-tethering domain (CTD) of IE1 bound to the nucleosome core particle, and discovered that IE1-CTD specifically interacts with the H2A-H2B acidic patch and impairs the compaction of higher-order chromatin structure. Our results suggest that IE1 loosens up the folding of host chromatin during hCMV infections.

  3. Characterization of an epitope of the human cytomegalovirus protein IE1 recognized by a CD4+ T cell clone.

    PubMed

    Gautier, N; Chavant, E; Prieur, E; Monsarrat, B; Mazarguil, H; Davrinche, C; Gairin, J E; Davignon, J L

    1996-05-01

    CD4+ T cells specific for human cytomegalovirus (HCMV) IE1 protein are potential effectors of the control of HCMV infection through cytokine production. Better knowledge of major histocompatibility complex (MHC)-peptide-T cell receptor (TcR) interactions in the CD4+ T cell response should result in a better design of immunizing peptides and is a prerequisite for the development of vaccines or anti-cytomegalovirus therapy. In this study, the recombinant protein comprising residues 86-491 encoded by exon 4 of IE1 (GST-e4) was cleaved by enzymatic digestion and analyzed by high pressure liquid chromatography-mass spectroscopy (HPLC-MS). We identified the 14-residue epitope 162-DKREMWMACIKELH-175 recognized by an HLA-DR8-restricted clone, BeA3. Synthetic elongated, truncated and di-Ala-substituted peptides of the 18-mer IE1 158-IVPEDKREMWMACIKELH-175 sequence were used to analyze the amino acid motifs involved in binding to HLA-DR8 and recognition by the BeA3 clone. Substitutions which abolished (MW --> AA), or decreased (RE --> AA and MA --> AA) T cell clone proliferation, cytokine production and cytotoxicity were identified. Loss of T cell function induced by the MW --> AA substitution was associated with poor HLA-DR8 binding. Decreased T cell function (RE --> AA and MA --> AA) was associated with good HLA-DR8 binding, which suggested that these motifs were involved in TcR binding. Other substitutions induced potentiation of the T cell clone response: the IV --> AA substitution induced stronger proliferation, but equivalent cytokine production, when compared with the reference peptide IE1 (158-175). CI --> AA substitution induced strong potentiation of HLA-DR8 binding, proliferation and interferon-gamma and interleukin-4 production, possibly due to the removal of negative effects of Cys, Ile, or both side chains. Cytotoxicity was not improved by any substitution. Our results show modulation of the CD4+ T cell response according to the peptide residues involved in the

  4. The Human Cytomegalovirus IE1 Protein Antagonizes PML Nuclear Body-Mediated Intrinsic Immunity via the Inhibition of PML De Novo SUMOylation.

    PubMed

    Schilling, Eva-Maria; Scherer, Myriam; Reuter, Nina; Schweininger, Johannes; Muller, Yves A; Stamminger, Thomas

    2017-02-15

    PML nuclear bodies (NBs) are accumulations of cellular proteins embedded in a scaffold-like structure built by SUMO-modified PML/TRIM19. PML and other NB proteins act as cellular restriction factors against human cytomegalovirus (HCMV); however, this intrinsic defense is counteracted by the immediate early protein 1 (IE1) of HCMV. IE1 directly interacts with the PML coiled-coil domain via its globular core region and disrupts NB foci by inducing a loss of PML SUMOylation. Here, we demonstrate that IE1 acts via abrogating the de novo SUMOylation of PML. In order to overcome reversible SUMOylation dynamics, we made use of a cell-based assay that combines inducible IE1 expression with a SUMO mutant resistant to SUMO proteases. Interestingly, we observed that IE1 expression did not affect preSUMOylated PML; however, it clearly prevented de novo SUMO conjugation. Consistent results were obtained by in vitro SUMOylation assays, demonstrating that IE1 alone is sufficient for this effect. Furthermore, IE1 acts in a selective manner, since K160 was identified as the main target lysine. This is strengthened by the fact that IE1 also prevents As2O3-mediated hyperSUMOylation of K160, thereby blocking PML degradation. Since IE1 did not interfere with coiled-coil-mediated PML dimerization, we propose that IE1 affects PML autoSUMOylation either by directly abrogating PML E3 ligase function or by preventing access to SUMO sites. Thus, our data suggest a novel mechanism for how a viral protein counteracts a cellular restriction factor by selectively preventing the de novo SUMOylation at specific lysine residues without affecting global protein SUMOylation. The human cytomegalovirus IE1 protein acts as an important antagonist of a cellular restriction mechanism that is mediated by subnuclear structures termed PML nuclear bodies. This function of IE1 is required for efficient viral replication and thus constitutes a potential target for antiviral strategies. In this paper, we further

  5. Anti-human cytomegalovirus activity of cytokines produced by CD4+ T-cell clones specifically activated by IE1 peptides in vitro.

    PubMed Central

    Davignon, J L; Castanié, P; Yorke, J A; Gautier, N; Clément, D; Davrinche, C

    1996-01-01

    The control of latent cytomegalovirus (CMV) infections by the immune system is poorly understood. We have previously shown that CD4+ T cells specific for the human CMV major regulatory protein IE1 are frequent in latently infected healthy blood donors. In order to learn about the possible role of these cells, we have developed IE1-specific CD4+ T-cell clones and, in this study, analyzed their epitope specificity and function in vitro. We measured their cytokine production when stimulated with specific IE1 peptides or whole recombinant IE1 protein. Their cytokine profiles, as deduced from gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and interleukin-4 (IL-4) and IL-6 production, were of the Th0- and Th1-like phenotypes. Supernatants from IE1-specific clones producing IFN-gamma and TNF-alpha were shown to inhibit CMV replication in U373 MG cells. This effect was due, as found by using cytokine-specific neutralizing antibodies, mostly to IFN-gamma, which was secreted at higher levels than TNF-alpha. To better assess the anti-CMV activity of cytokines, recombinant IFN-gamma and TNF-alpha were used and shown to have a synergistic effect on the inhibition of CMV replication and protein expression. Thus, IE1-specific CD4+ T cells display in vitro anti-CMV activity through cytokine secretion and may play a role in the control of in vivo latent infections. PMID:8642638

  6. Binding STAT2 by the acidic domain of human cytomegalovirus IE1 promotes viral growth and is negatively regulated by SUMO.

    PubMed

    Huh, Yong Ho; Kim, Young Eui; Kim, Eui Tae; Park, Jung Jin; Song, Moon Jung; Zhu, Hua; Hayward, Gary S; Ahn, Jin-Hyun

    2008-11-01

    The human cytomegalovirus (HCMV) 72-kDa immediate-early 1 (IE1) protein is thought to modulate cellular antiviral functions impacting on promyelocytic leukemia (PML) nuclear bodies and signal transducer and activator of transcription (STAT) signaling. IE1 consists of four distinct regions: an amino-terminal region required for nuclear localization, a large central hydrophobic region responsible for PML targeting and transactivation activity, an acidic domain, and a carboxyl-terminal chromatin tethering domain. We found that the acidic domain of IE1 is required for binding to STAT2. A mutant HCMV encoding IE1(Delta421-475) with the acidic domain deleted was generated. In mutant virus-infected cells, IE1(Delta421-475) failed to bind to STAT2. The growth of mutant virus was only slightly delayed at a high multiplicity of infection (MOI) but was severely impaired at a low MOI with low-level accumulation of viral proteins. When cells were pretreated with beta interferon, the mutant virus showed an additional 1,000-fold reduction in viral growth, even at a high MOI, compared to the wild type. The inhibition of STAT2 loading on the target promoter upon infection was markedly reduced with mutant virus. Furthermore, sumoylation of IE1 at this acidic domain was found to abolish the activity of IE1 to bind to STAT2 and repress the interferon-stimulated genes. Our results provide genetic evidence that IE1 binding to STAT2 requires the 55-amino-acid acidic domain and promotes viral growth by interfering with interferon signaling and demonstrate that this viral activity is negatively regulated by a cellular sumoylation pathway.

  7. Human Cytomegalovirus IE1 Protein Elicits a Type II Interferon-Like Host Cell Response That Depends on Activated STAT1 but Not Interferon-γ

    PubMed Central

    Knoblach, Theresa; Grandel, Benedikt; Seiler, Jana

    2011-01-01

    Human cytomegalovirus (hCMV) is a highly prevalent pathogen that, upon primary infection, establishes life-long persistence in all infected individuals. Acute hCMV infections cause a variety of diseases in humans with developmental or acquired immune deficits. In addition, persistent hCMV infection may contribute to various chronic disease conditions even in immunologically normal people. The pathogenesis of hCMV disease has been frequently linked to inflammatory host immune responses triggered by virus-infected cells. Moreover, hCMV infection activates numerous host genes many of which encode pro-inflammatory proteins. However, little is known about the relative contributions of individual viral gene products to these changes in cellular transcription. We systematically analyzed the effects of the hCMV 72-kDa immediate-early 1 (IE1) protein, a major transcriptional activator and antagonist of type I interferon (IFN) signaling, on the human transcriptome. Following expression under conditions closely mimicking the situation during productive infection, IE1 elicits a global type II IFN-like host cell response. This response is dominated by the selective up-regulation of immune stimulatory genes normally controlled by IFN-γ and includes the synthesis and secretion of pro-inflammatory chemokines. IE1-mediated induction of IFN-stimulated genes strictly depends on tyrosine-phosphorylated signal transducer and activator of transcription 1 (STAT1) and correlates with the nuclear accumulation and sequence-specific binding of STAT1 to IFN-γ-responsive promoters. However, neither synthesis nor secretion of IFN-γ or other IFNs seems to be required for the IE1-dependent effects on cellular gene expression. Our results demonstrate that a single hCMV protein can trigger a pro-inflammatory host transcriptional response via an unexpected STAT1-dependent but IFN-independent mechanism and identify IE1 as a candidate determinant of hCMV pathogenicity. PMID:21533215

  8. Characterization of Recombinant Human Cytomegaloviruses Encoding IE1 Mutants L174P and 1-382 Reveals that Viral Targeting of PML Bodies Perturbs both Intrinsic and Innate Immune Responses.

    PubMed

    Scherer, Myriam; Otto, Victoria; Stump, Joachim D; Klingl, Stefan; Müller, Regina; Reuter, Nina; Muller, Yves A; Sticht, Heinrich; Stamminger, Thomas

    2015-11-11

    PML is the organizer of cellular structures termed nuclear domain 10 (ND10) or PML-nuclear bodies (PML-NBs) that act as key mediators of intrinsic immunity against human cytomegalovirus (HCMV) and other viruses. The antiviral function of ND10 is antagonized by viral regulatory proteins such as the immediate early protein IE1 of HCMV. IE1 interacts with PML through its globular core domain (IE1CORE) and induces ND10 disruption in order to initiate lytic HCMV infection. Here, we investigate the consequences of a point mutation (L174P) in IE1CORE, which was shown to abrogate the interaction with PML, for lytic HCMV infection. We found that a recombinant HCMV encoding IE1-L174P displays a severe growth defect similar to that of an IE1 deletion virus. Bioinformatic modeling based on the crystal structure of IE1CORE suggested that insertion of proline into the highly alpha-helical domain severely affects its structural integrity. Consistently, L174P mutation abrogates the functionality of IE1CORE and results in degradation of the IE1 protein during infection. In addition, our data provide evidence that IE1CORE as expressed by a recombinant HCMV encoding IE1 1-382 not only is required to antagonize PML-mediated intrinsic immunity but also affects a recently described function of PML in innate immune signaling. We demonstrate a coregulatory role of PML in type I and type II interferon-induced gene expression and provide evidence that upregulation of interferon-induced genes is inhibited by IE1CORE. In conclusion, our data suggest that targeting PML by viral regulatory proteins represents a strategy to antagonize both intrinsic and innate immune mechanisms. PML nuclear bodies (PML-NBs), which represent nuclear multiprotein complexes consisting of PML and additional proteins, represent important cellular structures that mediate intrinsic resistance against many viruses, including human cytomegalovirus (HCMV). During HCMV infection, the major immediate early protein IE1 binds

  9. The essential role of guinea pig cytomegalovirus (GPCMV) IE1 and IE2 homologs in viral replication and IE1-mediated ND10 targeting.

    PubMed

    Hornig, Julia; Choi, K Yeon; McGregor, Alistair

    2017-04-01

    Guinea pig cytomegalovirus (GPCMV) immediate early proteins, IE1 and IE2, demonstrated structural and functional homologies with human cytomegalovirus (HCMV). GPCMV IE1 and IE2 co-localized in the nucleus with each other, the viral polymerase and guinea pig ND10 components (gpPML, gpDaxx, gpSp100, gpATRX). IE1 showed direct interaction with ND10 components by immunoprecipitation unlike IE2. Additionally, IE1 protein disrupted ND10 bodies. IE1 mutagenesis mapped the nuclear localization signal to the C-terminus and identified the core domain for gpPML interaction. Individual knockout of GPCMV GP122 or GP123 (IE2 and IE1 unique exons respectively) was lethal to the virus. However, an IE1 mutant (codons 234-474 deleted), was viable with attenuated viral growth kinetics and increased susceptibility to type I interferon (IFN-I). In HCMV, the IE proteins are important T cell target antigens. Consequently, characterization of the homologs in GPCMV provides a basis for their evaluation in candidate vaccines against congenital infection. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Two Polypyrimidine Tracts in Intron 4 of the Major Immediate Early Gene Are Critical for Gene Expression Switching from IE1 to IE2 and for Replication of Human Cytomegalovirus

    PubMed Central

    Hou, Wangheng; Torres, Lilith; Cruz-Cosme, Ruth; Arroyo, Fernando; Irizarry, Luis; Luciano, Dalia; Márquez, Arturo; Rivera, Leslie L.; Sala, Antonio L.; Luo, Min-hua

    2016-01-01

    ABSTRACT The human cytomegalovirus (HCMV) major immediate early (MIE) gene is essential for viral replication. The most abundant products encoded by the MIE gene include IE1 and IE2. Genes of IE1 and IE2 share the MIE promoter (MIEP), the first 3 exons, and the first 2 introns. IE1 is expressed earlier than IE2 after CMV infection or MIE gene transfection. In this study, we identified 2 polypyrimidine (Py) tracts in intron 4 (between exons 4 and 5) that are responsible for transcriptional switching from IE1 to IE2. The first Py is important and the second one is essential for the splicing and expression of IE2. In searching for the mechanisms of MIE gene switching from IE1 to IE2, we found that the second Py was required for the IE2's fourth intron to bind to a splicing factor such as U2AF65, as determined by an RNA electrophoretic mobility shift assay and a chromatin immunoprecipitation (ChIP) assay, while the first Py enhanced the binding of U2AF65 with the intron. An HCMV BACmid with the second Py mutated failed to produce any virus, while the HCMV with the first Py mutated replicated with a defective phenotype. Furthermore, we designed a small RNA (scRNAPy) that is complementary to the intron RNA covering the two Pys. The scRNAPy interfered with the interaction of U2AF65 with the intron and repressed the IE2 expression. Therefore, our studies implied that IE2 gene splicing might be an anti-CMV target. IMPORTANCE CMV is a ubiquitous herpesvirus and a significant cause of disease and death in the immunocompromised and elderly. Insights into its gene regulation will provide clues in designing anti-CMV strategies. The MIE gene is one of the earliest genes of CMV and is essential for CMV replication. It is known that the MIE gene needs to be spliced to produce more than two proteins; however, how MIE gene splicing is regulated remains elusive. In the present studies, we identified two Pys in intron 4 and found that the first Py is important and the second is

  11. Two Polypyrimidine Tracts in Intron 4 of the Major Immediate Early Gene Are Critical for Gene Expression Switching from IE1 to IE2 and for Replication of Human Cytomegalovirus.

    PubMed

    Hou, Wangheng; Torres, Lilith; Cruz-Cosme, Ruth; Arroyo, Fernando; Irizarry, Luis; Luciano, Dalia; Márquez, Arturo; Rivera, Leslie L; Sala, Antonio L; Luo, Min-Hua; Tang, Qiyi

    2016-08-15

    The human cytomegalovirus (HCMV) major immediate early (MIE) gene is essential for viral replication. The most abundant products encoded by the MIE gene include IE1 and IE2. Genes of IE1 and IE2 share the MIE promoter (MIEP), the first 3 exons, and the first 2 introns. IE1 is expressed earlier than IE2 after CMV infection or MIE gene transfection. In this study, we identified 2 polypyrimidine (Py) tracts in intron 4 (between exons 4 and 5) that are responsible for transcriptional switching from IE1 to IE2. The first Py is important and the second one is essential for the splicing and expression of IE2. In searching for the mechanisms of MIE gene switching from IE1 to IE2, we found that the second Py was required for the IE2's fourth intron to bind to a splicing factor such as U2AF65, as determined by an RNA electrophoretic mobility shift assay and a chromatin immunoprecipitation (ChIP) assay, while the first Py enhanced the binding of U2AF65 with the intron. An HCMV BACmid with the second Py mutated failed to produce any virus, while the HCMV with the first Py mutated replicated with a defective phenotype. Furthermore, we designed a small RNA (scRNAPy) that is complementary to the intron RNA covering the two Pys. The scRNAPy interfered with the interaction of U2AF65 with the intron and repressed the IE2 expression. Therefore, our studies implied that IE2 gene splicing might be an anti-CMV target. CMV is a ubiquitous herpesvirus and a significant cause of disease and death in the immunocompromised and elderly. Insights into its gene regulation will provide clues in designing anti-CMV strategies. The MIE gene is one of the earliest genes of CMV and is essential for CMV replication. It is known that the MIE gene needs to be spliced to produce more than two proteins; however, how MIE gene splicing is regulated remains elusive. In the present studies, we identified two Pys in intron 4 and found that the first Py is important and the second is required for the splicing

  12. Crystal structure of cytomegalovirus IE1 protein reveals targeting of TRIM family member PML via coiled-coil interactions.

    PubMed

    Scherer, Myriam; Klingl, Stefan; Sevvana, Madhumati; Otto, Victoria; Schilling, Eva-Maria; Stump, Joachim D; Müller, Regina; Reuter, Nina; Sticht, Heinrich; Muller, Yves A; Stamminger, Thomas

    2014-11-01

    PML nuclear bodies (PML-NBs) are enigmatic structures of the cell nucleus that act as key mediators of intrinsic immunity against viral pathogens. PML itself is a member of the E3-ligase TRIM family of proteins that regulates a variety of innate immune signaling pathways. Consequently, viruses have evolved effector proteins to modify PML-NBs; however, little is known concerning structure-function relationships of viral antagonists. The herpesvirus human cytomegalovirus (HCMV) expresses the abundant immediate-early protein IE1 that colocalizes with PML-NBs and induces their dispersal, which correlates with the antagonization of NB-mediated intrinsic immunity. Here, we delineate the molecular basis for this antagonization by presenting the first crystal structure for the evolutionary conserved primate cytomegalovirus IE1 proteins. We show that IE1 consists of a globular core (IE1CORE) flanked by intrinsically disordered regions. The 2.3 Å crystal structure of IE1CORE displays an all α-helical, femur-shaped fold, which lacks overall fold similarity with known protein structures, but shares secondary structure features recently observed in the coiled-coil domain of TRIM proteins. Yeast two-hybrid and coimmunoprecipitation experiments demonstrate that IE1CORE binds efficiently to the TRIM family member PML, and is able to induce PML deSUMOylation. Intriguingly, this results in the release of NB-associated proteins into the nucleoplasm, but not of PML itself. Importantly, we show that PML deSUMOylation by IE1CORE is sufficient to antagonize PML-NB-instituted intrinsic immunity. Moreover, co-immunoprecipitation experiments demonstrate that IE1CORE binds via the coiled-coil domain to PML and also interacts with TRIM5α We propose that IE1CORE sequesters PML and possibly other TRIM family members via structural mimicry using an extended binding surface formed by the coiled-coil region. This mode of interaction might render the antagonizing activity less susceptible to

  13. Immediate-Early (IE) gene regulation of cytomegalovirus: IE1- and pp71-mediated viral strategies against cellular defenses.

    PubMed

    Torres, Lilith; Tang, Qiyi

    2014-12-01

    Three crucial hurdles hinder studies on human cytomegalovirus (HCMV): strict species specificity, differences between in vivo and in vitro infection, and the complexity of gene regulation. Ever since the sequencing of the whole genome was first accomplished, functional studies on individual genes have been the mainstream in the CMV field. Gene regulation has therefore been elucidated in a more detailed fashion. However, viral gene regulation is largely controlled by both cellular and viral components. In other words, viral gene expression is determined by the virus-host interaction. Generally, cells respond to viral infection in a defensive pattern; at the same time, viruses try to counteract the cellular defense or else hide in the host (latency). Viruses evolve effective strategies against cellular defense in order to achieve replicative success. Whether or not they are successful, cellular defenses remain in the whole viral replication cycle: entry, immediate-early (IE) gene expression, early gene expression, DNA replication, late gene expression, and viral egress. Many viral strategies against cellular defense, and which occur in the immediate-early time of viral infection, have been documented. In this review, we will summarize the documented biological functions of IE1 and pp71 proteins, especially with regard to how they counteract cellular intrinsic defenses.

  14. Immediate–Early (IE) gene regulation of cytomegalovirus: IE1- and pp71-mediated viral strategies against cellular defenses

    PubMed Central

    Torres, Lilith; Tang, Qiyi

    2015-01-01

    Three crucial hurdles hinder studies on human cytomegalovirus (HCMV): strict species specificity, differences between in vivo and in vitro infection, and the complexity of gene regulation. Ever since the sequencing of the whole genome was first accomplished, functional studies on individual genes have been the mainstream in the CMV field. Gene regulation has therefore been elucidated in a more detailed fashion. However, viral gene regulation is largely controlled by both cellular and viral components. In other words, viral gene expression is determined by the virus–host interaction. Generally, cells respond to viral infection in a defensive pattern; at the same time, viruses try to counteract the cellular defense or else hide in the host (latency). Viruses evolve effective strategies against cellular defense in order to achieve replicative success. Whether or not they are successful, cellular defenses remain in the whole viral replication cycle: entry, immediate–early (IE) gene expression, early gene expression, DNA replication, late gene expression, and viral egress. Many viral strategies against cellular defense, and which occur in the immediate–early time of viral infection, have been documented. In this review, we will summarize the documented biological functions of IE1 and pp71 proteins, especially with regard to how they counteract cellular intrinsic defenses. PMID:25501994

  15. Inconsistent responses of cytomegalovirus-specific T cells to pp65 and IE-1 versus infected dendritic cells in organ transplant recipients.

    PubMed

    Lilleri, D; Zelini, P; Fornara, C; Comolli, G; Gerna, G

    2007-08-01

    CD4(+) and CD8(+) T cells specific for human cytomegalovirus (HCMV) and two immunodominant HCMV antigens (pp65 and IE-1) were monitored in 20 solid organ transplant recipients undergoing primary (n = 4) or reactivated (n = 16) HCMV infection during the first year after transplantation by using as a stimulator either HCMV-infected autologous dendritic cells (DCs) or pp65- or IE-1 peptide mixtures. Turnaround times for test performance were 7 days for infected DCs and 24 h for peptides. Using infected DCs, HCMV-specific T-cell restoration occurred in all patients for CD8(+) and in 18/20 (90%) for CD4(+) T-cell subpopulations, resulting in virus clearance from blood. Using peptide mixtures, T-cell responses were less frequently detected. In detail, 14 (70%) patients showed pp65-specific CD8(+) T cells and 10 (50%) patients IE-1-specific CD8(+) T cells, whereas pp65-specific CD4(+) T cells were detected in 14 (70%) patients, and IE-1-specific CD4(+) T cells in three (15%) patients only. Protection from HCMV infection was associated with the presence of a HCMV-specific T-cell response directed against multiple viral proteins, but not against pp65 or IE-1 only. In conclusion, the use of pp65 and IE-1 peptide mixtures for rapid monitoring of HCMV-specific T-cell responses in solid organ transplant recipients underestimates the actual T-cell immune response against HCMV.

  16. Protective CD8+ T-cell responses to cytomegalovirus driven by rAAV/GFP/IE1 loading of dendritic cells

    PubMed Central

    Yu, Yuefei; Pilgrim, Petra; Yan, Juqiang; Zhou, Wei; Jenkins, Marjorie; Gagliano, Nicoletta; Bumm, Klaus; Cannon, Martin; Milzani, Aldo; Dalle-Donne, Isabella; Kast, W Martin; Cobos, Everardo; Chiriva-Internati, Maurizio

    2008-01-01

    Background Recent studies demonstrate that recombinant adeno-associated virus (rAAV)-based antigen loading of dendritic cells (DCs) generates in vitro, significant and rapid cytotoxic T-lymphocyte (CTL) responses against viral antigens. Methods We used the rAAV system to induce specific CTLs against CVM antigens for the development of cytomegalovirus HCMV) gene therapy. As an extension of the versatility of the rAAV system, we incorporated immediate-early 1 (IE1), expressed in HCMV. Our rAAV vector induced a strong stimulation of CTLs directed against the HCMV antigen IE1. We then investigated the efficiency of the CTLs in killing IE1 targeted cells. Results A significant MHC Class I-restricted, anti-IE1-specific CTL killing was demonstrated against IE1 positive peripheral blood mononuclear cells (PBMC) after one, in vitro, stimulation. Conclusion In summary, single PBMC stimulation with rAAV/IE1 pulsed DCs induces strong antigen specific-CTL generation. CTLs were capable to lyse low doses of peptides pulsed into target cells. These data suggest that AAV-based antigen loading of DCs is highly effective for generating human CTL responses against HCMV antigens. PMID:18834548

  17. Expansion of cytomegalovirus pp65 and IE-1 specific cytotoxic T lymphocytes for cytomegalovirus-specific immunotherapy following allogeneic stem cell transplantation.

    PubMed

    Bao, Lei; Dunham, Kimberly; Stamer, Mindy; Mulieri, Kevin M; Lucas, Kenneth G

    2008-10-01

    Adoptive immunotherapy with antigen-specific cytotoxic T lymphocytes (CTLs) has proven effective in restoring cellular immunity to cytomegalovirus (CMV) and preventing viral reactivation after allogeneic stem cell transplantation (SCT). In an effort to develop a cost-effective, relatively rapid method of CMV CTL expansion, we investigated the use of a pool of overlapping CMV peptides. Because the possibility exists of vaccinating CMV-seronegative donors, and these individuals may have T cell responses predominantly against IE-1, commercially available peptide mixes for pp65 as well as IE-1 were used to stimulate CTLs from 10 seropositive donors. Of these 10 donors, 4 responded to pp65 only, 1 did not respond to either pp65 or IE-1, 4 responded to both pp65 and IE-1, and 1 responded to IE-1 only. These CMV- specific T cells included a mixture of CD4(+) and CD8(+) effectors, and specific cytotoxicity correlated with interferon-gamma production. The costs associated with a 28-day maintenance course of intravenous ganciclovir, cidofovir, foscarnet, and valganciclovir, as well as the preparation and shipping a single dose of CTLs, were determined. The price of generating CMV CTLs using this method was comparable to or less expensive than a 28-day maintenance course for these agents, not including the costs associated with drug administration, supportive care, and the treatment of drug-related complications. Considering the relative ease, low cost, and the fact that CTL administration can result in CMV-specific immune reconstitution, this option should be considered for patients with CMV reactivation or for prophylaxis in patients at high risk for infection.

  18. Ablation of the Regulatory IE1 Protein of Murine Cytomegalovirus Alters In Vivo Pro-inflammatory TNF-alpha Production during Acute Infection

    PubMed Central

    Wilhelmi, Vanessa; Lisnic, Vanda Juranic; Hsieh, Wei Yuan; Blanc, Mathieu; Livingston, Andrew; Busche, Andreas; Tekotte, Hille; Messerle, Martin; Auer, Manfred; Fraser, Iain; Jonjic, Stipan; Angulo, Ana; Reddehase, Matthias J.; Ghazal, Peter

    2012-01-01

    Little is known about the role of viral genes in modulating host cytokine responses. Here we report a new functional role of the viral encoded IE1 protein of the murine cytomegalovirus in sculpting the inflammatory response in an acute infection. In time course experiments of infected primary macrophages (MΦs) measuring cytokine production levels, genetic ablation of the immediate-early 1 (ie1) gene results in a significant increase in TNFα production. Intracellular staining for cytokine production and viral early gene expression shows that TNFα production is highly associated with the productively infected MΦ population of cells. The ie1- dependent phenotype of enhanced MΦ TNFα production occurs at both protein and RNA levels. Noticeably, we show in a series of in vivo infection experiments that in multiple organs the presence of ie1 potently inhibits the pro-inflammatory cytokine response. From these experiments, levels of TNFα, and to a lesser extent IFNβ, but not the anti-inflammatory cytokine IL10, are moderated in the presence of ie1. The ie1- mediated inhibition of TNFα production has a similar quantitative phenotype profile in infection of susceptible (BALB/c) and resistant (C57BL/6) mouse strains as well as in a severe immuno-ablative model of infection. In vitro experiments with infected macrophages reveal that deletion of ie1 results in increased sensitivity of viral replication to TNFα inhibition. However, in vivo infection studies show that genetic ablation of TNFα or TNFRp55 receptor is not sufficient to rescue the restricted replication phenotype of the ie1 mutant virus. These results provide, for the first time, evidence for a role of IE1 as a regulator of the pro-inflammatory response and demonstrate a specific pathogen gene capable of moderating the host production of TNFα in vivo. PMID:22952450

  19. Controlled crystal dehydration triggers a space-group switch and shapes the tertiary structure of cytomegalovirus immediate-early 1 (IE1) protein.

    PubMed

    Klingl, Stefan; Scherer, Myriam; Stamminger, Thomas; Muller, Yves A

    2015-07-01

    Cytomegalovirus immediate-early 1 (IE1) protein is a key viral effector protein that reprograms host cells. Controlled dehydration experiments with IE1 crystals not only extended their diffraction limit from 2.85 to 2.3 Å resolution but also triggered a monoclinic to tetragonal space-group transition with only minor alterations in the unit-cell parameters. An analysis of the pre-dehydration and post-dehydration crystal structures shows how dehydration rearranges the packing of IE1 molecules to meet the unit-cell constraints of the higher lattice symmetry. The transition from P21 to P43 reduces the number of copies in the asymmetric unit from four to two, and molecules previously related by noncrystallographic symmetry merge into identical crystallographic copies in the tetragonal space group. At the same time, dehydration considerably alters the tertiary structure of one of the two remaining IE1 chains in the asymmetric unit. It appears that this conformational switch is required to compensate for a transition that is assumed to be unfavourable, namely from a highly preferred to a rarely observed space group. At the same time, the dehydration-triggered molecular reshaping could reveal an inherent molecular flexibility that possibly informs on the biological function of IE1, namely on its binding to target proteins from the host cell.

  20. Cytomegalovirus (CMV) IE1- and pp65-specific CD8+ T cell responses broaden over time after primary CMV infection in infants.

    PubMed

    Gibson, Laura; Dooley, Sheryl; Trzmielina, Sonia; Somasundaran, Mohan; Fisher, Donna; Revello, Maria Grazia; Luzuriaga, Katherine

    2007-06-15

    Cytomegalovirus (CMV) infection remains a significant cause of morbidity and mortality in young children. We have previously shown that CD8+ T cell responses to CMV pp65 or IE1 protein were readily detectable in children with congenital or postnatal CMV infection. Here, we have further characterized the evolution of the peptide specificity of these responses in 7 infants<6 months of age at the start of the study. Thirteen pp65 and 15 IE1 peptides (median, 5 peptides/infant) were targeted, and most (61%) represented sequences not previously reported. Peptide specificity remained stable or broadened over time despite the clearance of CMV viremia. Loss of peptide recognition was not observed. Responses with the highest functional peptide avidity were not necessarily detected earliest. These data provide additional evidence that young infants can generate diverse CMV-specific CD8+ T cell responses but show that early responses may exhibit relatively focused peptide specificity and lower peptide avidity.

  1. [Infection by human cytomegalovirus].

    PubMed

    Sanbonmatsu Gámez, Sara; Ruiz, Mercedes Pérez; Navarro Marí, José María

    2014-02-01

    Prevalence of human cytomegalovirus infection is very high worldwide. Following primary infection, the virus remains latent, being able to cause recurrences either by reinfection with a new strain or by reactivation of the replication of the latent virus. The most severe disease is seen in congenital infection and in immunosuppressed patients, in whom the virus act as an opportunistic pathogen. Serological techniques are the methods of choice in primary infection and to determine the immune status against CMV in organ donor and receptor. Although well-standardized studies are lacking, the recent commercial availability of methods that measure cellular immune response are promising to predict the risk of CMV disease in immunosuppressed individuals. Molecular assays, that have gradually been substituting viral culture and/or antigen detection, are the most widely used methods for the diagnosis and control of CMV infection. Copyright © 2014 Elsevier España, S.L. All rights reserved.

  2. [Isolation of human cytomegalovirus in primary cytomegalic infection].

    PubMed

    Karazhas, N V; Khaustov, V I

    1994-01-01

    Cytomegalovirus was isolated from a patient with generalized cytomegaloviral infection. The strain was identified using tissue culture method, electron microscopy, and serologic tests. The virus was repeatedly passed in diploid human fibroblast cells and recorded as Vesna human cytomegalovirus strain.

  3. Human Cytomegalovirus Induces JC Virus DNA Replication in Human Fibroblasts

    NASA Astrophysics Data System (ADS)

    Heilbronn, Regine; Albrecht, Ingrid; Stephan, Sonja; Burkle, Alexander; Zur Hausen, Harald

    1993-12-01

    JC virus, a human papovavirus, is the causative agent of the demyelinating brain disease progressive multifocal leucoencephalopathy (PML). PML is a rare but fatal disease which develops as a complication of severe immunosuppression. Latent JC virus is harbored by many asymptomatic carriers and is transiently reactivated from the latent state upon immunosuppression. JC virus has a very restricted host range, with human glial cells being the only tissue in which it can replicate at reasonable efficiency. Evidence that latent human cytomegalovirus is harbored in the kidney similar to latent JC virus led to the speculation that during episodes of impaired immunocompetence, cytomegalovirus might serve as helper virus for JC virus replication in otherwise nonpermissive cells. We show here that cytomegalovirus infection indeed leads to considerable JC virus DNA replication in cultured human fibroblasts that are nonpermissive for the replication of JC virus alone. Cytomegalovirus-mediated JC virus replication is dependent on the JC virus origin of replication and T antigen. Ganciclovir-induced inhibition of cytomegalovirus replication is associated with a concomitant inhibition of JC virus replication. These results suggest that reactivation of cytomegalovirus during episodes of immunosuppression might lead to activation of latent JC virus, which would enhance the probability of subsequent PML development. Ganciclovir-induced repression of both cytomegalovirus and JC virus replication may form the rational basis for the development of an approach toward treatment or prevention of PML.

  4. Human cytomegalovirus: propagation, quantification, and storage.

    PubMed

    Britt, William J

    2010-08-01

    Human cytomegalovirus (HCMV) is the largest and perhaps the most structurally complex member of the family of human herpesviruses. It is the prototypic virus of the beta-herpesvirus subfamily. As with other cytomegaloviruses, HCMV is exquisitely species specific and undergoes lytic replication only in cells of human origin. In addition, its replication is limited almost entirely to primary cells and a limited number of transformed cell lines. Together with its prolonged replicative cycle of approximately 48 hr, the propagation and quantification of HCMV can present technical challenges. In this brief set of protocols, the propagation of laboratory strains of HCMV and their quantitation is described. In a third series of protocols, the concentration and gradient purification of HCMV for more specialized downstream applications is described.

  5. Transactivation of cellular genes involved in nucleotide metabolism by the regulatory IE1 protein of murine cytomegalovirus is not critical for viral replicative fitness in quiescent cells and host tissues.

    PubMed

    Wilhelmi, Vanessa; Simon, Christian O; Podlech, Jürgen; Böhm, Verena; Däubner, Torsten; Emde, Simone; Strand, Dennis; Renzaho, Angélique; Lemmermann, Niels A W; Seckert, Christof K; Reddehase, Matthias J; Grzimek, Natascha K A

    2008-10-01

    Despite its high coding capacity, murine CMV (mCMV) does not encode functional enzymes for nucleotide biosynthesis. It thus depends on cellular enzymes, such as ribonucleotide reductase (RNR) and thymidylate synthase (TS), to be supplied with deoxynucleoside triphosphates (dNTPs) for its DNA replication. Viral transactivation of these cellular genes in quiescent cells of host tissues is therefore a parameter of viral fitness relevant to pathogenicity. Previous work has shown that the IE1, but not the IE3, protein of mCMV transactivates RNR and TS gene promoters and has revealed an in vivo attenuation of the mutant virus mCMV-DeltaIE1. It was attractive to propose the hypothesis that lack of transactivation by IE1 and a resulting deficiency in the supply of dNTPs are the reasons for growth attenuation. Here, we have tested this hypothesis with the mutant virus mCMV-IE1-Y165C expressing an IE1 protein that selectively fails to transactivate RNR and TS in quiescent cells upon transfection while maintaining the capacity to disperse repressive nuclear domains (ND10). Our results confirm in vivo attenuation of mCMV-DeltaIE1, as indicated by a longer doubling time in host organs, whereas mCMV-IE1-Y165C replicated like mCMV-WT and the revertant virus mCMV-IE1-C165Y. Notably, the mutant virus transactivated RNR and TS upon infection of quiescent cells, thus indicating that IE1 is not the only viral transactivator involved. We conclude that transactivation of cellular genes of dNTP biosynthesis is ensured by redundancy and that attenuation of mCMV-DeltaIE1 results from the loss of other critical functions of IE1, with its function in the dispersal of ND10 being a promising candidate.

  6. Candidate cytomegalovirus strain for human vaccination.

    PubMed Central

    Plotkin, S A; Furukawa, T; Zygraich, N; Huygelen, C

    1975-01-01

    A strain of human cytomegalovirus called Towne was isolated in WI-38 human fibrolast cell cultures from the urine of an infected infant. It was then passaged 125 times in WI-38, including three clonings, and a pool was prepared in the same cell substrate for use as a potential live attenuated vaccine. The Towne virus has a broad antigenicity and cross-reacts with the AD-169 strain. Several markers of the Towne virus were found which differentiated it from fresh isolates. One of these was resistance of the former to trypsin. The Towne virus was tested for freedom from oncogenicity or other harmful effects in preparation for tests in humans. PMID:170203

  7. Diverse immune evasion strategies by human cytomegalovirus.

    PubMed

    Noriega, Vanessa; Redmann, Veronika; Gardner, Thomas; Tortorella, Domenico

    2012-12-01

    Members of the Herpesviridae family have the capacity to undergo both lytic and latent infection to establish a lifelong relationship with their host. Following primary infection, human cytomegalovirus (HCMV) can persist as a subclinical, recurrent infection for the lifetime of an individual. This quiescent portion of its life cycle is termed latency and is associated with periodic bouts of reactivation during times of immunosuppression, inflammation, or stress. In order to exist indefinitely and establish infection, HCMV encodes a multitude of immune modulatory mechanisms devoted to escaping the host antiviral response. HCMV has become a paradigm for studies of viral immune evasion of antigen presentation by both major histocompatibility complex (MHC) class I and II molecules. By restricting the presentation of viral antigens during both productive and latent infection, HCMV limits elimination by the human immune system. This review will focus on understanding how the virus manipulates the pathways of antigen presentation in order to modulate the host response to infection.

  8. Human immune responses to major human cytomegalovirus glycoprotein complexes.

    PubMed Central

    Liu, Y N; Kari, B; Gehrz, R C

    1988-01-01

    Sera from both human cytomegalovirus (HCMV)-seropositive adults and infants with congenital HCMV infection recognized two major HCMV glycoprotein complexes. However, proliferative responses of peripheral blood mononuclear cells to these complexes varied among seropositive adults and were not detected in any of the infants. Thus, these glycoproteins alone may not be sufficient to develop a subviral HCMV vaccine. Images PMID:2828655

  9. Monitoring cytomegalovirus IE-1 and pp65-specific CD4+ and CD8+ T-cell responses after allogeneic stem cell transplantation may identify patients at risk for recurrent CMV reactivations.

    PubMed

    Gratama, Jan W; Brooimans, Rik A; van der Holt, Bronno; Sintnicolaas, Kees; van Doornum, Gerard; Niesters, Hubertus G; Löwenberg, Bob; Cornelissen, Jan J

    2008-07-01

    We studied the recovery of CMV-specific CD4+ and CD8+ T-cell immunity in 52 recipients of allogeneic stem cell transplantation (SCT). The proportions of IFN-gamma-producing CD4+ and CD8+ T cells upon in vitro activation using peptide pools representing the CMV pp65 and IE-1 proteins were assessed at multiple time points post SCT, and correlated with the occurrence of CMV reactivation. In a retrospective analysis, recurrent CMV reactivations occurred in 9 patients and were associated with low pp65-specific CD4+ T-cell and low IE-1-specific CD8(+) T-cell reactivities, whereas patients without detectable CMV reactivation (n = 30) or a single reactivation (n = 13) showed a better recovery of these immune responses. CD4+ T-cell responses to IE-1 were infrequent in most patients, whereas CD8+ T-cell responses to pp65 occurred frequently, but did not correlate with protection against (recurrent) reactivation. Prospectively, CMV-specific T-cell responses could be studied prior to 14 reactivation episodes in 8 patients. CD4+ T-cell responses to IE-1 and pp65 were positive in only 1 and 2 episodes, respectively. CD8+ T-cell responses against IE-1 were positive in 4, but against pp65 in 12 episodes, again showing that CD8+ T-cell reactivity against pp65 did not prevent CMV reactivation. Thus, monitoring of particular CMV-specific CD4+ and CD8+ T-cell responses after allogeneic SCT may identify patients at risk for recurrent CMV reactivations. (c) 2008 Clinical Cytometry Society

  10. Positive Expression of Human Cytomegalovirus Phosphoprotein 65 in Atherosclerosis

    PubMed Central

    Wang, Zhe; Cai, Jun; Zhang, Mingming; Wang, Xiaojing; Chi, Hongjie; Feng, Haijun

    2016-01-01

    Previous studies showed that human cytomegalovirus (HCMV) is associated with atherosclerosis. However, local vascular atherosclerosis related HCMV infection and protein expression remain unclear. This study aimed to assess the relationship between HCMV infection and atherosclerosis. Formalin-fixed, paraffin-embedded peripheral artery specimens were obtained from 15 patients with atherosclerosis undergoing vascular surgery from 2008 to 2010 at Zhongnan Hospital, Wuhan University. Pathological analyses were carried out after hematoxylin and eosin (H&E) and Masson trichrome staining. In situ hybridization and immunohistochemistry with two different monoclonal antibodies were employed to detect HCMV nucleic acids and proteins, respectively. H&E and Masson trichrome staining showed homogeneous extracellular matrix in femoral artery, while smooth muscle fibers were interlaced with collagen fibers; in carotid artery, inflammatory cell infiltration, foam cell vascular change, cholesterol crystals, and layered collagen fibers were observed. In situ hybridization showed no expression of HCMV nucleic acids in all 15 cases. Immunohistochemical staining for protein immediate-early protein (IE1 72) was negative in all cases, while phosphoprotein 65 (pp65) expression was detected in 14 cases. A high rate of positive pp65 signals was found in patients with atherosclerosis, suggesting that local HCMV infection may be associated with the pathogenesis of atherosclerosis. Further studies on this relationship are warranted. PMID:27990427

  11. Human Cytomegalovirus Manipulation of Latently Infected Cells

    PubMed Central

    Sinclair, John H.; Reeves, Matthew B.

    2013-01-01

    Primary infection with human cytomegalovirus (HCMV) results in the establishment of a lifelong infection of the host which is aided by the ability of HCMV to undergo a latent infection. One site of HCMV latency in vivo is in haematopoietic progenitor cells, resident in the bone marrow, with genome carriage and reactivation being restricted to the cells of the myeloid lineage. Until recently, HCMV latency has been considered to be relatively quiescent with the virus being maintained essentially as a “silent partner” until conditions are met that trigger reactivation. However, advances in techniques to study global changes in gene expression have begun to show that HCMV latency is a highly active process which involves expression of specific latency-associated viral gene products which orchestrate major changes in the latently infected cell. These changes are argued to help maintain latent infection and to modulate the cellular environment to the benefit of latent virus. In this review, we will discuss these new findings and how they impact not only on our understanding of the biology of HCMV latency but also how they could provide tantalising glimpses into mechanisms that could become targets for the clearance of latent HCMV. PMID:24284875

  12. A Diverse Repertoire of CD4 T Cells Targets the Immediate-Early 1 Protein of Human Cytomegalovirus

    PubMed Central

    Ameres, Stefanie; Liang, Xiaoling; Wiesner, Martina; Mautner, Josef; Moosmann, Andreas

    2015-01-01

    T-cell responses to the immediate-early 1 (IE-1) protein of human cytomegalovirus (HCMV) are associated with protection from viral disease. Thus, IE-1 is a promising target for immunotherapy. CD8 T-cell responses to IE-1 are generally strong. In contrast, CD4 T-cell responses to IE-1 were described to be comparatively infrequent or undetectable in HCMV carriers, and information on their target epitopes and their function has been limited. To analyze the repertoire of IE-1-specific CD4 T cells, we expanded them from healthy donors with autologous IE-1-expressing mini-Epstein–Barr virus-transformed B-cell lines and established IE-1-specific CD4 T-cell clones. Clones from seven out of seven HCMV-positive donors recognized endogenously processed IE-1 epitopes restricted through HLA-DR, DQ, or DP. Three to seven IE-1 epitopes were recognized per donor. Cumulatively, about 27 different HLA/peptide class II complexes were recognized by 117 IE-1-specific clones. Our results suggest that a highly diversified repertoire of IE-1-specific CD4 T cells targeting multiple epitopes is usually present in healthy HCMV carriers. Therefore, multiepitope approaches to immunomonitoring and immunotherapy will make optimal use of this potentially important class of HCMV-specific effector cells. PMID:26635812

  13. Wnt modulating agents inhibit human cytomegalovirus replication.

    PubMed

    Kapoor, Arun; He, Ran; Venkatadri, Rajkumar; Forman, Michael; Arav-Boger, Ravit

    2013-06-01

    Infection 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 replication in 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.

  14. Wnt Modulating Agents Inhibit Human Cytomegalovirus Replication

    PubMed Central

    Kapoor, Arun; He, Ran; Venkatadri, Rajkumar; Forman, Michael

    2013-01-01

    Infection 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 replication in 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. PMID:23571549

  15. Activation of telomerase by human cytomegalovirus.

    PubMed

    Strååt, Klas; Liu, Cheng; Rahbar, Afsar; Zhu, Qingjun; Liu, Li; Wolmer-Solberg, Nina; Lou, Fenglan; Liu, Zhaoxu; Shen, Jie; Jia, Jihui; Kyo, Satoru; Björkholm, Magnus; Sjöberg, Jan; Söderberg-Nauclér, Cecilia; Xu, Dawei

    2009-04-01

    The mechanism by which human cytomegalovirus (HCMV) stimulates oncogenesis is unclear. Because cellular immortalization and transformation require telomerase activation by expression of the telomerase reverse transcriptase (hTERT) gene, we examined the role of HCMV in telomerase activation. Normal human diploid fibroblasts (HDFs) and human malignant glioma (MG) cell lines were infected with HCMV or transfected with expression vectors encoding HCMV immediate early (IE) antigen 72 or 86. hTERT expression and promoter activity and telomerase activity were evaluated using reverse transcription-polymerase chain reaction, a luciferase reporter assay, and a telomeric repeat amplification protocol, respectively. hTERT promoter occupancy by the transcription factor Sp1, IE antigens, and histone deacetylases (HDACs) was assessed by chromatin immunoprecipitation. hTERT and IE protein expression in human primary glioblastoma multiforme (GBM) was determined immunohistochemically. All statistical tests were two-sided. In telomerase and hTERT-negative HDFs, HCMV infection induced constitutive hTERT expression and telomerase activation. The hTERT promoter activity in HDFs and MG cell lines was statistically significantly enhanced by HCMV in a dose-dependent manner (mean luciferase activity [arbitrary units] in control HDFs and in HDFs infected with HCMV at multiplicities of infection [MOIs] of 0.1 = 6 and 521, respectively, difference = 515, 95% CI = 178 to 850; mean activity at MOI of 1 and 10 = 8828 and 59,923, respectively; P < .001 comparing control with HCMV-infected cells at all MOIs). Ectopic expression of HCMV IE-72 protein also stimulated hTERT promoter activity in HDFs. HCMV-mediated transactivation of the hTERT gene was dependent on the presence of Sp1-binding sites in the hTERT promoter and was accompanied by increases in Sp1 binding, acetylation of histone H3, and a reduction in HDAC binding at the core promoter. In specimens of GBM, HCMV IE and hTERT proteins were

  16. Human cytomegalovirus latent gene expression in granulocyte-macrophage progenitors in culture and in seropositive individuals.

    PubMed Central

    Kondo, K; Xu, J; Mocarski, E S

    1996-01-01

    Following infection with cytomegalovirus, human granulocyte-macrophage progenitors carry the viral genome but fail to support productive replication. Viral transcripts arise from a region encompassing the major regulatory gene locus; however, their structure differs significantly from productive phase transcripts. One class, sense transcripts, is encoded in the same direction as productive phase transcripts but uses two novel start sites in the ie1/ie2 promoter/enhancer region. These transcripts have the potential to encode a novel 94 aa protein. The other class, antisense transcript, is unspliced and complimentary to ie1 exons 2-4, and has the potential to encode novel 154 and 152 aa proteins. Consistent with a role in latency, these transcripts are present in bone marrow aspirates from naturally infected, healthy seropositive donors but are not present in seronegative controls. Sense latent transcripts are present in a majority of seropositive individuals. Consistent with the expression of latent transcripts, antibody to the 94 aa and 152 aa proteins is detectable in the serum of seropositive individuals. Thus, latent infection by cytomegalovirus is accompanied by the presence of latency-associated transcripts and expression of immunogenic proteins. Overall, these results suggest that bone marrow-derived myeloid progenitors are an important natural site of viral latency. Images Fig. 1 Fig. 2 Fig. 3 PMID:8855322

  17. Infrequency of cytomegalovirus genome in coronary arteriopathy of human heart allografts.

    PubMed Central

    Gulizia, J. M.; Kandolf, R.; Kendall, T. J.; Thieszen, S. L.; Wilson, J. E.; Radio, S. J.; Costanzo, M. R.; Winters, G. L.; Miller, L. L.; McManus, B. M.

    1995-01-01

    In heart transplantation, long-term engraftment success is severely limited by the rapid development of obliterative disease of the coronary arteries. Data from various groups have been suggestive of a pathogenetic role of herpesviruses, particularly human cytomegalovirus, in accelerated allograft coronary artery disease; however, results are not yet conclusive. This study examines the hypothesis that human cytomegalovirus infection of allograft tissues is related pathogenetically and directly to accelerated coronary artery disease. Using in situ DNA hybridization and polymerase chain reaction, we examined particular coronary artery segments from 41 human heart allografts (ranging from 4 days to greater than 4 years after transplantation; mean, 457 days) and 22 donor age- and gender-comparable, coronary site-matched trauma victims for presence of human cytomegalovirus DNA. Human cytomegalovirus genome was detected in 8 of 41 (19.5%) allografts and in 1 of 22 (4.5%) control hearts. This difference in positivity was not statistically significant (P = 0.10). In the human cytomegalovirus-positive hearts, viral genome was localized to perivascular myocardium or coronary artery media or adventitia. Human cytomegalovirus genome was not detected in arterial intima of any allograft or control heart, although human cytomegalovirus genome was readily identified within intima of small pulmonary arteries from lung tissue with human cytomegalovirus pneumonitis. By statistical analyses, the presence of human cytomegalovirus genome was not associated with the nature or digitized extent of transplant arteriopathy, evidence of rejection, allograft recipient or donor serological data suggestive of human cytomegalovirus infection, duration of allograft implantation, or causes of death or retransplantation. Thus, our data indicate a low frequency of detectable human cytomegalovirus genome in accelerated coronary artery disease and do not support a direct role for human cytomegalovirus

  18. Cytomegalovirus (CMV) infection

    MedlinePlus

    CMV mononucleosis; Cytomegalovirus; CMV; Human cytomegalovirus; HCMV ... infection is spread by: Blood transfusions Organ transplants ... viruses remain in your body for the rest of your life. If your ...

  19. A Phase 1 Study of 4 Live, Recombinant Human Cytomegalovirus Towne/Toledo Chimera Vaccines in Cytomegalovirus-Seronegative Men.

    PubMed

    Adler, Stuart P; Manganello, Anne-Marie; Lee, Ronzo; McVoy, Michael A; Nixon, Daniel E; Plotkin, Stanley; Mocarski, Edward; Cox, Josephine H; Fast, Patricia E; Nesterenko, Pavlo A; Murray, Susan E; Hill, Ann B; Kemble, George

    2016-11-01

    Human cytomegalovirus (HCMV) infection causes disease in newborns and transplant recipients. A HCMV vaccine (Towne) protects transplant recipients.  The genomes of Towne and the nonattenuated Toledo strain were recombined, yielding 4 Towne/Toledo chimera vaccines. Each of 36 HCMV-seronegative men received 1 subcutaneous dose of 10, 100, or 1000 plaque-forming units (PFU) in cohorts of 3. Safety and immunogenicity were evaluated over 12 weeks after immunization and for 52 weeks for those who seroconverted.  There were no serious local or systemic reactions. No subject had HCMV in urine or saliva. For chimera 3, none of 9 subjects seroconverted. For chimera 1, 1 of 9 seroconverted (the seroconverter received 100 PFU). For chimera 2, 3 subjects seroconverted (1 received 100 PFU, and 2 received 1000 PFU). For chimera 4, 7 subjects seroconverted (1 received 10 PFU, 3 received 100 PFU, and 3 received 1000 PFU). All 11 seroconverters developed low but detectable levels of neutralizing activity. CD4(+) T-cell responses were detectable in 1 subject (who received 100 PFU of chimera 4). Seven subjects receiving chimera 2 or 4 had detectable CD8(+) T-cell responses to IE1; 3 responded to 1-2 additional antigens.  The Towne/Toledo chimera vaccine candidates were well tolerated and were not excreted. Additional human trials of chimeras 2 and 4 are appropriate.  NCT01195571. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  20. Human embryonic stem cell lines model experimental human cytomegalovirus latency.

    PubMed

    Penkert, Rhiannon R; Kalejta, Robert F

    2013-05-28

    Herpesviruses are highly successful pathogens that persist for the lifetime of their hosts primarily because of their ability to establish and maintain latent infections from which the virus is capable of productively reactivating. Human cytomegalovirus (HCMV), a betaherpesvirus, establishes latency in CD34(+) hematopoietic progenitor cells during natural infections in the body. Experimental infection of CD34(+) cells ex vivo has demonstrated that expression of the viral gene products that drive productive infection is silenced by an intrinsic immune defense mediated by Daxx and histone deacetylases through heterochromatinization of the viral genome during the establishment of latency. Additional mechanistic details about the establishment, let alone maintenance and reactivation, of HCMV latency remain scarce. This is partly due to the technical challenges of CD34(+) cell culture, most notably, the difficulty in preventing spontaneous differentiation that drives reactivation and renders them permissive for productive infection. Here we demonstrate that HCMV can establish, maintain, and reactivate in vitro from experimental latency in cultures of human embryonic stem cells (ESCs), for which spurious differentiation can be prevented or controlled. Furthermore, we show that known molecular aspects of HCMV latency are faithfully recapitulated in these cells. In total, we present ESCs as a novel, tractable model for studies of HCMV latency.

  1. Murine cytomegalovirus resistant to antivirals has genetic correlates with human cytomegalovirus.

    PubMed

    Scott, G M; Ng, H-L; Morton, C J; Parker, M W; Rawlinson, W D

    2005-08-01

    Human cytomegalovirus (HCMV) resistance to antivirals is a significant clinical problem. Murine cytomegalovirus (MCMV) infection of mice is a well-described animal model for in vivo studies of CMV pathogenesis, although the mechanisms of MCMV antiviral susceptibility need elucidation. Mutants resistant to nucleoside analogues aciclovir, adefovir, cidofovir, ganciclovir, penciclovir and valaciclovir, and the pyrophosphate analogue foscarnet were generated by in vitro passage of MCMV (Smith) in increasing concentrations of antiviral. All MCMV antiviral resistant mutants contained DNA polymerase mutations identical or similar to HCMV DNA polymerase mutations known to confer antiviral resistance. Mapping of the mutations onto an MCMV DNA polymerase three-dimensional model generated using the Thermococcus gorgonarius Tgo polymerase crystal structure showed that the DNA polymerase mutations potentially confer resistance through changes in regions surrounding a catalytic aspartate triad. The ganciclovir-, penciclovir- and valaciclovir-resistant isolates also contained mutations within MCMV M97 identical or similar to recognized GCV-resistant mutations of HCMV UL97 protein kinase, and demonstrated cross-resistance to antivirals of the same class. This strongly suggests that MCMV M97 has a similar role to HCMV UL97 in the phosphorylation of nucleoside analogue antivirals. All MCMV mutants demonstrated replication-impaired phenotypes, with the lowest titre and plaque size observed for isolates containing mutations in both DNA polymerase and M97. These findings indicate DNA polymerase and protein kinase regions of potential importance for antiviral susceptibility and replication. The similarities between MCMV and HCMV mutations that arise under antiviral selective pressure increase the utility of MCMV as a model for in vivo studies of CMV antiviral resistance.

  2. DNA repair mechanisms and human cytomegalovirus (HCMV) infection.

    PubMed

    Smolarz, Beata; Wilczyński, Jan; Nowakowska, Dorota

    2015-05-01

    Herpesvirus infections, such as those induced by human cytomegalovirus (HCMV), induce specific DNA damages. DNA damages can lead to cell mutation, death, apoptosis and immune system activation. Various types of DNA damage are repaired through multiple repair pathways, such as base excision, nucleotide excision, homologous recombination and nonhomologous end joining. Changes in the activity of DNA repair proteins during viral infection can cause disturbances in the DNA repair system and change its mechanisms. This report reviews results from studies, assaying a DNA repair system in HCMV infection.

  3. Identification of Cellular Proteins that Interact with Human Cytomegalovirus Immediate-Early Protein 1 by Protein Array Assay

    PubMed Central

    Puerta Martínez, Francisco; Tang, Qiyi

    2013-01-01

    Human cytomegalovirus (HCMV) gene expression during infection is characterized as a sequential process including immediate-early (IE), early (E), and late (L)-stage gene expression. The most abundantly expressed gene at the IE stage of infection is the major IE (MIE) gene that produces IE1 and IE2. IE1 has been the focus of study because it is an important protein, not only for viral gene expression but also for viral replication. It is believed that IE1 plays important roles in viral gene regulation by interacting with cellular proteins. In the current study, we performed protein array assays and identified 83 cellular proteins that interact with IE1. Among them, seven are RNA-binding proteins that are important in RNA processing; more than half are nuclear proteins that are involved in gene regulations. Tumorigenesis-related proteins are also found to interact with IE1, implying that the role of IE1 in tumorigenesis might need to be reevaluated. Unexpectedly, cytoplasmic proteins, such as Golgi autoantigen and GGA1 (both related to the Golgi trafficking protein), are also found to be associated with IE1. We also employed a coimmunoprecipitation assay to test the interactions of IE1 and some of the proteins identified in the protein array assays and confirmed that the results from the protein array assays are reliable. Many of the proteins identified by the protein array assay have not been previously reported. Therefore, the functions of the IE1-protein interactions need to be further explored in the future. PMID:24385082

  4. Functionally inactivated dominant viral antigens of human cytomegalovirus delivered in replication incompetent adenovirus type 6 vectors as vaccine candidates.

    PubMed

    Tang, Aimin; Freed, Daniel C; Li, Fengsheng; Meschino, Steve; Prokop, Michael; Bett, Andrew; Casimiro, Danilo; Wang, Dai; Fu, Tong-Ming

    2017-05-11

    T cell immunity is critical in controlling human cytomegalovirus (HCMV) infection in transplant recipients, and T cells targeting viral immediate early proteins such as IE1, IE2 and pp65 have been speculated to be more effective against reactivation. Here we report efforts to construct replication incompetent adenovirus 6 vectors expressing these viral antigens as vaccine candidates. To reduce the potential liabilities of these viral proteins as vaccine antigens, we introduced mutations to inactivate their reported functions including their nuclear localization signals. The modifications greatly reduced their localization to the nuclei, thus limiting their interactions with cellular proteins important for cell cycle modulation and transactivation. The immunogenicity of modified pp65, IE1 and IE2 vaccines was comparable to their wild-type counterparts in mice and the immunogenicity of the modified antigens was demonstrated in non-human primates.

  5. Antiviral effects of IFIT1 in human cytomegalovirus-infected fetal astrocytes.

    PubMed

    Zhang, Li; Wang, Bin; Li, Ling; Qian, Dong-Meng; Yu, Hong; Xue, Mei-Lan; Hu, Ming; Song, Xu-Xia

    2017-04-01

    The prominent feature of human cytomegalovirus (HCMV) is cell tropism specificity for human fetal nervous system, which leads to severe fetal nervous system damage especially in first-trimester gestation. In this study, human astrocytes isolated from fetal brain were infected with HCMV AD169 and whole genome transcriptome profile was performed. The results showed that the gene expression of interferon stimulated genes (ISGs), chemokine and chemokine receptors were significantly up-regulated (P < 0.01). The antiviral replication effects of IFIT1 (Interferon-induced protein with tetratricopeptide repeats 1, Fc = 148.17) was investigated. Lentivirus with IFIT1 overexpression or knockdown was transduced into astrocytes, respectively. The viral mRNA, protein expression and HCMV titers were determined. The results showed that IE1, IE2, pp65, and viral titers were significantly decreased in IFIT1 overexpression group and enhanced in the knockdown group compared with control one (P < 0.01). Taken together, this study revealed IFIT1 played an important antiviral role in HCMV infected fetal astrocytes. The prominent feature of human cytomegalovirus (HCMV) is cellular tropism specificity for human fetal brain nervous system leading to severe fetal nervous damage especially in first-trimester gestation. In this study, human astrocytes isolated from first-trimester fetal brain were infected with HCMV AD169 and IFIT1 was studied for its antiviral replication effects. The results provided insights into the function of IFIT1 as a key factor in antiviral defense contributing to development of targeted therapeutics to fetal brain with HCMV infection. J. Med. Virol. 89:672-684, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  6. Association of Human Cytomegalovirus with Hodgkin’s Disease and Non-Hodgkin’s lymphomas

    PubMed

    Mehravaran, Hamide; Makvandi, Manoochehr; Samarbaf Zade, Alireza; Neisi, Niloofar; Kiani, Hadis; Radmehr, Hashem; Shahani, Toran; Hoseini, Seyedeh Zeinab; Ranjbari, Nastaran; Nahid Samiei, Rahil

    2017-03-01

    Background and Objective: The human cytomegalovirus (HCMV) can persist lifelong as a latent infection and may result in a series of disorders. Associations with both Hodgkin’s disease and non-Hodgkin´s lymphomas have been reported. Expression of the unique long (UL)138 gene of HCMV is linked with the viral latency phase while that of the immediate-early (IE)1 gene is typical of the viral replication phase in patients. This study conducted to determine the prevalence of CMV latent infection in histological tissue samples from patients with Hodgkin’s and Non-Hodgkin´s lymphomas. Material and Methods: A cross sectional study was carried out with a total of 50 paraffin embedded tissues blocks, including 25 samples for Hodgkin’s disease and 25 samples for non-Hodgkin´s lymphomas. After RNA extraction and cDNA preparation, detection of IE1 mRNA was conducted by RT-PCR and identification of mRNA UL138 was achieved by nested PCR. Results: Among 25 cases of Non-Hodgkin´s lymphoma, 5 (20%) were positive for UL 138 and 1 (4%) for both IE1 and UL 138. Among 25 cases of Hodgkin only 1 (4%) was positive for UL 138 and all were negative for IE1 .Conclusion: A relatively high 20% rate of expression of UL 138 was detected in patients with non-Hodgkin´s lymphoma, so that latent CMV infection may play a role in development of the disease. Creative Commons Attribution License

  7. Association of Human Cytomegalovirus with Hodgkin’s Disease and Non-Hodgkin’s lymphomas

    PubMed Central

    Mehravaran, Hamide; Makvandi, Manoochehr; Zade, Alireza Samarbaf; Neisi, Niloofar; Kiani, Hadis; Radmehr, Hashem; Shahani, Toran; Hoseini, Seyedeh Zeinab; Ranjbari, Nastaran; Samiei, Rahil Nahid

    2017-01-01

    Background and Objective: The human cytomegalovirus (HCMV) can persist lifelong as a latent infection and may result in a series of disorders. Associations with both Hodgkin’s disease and non-Hodgkin´s lymphomas have been reported. Expression of the unique long (UL)138 gene of HCMV is linked with the viral latency phase while that of the immediate-early (IE)1 gene is typical of the viral replication phase in patients. This study conducted to determine the prevalence of CMV latent infection in histological tissue samples from patients with Hodgkin’s and Non-Hodgkin´s lymphomas. Material and Methods: A cross sectional study was carried out with a total of 50 paraffin embedded tissues blocks, including 25 samples for Hodgkin’s disease and 25 samples for non-Hodgkin´s lymphomas. After RNA extraction and cDNA preparation, detection of IE1 mRNA was conducted by RT-PCR and identification of mRNA UL138 was achieved by nested PCR. Results: Among 25 cases of Non-Hodgkin´s lymphoma, 5 (20%) were positive for UL 138 and 1 (4%) for both IE1 and UL 138. Among 25 cases of Hodgkin only 1 (4%) was positive for UL 138 and all were negative for IE1 Conclusion: A relatively high 20% rate of expression of UL 138 was detected in patients with non-Hodgkin´s lymphoma, so that latent CMV infection may play a role in development of the disease. PMID:28440608

  8. Human Cytomegalovirus II. Lack of Relatedness to DNA of Herpes Simplex I and II, Epstein-Barr Virus, and Nonhuman Strains of Cytomegalovirus

    PubMed Central

    Huang, Eng-Shang; Pagano, Joseph S.

    1974-01-01

    The purified DNA of human cytomegalovirus, radiolabeled in vitro, was examined for homology to Epstein-Barr virus, herpes simplex type I and II, and simian and murine cytomegalovirus DNA by DNA-DNA reassociation kinetics analyses with the S1 enzyme differential digestion technique. Cross-matching of the DNAs showed no relatedness or less than 5% detectable homology. PMID:4362866

  9. Presentation of an Immunodominant Immediate-Early CD8+ T Cell Epitope Resists Human Cytomegalovirus Immunoevasion

    PubMed Central

    Ameres, Stefanie; Mautner, Josef; Schlott, Fabian; Neuenhahn, Michael; Busch, Dirk H.; Plachter, Bodo; Moosmann, Andreas

    2013-01-01

    Control of human cytomegalovirus (HCMV) depends on CD8+ T cell responses that are shaped by an individual's repertoire of MHC molecules. MHC class I presentation is modulated by a set of HCMV-encoded proteins. Here we show that HCMV immunoevasins differentially impair T cell recognition of epitopes from the same viral antigen, immediate-early 1 (IE-1), that are presented by different MHC class I allotypes. In the presence of immunoevasins, HLA-A- and HLA-B-restricted T cell clones were ineffective, but HLA-C*0702-restricted T cell clones recognized and killed infected cells. Resistance of HLA-C*0702 to viral immunoevasins US2 and US11 was mediated by the alpha3 domain and C-terminal region of the HLA heavy chain. In healthy donors, HLA-C*0702-restricted T cells dominated the T cell response to IE-1. The same HLA-C allotype specifically protected infected cells from attack by NK cells that expressed a corresponding HLA-C-specific KIR. Thus, allotype-specific viral immunoevasion allows HCMV to escape control by NK cells and HLA-A- and HLA-B-restricted T cells, while the virus becomes selectively vulnerable to an immunodominant population of HLA-C-restricted T cells. Our work identifies a T cell population that may be of particular efficiency in HCMV-specific immunotherapy. PMID:23717207

  10. Growth in Agarose of Human Cells Infected with Cytomegalovirus

    PubMed Central

    Lang, David J.; Montagnier, Luc; Latarjet, Raymond

    1974-01-01

    After infection by human cytomegalovirus (CMV), human diploid fibroblasts could grow in agarose medium for several generations. Clones of infected cells grew for weeks, although in every case they ultimately underwent lysis owing to the cytopathic effect of the virus. Virus was inoculated at high dilution and after UV irradiation in an effort to derive cells infected with noninfectious defective particles still capable of inducing cell stimulation. Dilute or irradiated virus occasionally yielded large colonies of replicating cells, although permanent transformation was not observed. One clone derived from UV-CMV-infected cells was passaged four times before undergoing lysis. During these passages the cells exhibited alterations in morphology and orientation. Images PMID:4367907

  11. Role of Human Cytomegalovirus Tegument Proteins in Virion Assembly

    PubMed Central

    Smith, Rebecca Marie; Kosuri, Srivenkat; Kerry, Julie Anne

    2014-01-01

    Like other herpesviruses, human cytomegalovirus (HCMV) contains a unique proteinaceous layer between the virion envelope and capsid, termed the tegument. Upon infection, the contents of the tegument layer are delivered to the host cell, along with the capsid and the viral genome, where they facilitate the initial stages of virus replication. The tegument proteins also play important roles in virion assembly and this dual nature makes them attractive potential targets for antiviral therapies. While our knowledge regarding tegument protein function during the initiation of infection has been the subject of intense study, their roles in assembly are much less well understood. In this review, we will focus on recent studies that highlight the functions of HCMV tegument proteins during assembly, and pose key questions for further investigation. PMID:24509811

  12. Human Cytomegalovirus Infection Upregulates the Mitochondrial Transcription and Translation Machineries

    PubMed Central

    Weekes, M. P.; Antrobus, R.; Rorbach, J.; van Haute, L.; Umrania, Y.; Smith, D. L.; Minczuk, M.; Lehner, P. J.; Sinclair, J. H.

    2016-01-01

    ABSTRACT Infection with human cytomegalovirus (HCMV) profoundly affects cellular metabolism. Like in tumor cells, HCMV infection increases glycolysis, and glucose carbon is shifted from the mitochondrial tricarboxylic acid cycle to the biosynthesis of fatty acids. However, unlike in many tumor cells, where aerobic glycolysis is accompanied by suppression of mitochondrial oxidative phosphorylation, HCMV induces mitochondrial biogenesis and respiration. Here, we affinity purified mitochondria and used quantitative mass spectrometry to determine how the mitochondrial proteome changes upon HCMV infection. We found that the mitochondrial transcription and translation systems are induced early during the viral replication cycle. Specifically, proteins involved in biogenesis of the mitochondrial ribosome were highly upregulated by HCMV infection. Inhibition of mitochondrial translation with chloramphenicol or knockdown of HCMV-induced ribosome biogenesis factor MRM3 abolished the HCMV-mediated increase in mitochondrially encoded proteins and significantly impaired viral growth under bioenergetically restricting conditions. Our findings demonstrate how HCMV manipulates mitochondrial biogenesis to support its replication. PMID:27025248

  13. Is human cytomegalovirus associated with breast cancer progression?

    PubMed Central

    2013-01-01

    Background It has been hypothesized that human cytomegalovirus (HCMV) may be associated with breast cancer progression. However, the role of HCMV infection in breast cancer remains controversial. We aimed to assess whether HCMV genes (UL122 and UL83) could be detected in breast carcinomas and reinvestigated their possible association with breast cancer progression. DNA from paraffin-embedded tissues was analyzed by real-time PCR. We investigated 20 fibroadenomas and 27 primary breast carcinomas (stages II, III, and IV). Findings Two carcinomas were positive for HCMV, one was positive for two TaqMan viral detection probes, and one was positive for a sole TaqMan viral detection probe (UL83), whereas the remainder of the samples was negative. Conclusions Samples studied showed no association between HCMV infection and breast cancer progression. PMID:23557440

  14. Human cytomegalovirus: bacterial artificial chromosome (BAC) cloning and genetic manipulation.

    PubMed

    Paredes, Anne M; Yu, Dong

    2012-02-01

    The understanding of human cytomegalovirus (HCMV) biology was long hindered by the inability to perform efficient viral genetic analysis. This hurdle was recently overcome when the genomes of multiple HCMV strains were cloned as infectious bacterial artificial chromosomes (BACs). The BAC system takes advantage of the single-copy F plasmid of E. coli that can stably carry large pieces of foreign DNA. In this system, a recombinant HCMV virus carrying a modified F plasmid is first generated in eukaryotic cells. Recombinant viral genomes are then isolated and recovered in E. coli as BAC clones. BAC-captured viral genomes can be manipulated using prokaryotic genetics, and recombinant virus can be reconstituted from BAC transfection in eukaryotic cells. The BAC reverse genetic system provides a reliable and efficient method to introduce genetic alterations into the viral genome in E.coli and subsequently analyze their effects on virus biology in eukaryotic cells.

  15. Alteration of lipid metabolism in cells infected with human cytomegalovirus.

    PubMed

    Sanchez, Veronica; Dong, Jennifer J

    2010-08-15

    The human cytomegalovirus (HCMV) envelope contains 12 virus-encoded glycoproteins and glycoprotein complexes but the lipid composition of the envelope has not been clearly defined. Given the specificity of the interactions between integral membrane proteins and lipids, it is likely that the lipid content of the virion envelope is regulated during infection. In an effort to determine the effects of HCMV infection on lipid metabolism, we have used PCR array technology to investigate how infection affects the expression of genes involved in lipoprotein signaling and cholesterol homeostasis pathways. Our results indicate that HCMV infection leads to down-regulation of the ABCA1 transporter. Decreased levels of ABCA1 appear to be the result of enhanced calpain-mediated cleavage in virus-infected cells. In addition, our data also show that HCMV infection inhibits the development of the foam cell phenotype in conditionally permissive THP-1 derived macrophages

  16. Human Cytomegalovirus Infection Upregulates the Mitochondrial Transcription and Translation Machineries.

    PubMed

    Karniely, S; Weekes, M P; Antrobus, R; Rorbach, J; van Haute, L; Umrania, Y; Smith, D L; Stanton, R J; Minczuk, M; Lehner, P J; Sinclair, J H

    2016-03-29

    Infection with human cytomegalovirus (HCMV) profoundly affects cellular metabolism. Like in tumor cells, HCMV infection increases glycolysis, and glucose carbon is shifted from the mitochondrial tricarboxylic acid cycle to the biosynthesis of fatty acids. However, unlike in many tumor cells, where aerobic glycolysis is accompanied by suppression of mitochondrial oxidative phosphorylation, HCMV induces mitochondrial biogenesis and respiration. Here, we affinity purified mitochondria and used quantitative mass spectrometry to determine how the mitochondrial proteome changes upon HCMV infection. We found that the mitochondrial transcription and translation systems are induced early during the viral replication cycle. Specifically, proteins involved in biogenesis of the mitochondrial ribosome were highly upregulated by HCMV infection. Inhibition of mitochondrial translation with chloramphenicol or knockdown of HCMV-induced ribosome biogenesis factor MRM3 abolished the HCMV-mediated increase in mitochondrially encoded proteins and significantly impaired viral growth under bioenergetically restricting conditions. Our findings demonstrate how HCMV manipulates mitochondrial biogenesis to support its replication. Human cytomegalovirus (HCMV), a betaherpesvirus, is a leading cause of morbidity and mortality during congenital infection and among immunosuppressed individuals. HCMV infection significantly changes cellular metabolism. Akin to tumor cells, in HCMV-infected cells, glycolysis is increased and glucose carbon is shifted from the tricarboxylic acid cycle to fatty acid biosynthesis. However, unlike in tumor cells, HCMV induces mitochondrial biogenesis even under aerobic glycolysis. Here, we have affinity purified mitochondria and used quantitative mass spectrometry to determine how the mitochondrial proteome changes upon HCMV infection. We find that the mitochondrial transcription and translation systems are induced early during the viral replication cycle

  17. Seroprevalence of Human Cytomegalovirus (HCMV) infection in pregnant women and outcomes of pregnancies with active infection.

    PubMed

    Mujtaba, Ghulam; Shaukat, Shahzad; Angez, Mehar; Alam, Muhammad Masroor; Hasan, Fariha; Zahoor Zaidi, Syed Sohail; Shah, Aamer Ali

    2016-08-01

    To determine the prevalence of cytomegalovirus in pregnant women and types of overt congenital infection in neonates. This cross-sectional study was conducted at the Pakistan Institute of Medical Sciences and Federal Government Services Hospital in Islamabad, Pakistan, from March 2010 to June 2011, and comprised blood samples of pregnant women. Seroprevalence of human cytomegalovirus, immunoglobulin G and immunoglobulin M was determined by enzyme-linked immunosorbent assay while its deoxyribonucleic acid was detected by nested polymerase chain reaction.The congenital human cytomegalovirus infection was also identified in newborn babies from actively infected pregnant women. SPSS 18 was used for data analysis. Of the 409 pregnant women enrolled, 399(97.55%) were seropositive for cytomegalovirus immunoglobulinG and 52(12.71%) for immunoglobulinM, while cytomegalovirus deoxyribonucleic acid was detected in 82(20%). Of the cytomegalovirus immunoglobulinM-positive women, sera of 40(80%) had immunoglobulinG avidity >50%. The remaining 12(23%) sera had avidity assay value <50%. Among the 82(20%) infected pregnant women, 70(85.4%) were successfully followed up. Among them, the virus was isolated from 41(58.5%) newborns babies, of which 15(21%) were symptomatic while 26(47.2%) were asymptomatic. Of the former, 4(26.6%) had hepatosplenomegaly. Human cytomegalovirus infection in pregnant women was the main reason of congenital defects among neonates.

  18. Human Cytomegalovirus: detection of congenital and perinatal infection in Argentina

    PubMed Central

    Distéfano, Angélica Lidia; Alonso, Alicia; Martin, Fabián; Pardon, Fabián

    2004-01-01

    Background Human cytomegalovirus (CMV) is one of the most commonly found agents of congenital infections. Primary maternal infection is associated with risk of symptomatic congenital diseases, and high morbidity is frequently associated with very low birth weight. Neonates with asymptomatic infection develop various sequelae during infancy. This is the first Argentine study performed in neonates with congenital and postnatal HCMV infection. The purpose of this study was to evaluate the performance of the polymerase chain reaction (PCR) technique with different pairs of primers, to detect cytomegalovirus isolated in tissue cultures and directly in urine and dried blood spot (DBS) specimens. Results were compared with IgM detection. Methods The study was performed between 1999 and 2001 on routine samples in the Laboratory. A total of 61 urine and 56 serum samples were selected from 61 newborns/infants, 33 patients whose samples were analyzed during the first two to three weeks of life were considered congenital infections; the remaining 28 patients whose samples were taken later than the third week were grouped as perinatal infections, although only in 4 the perinatal transmission of infection was determined unequivocally Cytomegalovirus diagnosis was made by isolating the virus from urine samples in human foreskin fibroblast cells. Three different primer pairs directed to IE, LA and gB genes were used for the HCMV PCR assay in viral isolates. Subsequently, PCR and nested PCR (nPCR) assays with gB primers were performed directly in urine and in 11 samples of dried blood spot (DBS) on Guthrie Card, these results were then compared with serology. Results The main clinical manifestations of the 33 patients with congenital infection were purpura, jaundice, hepatomegaly and anaemia. Three patients presented low birth weight as single symptom, 10, intracranial calcifications, and 2, kidney failure. In the 28 patients grouped as with perinatal infection, anaemia

  19. Limits and patterns of cytomegalovirus genomic diversity in humans

    PubMed Central

    Renzette, Nicholas; Pokalyuk, Cornelia; Gibson, Laura; Bhattacharjee, Bornali; Schleiss, Mark R.; Hamprecht, Klaus; Yamamoto, Aparecida Y.; Mussi-Pinhata, Marisa M.; Britt, William J.; Jensen, Jeffrey D.; Kowalik, Timothy F.

    2015-01-01

    Human cytomegalovirus (HCMV) exhibits surprisingly high genomic diversity during natural infection although little is known about the limits or patterns of HCMV diversity among humans. To address this deficiency, we analyzed genomic diversity among congenitally infected infants. We show that there is an upper limit to HCMV genomic diversity in these patient samples, with ∼25% of the genome being devoid of polymorphisms. These low diversity regions were distributed across 26 loci that were preferentially located in DNA-processing genes. Furthermore, by developing, to our knowledge, the first genome-wide mutation and recombination rate maps for HCMV, we show that genomic diversity is positively correlated with these two rates. In contrast, median levels of viral genomic diversity did not vary between putatively single or mixed strain infections. We also provide evidence that HCMV populations isolated from vascular compartments of hosts from different continents are genetically similar and that polymorphisms in glycoproteins and regulatory proteins are enriched in these viral populations. This analysis provides the most highly detailed map of HCMV genomic diversity in human hosts to date and informs our understanding of the distribution of HCMV genomic diversity within human hosts. PMID:26150505

  20. Human Cytomegalovirus Carries a Cell-Derived Phospholipase A2 Required for Infectivity

    PubMed Central

    Allal, Cuider; Buisson-Brenac, Claire; Marion, Vincent; Claudel-Renard, Clotilde; Faraut, Thomas; Dal Monte, Paola; Streblow, Daniel; Record, Michel

    2004-01-01

    Human cytomegalovirus (HCMV) is known to carry host cell-derived proteins and mRNAs whose role in cell infection is not understood. We have identified a phospholipase A2 (PLA2) activity borne by HCMV by using an assay based on the hydrolysis of fluorescent phosphatidylcholine. This activity was found in all virus strains analyzed and in purified strains. It was calcium dependent and was sensitive to inhibitors of cytosolic PLA2 (cPLA2) but not to inhibitors of soluble PLA2 or calcium-independent PLA2. No other phospholipase activity was detected in the virus. Purified virus was found to contain human cellular cPLA2α, as detected by monoclonal antibody. No homology with PLA2 was found in the genome of HCMV, indicating that HCMV does not code for a PLA2. Decreased de novo expression of immediate-early proteins 1 and 2 (IE1 and IE2), tegument phosphoprotein pp65, and virus production was observed when HCMV was treated with inhibitors of cPLA2. Cell entry of HCMV was not altered by those inhibitors, suggesting the action of cPLA2 was postentry. Together, our results indicate a selective sorting of a cell-derived cPLA2 during HCMV maturation, which is further required for infectivity. PMID:15220446

  1. BST2/Tetherin Enhances Entry of Human Cytomegalovirus

    PubMed Central

    Viswanathan, Kasinath; Smith, M. Shane; Malouli, Daniel; Mansouri, Mandana; Nelson, Jay A.; Früh, Klaus

    2011-01-01

    Interferon-induced BST2/Tetherin prevents budding of vpu-deficient HIV-1 by tethering mature viral particles to the plasma membrane. BST2 also inhibits release of other enveloped viruses including Ebola virus and Kaposi's sarcoma associated herpesvirus (KSHV), indicating that BST2 is a broadly acting antiviral host protein. Unexpectedly however, recovery of human cytomegalovirus (HCMV) from supernatants of BST2-expressing human fibroblasts was increased rather than decreased. Furthermore, BST2 seemed to enhance viral entry into cells since more virion proteins were released into BST2-expressing cells and subsequent viral gene expression was elevated. A significant increase in viral entry was also observed upon induction of endogenous BST2 during differentiation of the pro-monocytic cell line THP-1. Moreover, treatment of primary human monocytes with siRNA to BST2 reduced HCMV infection, suggesting that BST2 facilitates entry of HCMV into cells expressing high levels of BST2 either constitutively or in response to exogenous stimuli. Since BST2 is present in HCMV particles we propose that HCMV entry is enhanced via a reverse-tethering mechanism with BST2 in the viral envelope interacting with BST2 in the target cell membrane. Our data suggest that HCMV not only counteracts the well-established function of BST2 as inhibitor of viral egress but also employs this anti-viral protein to gain entry into BST2-expressing hematopoietic cells, a process that might play a role in hematogenous dissemination of HCMV. PMID:22072961

  2. Human antibody technology and the development of antibodies against cytomegalovirus.

    PubMed

    Ohlin, Mats; Söderberg-Nauclér, Cecilia

    2015-10-01

    Cytomegalovirus (CMV) is a virus that causes chronic infections in a large set of the population. It may cause severe disease in immunocompromised individuals, is linked to immunosenescence and implied to play an important role in the pathogenesis of cardiovascular diseases and cancer. Modulation of the immune system's abilities to manage the virus represent a highly viable therapeutic option and passive immunotherapy with polyclonal antibody preparations is already in clinical use. Defined monoclonal antibodies offer many advantages over polyclonal antibodies purified from serum. Human CMV-specific monoclonal antibodies have consequently been thoroughly investigated with respect to their potential in the treatment of diseases caused by CMV. Recent advances in human antibody technology have substantially expanded the breadth of antibodies for such applications. This review summarizes the fundamental basis for treating CMV disease by use of antibodies, the basic technologies to be used to develop such antibodies, and relevant human antibody specificities available to target this virus. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Antiviral activity of a phosphorothioate oligonucleotide complementary to RNA of the human cytomegalovirus major immediate-early region.

    PubMed Central

    Azad, R F; Driver, V B; Tanaka, K; Crooke, R M; Anderson, K P

    1993-01-01

    Phosphorothioate oligonucleotides complementary to mRNA of the human cytomegalovirus (HCMV) DNA polymerase gene or to RNA transcripts of the major immediate-early regions 1 and 2 (IE1 and IE2) of HCMV were evaluated for antiviral activity in a 96-well immunoassay with primary human dermal fibroblasts as host cells. Oligonucleotides complementary to RNA of the IE2 region exhibited the most potent antiviral activity. One of these oligonucleotides, ISIS 2922, was at least 30-fold more potent than the nucleoside analog, ganciclovir, with a 50% effective concentration of 0.37 microM in the 96-well immunoassay. In an infectious virus yield reduction assay, ISIS 2922 and ganciclovir reduced production of infectious virus by 2 log units at concentrations of 2.2 and 36 microM, respectively. A control oligonucleotide showed no inhibition of virus production at concentrations as high as 3 microM. ISIS 2922 reduced IE protein synthesis in HCMV-infected cells in a dose-dependent manner which correlated with antiviral activity. The antiviral activity of ISIS 2922 was not due to oligonucleotide-induced cytotoxicity since effects on cell viability or proliferation were observed only at concentrations well in excess of effective antiviral concentrations. The specificity and potency of ISIS 2922 suggest that it may be useful for the treatment of cytomegalovirus disease in humans. Images PMID:8239610

  4. Cytomegalovirus Replicates in Differentiated but not in Undifferentiated Human Embryonal Carcinoma Cells

    NASA Astrophysics Data System (ADS)

    Gonczol, Eva; Andrews, Peter W.; Plotkin, Stanley A.

    1984-04-01

    To study the mode of action of human cytomegalovirus, an important teratogenic agent in human populations, the susceptibility of a pluripotent human embryonal carcinoma cell line to the virus was investigated. Viral antigens were not expressed nor was infectious virus produced by human embryonal carcinoma cells after infection, although the virus was able to penetrate these cells. In contrast, retinoic acid-induced differentiated derivatives of embryonal carcinoma cells were permissive for antigen expression and infectious virus production. Replication of human cytomegalovirus in human teratocarcinoma cells may therefore depend on cellular functions associated with differentiation.

  5. Human Cytomegalovirus UL97 Phosphorylates the Viral Nuclear Egress Complex

    PubMed Central

    Sharma, Mayuri; Bender, Brian J.; Kamil, Jeremy P.; Lye, Ming F.; Pesola, Jean M.; Reim, Natalia I.; Hogle, James M.

    2014-01-01

    ABSTRACT Herpesvirus nucleocapsids exit the host cell nucleus in an unusual process known as nuclear egress. The human cytomegalovirus (HCMV) UL97 protein kinase is required for efficient nuclear egress, which can be explained by its phosphorylation of the nuclear lamina component lamin A/C, which disrupts the nuclear lamina. We found that a dominant negative lamin A/C mutant complemented the replication defect of a virus lacking UL97 in dividing cells, validating this explanation. However, as complementation was incomplete, we investigated whether the HCMV nuclear egress complex (NEC) subunits UL50 and UL53, which are required for nuclear egress and recruit UL97 to the nuclear rim, are UL97 substrates. Using mass spectrometry, we detected UL97-dependent phosphorylation of UL50 residue S216 (UL50-S216) and UL53-S19 in infected cells. Moreover, UL53-S19 was specifically phosphorylated by UL97 in vitro. Notably, treatment of infected cells with the UL97 inhibitor maribavir or infection with a UL97 mutant led to a punctate rather than a continuous distribution of the NEC at the nuclear rim. Alanine substitutions in both UL50-S216 and UL53-S19 resulted in a punctate distribution of the NEC in infected cells and also decreased virus production and nuclear egress in the absence of maribavir. These results indicate that UL97 phosphorylates the NEC and suggest that this phosphorylation modulates nuclear egress. Thus, the UL97-NEC interaction appears to recruit UL97 to the nuclear rim both for disruption of the nuclear lamina and phosphorylation of the NEC. IMPORTANCE Human cytomegalovirus (HCMV) causes birth defects and it can cause life-threatening diseases in immunocompromised patients. HCMV assembles in the nucleus and then translocates to the cytoplasm in an unusual process termed nuclear egress, an attractive target for antiviral therapy. A viral enzyme, UL97, is important for nuclear egress. It has been proposed that this is due to its role in disruption of the

  6. Human cytomegalovirus replicates in gamma-irradiated fibroblasts

    SciTech Connect

    Shanley, J.D.

    1986-12-01

    Because of the unique interdependence of human cytomegalovirus (HCMV) and the physiological state of the host cell, we evaluated the ability of human foreskin fibroblasts (HFF), exposed to gamma radiation, to support HCMV growth. Irradiation of HFF with 2,500 rADS prevented cellular proliferation and suppressed cellular DNA, but not RNA or protein synthesis. Treatment of HFF cells with 2,500 rADS 6 or 48 hours prior to infection did not alter the time course or virus yield during HCMV replication. Virus plaquing efficiency in irradiated cells was comparable to that of nonirradiated cells. As judged by thymidine incorporation and BUdR inhibition of virus replication, HCMV infection induced both thymidine kinase activity and host cell DNA synthesis in irradiated cells. In addition, virus could be recovered from HFF exposed to radiation 0-2 days after infection with HCMV. These studies indicate that the damage to cells by gamma irradiation does not alter the capacity of host cells to support HCMV replication.

  7. Absence of human cytomegalovirus infection in childhood brain tumors

    PubMed Central

    Sardi, Iacopo; Lucchesi, Maurizio; Becciani, Sabrina; Facchini, Ludovica; Guidi, Milena; Buccoliero, Anna Maria; Moriondo, Maria; Baroni, Gianna; Stival, Alessia; Farina, Silvia; Genitori, Lorenzo; de Martino, Maurizio

    2015-01-01

    Human cytomegalovirus (HCMV) is a common human pathogen which induces different clinical manifestations related to the age and the immune conditions of the host. HCMV infection seems to be involved in the pathogenesis of adult glioblastomas. The aim of our study was to detect the presence of HCMV in high grade gliomas and other pediatric brain tumors. This hypothesis might have important therapeutic implications, offering a new target for adjuvant therapies. Among 106 pediatric patients affected by CNS tumors we selected 27 patients with a positive HCMV serology. The serological analysis revealed 7 patients with positive HCMV IGG (≥14 U/mL), whom had also a high HCMV IgG avidity, suggesting a more than 6 months-dated infection. Furthermore, HCMV IGM were positive (≥22 U/mL) in 20 patients. Molecular and immunohistochemical analyses were performed in all the 27 samples. Despite a positive HCMV serology, confirmed by ELISA, no viral DNA was shown at the PCR analysis in the patients’ neoplastic cells. At immunohistochemistry, no expression of HCMV antigens was observed in tumoral cells. Our results are in agreement with recent results in adults which did not evidence the presence of HCMV genome in glioblastoma lesions. We did not find any correlation between HCMV infection and pediatric CNS tumors. PMID:26396923

  8. Resolution of hydrops secondary to cytomegalovirus after maternal and fetal treatment with human cytomegalovirus hyperimmune globulin.

    PubMed

    Moxley, Katherine; Knudtson, Eric J

    2008-02-01

    Congenital cytomegalovirus (CMV) is a common infection with limited treatment options. Vertical transmission can lead to fetal death or long-term neurologic injury. We present a case wherein fetal hydrops resolved after maternal and fetal intravenous administration of CMV hyperimmune globulin. A 20-year-old gravida 3, para 0 was referred for Level II ultrasonography secondary to hydrops fetalis. Amniocentesis demonstrated in utero CMV infection. Resolution of hydrops occurred after the administration of CMV hyperimmune globulin to the patient and then to her fetus. Resolution of hydrops secondary to congenital CMV was temporally related to the administration of maternal and fetal hyperimmune globulin.

  9. Impact of Persistent Cytomegalovirus Infection on Dynamic Changes in Human Immune System Profile

    PubMed Central

    Vescovini, Rosanna; Telera, Anna Rita; Pedrazzoni, Mario; Abbate, Barbara; Rossetti, Pietro; Verzicco, Ignazio; Arcangeletti, Maria Cristina; Medici, Maria Cristina; Calderaro, Adriana; Volpi, Riccardo; Sansoni, Paolo; Fagnoni, Francesco Fausto

    2016-01-01

    Human cytomegalovirus (HCMV) imprints the immune system after primary infection, however its effect during chronic infection still needs to be deciphered. In this study we report the variation of blood cell count along with anti-HCMV IgG and T cell responses to pp-65 and IE-1 antigens, that occurred after an interval of five years in a cohort of 25 seropositive healthy adults. We found increased anti-viral IgG antibody responses and intracellular interferon-gamma secreting CD8+ T cell responses to pp-65: a result consistent with memory inflation. With the only exception of shortage in naive CD8+ T cells most memory T cell subsets as well as total CD8+ T cells, T cells, lymphocytes, monocytes and leukocytes had increased. By contrast, none of the cell types tested were found to have increased in 14 subjects stably seronegative. Rather, in addition to a shortage in naive CD8+ T cells, also memory T cell subsets and most other cell types decreased, either in a statistically significant or non-significant manner. The trend of T cell pool representation with regard to CD4/CD8 ratio was in the opposing directions depending on HCMV serology. Globally, this study demonstrates different dynamic changes of most blood cell types depending on presence or absence of HCMV infection. Therefore, HCMV plays a continual role in modulating homeostasis of blood T cells and a broader expanding effect on other cell populations of lymphoid and myeloid origin. PMID:26990192

  10. Viperin Regulates Cellular Lipid Metabolism during Human Cytomegalovirus Infection

    PubMed Central

    Seo, Jun-Young; Cresswell, Peter

    2013-01-01

    Human cytomegalovirus (HCMV) has been shown to induce increased lipogenesis in infected cells, and this is believed to be required for proper virion envelopment. We show here that this increase is a consequence of the virus-induced redistribution of the host protein viperin to mitochondria and its capacity to interact with and block the function of the mitochondrial trifunctional protein (TFP), the enzyme that mediates fatty acid-β-oxidation. The resulting decrease in cellular ATP levels activates the enzyme AMP-activated protein kinase (AMPK), which induces expression of the glucose transporter GLUT4, resulting in increased glucose import and translocation to the nucleus of the glucose-regulated transcription factor ChREBP. This induces increased transcription of genes encoding lipogenic enzymes, increased lipid synthesis and lipid droplet accumulation, and generation of the viral envelope. Viperin-dependent lipogenesis is required for optimal production of infectious virus. We show that all of these metabolic outcomes can be replicated by direct targeting of viperin to mitochondria in the absence of HCMV infection, and that the motif responsible for Fe-S cluster binding by viperin is essential. The data indicate that viperin is the major effector underlying the ability of HCMV to regulate cellular lipid metabolism. PMID:23935494

  11. Resistance to antivirals in human cytomegalovirus: mechanisms and clinical significance.

    PubMed

    Pérez, J L

    1997-09-01

    Long term therapies needed for managing human cytomegalovirus (HCMV) infections in immunosupressed patients provided the background for the emergence of the resistance to antivirals active against HCMV. In addition, laboratory selected mutants have also been readily achieved. Both clinical and laboratory resistant strains share the same determinants of resistance. Ganciclovir resistance may be due to a few mutations in the HCMV UL97 gene and/or viral DNA pol gene, the former being responsible for about 70% of clinical resistant isolates. Among them, V464, V594, S595 and F595 are the most frequent mutations. Because of their less extensive clinical use, much less is known about resistance to foscarnet and cidofovir (formerly, HPMPC) but in both cases, it has been associated to mutations in the DNA pol. Ganciclovir resistant strains showing DNA pol mutations are cross-resistant to cidofovir and their corresponding IC50 are normally higher than those from strains harboring only mutations at the UL97 gene. To date, foscarnet resistance seems to be independent of both ganciclovir and cidofovir resistance.

  12. Human cytomegalovirus US3 and UL36-38 immediate-early proteins regulate gene expression.

    PubMed Central

    Colberg-Poley, A M; Santomenna, L D; Harlow, P P; Benfield, P A; Tenney, D J

    1992-01-01

    We have established the ability of the human cytomegalovirus (HCMV) UL36-38 and US3 immediate-early (IE) gene products to alter gene expression in human cells by using transient transfection assays. The cellular heat shock protein 70 (hsp70) promoter was transactivated following cotransfection with the HCMV IE regions in nonpermissive HeLa cells by UL36-38, US3, or IE1 and in permissive human diploid fibroblasts (HFF) by IE1 or IE2. Moreover, hsp70 expression was synergistically increased in HeLa cells cotransfected with US3 and UL36, with US3 and UL37, or with US3 and UL37x1. The synergistic transactivation of hsp70 expression by US3 and UL36-38 was not observed in HFF cells. Synergy was also not observed in HeLa cells between US3 and UL38, an early gene product encoded by the UL36-38 IE locus. Synergistic transactivation of hsp70 expression in HeLa cells required the syntheses of UL36-38 and US3 IE proteins, since nonsense mutants were not functional. hsp70 expression increased with increasing amounts of transfected US3 and UL37 DNA and occurred at the level of stable hsp70-promoted RNA. In contrast to the broad hsp70 response, promoters from the HCMV UL112 early gene and another cellular gene, brain creatine kinase, both responded strongly only to singly transfected IE2 in HeLa cells. Nevertheless, IE2 transactivation of the UL112 promoter was further stimulated by cotransfection of IE1 or of UL36-38 in both HeLa and HFF cells. Thus, different patterns of promoter transactivation and interactions between HCMV IE gene products in transactivation were found in HFF cells and in HeLa cells. These results establish the ability of the HCMV US3 and UL36-38 proteins to alter cellular and viral gene expression and are consistent with involvement of cellular transcription factors in HCMV IE regulation of gene expression. Images PMID:1370097

  13. Inhibition of Human Cytomegalovirus pUL89 Terminase Subunit Blocks Virus Replication and Genome Cleavage.

    PubMed

    Wang, Yan; Mao, Lili; Kankanala, Jayakanth; Wang, Zhengqiang; Geraghty, Robert J

    2017-02-01

    The human cytomegalovirus terminase complex cleaves concatemeric genomic DNA into unit lengths during genome packaging and particle assembly. This process is an attractive drug target because cleavage of concatemeric DNA is not required in mammalian cell DNA replication, indicating that drugs targeting the terminase complex could be safe and selective. One component of the human cytomegalovirus terminase complex, pUL89, provides the endonucleolytic activity for genome cleavage, and the domain responsible is reported to have an RNase H-like fold. We hypothesize that the pUL89 endonuclease activity is inhibited by known RNase H inhibitors. Using a novel enzyme-linked immunosorbent assay (ELISA) format as a screening assay, we found that a hydroxypyridonecarboxylic acid compound, previously reported to be an inhibitor of human immunodeficiency virus RNase H, inhibited pUL89 endonuclease activity at low-micromolar concentrations. Further characterization revealed that this pUL89 endonuclease inhibitor blocked human cytomegalovirus replication at a relatively late time point, similarly to other reported terminase complex inhibitors. Importantly, this inhibitor also prevented the cleavage of viral genomic DNA in infected cells. Taken together, these results substantiate our pharmacophore hypothesis and validate our ligand-based approach toward identifying novel inhibitors of pUL89 endonuclease. Human cytomegalovirus infection in individuals lacking a fully functioning immune system, such as newborns and transplant patients, can have severe and debilitating consequences. The U.S. Food and Drug Administration-approved anti-human cytomegalovirus drugs mainly target the viral polymerase, and resistance to these drugs has appeared. Therefore, anti-human cytomegalovirus drugs from novel targets are needed for use instead of, or in combination with, current polymerase inhibitors. pUL89 is a viral ATPase and endonuclease and is an attractive target for anti-human cytomegalovirus

  14. The host ubiquitin-dependent segregase VCP/p97 is required for the onset of human cytomegalovirus replication

    PubMed Central

    Lin, Yao-Tang; Grey, Finn

    2017-01-01

    The human cytomegalovirus major immediate early proteins IE1 and IE2 are critical drivers of virus replication and are considered pivotal in determining the balance between productive and latent infection. IE1 and IE2 are derived from the same primary transcript by alternative splicing and regulation of their expression likely involves a complex interplay between cellular and viral factors. Here we show that knockdown of the host ubiquitin-dependent segregase VCP/p97, results in loss of IE2 expression, subsequent suppression of early and late gene expression and, ultimately, failure in virus replication. RNAseq analysis showed increased levels of IE1 splicing, with a corresponding decrease in IE2 splicing following VCP knockdown. Global analysis of viral transcription showed the expression of a subset of viral genes is not reduced despite the loss of IE2 expression, including UL112/113. Furthermore, Immunofluorescence studies demonstrated that VCP strongly colocalised with the viral replication compartments in the nucleus. Finally, we show that NMS-873, a small molecule inhibitor of VCP, is a potent HCMV antiviral with potential as a novel host targeting therapeutic for HCMV infection. PMID:28494016

  15. In silico pattern-based analysis of the human cytomegalovirus genome.

    PubMed

    Rigoutsos, Isidore; Novotny, Jiri; Huynh, Tien; Chin-Bow, Stephen T; Parida, Laxmi; Platt, Daniel; Coleman, David; Shenk, Thomas

    2003-04-01

    More than 200 open reading frames (ORFs) from the human cytomegalovirus genome have been reported as potentially coding for proteins. We have used two pattern-based in silico approaches to analyze this set of putative viral genes. With the help of an objective annotation method that is based on the Bio-Dictionary, a comprehensive collection of amino acid patterns that describes the currently known natural sequence space of proteins, we have reannotated all of the previously reported putative genes of the human cytomegalovirus. Also, with the help of MUSCA, a pattern-based multiple sequence alignment algorithm, we have reexamined the original human cytomegalovirus gene family definitions. Our analysis of the genome shows that many of the coded proteins comprise amino acid combinations that are unique to either the human cytomegalovirus or the larger group of herpesviruses. We have confirmed that a surprisingly large portion of the analyzed ORFs encode membrane proteins, and we have discovered a significant number of previously uncharacterized proteins that are predicted to be G-protein-coupled receptor homologues. The analysis also indicates that many of the encoded proteins undergo posttranslational modifications such as hydroxylation, phosphorylation, and glycosylation. ORFs encoding proteins with similar functional behavior appear in neighboring regions of the human cytomegalovirus genome. All of the results of the present study can be found and interactively explored online (http://cbcsrv.watson.ibm.com/virus/).

  16. In Silico Pattern-Based Analysis of the Human Cytomegalovirus Genome

    PubMed Central

    Rigoutsos, Isidore; Novotny, Jiri; Huynh, Tien; Chin-Bow, Stephen T.; Parida, Laxmi; Platt, Daniel; Coleman, David; Shenk, Thomas

    2003-01-01

    More than 200 open reading frames (ORFs) from the human cytomegalovirus genome have been reported as potentially coding for proteins. We have used two pattern-based in silico approaches to analyze this set of putative viral genes. With the help of an objective annotation method that is based on the Bio-Dictionary, a comprehensive collection of amino acid patterns that describes the currently known natural sequence space of proteins, we have reannotated all of the previously reported putative genes of the human cytomegalovirus. Also, with the help of MUSCA, a pattern-based multiple sequence alignment algorithm, we have reexamined the original human cytomegalovirus gene family definitions. Our analysis of the genome shows that many of the coded proteins comprise amino acid combinations that are unique to either the human cytomegalovirus or the larger group of herpesviruses. We have confirmed that a surprisingly large portion of the analyzed ORFs encode membrane proteins, and we have discovered a significant number of previously uncharacterized proteins that are predicted to be G-protein-coupled receptor homologues. The analysis also indicates that many of the encoded proteins undergo posttranslational modifications such as hydroxylation, phosphorylation, and glycosylation. ORFs encoding proteins with similar functional behavior appear in neighboring regions of the human cytomegalovirus genome. All of the results of the present study can be found and interactively explored online (http://cbcsrv.watson.ibm.com/virus/). PMID:12634390

  17. Human cytomegalovirus function inhibits replication of herpes simplex virus

    SciTech Connect

    Cockley, K.D.; Shiraki, K.; Rapp, F.

    1988-01-01

    Human embryonic lung (HEL) cells infected with human cytomegalovirus (HCMV) restricted the replication of herpes simplex virus type 1 (HSV-1). A delay in HSV replication of 15 h as well as a consistent, almost 3 log inhibition of HSV replication in HCMV-infected cell cultures harvested 24 to 72 h after superinfection were observed compared with controls infected with HSV alone. Treatment of HCMV-infected HEL cells with cycloheximide (100 ..mu..g/ml) for 3 or 24 h was demonstrated effective in blocking HCMV protein synthesis, as shown by immunoprecipitation with HCMV antibody-positive polyvalent serum. Cycloheximide treatment of HCMV-infected HEL cells and removal of the cycloheximide block before superinfection inhibited HSV-1 replication more efficiently than non-drug-treated superinfected controls. HCMV DNA-negative temperature-sensitive mutants restricted HSV as efficiently as wild-type HCMV suggesting that immediate-early and/or early events which occur before viral DNA synthesis are sufficient for inhibition of HSV. Inhibition of HSV-1 in HCMV-infected HEL cells was unaffected by elevated temperature (40.5/sup 0/C). However, prior UV irradiation of HCMV removed the block to HSV replication, demonstrating the requirement for an active HCMV genome. HSV-2 replication was similarly inhibited in HCMV-infected HEL cells. Superinfection of HCMV-infected HEL cells with HSV-1 labeled with (/sup 3/H)thymidine provided evidence that the labeled virus could penetrate to the nucleus of cells after superinfection. Evidence for penetration of superinfecting HSV into HCMV-infected cells was also provided by blot hybridization of HSV DNA synthesized in cells infected with HSV alone versus superinfected cell cultures at 0 and 48 h after superinfection.

  18. Detection of human cytomegalovirus in normal and neoplastic breast epithelium

    PubMed Central

    2010-01-01

    Introduction Human cytomegalovirus (HCMV) establishes a persistent life-long infection, and can cause severe pathology in the fetus and the immunocompromised host[1]. Breast milk is the primary route of transmission in humans worldwide, and breast epithelium is thus a likely site of persistent infection and/or reactivation, though this phenomenon has not previously been demonstrated. Increasing evidence indicates HCMV infection can modulate signaling pathways associated with oncogenesis. We hypothesized that persistent HCMV infection occurs in normal adult breast epithelium and that persistent viral expression might be associated with normal and neoplastic ductal epithelium. Methods Surgical biopsy specimens of normal breast (n = 38) breast carcinoma (n = 39) and paired normal breast from breast cancer patients (n = 21) were obtained. Specimens were evaluated by immunohistochemistry, in situ hybridization, PCR and DNA sequencing for evidence of HCMV antigens and nucleic acids. Results We detected HCMV expression specifically in glandular epithelium in 17/27 (63%) of normal adult breast cases evaluated. In contrast, HCMV expression was evident in the neoplastic epithelium of 31/32 (97%) patients with ductal carcinoma in situ (DCIS) and infiltrating ductal carcinoma (IDC) cases evaluated (p = 0.0009). Conclusions These findings are the first to demonstrate that persistent HCMV infection occurs in breast epithelium in a significant percentage of normal adult females. HCMV expression was also evident in neoplastic breast epithelium in a high percentage of normal and neoplastic breast tissues obtained from breast cancer patients, raising the possibility that viral infection may be involved in the neoplastic process. PMID:21429243

  19. Direct Quantification of Human Cytomegalovirus Immediate-Early and Late mRNA Levels in Blood of Lung Transplant Recipients by Competitive Nucleic Acid Sequence-Based Amplification

    PubMed Central

    Greijer, Astrid E.; Verschuuren, Erik A. M.; Harmsen, Martin C.; Dekkers, Chantal A. J.; Adriaanse, Henriëtte M. A.; The, T. Hauw; Middeldorp, Jaap M.

    2001-01-01

    The dynamics of active human cytomegalovirus (HCMV) infection was monitored by competitive nucleic acid sequence-based amplification (NASBA) assays for quantification of IE1 (UL123) and pp67 (UL65) mRNA expression levels in the blood of patients after lung transplantation. RNA was isolated from 339 samples of 13 lung transplant recipients and analyzed by the quantitative IE1 and pp67 NASBA in parallel with pp65 antigenemia and serology. Rapid increases in IE1 RNA exceeding 104 copies per 100 μl of blood were associated with active infection, whereas lower levels were suggestive for abortive, subclinical viral activity. Any positive value for pp67 RNA was indicative for active infection, and quantification of pp67 mRNA did not give additional diagnostic information. The onset of IE1-positive NASBA preceded pp67 NASBA and was earlier than the pp65 antigenemia assay, confirming previous studies with qualitative NASBA. Effective antiviral treatment was reflected by a rapid disappearance of pp67 mRNA, whereas IE1 mRNA remained detectable for longer periods. Quantification of IE1 might be relevant to monitor progression of HCMV infection but should be validated in prospective studies. PMID:11136779

  20. Association between human cytomegalovirus and onset of epilepsy

    PubMed Central

    Lei, Hong-Yan; Yang, Dai-Qun; Li, Yu-Xin; Wang, Li-Quan; Zheng, Mei

    2015-01-01

    Objective: To explore the association between human cytomegalovirus (HCMV) and epilepsy. Methods: Epilepsy patients (n = 112) in neurology clinic of our hospital during January 2012 and December 2014 were allocated to the case groups, including intractable epilepsy group (n = 96) and non-intractable epilepsy group (n = 16). Healthy individual (n = 120) who received physical examination during the same period were allocated to the control group. The expression of serum HCMV late gene pp67-RNA was detected by reverse transcription-polymerase chain reaction (RT-PCR). The expressions of serum HCMV immunoglobulin G (IgG), immunoglobulin M (IgM) and interleukin-6 (IL-6) were detected by enzyme-linked immunosorbent assay (ELISA). Serum hypersensitive c-reactive protein (hs-CRP) was detected by latex-enhanced immunoturbidimetry. The electroencephalogram (EEG) of refractory epilepsy group, non-refractory epilepsy group and control group were recorded. Results: The expression of pp67-mRNA was significantly higher in intractable epilepsy group than non-intractable epilepsy group (P < 0.05) and control group (P < 0.001). The HCMV-IgG positive rate and HCMV-IgM positive rate were significantly higher in intractable epilepsy group than control group (both P < 0.001). The HCMV-IgM positive rate was significantly higher in intractable epilepsy group than non-intractable epilepsy group (P < 0.001). The HCMV-IgM positive rate was significantly higher in non-intractable epilepsy group than control group (P < 0.001). The hs-CRP and IL-6 levels presented descending trends respectively in intractable epilepsy group, non-intractable epilepsy group and control group (all P < 0.001). Conclusion: HCMV was prominently expressed in epilepsy and might contribute to the development of epilepsy. PMID:26884973

  1. Persistent Cytomegalovirus Infection in Amniotic Membranes of the Human Placenta.

    PubMed

    Tabata, Takako; Petitt, Matthew; Fang-Hoover, June; Zydek, Martin; Pereira, Lenore

    2016-11-01

    Human cytomegalovirus (HCMV) is the leading viral cause of birth defects, including microcephaly, neurological deficits, hearing impairment, and vision loss. We previously reported that epithelial cells in amniotic membranes of placentas from newborns with intrauterine growth restriction and underlying congenital HCMV infection contain viral proteins in cytoplasmic vesicles. Herein, we immunostained amniotic membranes from 51 placentas from symptomatic and asymptomatic congenital infection with HCMV DNA in amniotic fluid and/or newborn saliva, intrauterine growth restriction, preterm deliveries, and controls. We consistently observed HCMV proteins in amniotic epithelial cells (AmEpCs) from infected placentas, sometimes with aberrant morphology. Primary AmEpCs isolated from mid-gestation placentas infected with pathogenic VR1814 proliferated and released infectious progeny for weeks, producing higher virus titers than late-gestation cells that varied by donor. In contrast to intact virion assembly compartments in differentiated retinal pigment epithelial cells, infected AmEpCs made dispersed multivesicular bodies. Primary AmEpCs and explants of amniochorionic membranes from mid-gestation placentas formed foci of infection, and interferon-β production was prolonged. Infected AmEpCs up-regulated anti-apoptotic proteins survivin and Bcl-xL by mechanisms dependent and independent of the activated STAT3. Amniotic membranes naturally expressed both survivin and Bcl-xL, indicating that fetal membranes could foster persistent viral infection. Our results suggest strengthening innate immune responses and reducing viral functions could suppress HCMV infection in the fetal compartment. Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  2. Human fetal inner ear involvement in congenital cytomegalovirus infection

    PubMed Central

    2013-01-01

    Background Congenital cytomegalovirus (CMV) infection is a leading cause of sensorineural hearing loss (SNHL). The mechanisms of pathogenesis of CMV-related SNHL are still unclear. The aim is to study congenital CMV-related damage in the fetal inner ear, in order to better understand the underlying pathophysiology behind CMV-SNHL. Results We studied inner ears and brains of 20 human fetuses, all at 21 week gestational age, with a high viral load in the amniotic fluid, with and without ultrasound (US) brain abnormalities. We evaluated histological brain damage, inner ear infection, local inflammatory response and tissue viral load. Immunohistochemistry revealed that CMV was positive in 14/20 brains (70%) and in the inner ears of 9/20 fetuses (45%). In the cases with inner ear infection, the marginal cell layer of the stria vascularis was always infected, followed by infection in the Reissner’s membrane. The highest tissue viral load was observed in the inner ear with infected Organ of Corti. Vestibular labyrinth showed CMV infection of sensory cells in the utricle and in the crista ampullaris. US cerebral anomalies were detected in 6 cases, and in all those cases, the inner ear was always involved. In the other 14 cases with normal brain scan, histological brain damage was present in 8 fetuses and 3 of them presented inner ear infection. Conclusions CMV-infection of the marginal cell layer of the stria vascularis may alter potassium and ion circulation, dissipating the endocochlear potential with consequent SNHL. Although abnormal cerebral US is highly predictive of brain and inner ear damage, normal US findings cannot exclude them either. PMID:24252374

  3. Two novel human cytomegalovirus NK cell evasion functions target MICA for lysosomal degradation.

    PubMed

    Fielding, Ceri A; Aicheler, Rebecca; Stanton, Richard J; Wang, Eddie C Y; Han, Song; Seirafian, Sepehr; Davies, James; McSharry, Brian P; Weekes, Michael P; Antrobus, P Robin; Prod'homme, Virginie; Blanchet, Fabien P; Sugrue, Daniel; Cuff, Simone; Roberts, Dawn; Davison, Andrew J; Lehner, Paul J; Wilkinson, Gavin W G; Tomasec, Peter

    2014-05-01

    NKG2D plays a major role in controlling immune responses through the regulation of natural killer (NK) cells, αβ and γδ T-cell function. This activating receptor recognizes eight distinct ligands (the MHC Class I polypeptide-related sequences (MIC) A andB, and UL16-binding proteins (ULBP)1-6) induced by cellular stress to promote recognition cells perturbed by malignant transformation or microbial infection. Studies into human cytomegalovirus (HCMV) have aided both the identification and characterization of NKG2D ligands (NKG2DLs). HCMV immediate early (IE) gene up regulates NKGDLs, and we now describe the differential activation of ULBP2 and MICA/B by IE1 and IE2 respectively. Despite activation by IE functions, HCMV effectively suppressed cell surface expression of NKGDLs through both the early and late phases of infection. The immune evasion functions UL16, UL142, and microRNA(miR)-UL112 are known to target NKG2DLs. While infection with a UL16 deletion mutant caused the expected increase in MICB and ULBP2 cell surface expression, deletion of UL142 did not have a similar impact on its target, MICA. We therefore performed a systematic screen of the viral genome to search of addition functions that targeted MICA. US18 and US20 were identified as novel NK cell evasion functions capable of acting independently to promote MICA degradation by lysosomal degradation. The most dramatic effect on MICA expression was achieved when US18 and US20 acted in concert. US18 and US20 are the first members of the US12 gene family to have been assigned a function. The US12 family has 10 members encoded sequentially through US12-US21; a genetic arrangement, which is suggestive of an 'accordion' expansion of an ancestral gene in response to a selective pressure. This expansion must have be an ancient event as the whole family is conserved across simian cytomegaloviruses from old world monkeys. The evolutionary benefit bestowed by the combinatorial effect of US18 and US20 on MICA

  4. Bioactive Molecules Released From Cells Infected with the Human Cytomegalovirus

    PubMed Central

    Luganini, Anna; Terlizzi, Maria E.; Gribaudo, Giorgio

    2016-01-01

    Following primary infection in humans, the human cytomegalovirus (HCMV) persists in a latent state throughout the host’s lifetime despite a strong and efficient immune response. If the host experiences some form of immune dysregulation, such as immunosuppression or immunodeficiency, HCMV reactivates, thereby emerging from latency. Thus, in the absence of effective functional immune responses, as occurs in immunocompromised or immunoimmature individuals, both HCMV primary infections and reactivations from latency can cause significant morbidity and mortality. However, even in immunocompetent hosts, HCMV represents a relevant risk factor for the development of several chronic inflammatory diseases and certain forms of neoplasia. HCMV infection may shift between the lytic and latent state, regulated by a delicate and intricate balance between virus-mediated immunomodulation and host immune defenses. Indeed, HCMV is a master in manipulating innate and adaptive host defense pathways, and a large portion of its genome is devoted to encoding immunomodulatory proteins; such proteins may thus represent important virulence determinants. However, the pathogenesis of HCMV-related diseases is strengthened by the activities of bioactive molecules, of both viral and cellular origin, that are secreted from infected cells and collectively named as the secretome. Here, we review the state of knowledge on the composition and functions of HCMV-derived secretomes. In lytic infections of fibroblasts and different types of endothelial cells, the majority of HCMV-induced secreted proteins act in a paracrine fashion to stimulate the generation of an inflammatory microenvironment around infected cells; this may lead to vascular inflammation and angiogenesis that, in turn, foster HCMV replication and its dissemination through host tissues. Conversely, the HCMV secretome derived from latently infected hematopoietic progenitor cells induces an immunosuppressive extracellular environment that

  5. Burden of disease associated with human cytomegalovirus and prospects for elimination by universal immunisation.

    PubMed

    Griffiths, Paul D

    2012-10-01

    Cytomegalovirus is the most frequent cause of intrauterine infection and the commonest infectious agent to affect allograft recipients, yet the virus is acknowledged rarely as an occupational hazard for women of childbearing age or as a nosocomial infection. The potential role of cytomegalovirus in hastening the death of patients with AIDS, elderly people, individuals admitted to intensive-care units, and the general population is not emphasised. Development of vaccines against this important human pathogen has been delayed by reluctance to initiate proof-of-concept studies, but after recent trials, protection is a distinct possibility. Cytomegalovirus deserves to be eliminated from selected populations by means of universal immunisation as soon as suitable vaccines become licensed. This action should control disease in neonates and transplant recipients and could provide substantial additional benefits if other disease associations prove to be causal.

  6. Complete Genome Sequence of a Human Cytomegalovirus Strain AD169 Bacterial Artificial Chromosome Clone

    PubMed Central

    Ostermann, Eleonore; Spohn, Michael; Indenbirken, Daniela

    2016-01-01

    The complete sequence of the human cytomegalovirus strain AD169 (variant ATCC) cloned as a bacterial artificial chromosome (AD169-BAC, also known as HB15 or pHB15) was determined. The viral genome has a length of 230,290 bp and shows 52 nucleotide differences compared to a previously sequenced AD169varATCC clone. PMID:27034483

  7. 76 FR 69743 - The Development and Evaluation of Human Cytomegalovirus Vaccines; Public Workshop

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-09

    ... Vaccines; Public Workshop AGENCY: Food and Drug Administration, HHS. ACTION: Notice of public workshop. The... Prevention, and the National Vaccine Program Office are announcing a public workshop entitled ``The Development and Evaluation of Human Cytomegalovirus Vaccines.'' The purpose of the public workshop is...

  8. Detection of Human Cytomegalovirus DNA by Real-Time Quantitative PCR

    PubMed Central

    Nitsche, Andreas; Steuer, Nina; Schmidt, Christian Andreas; Landt, Olfert; Ellerbrok, Heinz; Pauli, Georg; Siegert, Wolfgang

    2000-01-01

    A real-time PCR assay was developed to quantify human cytomegalovirus (CMV) DNA. This assay was used to demonstrate a higher CMV DNA load in plasma of bone marrow transplant patients than in that of blood donors. The CMV load was higher in CMV antigen-positive patients than in antigen-negative patients. PMID:10878073

  9. Clinical evaluation of a chemiluminescence immunoassay for determination of immunoglobulin g avidity to human cytomegalovirus.

    PubMed

    Revello, Maria Grazia; Gorini, Giovanna; Gerna, Giuseppe

    2004-07-01

    Clinical evaluation of a novel fully automated chemiluminescence immunoassay for determination of immunoglobulin G avidity to human cytomegalovirus (HCMV) showed 92.8% sensitivity and 84.7% specificity in detecting a recent (< or =90 days) primary HCMV infection. The assay appears useful for accurately diagnosing recent primary HCMV infections.

  10. CMV pp65 and IE-1 T cell epitopes recognized by healthy subjects.

    PubMed

    Slezak, Stefanie L; Bettinotti, Maria; Selleri, Silvia; Adams, Sharon; Marincola, Francesco M; Stroncek, David F

    2007-03-28

    Adoptive immune and vaccine therapies have been used to prevent cytomegalovirus (CMV) disease in recipients of hematopoietic progenitor cell transplants, but the nature of T cell responses to CMV have not been completely characterized. Peptide pools and individual peptides derived from the immune-dominant CMV proteins pp65 and IE-1 and antigen-specific, cytokine flow cytometry were used to characterize the prevalence and frequency of CD4+ and CD8+ memory T cells in 20 healthy CMV-seropositive subjects. CD8+ T cell responses to pp65 were detected in 35% of subjects and to IE-1 in 40% of subjects. CD4+ T cell responses to pp65 were detected in 50% of subjects, but none were detected to IE-1. Several new IE-1 HLA class I epitopes were identified, including 4 restricted to HLA-C antigens. One region of IE-1 spanning amino acids 300 to 327 was rich in class I epitopes. The HLA class I restrictions of IE-1 peptides were more promiscuous than those of pp65 peptides. Since naturally occurring CD4+ and CD8+ T cell responses to pp65 were detectable in many subjects, but only CD8+ T cell responses to IE-1 were detected, pp65 may be better than IE-1 for use in vaccine and adoptive immune therapies.

  11. Human Cytomegalovirus Immediate-Early 1 Protein Rewires Upstream STAT3 to Downstream STAT1 Signaling Switching an IL6-Type to an IFNγ-Like Response

    PubMed Central

    Lukas, Simone; Zenger, Marion; Reitberger, Tobias; Danzer, Daniela; Übner, Theresa; Munday, Diane C.; Paulus, Christina

    2016-01-01

    The human cytomegalovirus (hCMV) major immediate-early 1 protein (IE1) is best known for activating transcription to facilitate viral replication. Here we present transcriptome data indicating that IE1 is as significant a repressor as it is an activator of host gene expression. Human cells induced to express IE1 exhibit global repression of IL6- and oncostatin M-responsive STAT3 target genes. This repression is followed by STAT1 phosphorylation and activation of STAT1 target genes normally induced by IFNγ. The observed repression and subsequent activation are both mediated through the same region (amino acids 410 to 445) in the C-terminal domain of IE1, and this region serves as a binding site for STAT3. Depletion of STAT3 phenocopies the STAT1-dependent IFNγ-like response to IE1. In contrast, depletion of the IL6 receptor (IL6ST) or the STAT kinase JAK1 prevents this response. Accordingly, treatment with IL6 leads to prolonged STAT1 instead of STAT3 activation in wild-type IE1 expressing cells, but not in cells expressing a mutant protein (IE1dl410-420) deficient for STAT3 binding. A very similar STAT1-directed response to IL6 is also present in cells infected with a wild-type or revertant hCMV, but not an IE1dl410-420 mutant virus, and this response results in restricted viral replication. We conclude that IE1 is sufficient and necessary to rewire upstream IL6-type to downstream IFNγ-like signaling, two pathways linked to opposing actions, resulting in repressed STAT3- and activated STAT1-responsive genes. These findings relate transcriptional repressor and activator functions of IE1 and suggest unexpected outcomes relevant to viral pathogenesis in response to cytokines or growth factors that signal through the IL6ST-JAK1-STAT3 axis in hCMV-infected cells. Our results also reveal that IE1, a protein considered to be a key activator of the hCMV productive cycle, has an unanticipated role in tempering viral replication. PMID:27387064

  12. Human cytomegalovirus and transplantation: drug development and regulatory issues.

    PubMed

    McIntosh, Megan; Hauschild, Benjamin; Miller, Veronica

    2016-07-01

    Cytomegalovirus (CMV) infection is highly prevalent worldwide and can cause serious disease among immunocompromised individuals, including persons with HIV and transplant recipients on immunosuppressive therapies. It can also result in congenital cytomegalovirus when women are infected during pregnancy. Treatment and prevention of CMV in solid organ and haematopoietic stem cell transplant recipients is accomplished in one of three ways: (1) prophylactic therapy to prevent CMV viraemia; (2) pre-emptive therapy for those with low levels of replicating virus; and (3) treatment for established disease. Despite the high prevalence of CMV, there are few available approved drug therapies, and those that are available are hampered by toxicity and less-than-optimal efficacy. New therapies are being developed and tested; however, inconsistency in standardisation of virus levels and questions about potential endpoints in clinical trials present regulatory hurdles that must be addressed. This review covers the current state of CMV therapy, drugs currently under investigation, and clinical trial issues and questions that are in need of resolution.

  13. An Fc receptor for human immunoglobulin G is located within the tegument of human cytomegalovirus.

    PubMed Central

    Stannard, L M; Hardie, D R

    1991-01-01

    Immunogold electron microscopy has demonstrated that human immunoglobulin G (IgG) can bind to the tegument of human cytomegalovirus virions by the Fc portion of the molecule. This binding was inhibited by preincubation of the Fc probes with protein A. Treatment of AD169 virions with Triton X-100 allowed release of the Fc-binding proteins, which were precipitated and characterized by polyacrylamide gel electrophoresis (PAGE). Polypeptides of approximately 69 and 33 kDa were recovered and shown by immunoblotting to retain their capacity to bind Fc-gold after separation under both reducing and nonreducing conditions. The combined results of blocking experiments, PAGE of precipitates, and Western blots (immunoblots) indicate that the tegument proteins which bind IgG-Fc are identical to those which bind beta 2 microglobulin. Images PMID:1851889

  14. Virion basic phosphoprotein from human cytomegalovirus contains O-linked N-acetylglucosamine.

    PubMed Central

    Benko, D M; Haltiwanger, R S; Hart, G W; Gibson, W

    1988-01-01

    A 149-kDa virion protein of human strains of cytomegalovirus is the principal acceptor for galactose added in vitro by bovine milk galactosyltransferase. Peptide comparisons with other biochemical characteristics of the galactosylated protein identified it as the virus-encoded basic phosphoprotein. This protein is an abundant constituent of the virion and is located in the tegument region, between the capsid and the envelope, rather than in the envelope layer with the recognized virion glycoproteins. The galactosylated carbohydrate was resistant to a commercial preparation of endoglycosidase F but was sensitive to removal by alkali-induced beta-elimination, indicating an O-linkage to the protein. Chromatographic and electrophoretic determinations identified the beta-eliminated material as the alditol of Gal beta 1-4GlcNAc, establishing that the human cytomegalovirus virion basic phosphoprotein contains single O-linked residues of N-acetylglucosamine. Images PMID:2833746

  15. Receptor expression and responsiveness of human peripheral blood mononuclear cells to a human cytomegalovirus encoded CC chemokine.

    PubMed

    Zheng, Qi; Xu, Jun; Gao, Huihui; Tao, Ran; Li, Wei; Shang, Shiqiang; Gu, Weizhong

    2015-01-01

    Human cytomegalovirus is a ubiquitous pathogen that infects the majority of the world's population. After long period of time co-evolving with human being, this pathogen has developed several strategies to evade host immune surveillance. One of the major trick is encoding homologous to those of the host organism or stealing host cellular genes that have significant functions in immune system. To date, we have found several viral immune analogous which include G protein coupled receptor, class I major histocompatibility complex and chemokine. Chemokine is a small group of molecules which is defined by the presence of four cysteines in highly conserved region. The four kinds of chemokines (C, CC, CXC, and CX3C) are classified based on the arrangement of 1 or 2 N-terminal cysteine residues. UL128 protein is one of the analogous that encoded by human cytomegalovirus that has similar amino acid sequences to the human CC chemokine. It has been proved to be one of the essential particles that involved in human cytomegalovirus entry into epithelial/endothelial cells as well as macrophages. It is also the target of potent neutralizing antibodies in human cytomegalovirus-seropositive individuals. We had demonstrated the chemotactic trait of UL128 protein in our previous study. Recombinant UL128 in vitro has the ability to attract monocytes to the infection region and enhances peripheral blood mononuclear cell proliferation by activating the MAPK/ERK signaling pathway. However, the way that this viral encoded chemokine interacting with peripheral blood mononuclear cells and the detailed mechanism that involving the virus entry into host cells keeps unknown. Here we performed in vitro investigation into the effects of UL128 protein on peripheral blood mononuclear cell's activation and receptor binding, which may help us further understand the immunomodulatory function of UL128 protein as well as human cytomegalovirus diffusion mechanism.

  16. Estrogen-related receptor α is required for efficient human cytomegalovirus replication

    PubMed Central

    Hwang, Jesse; Purdy, John G.; Wu, Kai; Rabinowitz, Joshua D.; Shenk, Thomas

    2014-01-01

    An shRNA-mediated screen of the 48 human nuclear receptor genes identified multiple candidates likely to influence the production of human cytomegalovirus in cultured human fibroblasts, including the estrogen-related receptor α (ERRα), an orphan nuclear receptor. The 50-kDa receptor and a 76-kDa variant were induced posttranscriptionally following infection. Genetic and pharmacological suppression of the receptor reduced viral RNA, protein, and DNA accumulation, as well as the yield of infectious progeny. In addition, RNAs encoding multiple metabolic enzymes, including enzymes sponsoring glycolysis (enolase 1, triosephosphate isomerase 1, and hexokinase 2), were reduced when the function of ERRα was inhibited in infected cells. Consistent with the effect on RNAs, a substantial number of metabolites, which are normally induced by infection, were either not increased or were increased to a reduced extent in the absence of normal ERRα activity. We conclude that ERRα is needed for the efficient production of cytomegalovirus progeny, and we propose that the nuclear receptor contributes importantly to the induction of a metabolic environment that supports optimal cytomegalovirus replication. PMID:25512541

  17. Host cellular annexin II is associated with cytomegalovirus particles isolated from cultured human fibroblasts.

    PubMed Central

    Wright, J F; Kurosky, A; Pryzdial, E L; Wasi, S

    1995-01-01

    A significant amount of host cellular annexin II was found to be associated with human cytomegalovirus isolated from cultured human fibroblasts (approximately 1,160 molecules per virion). This composition was established by four different analytical approaches that included (i) Western blot (immunoblot) analysis of gradient-purified virions with a monoclonal antibody specific for annexin II, (ii) peptide mapping and sequence analysis of virus-associated proteins and proteins dissociated from virus following EDTA treatment, (iii) electron microscopic immunocytochemistry of gradient-purified virions, and (iv) labeling of virus-associated proteins by lactoperoxidase-catalyzed radioiodination. These results indicated that annexin II was primarily localized to the viral surface, where it bound in a divalent cation-dependent manner. In functional experiments, a rabbit antiserum raised against annexin II inhibited cytomegalovirus plaque formation in human foreskin fibroblast monolayers in a concentration-dependent manner. Cumulatively, these studies demonstrate an association of host annexin II with cytomegalovirus particles and provide evidence for the involvement of this cellular protein in virus infectivity. PMID:7609045

  18. Human cytomegalovirus infection and colorectal cancer risk: a meta-analysis

    PubMed Central

    Bai, Bingjun; Wang, Xingxing; Chen, Engeng; Zhu, Hongbo

    2016-01-01

    Human cytomegalovirus infection (HCMV) has been recently considered as a factor for tumorigenesis. The current study used meta-analytical techniques to explore the prevalence of HCMV in tumor tissues and the relationship between human cytomegalovirus and colorectal cancer (CRC) risk. 11 studies detecting HCMV DNA in tumor tissues were included in meta-analysis. The prevalence rate and odds ratio (OR) were two main parameters. The overall prevalence of human cytomegalovirus DNA in tumor tissues were 27.5% (95% CI = 17.2%−37.8%). Binary logistic regression showed that the studies reported before 2010 involving formalin-fixed specimens from patients in developed region represented a lower proportion of HCMV. The tumor tissues had a significantly higher rate of virus infection compared with normal tissues (OR = 6.59, 95% CI = 4.48−9.69, I2 = 0%, P = 0.71). Subgroup analysis revealed the prevalence of the virus didn't differ in patients with different tumor stages, in tumor cells with different histologic grades, also in different kinds of specimen (polyp and adenocarcinoma). The results of current study suggested a statistically association between the virus infection and an increased risk of colorectal cancer. PMID:27732934

  19. Transformation of NIH 3T3 cells with cloned fragments of human cytomegalovirus strain AD169.

    PubMed Central

    Nelson, J A; Fleckenstein, B; Galloway, D A; McDougall, J K

    1982-01-01

    NIH 3T3 cells were transfected with restriction endonuclease and cloned human cytomegalovirus DNA fragments to identify the transforming region(s). Cleavage of human cytomegalovirus strain AD169 DNA with XbaI and HindIII left a transforming region intact whereas EcoRI inactivated this function. Transfection of cells with cosmids containing human cytomegalovirus DNA spanning the entire genome resulted in transformation by one cosmid, pCM1058, with the AD169 HindIII DNA fragments E, R, T, and a'. Cells were selected for their growth in 1.2% methylcellulose. The clones isolated had a significant replating efficiency and were oncogenic in BALB/c nu/nu mice. Transfection of cosmids and plasmids containing subsets of the viral sequences in pCM1058 identified a common region possessed by all of the transforming recombinant molecules. This region was in the HindIII E fragment with the left boundary defined by the EcoRI d-R junction and the right boundary defined by the HindIII E-T junction. Further mapping and transfection experiments determined that the transforming region was contained without a 2.9-kilobase fragment between map units 0.123 and 0.14 on the prototype molecule of the AD169 strain. Images PMID:6287019

  20. Modulation of the Host Environment by Human Cytomegalovirus with Viral Interleukin 10 in Peripheral Blood.

    PubMed

    Young, Vivian P; Mariano, Margarette C; Tu, Carolyn C; Allaire, Kathryn M; Avdic, Selmir; Slobedman, Barry; Spencer, Juliet V

    2017-03-15

    Human cytomegalovirus (HCMV) is a herpesvirus with both lytic and latent life cycles. Human cytomegalovirus encodes 2 viral cytokines that are orthologs of human cellular interleukin 10 (cIL-10). Both cytomegalovirus interleukin 10 (cmvIL-10) and Latency-associated cytomegalovirus interleukin 10 (LAcmvIL-10) (collectively vIL-10) are expressed during lytic infection and cause immunosuppressive effects that impede virus clearance. LAcmvIL-10 is also expressed during latent infection of myeloid progenitor cells and monocytes and facilitates persistence. Here, we investigated whether vIL-10 could be detected during natural infection. Plasma from healthy blood donors was tested by enzyme-linked immunosorbent assay for anti-HCMV immunoglobulin G and immunoglobulin M and for cIL-10 and vIL-10 levels using a novel vIL-10 assay that detects cmvIL-10 and LAcmvIL-10, with no cross-reactivity to cIL-10. vIL-10 was evident in HCMV+ donors (n = 19 of 26), at levels ranging 31-547 pg/mL. By comparison, cIL-10 was detected at lower levels ranging 3-69 pg/mL. There was a strong correlation between vIL-10 and cIL-10 levels (P = .01). Antibodies against vIL-10 were also detected and neutralized vIL-10 activity. vIL-10 was detected in peripheral blood of healthy blood donors. These findings suggest that vIL-10 may play a key role in sensing or modifying the host environment during latency and, therefore, may be a potential target for intervention strategies.

  1. Molecular Detection of Human Cytomegalovirus (HCMV) Among Infants with Congenital Anomalies in Khartoum State, Sudan

    PubMed Central

    Ebrahim, Maha G.; Ali, Aisha S.; Mustafa, Mohamed O.; Musa, Dalal F.; El Hussein, Abdel Rahim M.; Elkhidir, Isam M.; Enan, Khalid A.

    2015-01-01

    Human Cytomegalovirus (HCMV) infection still represents the most common potentially serious viral complication in humans and is a major cause of congenital anomalies in infants. This study is aimed to detect HCMV in infants with congenital anomalies. Study subjects consisted of infants born with neural tube defect, hydrocephalus and microcephaly. Fifty serum specimens (20 males, 30 females) were collected from different hospitals in Khartoum State. The sera were investigated for cytomegalovirus specific immunoglobin M (IgM) antibodies using enzyme-linked immunosorbent assay (ELISA), and for Cytomegalovirus DNA using polymerase chain reaction (PCR). Out of the 50 sera tested, one patient’s (2%) sample showed HCMV IgM, but with no detectable DNA, other 4(8.2 %) sera were positive for HCMV DNA but with no detectable IgM. Various diagnostic techniques should be considered to evaluate HCMV disease and routine screening for HCMV should be introduced for pregnant women in this setting. It is vital to initiate further research work with many samples from different area to assess prevalence and characterize HCMV and evaluate its maternal health implications. PMID:26862356

  2. An intein-mediated modulation of protein stability system and its application to study human cytomegalovirus essential gene function

    PubMed Central

    Pan, Deng; Xuan, Baoqin; Sun, Yamei; Huang, Shaowu; Xie, Maorong; Bai, Yadan; Xu, Wenjia; Qian, Zhikang

    2016-01-01

    Functional analysis of the essential proteins encoded by human cytomegalovirus (HCMV) is hindered by the lack of complementing systems. To overcome this difficulty, we have established a novel approach, termed the intein-mediated modulation of protein stability (imPS), in which a destabilizing domain and part of a split intein are fused to the essential protein. The growth of the mutant virus can then be regulated by the degradation and splicing of the protein. We found that an ultrafast gp41-1 split intein was able to rescue or degrade the protein of interest (POI) by removing or adding a strong degron through protein splicing. As a result, the function of the POI was turned on or off during the process. Using HCMV essential gene IE1/IE2, we confirmed that imPS worked remarkably well in conditionally regulating protein stability during viral infection. This conditional approach is likely to be applicable for dissecting the gene functions of HCMV or other viruses. PMID:27188239

  3. Human Cytomegalovirus Infection Enhances NK Cell Activity In Vitro

    PubMed Central

    Tschan-Plessl, Astrid; Stern, Martin; Schmied, Laurent; Retière, Christelle; Hirsch, Hans H.; Garzoni, Christian; van Delden, Christian; Boggian, Katia; Mueller, Nicolas J.; Berger, Christoph; Villard, Jean; Manuel, Oriol; Meylan, Pascal; Terszowski, Grzegorz

    2016-01-01

    Background Occurring frequently after solid organ and hematopoietic stem cell transplantation, cytomegalovirus (CMV) replication remains a relevant cause of mortality and morbidity in affected patients. Despite these adverse effects, an increased alloreactivity of natural killer (NK) cells after CMV infection has been assumed, but the underlying physiopathological mechanisms have remained elusive. Methods We used serial analyses of NK cells before and after CMV infection in kidney transplant recipients as an in vivo model for CMV primary infection to explore the imprint of CMV infection using every patient as their own control: We analyzed NK cell phenotype and function in 47 CMV seronegative recipients of CMV seropositive kidney grafts, who developed CMV primary infection posttransplant. Seronegative recipients of seronegative kidney grafts served as controls. Results We observed a significant increase of NKG2C expressing NK cells after CMV infection (mean increase, 17.5%; 95% confidence interval [95% CI], 10.2-24.9, P < 0.001), whereas cluster of differentiation (CD)57 expressing cells decreased (mean decrease, 14.1%; 95% CI, 8.0-20.2; P < 0.001). Analysis of killer immunoglobulin-like receptor (KIR) expression showed an increase of cells expressing KIR2DL1 as their only inhibitory KIR in patients carrying the cognate ligand HLA-C2 (mean increase, 10.0%; 95% CI, 1.7-18.3; P = 0.018). In C2-negative individuals, KIR2DL1 expression decreased (mean decrease, 3.9%; 95% CI, 1.6-6.2; P = 0.001). As for activating KIR, there was no conclusive change pattern. Most importantly, we observed a significantly higher NK cell degranulation and IFNγ production in response to different target cells (target K562, CD107a: mean increase, 9.9%; 95% CI, 4.8-15.0; P < 0.001; IFNγ: mean increase, 6.6%; 95% CI, 1.6-11.1; P < 0.001; target MRC-5, CD107a: mean increase, 6.9%; 95% CI, 0.7-13.1; P = 0.03; IFNγ: mean increase, 4.8%; 95% CI, 1.7-7.8; P = 0.002). Conclusions We report

  4. Probable neuroimmunological link between Toxoplasma and cytomegalovirus infections and personality changes in the human host

    PubMed Central

    Novotná, Martina; Hanusova, Jitka; Klose, Jiří; Preiss, Marek; Havlicek, Jan; Roubalová, Kateřina; Flegr, Jaroslav

    2005-01-01

    Background Recently, a negative association between Toxoplasma-infection and novelty seeking was reported. The authors suggested that changes of personality trait were caused by manipulation activity of the parasite, aimed at increasing the probability of transmission of the parasite from an intermediate to a definitive host. They also suggested that low novelty seeking indicated an increased level of the neurotransmitter dopamine in the brain of infected subjects, a phenomenon already observed in experimentally infected rodents. However, the changes in personality can also be just a byproduct of any neurotropic infection. Moreover, the association between a personality trait and the toxoplasmosis can even be caused by an independent correlation of both the probability of Toxoplasma-infection and the personality trait with the third factor, namely with the size of living place of a subject. To test these two alternative hypotheses, we studied the influence of another neurotropic pathogen, the cytomegalovirus, on the personality of infected subjects, and reanalyzed the original data after the effect of the potential confounder, the size of living place, was controlled. Methods In the case-control study, 533 conscripts were tested for toxoplasmosis and presence of anti-cytomegalovirus antibodies and their novelty seeking was examined with Cloninger's TCI questionnaire. Possible association between the two infections and TCI dimensions was analyzed. Results The decrease of novelty seeking is associated also with cytomegalovirus infection. After the size of living place was controlled, the effect of toxoplasmosis on novelty seeking increased. Significant difference in novelty seeking was observed only in the largest city, Prague. Conclusion Toxoplasma and cytomegalovirus probably induce a decrease of novelty seeking. As the cytomegalovirus spreads in population by direct contact (not by predation as with Toxoplasma), the observed changes are the byproduct of brain

  5. Examination of brains of AIDS cases for human immunodeficiency virus and human cytomegalovirus nucleic acids.

    PubMed Central

    Walker, D G; Itagaki, S; Berry, K; McGeer, P L

    1989-01-01

    The role of direct virus infection as a determining factor in acquired immunodeficiency syndrome (AIDS) dementia was investigated using in situ hybridisation for human immunodeficiency virus (HIV) and human cytomegalovirus (HCMV). Four of the five AIDS dementia patients in this series demonstrated HIV infected cells distributed in widely different parts of the brain, but only one case showed HCMV infected cells. The greater abundance of HIV was in subcortical white matter in nodular areas consisting of monocyte/macrophage infiltrates. The cells were occasionally arranged as a multinucleated syncitium. In two cases, a few large cells with the appearance of neurons were positive for HIV hybridisation. By appropriate treatment with ribonuclease, it was shown that hybridisation was primarily to HIV RNA. HCMV infected cells were observed in small numbers in only one of the positive cases, suggesting that HCMV is not a determining factor in AIDS dementia. HCMV positive cells were located in the grey matter, with an appearance suggestive of neurons. Cells expressing the MHC-class II antigen HLA-DR, a marker of reactive microglia and macrophages, were observed to be extensive in affected brain sections in the one case examined. These cells were present in greater number than HIV infected cells. In this case, extensive numbers of HIV infected cells were noticed along the peripheral margin of the substantia innominata. This could indicate infection in this case of a critical brain region from the cerebrospinal fluid. Images PMID:2543795

  6. Systematic MicroRNA Analysis Identifies ATP6V0C as an Essential Host Factor for Human Cytomegalovirus Replication

    PubMed Central

    Pavelin, Jon; Reynolds, Natalie; Chiweshe, Stephen; Wu, Guanming; Tiribassi, Rebecca; Grey, Finn

    2013-01-01

    Recent advances in microRNA target identification have greatly increased the number of putative targets of viral microRNAs. However, it is still unclear whether all targets identified are biologically relevant. Here, we use a combined approach of RISC immunoprecipitation and focused siRNA screening to identify targets of HCMV encoded human cytomegalovirus that play an important role in the biology of the virus. Using both a laboratory and clinical strain of human cytomegalovirus, we identify over 200 putative targets of human cytomegalovirus microRNAs following infection of fibroblast cells. By comparing RISC-IP profiles of miRNA knockout viruses, we have resolved specific interactions between human cytomegalovirus miRNAs and the top candidate target transcripts and validated regulation by western blot analysis and luciferase assay. Crucially we demonstrate that miRNA target genes play important roles in the biology of human cytomegalovirus as siRNA knockdown results in marked effects on virus replication. The most striking phenotype followed knockdown of the top target ATP6V0C, which is required for endosomal acidification. siRNA knockdown of ATP6V0C resulted in almost complete loss of infectious virus production, suggesting that an HCMV microRNA targets a crucial cellular factor required for virus replication. This study greatly increases the number of identified targets of human cytomegalovirus microRNAs and demonstrates the effective use of combined miRNA target identification and focused siRNA screening for identifying novel host virus interactions. PMID:24385903

  7. Severe Thrombocytopenia and Acute Cytomegalovirus Colitis during Primary Human Immunodeficiency Virus Infection

    PubMed Central

    Furuhata, Masanori; Yanagisawa, Naoki; Nishiki, Shingo; Sasaki, Shugo; Suganuma, Akihiko; Imamura, Akifumi; Ajisawa, Atsushi

    2016-01-01

    We herein report the case of a 25-year-old man who was referred to our hospital due to acute cytomegalovirus (CMV) colitis. The initial blood tests showed that the patient had concurrent primary human immunodeficiency virus (HIV) infection and severe thrombocytopenia. Raltegravir-based antiretroviral therapy (ART) was initiated without the use of ganciclovir or corticosteroids and resulted in a rapid clinical improvement. Platelet transfusions were only necessary for a short period, and subsequent colonoscopy revealed a completely healed ulcer. This case implies that ART alone could be effective for treating severe thrombocytopenia during primary HIV and CMV coinfection. PMID:27980271

  8. Detection of Human Cytomegalovirus and Epstein-Barr Virus in Coronary Atherosclerotic Tissue

    PubMed Central

    Imbronito, Ana Vitória; Marcelino, Silvia Linardi; Grande, Sabrina Rosa; Nunes, Fabio Daumas; Romito, Giuseppe Alexandre

    2010-01-01

    Previous studies indicated that patients with atherosclerosis are predominantly infected by human cytomegalovirus (HCMV), but rarely infected by type 1 Epstein-Barr virus (EBV-1). In this study, atheromas of 30 patients who underwent aortocoronary bypass surgery with coronary endartherectomy were tested for the presence of these two viruses. HCMV occurred in 93.3% of the samples and EBV-1 was present in 50% of them. Concurrent presence of both pathogens was detected in 43.3% of the samples. PMID:24031529

  9. The DNA damage response induced by infection with human cytomegalovirus and other viruses.

    PubMed

    Xiaofei, E; Kowalik, Timothy F

    2014-05-23

    Viruses use different strategies to overcome the host defense system. Recent studies have shown that viruses can induce DNA damage response (DDR). Many of these viruses use DDR signaling to benefit their replication, while other viruses block or inactivate DDR signaling. This review focuses on the effects of DDR and DNA repair on human cytomegalovirus (HCMV) replication. Here, we review the DDR induced by HCMV infection and its similarities and differences to DDR induced by other viruses. As DDR signaling pathways are critical for the replication of many viruses, blocking these pathways may represent novel therapeutic opportunities for the treatment of certain infectious diseases. Lastly, future perspectives in the field are discussed.

  10. Human cytomegalovirus latency-associated protein LUNA is expressed during HCMV infections in vivo.

    PubMed

    Bego, Mariana G; Keyes, Lisa R; Maciejewski, Jarek; St Jeor, Stephen C

    2011-10-01

    Human cytomegalovirus (HCMV) latency is poorly understood. We previously described a novel HCMV latency-associated transcript, UL81-82ast, coding for a protein designated LUNA (latency unique natural antigen). The aim of this study was to confirm the presence of LUNA in HCMV-seropositive donors. Standard co-immunoprecipitation and ELISA assays were used to detect antibodies against the LUNA protein in the sera of HCMV-seropositive donors. Specific antibodies against LUNA were detected in all HCMV-seropositive donors but in none of the seronegative donors. These data confirm that LUNA is expressed during in vivo infections and is capable of eliciting an immune response.

  11. Clinical and biologic aspects of human cytomegalovirus resistance to antiviral drugs.

    PubMed

    Baldanti, Fausto; Lurain, Nell; Gerna, Giuseppe

    2004-05-01

    The emergence of human cytomegalovirus (HCMV) drug resistant strains is a life-threatening condition in immunocompromised individuals with active HCMV infection. HCMV drug resistance represented a major problem in patients with acquired immunodeficiency syndrome until the recent introduction of highly active antiretroviral combination therapy, which dramatically decreased the incidence in this clinical setting. However, HCMV resistance to antiviral drugs is now an emerging problem in the transplantation setting. The molecular mechanisms of HCMV drug resistance have been elucidated and rely on the selection during treatment of HCMV strains harboring mutations in two key viral genes: UL97 coding for a viral phosphotransferase and UL54 coding for the viral DNA polymerase.

  12. Human cytomegalovirus resistance to antiviral drugs: diagnosis, monitoring and clinical impact.

    PubMed

    Baldanti, Fausto; Gerna, Giuseppe

    2003-09-01

    The incidence of human cytomegalovirus (HCMV) disease in AIDS patients decreased dramatically after the introduction, a few years ago, of highly active antiretroviral combination therapy. As a consequence, the emergence of drug-resistant HCMV strains is no longer a major problem in HIV-infected individuals. However, HCMV resistance to antiviral drugs is presently recognized as an emerging problem in transplantation settings. The mechanisms of HCMV drug resistance will be analysed along with the clinical features relevant to the emergence of drug-resistant HCMV strains during antiviral treatment of patients receiving either solid organ or haematopoietic stem cell transplantation.

  13. CYTOMEGALOVIRUS RETINITIS ASSOCIATED WITH OCCLUSIVE VASCULOPATHY IN AN ELDERLY, HUMAN IMMUNODEFICIENCY VIRUS-NEGATIVE MAN.

    PubMed

    Moussa, Kareem; Doan, Thuy; Stewart, Jay M; Shantha, Jessica; Gonzales, John; Acharya, Nisha; Cunningham, Emmett T

    2017-09-20

    To present a case of cytomegalovirus (CMV) retinitis associated with occlusive vasculopathy presenting as sudden unilateral loss of vision in a human immunodeficiency virus-negative elderly man. Clinical case report and literature review. An 84-year-old Chinese man with diabetes mellitus and primary open-angle glaucoma was seen in consultation by our uveitis service for evaluation of sudden vision loss in the right eye. Examination revealed an occlusive retinal vasculopathy. An extensive diagnostic workup was performed, including fluorescein angiography, serologic testing for infectious etiologies including syphilis and tuberculosis and a temporal artery biopsy. The patient was treated with high-dose oral prednisone, after which the biopsy returned negative for giant-cell arteritis. Three weeks after initial presentation, the patient was noted to have a new area of retinitis in the temporal periphery. An anterior chamber paracentesis was performed, and the fluid was sent for directed polymerase chain reaction testing, which returned positive for CMV. Human immunodeficiency virus testing was negative. He was treated with oral valganciclovir and intravitreal foscarnet injections and the infection subsequently resolved. Cytomegalovirus infection can be associated with occlusive vasculopathy in human immunodeficiency virus-negative individuals. The diagnosis of CMV retinitis should be considered in patients with human immunodeficiency virus-negative who have other conditions that may compromise immune function, particularly advanced age, diabetes mellitus, malignancy, or use of immunosuppressive agents.

  14. Recombinant Human Cytomegalovirus (HCMV) RL13 Binds Human Immunoglobulin G Fc

    PubMed Central

    Cortese, Mirko; Calò, Stefano; D'Aurizio, Romina; Lilja, Anders; Pacchiani, Nicola; Merola, Marcello

    2012-01-01

    The human cytomegalovirus (HCMV) protein RL13 has recently been described to be present in all primary isolates but rapidly mutated in culture adapted viruses. Although these data suggest a crucial role for this gene product in HCMV primary infection, no function has so far been assigned to this protein. Working with RL13 expressed in isolation in transfected human epithelial cells, we demonstrated that recombinant RL13 from the clinical HCMV isolates TR and Merlin have selective human immunoglobulin (Ig)-binding properties towards IgG1 and IgG2 subtypes. An additional Fc binding protein, RL12, was also identified as an IgG1 and IgG2 binding protein but not further characterized. The glycoprotein RL13 trafficked to the plasma membrane where it bound and internalized exogenous IgG or its constant fragment (Fcγ). Analysis of RL13 ectodomain mutants suggested that the RL13 Ig-like domain is responsible for the Fc binding activity. Ligand-dependent internalization relied on a YxxL endocytic motif located in the C-terminal tail of RL13. Additionally, we showed that the tyrosine residue could be replaced by phenylalanine but not by alanine, indicating that the internalization signal was independent from phosphorylation events. In sum, RL13 binds human IgG and may contribute to HCMV immune evasion in the infected host, but this function does not readily explain the instability of the RL13 gene during viral propagation in cultured cells. PMID:23226246

  15. Sequestration of human cytomegalovirus by human renal and mammary epithelial cells

    SciTech Connect

    Twite, Nicolas; Andrei, Graciela; Kummert, Caroline; Donner, Catherine; Perez-Morga, David; De Vos, Rita; Snoeck, Robert; Marchant, Arnaud

    2014-07-15

    Urine and breast milk represent the main routes of human cytomegalovirus (HCMV) transmission but the contribution of renal and mammary epithelial cells to viral excretion remains unclear. We observed that kidney and mammary epithelial cells were permissive to HCMV infection and expressed immediate early, early and late antigens within 72 h of infection. During the first 24 h after infection, high titers of infectious virus were measured associated to the cells and in culture supernatants, independently of de novo synthesis of virus progeny. This phenomenon was not observed in HCMV-infected fibroblasts and suggested the sequestration and the release of HCMV by epithelial cells. This hypothesis was supported by confocal and electron microscopy analyses. The sequestration and progressive release of HCMV by kidney and mammary epithelial cells may play an important role in the excretion of the virus in urine and breast milk and may thereby contribute to HCMV transmission. - Highlights: • Primary renal and mammary epithelial cells are permissive to HCMV infection. • HCMV is sequestered by epithelial cells and this phenomenon does not require viral replication. • HCMV sequestration by epithelial cells is reduced by antibodies and IFN-γ.

  16. Promoter-specific trans activation and repression by human cytomegalovirus immediate-early proteins involves common and unique protein domains.

    PubMed Central

    Stenberg, R M; Fortney, J; Barlow, S W; Magrane, B P; Nelson, J A; Ghazal, P

    1990-01-01

    trans activation of promoters by viral regulatory proteins provides a useful tool to study coordinate control of gene expression. Immediate-early (IE) regions 1 and 2 of human cytomegalovirus (CMV) code for a series of proteins that originate from differentially spliced mRNAs. These IE proteins are proposed to regulate the temporal expression of the viral genome. To examine the structure and function of the IE proteins, we used linker insertion mutagenesis of the IE gene region as well as cDNA expression vector cloning of the abundant IE mRNAs. We showed that IE1 and IE2 proteins of CMV exhibit promoter-specific differences in their modes of action by either trans activating early and IE promoters or repressing the major IE promoter (MIEP). Transient cotransfection experiments with permissive human cells revealed a synergistic interaction between the 72- and the 86-kilodalton (kDa) IE proteins in trans activating an early promoter. In addition, transfection studies revealed that the 72-kDa protein was capable of trans activating the MIEP. In contrast, the 86-kDa protein specifically repressed the MIEP and this repression was suppressed by the 72-kDa protein. Furthermore, observations based on the primary sequence structure revealed a modular arrangement of putative regulatory motifs that could either potentiate or repress gene expression. These modular domains are either shared or unique among the IE proteins. From these data, we propose a model for IE protein function in the coordinate control of CMV gene expression. Images PMID:2157043

  17. Sumoylation of the major immediate-early IE2 protein of human cytomegalovirus Towne strain is not required for virus growth in cultured human fibroblasts.

    PubMed

    Lee, Hye-Ra; Ahn, Jin-Hyun

    2004-08-01

    Sumoylation of the major immediate-early IE2 protein of human cytomegalovirus has been shown to increase transactivation activity in target reporter gene assays. This study examined the role of IE2 sumoylation in viral infection. A Towne strain-based bacterial artificial chromosome clone was generated encoding a mutated form of the IE2 protein with Lys-->Arg substitutions at positions 175 and 180, the two major sumoylation sites. When human fibroblast (HF) cells were infected with the reconstituted mutant virus, (i) viral growth kinetics, (ii) the accumulation of IE1 (UL123), IE2 (UL122), p52 (UL44) and pp65 (UL83) proteins and (iii) the relocalization of the cellular small ubiquitin-like modifier (SUMO)-1, p53 and proliferating cell nuclear antigen proteins into viral DNA replication compartments were comparable with those of the wild-type and the revertant virus. The data demonstrate that sumoylation of IE2 is not essential for virus growth in cultured HF cells.

  18. Congenital cytomegalovirus

    MedlinePlus

    ... Churchill Livingstone; 2014:chap 140. Swanson EC. Congenital cytomegalovirus infection: new prospects for prevention and therapy. Pediatr Clin ... and the A.D.A.M. Editorial team. Cytomegalovirus Infections Read more Latest Health News Read more Health ...

  19. Analysis of human cytomegalovirus replication in primary cultured human corneal endothelial cells

    PubMed Central

    Hosogai, Mayumi; Shima, Nobuyuki; Nakatani, Yoko; Inoue, Teruki; Iso, Tatsuya; Yokoo, Hideaki; Yorifuji, Hiroshi; Akiyama, Hideo; Kishi, Shoji; Isomura, Hiroki

    2015-01-01

    Background/aims Since the first case of human cytomegalovirus (HCMV)-induced corneal endotheliitis in which HCMV DNA was detected from the patient's aqueous humour using PCR, the clinical evidence for HCMV endotheliitis has been accumulating. However, it remains to be confirmed whether HCMV can efficiently replicate in corneal endothelial cells. We, therefore, sought to determine whether primary cultured human corneal endothelial cells (HCECs) could support HCMV replication. Methods Human foreskin fibroblasts (HFFs) have been shown to be fully permissive for HCMV replication, and are commonly used as an in vitro model for HCMV lytic replication. Therefore, primary cultured HCECs or HFFs were infected with the vascular endotheliotropic HCMV strain TB40/E or laboratory strain Towne. We then compared viral mRNA and protein expression, genome replication and growth between the TB40/E-infected and Towne-infected HCECs and HFFs. Results When HCECs were infected with TB40/E or Towne, rounded cells resembling owl's eyes as well as viral antigens were detected. Viral mRNA synthesis and protein expression proceeded efficiently in the HCECs and HFFs infected with TB40/E or Towne at a high multiplicity of infection (MOI). Similarly, the viral genome was also effectively replicated, with UL44—a viral DNA polymerase processivity factor—foci observed in the nuclei of HCECs. HCECs produced a substantial number of infectious virions after infection with TB40/E at both a high and low MOI. Conclusions Primary cultured HCECs could efficiently support HCMV replication after infection at both a high and low MOI. PMID:26261231

  20. A Small Covalent Allosteric Inhibitor of Human Cytomegalovirus DNA Polymerase Subunit Interactions.

    PubMed

    Chen, Han; Coseno, Molly; Ficarro, Scott B; Mansueto, My Sam; Komazin-Meredith, Gloria; Boissel, Sandrine; Filman, David J; Marto, Jarrod A; Hogle, James M; Coen, Donald M

    2017-02-10

    Human cytomegalovirus DNA polymerase comprises a catalytic subunit, UL54, and an accessory subunit, UL44, the interaction of which may serve as a target for the development of new antiviral drugs. Using a high-throughput screen, we identified a small molecule, (5-((dimethylamino)methylene-3-(methylthio)-6,7-dihydrobenzo[c]thiophen-4(5H)-one), that selectively inhibits the interaction of UL44 with a UL54-derived peptide in a time-dependent manner, full-length UL54, and UL44-dependent long-chain DNA synthesis. A crystal structure of the compound bound to UL44 revealed a covalent reaction with lysine residue 60 and additional noncovalent interactions that cause steric conflicts that would prevent the UL44 connector loop from interacting with UL54. Analyses of the reaction of the compound with model substrates supported a resonance-stabilized conjugation mechanism, and substitution of the lysine reduced the ability of the compound to inhibit UL44-UL54 peptide interactions. This novel covalent inhibitor of polymerase subunit interactions may serve as a starting point for new, needed drugs to treat human cytomegalovirus infections.

  1. Presence of antibodies to human cytomegalovirus in patients with different forms of cancer and in other categories of subjects.

    PubMed

    Stoian, M; Hozoc, M; Iosipenco, M; Bolocan, J; Nastac, E

    1982-01-01

    Complement fixing (CF) antibodies to the AD--129 strain of human cytomegalovirus (HCMV) were detected in patients with different forms of cancer, as well as in blood donors, in a proportion of 16.6% and 73.2%, respectively. The prevalence of CF antibodies to HCMV, strain AT--129, in the population of Romania is similar to that reported in other countries.

  2. Infection and upregulation of proinflammatory cytokines in human brain vascular pericytes by human cytomegalovirus

    PubMed Central

    2012-01-01

    Background Congenital human cytomegalovirus (HCMV) infections can result in CNS abnormalities in newborn babies including vision loss, mental retardation, motor deficits, seizures, and hearing loss. Brain pericytes play an essential role in the development and function of the blood–brain barrier yet their unique role in HCMV dissemination and neuropathlogy has not been reported. Methods Primary human brain vascular pericytes were exposed to a primary clinical isolate of HCMV designated ‘SBCMV’. Infectivity was analyzed by microscopy, immunofluorescence, Western blot, and qRT-PCR. Microarrays were performed to identify proinflammatory cytokines upregulated after SBCMV exposure, and the results validated by real-time quantitative polymerase chain reaction (qPCR) methodology. In situ cytokine expression of pericytes after exposure to HCMV was examined by ELISA and in vivo evidence of HCMV infection of brain pericytes was shown by dual-labeled immunohistochemistry. Results HCMV-infected human brain vascular pericytes as evidenced by several markers. Using a clinical isolate of HCMV (SBCMV), microscopy of infected pericytes showed virion production and typical cytomegalic cytopathology. This finding was confirmed by the expression of major immediate early and late virion proteins and by the presence of HCMV mRNA. Brain pericytes were fully permissive for CMV lytic replication after 72 to 96 hours in culture compared to human astrocytes or human brain microvascular endothelial cells (BMVEC). However, temporal transcriptional expression of pp65 virion protein after SBCMV infection was lower than that seen with the HCMV Towne laboratory strain. Using RT-PCR and dual-labeled immunofluorescence, proinflammatory cytokines CXCL8/IL-8, CXCL11/ITAC, and CCL5/Rantes were upregulated in SBCMV-infected cells, as were tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1beta), and interleukin-6 (IL-6). Pericytes exposed to SBCMV elicited higher levels of IL-6

  3. The life cycle and pathogenesis of human cytomegalovirus infection: lessons from proteomics

    PubMed Central

    Beltran, Pierre M. Jean; Cristea, Ileana M.

    2015-01-01

    Viruses have co-evolved with their hosts, acquiring strategies to subvert host cellular pathways for effective viral replication and spread. Human cytomegalovirus (HCMV), a widely-spread β-herpesvirus, is a major cause of birth defects and opportunistic infections in HIV-1/AIDS patients. HCMV displays an intricate system-wide modulation of the human cell proteome. An impressive array of virus–host protein interactions occurs throughout the infection. To investigate the virus life cycle, proteomics has recently become a significant component of virology studies. Here, we review the mass spectrometry-based proteomics approaches used in HCMV studies, as well as their contribution to understanding the HCMV life cycle and the virus-induced changes to host cells. The importance of the biological insights gained from these studies clearly demonstrate the impact that proteomics has had and can continue to have on understanding HCMV biology and identifying new therapeutic targets. PMID:25327590

  4. Partial Functional Complementation between Human and Mouse Cytomegalovirus Chemokine Receptor Homologues▿

    PubMed Central

    Farrell, Helen E.; Abraham, Alexander M.; Cardin, Rhonda D.; Sparre-Ulrich, Alexander H.; Rosenkilde, Mette M.; Spiess, Katja; Jensen, Tine H.; Kledal, Thomas N.; Davis-Poynter, Nicholas

    2011-01-01

    The human cytomegalovirus (CMV) proteins US28 and UL33 are homologous to chemokine receptors (CKRs). Knockout of the mouse CMV M33 protein (UL33 homologue) results in substantial attenuation of salivary gland infection/replication and reduced efficiency of reactivation from tissue explants. M33-mediated G protein-coupled signaling is critical for the salivary gland phenotype. In this report, we demonstrate that US28 and (to a lesser degree) UL33 restore reactivation from tissue explants and partially restore replication in salivary glands (compared to a signaling-deficient M33 mutant). These studies provide a novel small animal model for evaluation of therapies targeting the human CMV CKRs. PMID:21490099

  5. [Possible role of cytomegalovirus infection in the pathogenesis of human vascular diseases].

    PubMed

    Yonemitsu, Y; Komori, K; Sueishi, K; Sugimachi, K

    1998-01-01

    In order to evaluate the pathogenic role of human cytomegalovirus(CMV) infection in human vascular disease, we first examined the role of CMV immediate early gene (CMV-IE) expression in vascular smooth muscle cell (VSMC) proliferation. The in vitro IE gene transfer stimulated VSMC proliferation. The in vivo IE gene transfer showed neointimal thickening while the control arteries did not. In the wall of "so-called" inflammatory abdominal aortic aneurysm (IAAA), CMV infected cells were more frequently encountered than in that of AA and control cases. CMV infected cells were largely identified as macrophages or fibroblasts, and these cells frequently expressed CMV-IE gene. These findings thus suggest that the persistent expression of CMV-IE gene in the vessel wall may play a role in the vascular cellular responses, including progression of atherosclerosis or vasculitis, in vivo.

  6. Inhibition of ganciclovir-resistant human cytomegalovirus replication by Kampo (Japanese herbal medicine).

    PubMed

    Murayama, Tsugiya; Yamaguchi, Nobuo; Iwamoto, Kozo; Eizuru, Yoshito

    2006-01-01

    We examined the effect of Kampo on the replication of ganciclovir (GCV)-resistant human cytomegalovirus (HCMV) in the human embryonic fibroblast cell line MRC-5. Treatment of HCMV-infected cells with Sho-seiryu-to (SST; Xiao-Qing-Long-Tang in Chinese) resulted in the inhibition of viral replication without affecting the cell growth. SST treatment decreased the synthesis of viral DNA, but had no virucidal effect on cell-free HCMV. However, the inhibitory effect of SST on HCMV replication was ablated by anti-interferon-beta (IFN-beta) antibody suggesting that SST inhibits the replication of GCV-resistant HCMV through the induction of IFN-beta. These results suggest that SST is a novel compund with potential as an anti-HCMV.

  7. Modulation of Human Mesenchymal Stem Cell Immunogenicity through Forced Expression of Human Cytomegalovirus US Proteins

    PubMed Central

    Soland, Melisa A.; Bego, Mariana G.; Colletti, Evan; Porada, Christopher D.; Zanjani, Esmail D.; St. Jeor, Stephen; Almeida-Porada, Graça

    2012-01-01

    Background Mesenchymal stem cells (MSC) are promising candidates for cell therapy, as they migrate to areas of injury, differentiate into a broad range of specialized cells, and have immunomodulatory properties. However, MSC are not invisible to the recipient's immune system, and upon in vivo administration, allogeneic MSC are able to trigger immune responses, resulting in rejection of the transplanted cells, precluding their full therapeutic potential. Human cytomegalovirus (HCMV) has developed several strategies to evade cytotoxic T lymphocyte (CTL) and Natural Killer (NK) cell recognition. Our goal is to exploit HCMV immunological evasion strategies to reduce MSC immunogenicity. Methodology/Principal Findings We genetically engineered human MSC to express HCMV proteins known to downregulate HLA-I expression, and investigated whether modified MSC were protected from CTL and NK attack. Flow cytometric analysis showed that amongst the US proteins tested, US6 and US11 efficiently reduced MSC HLA-I expression, and mixed lymphocyte reaction demonstrated a corresponding decrease in human and sheep mononuclear cell proliferation. NK killing assays showed that the decrease in HLA-I expression did not result in increased NK cytotoxicity, and that at certain NK∶MSC ratios, US11 conferred protection from NK cytotoxic effects. Transplantation of MSC-US6 or MSC-US11 into pre-immune fetal sheep resulted in increased liver engraftment when compared to control MSC, as demonstrated by qPCR and immunofluorescence analyses. Conclusions and Significance These data demonstrate that engineering MSC to express US6 and US11 can be used as a means of decreasing recognition of MSC by the immune system, allowing higher levels of engraftment in an allogeneic transplantation setting. Since one of the major factors responsible for the failure of allogeneic-donor MSC to engraft is the mismatch of HLA-I molecules between the donor and the recipient, MSC-US6 and MSC-US11 could constitute an

  8. Identification of HLA-A*2402-restricted HCMV immediate early-1 (IE-1) epitopes as targets for CD8+ HCMV-specific cytotoxic T lymphocytes.

    PubMed

    Lim, Jong-Baeck; Kim, Hyun Ok; Jeong, Seok Hoon; Ha, Joo Eun; Jang, Sunphil; Lee, Sang-Guk; Lee, Kyungwon; Stroncek, David

    2009-08-23

    To identify novel HLA-A*2402-restricted human cytomegalovirus (HCMV) immediate early-1 (IE-1) epitopes for adoptive immunotherapy, we explored 120 overlapping 15-amino acid spanning IE-1. These peptides were screened by measuring the frequency of polyclonal CD8+ T cells producing intracellular interferon-gamma (IFN-gamma) using flow cytometry and the epitopes were validated with a HCMV-infected target Cr release cytotoxicity assay. Initial screening was performed with 12 mini-pools of 10 consecutive peptides made from 120 overlapping peptides15-amino acids in length that spanned IE-1. When peripheral blood mononuclear cells (PBMCs) from HLA-A*2402 HCMV-seropositive donors were sensitized with each of the 12 mini-pools, mini-pools 1 and 2 induced the highest frequency of CD8+ cytotoxic T lymphocytes (CTLs) producing IFN-gamma. When PBMCs were stimulated with each of the twenty peptides belonging to mini-pools 1 and 2, peptides IE-11-15MESSAKRKMDPDNPD and IE-15-19AKRKMDPDNPDEGPS induced the greatest quantities of IFN-gamma production and cytotoxicity of HLA-matched HCMV-infected fibroblasts. To determine the exact HLA-A*2402-restricted epitopes within the two IE-1 proteins, we synthesized a total of twenty-one overlapping 9- or 10 amino acid peptides spanning IE-11-15 and IE-15-19. Peptide IE-13-12SSAKRKMDPD induced the greatest quantities of IFN-gamma production and target cell killing by CD8+ CTLs. HCMV IE-13-12SSAKRKMDPD is a HLA-A*2402-restricted HCMV IE-1 epitope that can serve as a common target for CD8+ HCMV-specific CTLs.

  9. Antitumor-promoting activity of lignans: inhibition of human cytomegalovirus IE gene expression.

    PubMed

    Pusztai, Rozália; Abrantes, Marta; Serly, Julianna; Sherly, Julia; Duarte, Noélia; Molnar, Joseph; Ferreira, Maria-José U

    2010-02-01

    Chemoprevention is a promising new approach to cancer prevention. Since the beginning of chemoprevention studies, short-term in vitro models used in the study of carcinogenesis have been applied in the identification of antitumor-promoting agents. The lignans threo-4,4'-dihydroxy-3-methoxylignan, (-)-dihydroguaiaretic acid, 4'-hydroxy-3,3',4-trimethoxylignan, 3,3',4,4'-tetramethoxylignan, 4,4'-diacetyl-3,3'-dimethoxylignan, talaumidin, heliobuphthalmin, (-)-dihydro-cubebin, and hinokinin were evaluated for their ability to inhibit human cytomegalovirus (HCMV) IE-antigen expression in lung cancer cells (A549). Most of the evaluated compounds reduced IE-antigen expression of HCMV, the best result being obtained with 4,4'-dihydroxy-3-methoxylignan. However, a dose-dependent significant increase of IE-antigen expression was found for the derivative (-)-dihydrocubebin. The results of this study suggest that some of these lignans might be valuable as potential cancer chemopreventive agents.

  10. [HUMAN CYTOMEGALOVIRUS INFECTION AND SPONTANEOUS ABORTION IN PREGNANT WOMEN OF I AND II TRIMESTER].

    PubMed

    Cheshik, S G; Kisteneva, L B

    2016-01-01

    The goal of this work was the evaluation of the frequency of human CMV infection among the women, whose pregnancy ended in miscarriage, detection of active forms of infection and treatment before pregnancy. Virological and sero-immunological techniques were used. A total of 116 women who had miscarriages before the 28 week of pregnancy were submitted to the CMV test. 109 women (94.0%) demonstrated positive results. 49 women (42.2%) had active form of the cytomegalovirus infection. 13 women (26.5%) had the recurrent form and 36 patients (73.5%) had the persistent form of CMV infection (stage of productive replication). All the women with active CMVI were treated before the next pregnancy. Immunomodulatory therapy for the treatment was used.

  11. The DNA Damage Response Induced by Infection with Human Cytomegalovirus and Other Viruses

    PubMed Central

    E, Xiaofei; Kowalik, Timothy F.

    2014-01-01

    Viruses use different strategies to overcome the host defense system. Recent studies have shown that viruses can induce DNA damage response (DDR). Many of these viruses use DDR signaling to benefit their replication, while other viruses block or inactivate DDR signaling. This review focuses on the effects of DDR and DNA repair on human cytomegalovirus (HCMV) replication. Here, we review the DDR induced by HCMV infection and its similarities and differences to DDR induced by other viruses. As DDR signaling pathways are critical for the replication of many viruses, blocking these pathways may represent novel therapeutic opportunities for the treatment of certain infectious diseases. Lastly, future perspectives in the field are discussed. PMID:24859341

  12. Interplay between Human Cytomegalovirus and Intrinsic/Innate Host Responses: A Complex Bidirectional Relationship

    PubMed Central

    Rossini, Giada; Cerboni, Cristina; Santoni, Angela; Landini, Maria Paola; Landolfo, Santo; Gatti, Deborah; Gribaudo, Giorgio; Varani, Stefania

    2012-01-01

    The interaction between human cytomegalovirus (HCMV) and its host is a complex process that begins with viral attachment and entry into host cells, culminating in the development of a specific adaptive response that clears the acute infection but fails to eradicate HCMV. We review the viral and cellular partners that mediate early host responses to HCMV with regard to the interaction between structural components of virions (viral glycoproteins) and cellular receptors (attachment/entry receptors, toll-like receptors, and other nucleic acid sensors) or intrinsic factors (PML, hDaxx, Sp100, viperin, interferon inducible protein 16), the reactions of innate immune cells (antigen presenting cells and natural killer cells), the numerous mechanisms of viral immunoevasion, and the potential exploitation of events that are associated with early phases of virus-host interplay as a therapeutic strategy. PMID:22701276

  13. Viral affects on metabolism: changes in glucose and glutamine utilization during human cytomegalovirus infection

    PubMed Central

    Yu, Yongjun; Clippinger, Amy J.; Alwine, James C.

    2011-01-01

    Human cytomegalovirus (HCMV) infection causes dramatic alterations of intermediary metabolism, similar to those found in tumor cells. In infected cells, glucose carbon is not completely broken down by the tricarboxylic acid (TCA) cycle for energy; instead it is used biosynthetically. This process requires increased glucose uptake, increased glycolysis and the diversion of glucose carbon, in the form of citrate, from the TCA cycle for use in HCMV-induced fatty acid biosynthesis. The diversion of citrate from the TCA cycle (cataplerosis) requires induction of enzymes to promote glutaminolysis, the conversion of glutamine to -ketoglutarate in order to maintain the TCA cycle (anaplerosis) and ATP production. Such changes could result in heretofore uncharacterized pathogenesis, potentially implicating HCMV as a subtle co-factor in many maladies, including oncogenesis. Recognition of the effects of HCMV, and other viruses, on host cell metabolism will provide new understanding of viral pathogenesis and novel avenues for antiviral therapy. PMID:21570293

  14. Latency-Associated Degradation Of The MRP1 Drug Transporter During Latent Human Cytomegalovirus Infection‡

    PubMed Central

    Weekes, Michael P.; Tan, Shireen Y. L.; Poole, Emma; Talbot, Suzanne; Antrobus, Robin; Smith, Duncan L.; Montag, Christina; Gygi, Steven P.; Sinclair, John H.; Lehner, Paul J.

    2013-01-01

    Reactivation of latent human cytomegalovirus (HCMV) infection following transplantation is associated with high morbidity and mortality. In vivo, myeloid cells and their progenitors are an important site of HCMV latency, whose establishment and/or maintenance requires expression of UL138. Using SILAC (stable isotope labeling by amino acids in cell culture)-based mass spectrometry, we found a dramatic UL138-mediated loss of cell surface Multidrug Resistance-associated Protein-1 (MRP1), and reduction of substrate export by this transporter. Latency-associated loss of MRP1 and accumulation of the cytotoxic drug vincristine, an MRP1 substrate, depleted virus from naturally latent CD14+ and CD34+ progenitors, all in vivo sites of latency. The UL138-mediated loss of MRP1 provides a marker for detecting latent HCMV infection and a therapeutic target for eliminating latently-infected cells prior to transplantation. PMID:23580527

  15. Characteristics and functions of human cytomegalovirus UL128 gene/protein.

    PubMed

    Tao, R; Xu, J; Gao, H -H; Zhao, W -T; Shang, S -Q

    2014-01-01

    Human cytomegalovirus (HCMV) ORF UL128 protein is highly conserved among viral field isolates and functions in two different molecular forms, monomeric UL128 protein and in a complex with glycoproteins gH, gL, UL130, and UL131A protein. Monomeric UL128 protein works as soluble chemokine analogue to attract peripheral blood mononuclear cells (PBMCs) and selectively induces expression of interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) in PBMCs. The gH/gL/UL128/UL130/UL131A complex is indispensable for entry into both endothelial and epithelial cells. In conclusion, UL128 plays an important role in HCMV infection.

  16. Knowledge of Human Cytomegalovirus Infection and Prevention in Pregnant Women: A Baseline, Operational Survey

    PubMed Central

    Micieli, Mariella; Votino, Carmela; Visconti, Federica; Quaresima, Paola; Torti, Carlo

    2017-01-01

    Currently, the only efficient way to prevent human Cytomegalovirus (HCMV) infection in pregnancy is primary prophylaxis through hygienic measures. So, we evaluated knowledge of HCMV and its prevention in a group of pregnant women. An anonymous questionnaire with multiple-choice answers was administered to all pregnant women who were followed up at the Obstetrics and Gynecology Unit of “Pugliese-Ciaccio Hospital,” a third-level hospital in Catanzaro (Southern Italy), from November 2015 to March 2016. Previously prescribed serology results for HCMV were also evaluated. Three hundred and fifty women participated in the study and the results clearly demonstrated that knowledge of pregnant women about HCMV is poor. Moreover, prescribed screening procedures need to be optimized, since one out of three pregnant women has not been tested for HCMV or the screening was not performed adequately. For this reason, it is important to implement informative campaign in both pregnant women and providing physicians. PMID:28831237

  17. Murine Cytomegalovirus Abortively Infects Human Dendritic Cells, Leading to Expression and Presentation of Virally Vectored Genes

    PubMed Central

    Wang, Xiuqing; Messerle, Martin; Sapinoro, Ramil; Santos, Kathlyn; Hocknell, Peter K.; Jin, Xia; Dewhurst, Stephen

    2003-01-01

    Dendritic cells (DC) are potent antigen-presenting cells that play a crucial role in antigen-specific immune responses. Thus, the targeting of exogenous antigens to DC has become a popular approach for cancer immunotherapy and vaccine development. In this report, we studied the interplay between murine cytomegalovirus (MCMV) and human monocyte-derived DC. The results showed that an enhanced green fluorescent protein (EGFP)-encoding, replication-competent MCMV vector underwent abortive infection in human DC; this was accompanied by the efficient expression of EGFP. Infection of human DC by this vector resulted in a modest increase in the expression of cell surface proteins associated with DC maturation and has no significant effect on the immunostimulatory function of the cells, as reflected by their ability to support T-cell proliferation in a mixed-lymphocyte reaction. Finally, an MCMV vector encoding the human immunodeficiency virus type 1 (HIV-1) gp120 envelope glycoprotein was constructed and used to infect cultured human DC. The infected DC were shown to be capable of stimulating the expansion of autologous, gp120-specific, class I-restricted T lymphocytes from an HIV-1-negative donor, as determined by tetramer staining and enzyme-linked immunospot analysis. Taken together, these results suggest that MCMV may have potential utility as a vector for human vaccine development. PMID:12805417

  18. Evaluating Human T-Cell Therapy of Cytomegalovirus Organ Disease in HLA-Transgenic Mice

    PubMed Central

    Thomas, Simone; Klobuch, Sebastian; Podlech, Jürgen; Plachter, Bodo; Hoffmann, Petra; Renzaho, Angelique; Theobald, Matthias

    2015-01-01

    Reactivation of human cytomegalovirus (HCMV) can cause severe disease in recipients of hematopoietic stem cell transplantation. Although preclinical research in murine models as well as clinical trials have provided 'proof of concept' for infection control by pre-emptive CD8 T-cell immunotherapy, there exists no predictive model to experimentally evaluate parameters that determine antiviral efficacy of human T cells in terms of virus control in functional organs, prevention of organ disease, and host survival benefit. We here introduce a novel mouse model for testing HCMV epitope-specific human T cells. The HCMV UL83/pp65-derived NLV-peptide was presented by transgenic HLA-A2.1 in the context of a lethal infection of NOD/SCID/IL-2rg-/- mice with a chimeric murine CMV, mCMV-NLV. Scenarios of HCMV-seropositive and -seronegative human T-cell donors were modeled by testing peptide-restimulated and T-cell receptor-transduced human T cells, respectively. Upon transfer, the T cells infiltrated host tissues in an epitope-specific manner, confining the infection to nodular inflammatory foci. This resulted in a significant reduction of viral load, diminished organ pathology, and prolonged survival. The model has thus proven its potential for a preclinical testing of the protective antiviral efficacy of HCMV epitope-specific human T cells in the evaluation of new approaches to an immunotherapy of CMV disease. PMID:26181057

  19. Human cytomegalovirus and mucoepidermoid carcinoma of salivary glands: cell-specific localization of active viral and oncogenic signaling proteins is confirmatory of a causal relationship.

    PubMed

    Melnick, Michael; Sedghizadeh, Parish P; Allen, Carl M; Jaskoll, Tina

    2012-02-01

    Human cytomegalovirus (hCMV) infection is common. Although still controversial, there is growing evidence that active hCMV infection is associated with a variety of malignancies, including brain, breast, lung, colon, and prostate. Given that hCMV is frequently resident in salivary gland (SG) ductal epithelium, we hypothesized that hCMV would be important to the pathogenesis of SG mucoepidermoid carcinoma (MEC). This was initially supported by our finding that purified CMV induces malignant transformation in SG cells in an in vitro mouse model, and utilizes a pathogenic pathway previously reported for human MEC. Here we present the histologic and molecular characterizations of 39 human SG MECs selected randomly from a repository of cases spanning 2004-2011. Serial sections were obtained from formalin-fixed, paraffin embedded, tissue blocks from previous incisional or excisional biopsies. Immunohistochemical assays were performed for active hCMV proteins (IE1 and pp65) and the activated COX/AREG/EGFR/ERK signaling pathway. All four prospective causal criteria for viruses and cancer are fully satisfied: (1) protein markers for active hCMV are present in 97% of MECs; (2) markers of active hCMV are absent in non-neoplastic SG tissues; (3) hCMV-specific proteins (IE1, pp65) are in specific cell types and expression is positively correlated with severity; (4) hCMV correlates and colocalizes with an upregulation and activation of an established oncogenic signaling pathway (COX/AREG/EGFR/ERK). Thus, the evidential support reported here and previously in a mouse model is strongly confirmatory of a causal relationship between hCMV and SG mucoepidermoid carcinoma. To our knowledge, this is the first demonstration of hCMV's role in human oncogenesis that fully responds to all of Koch's Postulates as revised for viruses and cancer. In the absence of any contrary evidence, hCMV can reasonably be designated an "oncovirus."

  20. Cytomegalovirus Survival and Transferability and the Effectiveness of Common Hand-Washing Agents against Cytomegalovirus on Live Human Hands

    PubMed Central

    Stowell, Jennifer D.; Forlin-Passoni, Daniela; Radford, Kay; Bate, Sheri L.; Dollard, Sheila C.; Bialek, Stephanie R.; Cannon, Michael J.

    2014-01-01

    Congenital cytomegalovirus (CMV) transmission can occur when women acquire CMV while pregnant. Infection control guidelines may reduce risk for transmission. We studied the duration of CMV survival after application of bacteria to the hands and after transfer from the hands to surfaces and the effectiveness of cleansing with water, regular and antibacterial soaps, sanitizer, and diaper wipes. Experiments used CMV AD169 in saliva at initial titers of 1 × 105 infectious particles/ml. Samples from hands or surfaces (points between 0 and 15 min) were placed in culture and observed for at least 2 weeks. Samples were also tested using CMV real-time PCR. After application of bacteria to the hands, viable CMV was recovered from 17/20 swabs at 0 min, 18/20 swabs at 1 min, 5/20 swabs at 5 min, and 4/20 swabs at 15 min. After transfer, duration of survival was at least 15 min on plastic (1/2 swabs), 5 min on crackers and glass (3/4 swabs), and 1 min or less on metal and cloth (3/4 swabs); no viable virus was collected from wood, rubber, or hands. After cleansing, no viable virus was recovered using water (0/22), plain soap (0/20), antibacterial soap (0/20), or sanitizer (0/22). Viable CMV was recovered from 4/20 hands 10 min after diaper wipe cleansing. CMV remains viable on hands for sufficient times to allow transmission. CMV may be transferred to surfaces with reduced viability. Hand-cleansing methods were effective at eliminating viable CMV from hands. PMID:24185855

  1. Cytomegalovirus survival and transferability and the effectiveness of common hand-washing agents against cytomegalovirus on live human hands.

    PubMed

    Stowell, Jennifer D; Forlin-Passoni, Daniela; Radford, Kay; Bate, Sheri L; Dollard, Sheila C; Bialek, Stephanie R; Cannon, Michael J; Schmid, D Scott

    2014-01-01

    Congenital cytomegalovirus (CMV) transmission can occur when women acquire CMV while pregnant. Infection control guidelines may reduce risk for transmission. We studied the duration of CMV survival after application of bacteria to the hands and after transfer from the hands to surfaces and the effectiveness of cleansing with water, regular and antibacterial soaps, sanitizer, and diaper wipes. Experiments used CMV AD169 in saliva at initial titers of 1 × 10(5) infectious particles/ml. Samples from hands or surfaces (points between 0 and 15 min) were placed in culture and observed for at least 2 weeks. Samples were also tested using CMV real-time PCR. After application of bacteria to the hands, viable CMV was recovered from 17/20 swabs at 0 min, 18/20 swabs at 1 min, 5/20 swabs at 5 min, and 4/20 swabs at 15 min. After transfer, duration of survival was at least 15 min on plastic (1/2 swabs), 5 min on crackers and glass (3/4 swabs), and 1 min or less on metal and cloth (3/4 swabs); no viable virus was collected from wood, rubber, or hands. After cleansing, no viable virus was recovered using water (0/22), plain soap (0/20), antibacterial soap (0/20), or sanitizer (0/22). Viable CMV was recovered from 4/20 hands 10 min after diaper wipe cleansing. CMV remains viable on hands for sufficient times to allow transmission. CMV may be transferred to surfaces with reduced viability. Hand-cleansing methods were effective at eliminating viable CMV from hands.

  2. Convallatoxin-Induced Reduction of Methionine Import Effectively Inhibits Human Cytomegalovirus Infection and Replication.

    PubMed

    Cohen, Tobias; Williams, John D; Opperman, Timothy J; Sanchez, Roberto; Lurain, Nell S; Tortorella, Domenico

    2016-12-01

    Cytomegalovirus (CMV) is a ubiquitous human pathogen that increases the morbidity and mortality of immunocompromised individuals. The current FDA-approved treatments for CMV infection are intended to be virus specific, yet they have significant adverse side effects, including nephrotoxicity and hematological toxicity. Thus, there is a medical need for safer and more effective CMV therapeutics. Using a high-content screen, we identified the cardiac glycoside convallatoxin as an effective compound that inhibits CMV infection. Using a panel of cardiac glycoside variants, we assessed the structural elements critical for anti-CMV activity by both experimental and in silico methods. Analysis of the antiviral effects, toxicities, and pharmacodynamics of different variants of cardiac glycosides identified the mechanism of inhibition as reduction of methionine import, leading to decreased immediate-early gene translation without significant toxicity. Also, convallatoxin was found to dramatically reduce the proliferation of clinical CMV strains, implying that its mechanism of action is an effective strategy to block CMV dissemination. Our study has uncovered the mechanism and structural elements of convallatoxin, which are important for effectively inhibiting CMV infection by targeting the expression of immediate-early genes. Cytomegalovirus is a highly prevalent virus capable of causing severe disease in certain populations. The current FDA-approved therapeutics all target the same stage of the viral life cycle and induce toxicity and viral resistance. We identified convallatoxin, a novel cell-targeting antiviral that inhibits CMV infection by decreasing the synthesis of viral proteins. At doses low enough for cells to tolerate, convallatoxin was able to inhibit primary isolates of CMV, including those resistant to the anti-CMV drug ganciclovir. In addition to identifying convallatoxin as a novel antiviral, limiting mRNA translation has a dramatic impact on CMV infection

  3. Human cytomegalovirus immediate early proteins promote degradation of connexin 43 and disrupt gap junction communication: implications for a role in gliomagenesis.

    PubMed

    Khan, Zahidul; Yaiw, Koon-Chu; Wilhelmi, Vanessa; Lam, Hoyin; Rahbar, Afsar; Stragliotto, Giuseppe; Söderberg-Nauclér, Cecilia

    2014-01-01

    A lack of gap junctional intercellular communication (GJIC) is common in cancer. Many oncogenic viruses have been shown to downregulate the junctional protein connexin 43 (Cx43) and reduce GJIC. Human cytomegalovirus (HCMV) is a ubiquitous, species-specific betaherpesvirus that establishes life-long latency after primary infection. It encodes two viral gene products, immediate early (IE) proteins IE1 and IE2, which are crucial in viral replication and pathogenesis of many diseases. Emerging evidence demonstrates that HCMV DNA and proteins are highly prevalent in glioblastoma multiforme (GBM) and in other tumors, but HCMV's role in tumorigenesis remains obscure. In the present study, we examined the effects of HCMV infection on Cx43 expression and GJIC as well as the viral mechanism mediating the effects in human GBM cells and tissue samples. We found that HCMV downregulated Cx43 protein, resulting in disruption of functional GJIC as assayed by fluorescent dye transfer assay. We show that both HCMV-IE72 and IE86 mediate downregulation of Cx43 by silencing RNA targeting either IE72 or IE86 coupled with ganciclovir. This finding was further validated by transfection with expression vectors encoding IE72 or IE86, and we show that viral-mediated Cx43 depletion involved proteasomal degradation. Importantly, we also observed that the Cx43 protein levels and IE staining correlated inversely in 10 human GBM tissue specimens. Thus, HCMV regulates Cx43 expression and GJIC, which may contribute to gliomagenesis.

  4. Interactions between human immunodeficiency virus type 1 and human cytomegalovirus in human term syncytiotrophoblast cells coinfected with both viruses.

    PubMed Central

    Tóth, F D; Mosborg-Petersen, P; Kiss, J; Aboagye-Mathiesen, G; Hager, H; Juhl, C B; Gergely, L; Zdravkovic, M; Aranyosi, J; Lampé, L

    1995-01-01

    Human cytomegalovirus (HCMV) and human immunodeficiency virus type 1 (HIV-1) may interact in the pathogenesis of AIDS. The placental syncytiotrophoblast layer serves as the first line of defense of the fetus against viruses. We analyzed the patterns of replication of HIV-1 and HCMV in singly an dually infected human term syncytiotrophoblast cells cultured in vitro. Syncytiotrophoblast cells exhibited restricted permissiveness for HIV-1, while HCMV replication was restricted at the level of immediate-early and early gene products in the singly infected cells. We found that the syncytiotrophoblasts as an overlapping cell population could be coinfected with HIV-1 and HCMV. HIV-1 replication was markedly upregulated by previous or simultaneous infection of the cells with HCMV, whereas prior HIV-1 infection of the cells converted HCMV infection from a nonpermissive to a permissive one. No simultaneous enhancement of HCMV and HIV-1 expression was observed in the dually infected cell cultures. Major immediate-early proteins of HCMV were necessary for enhancement of HIV-1 replication, and interleukin-6 production induced by HCMV and further increased by replicating HIV-1 synergized with these proteins to produce this effect. Permissive replication cycle of HCMV was induced by the HIV-1 tat gene product. We were unable to detect HIV-1 (HCMV) or HCMV (HIV-1) pseudotypes in supernatant fluids from dually infected cell cultures. Our results suggest that interactions between HIV-1 and HCMV in coinfected syncytiotrophoblast cells may contribute to the transplacental transmission of both viruses. PMID:7884869

  5. Enhanced cytomegalovirus infection in human trabecular meshwork cells and its implication in glaucoma pathogenesis

    PubMed Central

    Choi, Jin A; Kim, Ju-Eun; Noh, Seung-Jun; Kyoung Kim, Eun; Park, Chan Kee; Paik, Soon-Young

    2017-01-01

    Cytomegalovirus (CMV) is one of the infectious causes of hypertensive anterior uveitis, which is characterized by recurrent episodes of elevated intraocular pressure (IOP) and mild anterior uveitis. Despite the potentially vision-threatening complications of this disease, the underlying mechanisms remain largely undefined. We aimed to investigate whether human trabecular meshwork (TM) cells, the key cell type that regulates IOP, could support CMV replication, as well as demonstrate the relevant pathological changes in TM. When human TM cells were infected with CMV AD169, immediate early antigens were detected 1 day post-infection (dpi); cytopathic changes including rounding, a ballooned appearance with disorganization, and a decreased number of stress fibers were noted in TM cells. The marked increase in viral DNA accumulation was observed most notably at 5 and 7 dpi, suggesting that the active viral infection in human TM cells could be the key mechanism underlying the elevation of IOP in anterior viral uveitis. Notably, CMV infection enhanced the production of transforming growth factor (TGF)-β1, an upstream molecule that increases the resistance of the outflow pathway in human TM cells. The increase of TGF-β1 was countervailed by additional treatment with corticosteroids. Our results provide a pathogenic mechanism for IOP elevation in viral anterior uveitis. PMID:28240260

  6. Adenovirus E1A/E1B Transformed Amniotic Fluid Cells Support Human Cytomegalovirus Replication

    PubMed Central

    Krömmelbein, Natascha; Wiebusch, Lüder; Schiedner, Gudrun; Büscher, Nicole; Sauer, Caroline; Florin, Luise; Sehn, Elisabeth; Wolfrum, Uwe; Plachter, Bodo

    2016-01-01

    The human cytomegalovirus (HCMV) replicates to high titers in primary human fibroblast cell cultures. A variety of primary human cells and some tumor-derived cell lines do also support permissive HCMV replication, yet at low levels. Cell lines established by transfection of the transforming functions of adenoviruses have been notoriously resistant to HCMV replication and progeny production. Here, we provide first-time evidence that a permanent cell line immortalized by adenovirus type 5 E1A and E1B (CAP) is supporting the full HCMV replication cycle and is releasing infectious progeny. The CAP cell line had previously been established from amniotic fluid cells which were likely derived from membranes of the developing fetus. These cells can be grown under serum-free conditions. HCMV efficiently penetrated CAP cells, expressed its immediate-early proteins and dispersed restrictive PML-bodies. Viral DNA replication was initiated and viral progeny became detectable by electron microscopy in CAP cells. Furthermore, infectious virus was released from CAP cells, yet to lower levels compared to fibroblasts. Subviral dense bodies were also secreted from CAP cells. The results show that E1A/E1B expression in transformed cells is not generally repressive to HCMV replication and that CAP cells may be a good substrate for dense body based vaccine production. PMID:26848680

  7. Host protein Snapin interacts with human cytomegalovirus pUL130 and affects viral DNA replication.

    PubMed

    Wang, Guili; Ren, Gaowei; Cui, Xin; Lu, Zhitao; Ma, Yanpin; Qi, Ying; Huang, Yujing; Liu, Zhongyang; Sun, Zhengrong; Ruan, Qiang

    2016-06-01

    The interplay between the host and Human cytomegalovirus (HCMV) plays a pivotal role in the outcome of an infection. HCMV growth in endothelial and epithelial cells requires expression of viral proteins UL128, UL130, and UL131 proteins (UL128-131), of which UL130 is the largest gene and the only one that is not interrupted by introns.Mutation of the C terminus of the UL130 protein causes reduced tropism of endothelial cells (EC). However, very few host factors have been identified that interact with the UL130 protein. In this study, HCMV UL130 protein was shown to directly interact with the human protein Snapin in human embryonic kidney HEK293 cells by Yeast two-hybrid screening, in vitro glutathione S-transferase (GST) pull-down, and co-immunoprecipitation. Additionally, heterologous expression of protein UL130 revealed co-localization with Snapin in the cell membrane and cytoplasm of HEK293 cells using fluorescence confocal microscopy. Furthermore, decreasing the level of Snapin via specific small interfering RNAs decreased the number of viral DNA copies and titer inHCMV-infected U373-S cells. Taken together, these results suggest that Snapin, the pUL130 interacting protein, has a role in modulating HCMV DNA synthesis.

  8. Tumor control by human cytomegalovirus in a murine model of hepatocellular carcinoma

    PubMed Central

    Kumar, Amit; Coquard, Laurie; Pasquereau, Sébastien; Russo, Laetitia; Valmary-Degano, Séverine; Borg, Christophe; Pothier, Pierre; Herbein, Georges

    2016-01-01

    Although viruses can cause cancer, other studies reported the regression of human tumors upon viral infections. We investigated the cytoreductive potential of human cytomegalovirus (HCMV) in a murine model of human hepatocellular carcinoma (HCC) in severe-immunodeficient mice. Infection of HepG2 cells with HCMV resulted in the absence of tumor or in a limited tumor growth following injection of cells subcutaneously. By contrast all mice injected with uninfected HepG2 cells and with HepG2 cells infected with UV-treated HCMV did develop tumors without any significant restriction. Analysis of tumors indicated that in mice injected with HCMV-infected-HepG2 cells, but not in controls, a restricted cellular proliferation was observed parallel to a limited activation of the STAT3-cyclin D1 axis, decreased formation of colonies in soft agar, and activation of the intrinsic apoptotic pathway. We conclude that HCMV can provide antitumoral effects in a murine model of HCC which requires replicative virus at some stages that results in limitation of tumor cell proliferation and enhanced apoptosis mediated through the intrinsic caspase pathway. PMID:27626063

  9. Long and Short Isoforms of the Human Cytomegalovirus UL138 Protein Silence IE Transcription and Promote Latency.

    PubMed

    Lee, Song Hee; Caviness, Katie; Albright, Emily R; Lee, Jeong-Hee; Gelbmann, Christopher B; Rak, Mike; Goodrum, Felicia; Kalejta, Robert F

    2016-10-15

    The UL133-138 locus present in clinical strains of human cytomegalovirus (HCMV) encodes proteins required for latency and reactivation in CD34(+) hematopoietic progenitor cells and virion maturation in endothelial cells. The encoded proteins form multiple homo- and hetero-interactions and localize within secretory membranes. One of these genes, UL136 gene, is expressed as at least five different protein isoforms with overlapping and unique functions. Here we show that another gene from this locus, the UL138 gene, also generates more than one protein isoform. A long form of UL138 (pUL138-L) initiates translation from codon 1, possesses an amino-terminal signal sequence, and is a type one integral membrane protein. Here we identify a short protein isoform (pUL138-S) initiating from codon 16 that displays a subcellular localization similar to that of pUL138-L. Reporter, short-term transcription, and long-term virus production assays revealed that both pUL138-L and pUL138-S are able to suppress major immediate early (IE) gene transcription and the generation of infectious virions in cells in which HCMV latency is studied. The long form appears to be more potent at silencing IE transcription shortly after infection, while the short form seems more potent at restricting progeny virion production at later times, indicating that both isoforms of UL138 likely cooperate to promote HCMV latency. Latency allows herpesviruses to persist for the lives of their hosts in the face of effective immune control measures for productively infected cells. Controlling latent reservoirs is an attractive antiviral approach complicated by knowledge deficits for how latently infected cells are established, maintained, and reactivated. This is especially true for betaherpesviruses. The functional consequences of HCMV UL138 protein expression during latency include repression of viral IE1 transcription and suppression of virus replication. Here we show that short and long isoforms of UL138

  10. Human Cytomegalovirus US28 Is Important for Latent Infection of Hematopoietic Progenitor Cells

    PubMed Central

    Humby, Monica S.

    2015-01-01

    ABSTRACT Human cytomegalovirus (HCMV) resides latently in hematopoietic progenitor cells (HPCs). During latency, only a subset of HCMV genes is transcribed, including one of the four virus-encoded G protein-coupled receptors (GPCRs), US28. Although US28 is a multifunctional lytic protein, its function during latency has remained undefined. We generated a panel of US28 recombinant viruses in the bacterial artificial chromosome (BAC)-derived clinical HCMV strain TB40/E-mCherry. We deleted the entire US28 open reading frame (ORF), deleted all four of the viral GPCR ORFs, or deleted three of the HCMV GPCRs but not the US28 wild-type protein. Using these recombinant viruses, we assessed the requirement for US28 during latency in the Kasumi-3 in vitro latency model system and in primary ex vivo-cultured CD34+ HPCs. Our data suggest that US28 is required for latency as infection with viruses lacking the US28 ORF alone or in combination with the remaining HCMV-encoded GPCR results in transcription from the major immediate early promoter, the production of extracellular virions, and the production of infectious virus capable of infecting naive fibroblasts. The other HCMV GPCRs are not required for this phenotype as a virus expressing only US28 but not the remaining virus-encoded GPCRs is phenotypically similar to that of wild-type latent infection. Finally, we found that US28 copurifies with mature virions and is expressed in HPCs upon virus entry although its expression at the time of infection does not complement the US28 deletion latency phenotype. This work suggests that US28 protein functions to promote a latent state within hematopoietic progenitor cells. IMPORTANCE Human cytomegalovirus (HCMV) is a widespread pathogen that, once acquired, remains with its host for life. HCMV remains latent, or quiescent, in cells of the hematopoietic compartment and upon immune challenge can reactivate to cause disease. HCMV-encoded US28 is one of several genes expressed during

  11. Cell Surface THY-1 Contributes to Human Cytomegalovirus Entry via a Macropinocytosis-Like Process.

    PubMed

    Li, Qingxue; Fischer, Elizabeth; Cohen, Jeffrey I

    2016-11-01

    Previously we showed that THY-1 has a critical role in the initial stage of infection of certain cell types with human cytomegalovirus (HCMV) and that THY-1 is important for HCMV-mediated activation of phosphatidylinositol 3-kinase (PI3K)/Akt during virus entry. THY-1 is known to interact with integrins and is a major cargo protein of clathrin-independent endocytic vesicles. Since macropinocytosis involves integrin signaling, is PI3K/Akt dependent, and is a clathrin-independent endocytic process, we determined whether THY-1 has a role in HCMV entry by macropinocytosis. Using electron microscopy in two cell lines that support HCMV infection in a THY-1-dependent manner, we found that HCMV enters these cells by a macropinocytosis-like process. THY-1 associated with HCMV virions on the cell surface and colocalized with virus inside macropinosomes. 5-(N-Ethyl-N-isopropyl)amiloride (EIPA) and soluble THY-1 blocked HCMV infection in the cell lines by ≥80% and 60%, respectively. HCMV entry into the cells triggered increased influx of extracellular fluid, a marker of macropinocytosis, and this increased fluid uptake was inhibited by EIPA and by soluble THY-1. Blocking actin depolymerization, Na(+)/H(+) exchange, PI3K, and Pak1 kinase, which are critical for macropinocytosis, impaired HCMV infection. Neither internalized HCMV virions nor THY-1 in virus-infected cells colocalized with transferrin as determined by confocal microscopy, indicating that clathrin-mediated endocytosis was not involved in THY-1-associated virus entry. These results suggest that HCMV has adapted to utilize THY-1, a cargo protein of clathrin-independent endocytotic vesicles, to facilitate efficient entry into certain cell types by a macropinocytosis-like process. Human cytomegalovirus (HCMV) infects over half of the population and is the most common infectious cause of birth defects. The virus is the most important infection occurring in transplant recipients. The mechanism of how HCMV enters cells

  12. Protein-Protein Interactions Suggest Novel Activities of Human Cytomegalovirus Tegument Protein pUL103

    PubMed Central

    Ortiz, Daniel A.; Glassbrook, James E.

    2016-01-01

    ABSTRACT Human cytomegalovirus (HCMV) is an enveloped double-stranded DNA virus that causes severe disease in newborns and immunocompromised patients. During infection, the host cell endosecretory system is remodeled to form the cytoplasmic virion assembly complex (cVAC). We and others previously identified the conserved, multifunctional HCMV virion tegument protein pUL103 as important for cVAC biogenesis and efficient secondary envelopment. To help define its mechanisms of action and predict additional functions, we used two complementary methods, coimmunoprecipitation (co-IP) and proximity biotinylation (BioID), to identify viral and cellular proteins that interact with pUL103. By using the two methods in parallel and applying stringent selection criteria, we identified potentially high-value interactions of pUL103 with 13 HCMV and 18 cellular proteins. Detection of the previously identified pUL103-pUL71 interaction, as well as verification of several interactions by reverse co-IP, supports the specificity of our screening process. As might be expected for a tegument protein, interactions were identified that suggest distinct roles for pUL103 across the arc of lytic infection, including interactions with proteins involved in cellular antiviral responses, nuclear activities, and biogenesis and transport of cytoplasmic vesicles. Further analysis of some of these interactions expands our understanding of the multifunctional repertoire of pUL103: we detected HCMV pUL103 in nuclei of infected cells and identified an ALIX-binding domain within the pUL103 sequence. IMPORTANCE Human cytomegalovirus (HCMV) is able to reconfigure the host cell machinery to establish a virion production factory, the cytoplasmic virion assembly complex (cVAC). cVAC biogenesis and operation represent targets for development of novel HCMV antivirals. We previously showed that the HCMV tegument protein pUL103 is required for cVAC biogenesis. Using pUL103 as bait, we investigated viral and

  13. Effects of Jinye Baidu Granule on fetal growth and development with maternal active human cytomegalovirus infection.

    PubMed

    Jiang, Hong; Chen, Su-hua; Wen, Liang-zhen

    2006-12-01

    To evaluate the effects of Jinye Baidu Granule ( JYBDG), a traditional Chinese medicine compound prescription, on fetal growth and development with maternal active human cytomegalovirus infection. A prospective, randomized and controlled trial was adopted during January 1996 to June 2002. From the pregnant women with an abnormal pregnant history, 240 cases were screened to be infected by human cytomegalovirus (HCMV) by enzyme-linked immunoabsorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR). They were assigned according to the random number table to two groups. The 122 cases in the treatment group were administrated with JYBDG, one package each time, three times a day for two continuous weeks, while the other 118 in the control group did not receive any treatment. The negative conversion rate of both HCMV-IgM and HCMV late mRNA, the positive rate of HCMV-DNA in placenta and the intrauterine transmission rate between the two groups were compared, and fetal growth and development in partial fetuses were also observed. The negative conversion rate of both HCMV-lgM and HCMV late mRNA, the positive rate of HCMV-DNA in placenta and the intrauterine transmission rate in the treatment group were 77. 05% (94/122), 48. 98% (48/98) and 21.74% (10/46) respectively, while those in the control group were 38. 14% (45/118), 67.50% (54/80) and 52.63% (20/38) respectively, all showing significant difference between the two groups (P<0. 01). Totally 35 normal infants and 11 abnormal infants were born in the treatment group, and the number in the control group was 20 and 18 respectively, and comparison between the two groups showed significant difference (P<0.01). Six months of child birth, the scores of both mental development index (MDI) and psychomotor development index (PDI) of infants were higher in the treatment group (20 cases) than those in the control group (20 cases), but there was no significant difference between the two groups (P>0

  14. [Detection of DNA human cytomegalovirus of a molecular methods: hybrid capture DNA CMV by immunocompromised].

    PubMed

    Mhiri, Leila; Arrouji, Zakia; Slim, Amine; Ben Redjeb, Saida

    2006-10-01

    Human cytomegalovirus (HCMV), a member of the beta-virus herpes family, is a ubiquitous human pathogen. After a primary infection, HCMV establishes life latency. HCMV rarely causes symptomatic disease in an immunocompetent host, however, it is a major cause of infectious morbidity and mortality in immunocompromised individuals and developing fetuses. The HCMV genome consists of 240 kbp of double stranded DNA. Early diagnosis molecular of CMV infection is important. The objective of this study was to develop a molecular methods: Quantitative Hybrid capture for the detection of DNA CMV. We present results for 200 immunocompromised collected from 1999 to 2003 (122 men and 78 women, whom mean age was 35 years). Our results showed that 25% of women and 36% of men were positif for hybrid capture DNA CMV. This simple test (cold probe) provide quantitative and fast results. Also the efficacity of anti-CMV therapy can be followed. More over, in contrary with pp65-antigenemia assay and CMV PCR, this test can be managed on biopsy sample.

  15. Human Cytomegalovirus DNA Quantification and Gene Expression in Gliomas of Different Grades.

    PubMed

    Stangherlin, Lucas Matheus; Castro, Fabiane Lucy Ferreira; Medeiros, Raphael Salles Scortegagna; Guerra, Juliana Mariotti; Kimura, Lidia Midori; Shirata, Neuza Kazumi; Nonogaki, Suely; Dos Santos, Claudia Januário; Carlan Silva, Maria Cristina

    2016-01-01

    Gliomas are the most common type of primary brain tumors. The most aggressive type, Glioblastoma multiforme (GBM), is one of the deadliest human diseases, with an average survival at diagnosis of about 1 year. Previous evidence suggests a link between human cytomegalovirus (HCMV) and gliomas. HCMV has been shown to be present in these tumors and several viral proteins can have oncogenic properties in glioma cells. Here we have investigated the presence of HCMV DNA, RNA and proteins in fifty-two gliomas of different grades of malignancy. The UL83 viral region, the early beta 2.7 RNA and viral protein were detected in 73%, 36% and 57% by qPCR, ISH and IHC, respectively. Positivity of the viral targets and viral load was independent of tumor type or grade suggesting no correlation between viral presence and tumor progression. Our results demonstrate high prevalence of the virus in gliomas from Brazilian patients, contributing to a better understanding of the association between HCMV infection and gliomas worldwide and supporting further investigations of the virus oncomodulatory properties.

  16. ACSS2-mediated acetyl-CoA synthesis from acetate is necessary for human cytomegalovirus infection.

    PubMed

    Vysochan, Anna; Sengupta, Arjun; Weljie, Aalim M; Alwine, James C; Yu, Yongjun

    2017-02-21

    Recent studies have shown that human cytomegalovirus (HCMV) can induce a robust increase in lipid synthesis which is critical for the success of infection. In mammalian cells the central precursor for lipid biosynthesis, cytosolic acetyl CoA (Ac-CoA), is produced by ATP-citrate lyase (ACLY) from mitochondria-derived citrate or by acetyl-CoA synthetase short-chain family member 2 (ACSS2) from acetate. It has been reported that ACLY is the primary enzyme involved in making cytosolic Ac-CoA in cells with abundant nutrients. However, using CRISPR/Cas9 technology, we have shown that ACLY is not essential for HCMV growth and virally induced lipogenesis. Instead, we found that in HCMV-infected cells glucose carbon can be used for lipid synthesis by both ACLY and ACSS2 reactions. Further, the ACSS2 reaction can compensate for the loss of ACLY. However, in ACSS2-KO human fibroblasts both HCMV-induced lipogenesis from glucose and viral growth were sharply reduced. This reduction suggests that glucose-derived acetate is being used to synthesize cytosolic Ac-CoA by ACSS2. Previous studies have not established a mechanism for the production of acetate directly from glucose metabolism. Here we show that HCMV-infected cells produce more glucose-derived pyruvate, which can be converted to acetate through a nonenzymatic mechanism.

  17. Bacterial Muramyl Dipeptide (MDP) Restricts Human Cytomegalovirus Replication via an IFN-β-Dependent Pathway.

    PubMed

    Kapoor, Arun; Fan, Yi-Hsin; Arav-Boger, Ravit

    2016-02-02

    We recently reported that induction of NOD2 by human Cytomegalovirus (HCMV) resulted in virus inhibition and upregulation of antiviral and inflammatory cytokines. Here we investigated the effects of muramyl dipeptide (MDP), a bacterial cell wall component that activates NOD2, on HCMV replication and antiviral responses. HCMV infection of human foreskin fibroblasts induced NOD2, the downstream receptor-interacting serine/threonine-protein kinase 2 (RIPK2), resulting in phosphorylation of TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3). MDP treatment following infection at low multiplicity (MOI = 0.1 PFU/cell) inhibited HCMV in a dose-dependent manner and further induced phosphorylation of TBK1, IRF3 and expression of IFN-β. None of these effects of MDP were observed following infection at multiplicity of 1. In infected NOD2 knocked-down cells MDP did not induce IFN-β, irrespective of MOI. Treatment with MDP before infection also inhibited HCMV, an effect augmented with treatment duration. Treatment with an IFN-β receptor blocking antibody or knockdown of IFN-β significantly attenuated the inhibitory effect of MDP on HCMV. MDP treatment before or after infection with herpesvirus 1 did not inhibit its replication. Summarized, NOD2 activation exerts anti-HCMV activities predominantly via IFN-β. Since MDP is a bacterial cell wall component, ongoing microbial exposure may influence HCMV replication.

  18. Human Cytomegalovirus Encoded Homologs of Cytokines, Chemokines and their Receptors: Roles in Immunomodulation

    PubMed Central

    McSharry, Brian P.; Avdic, Selmir; Slobedman, Barry

    2012-01-01

    Human cytomegalovirus (HCMV), the largest human herpesvirus, infects a majority of the world’s population. Like all herpesviruses, following primary productive infection, HCMV establishes a life-long latent infection, from which it can reactivate years later to produce new, infectious virus. Despite the presence of a massive and sustained anti-HCMV immune response, productively infected individuals can shed virus for extended periods of time, and once latent infection is established, it is never cleared from the host. It has been proposed that HCMV must therefore encode functions which help to evade immune mediated clearance during productive virus replication and latency. Molecular mimicry is a strategy used by many viruses to subvert and regulate anti-viral immunity and HCMV has hijacked/developed a range of functions that imitate host encoded immunomodulatory proteins. This review will focus on the HCMV encoded homologs of cellular cytokines/chemokines and their receptors, with an emphasis on how these virus encoded homologs may facilitate viral evasion of immune clearance. PMID:23202490

  19. Multipotent mesenchymal stromal cells are fully permissive for human cytomegalovirus infection.

    PubMed

    Qiao, Guan-Hua; Zhao, Fei; Cheng, Shuang; Luo, Min-Hua

    2016-06-01

    Congenital human cytomegalovirus (HCMV) infection is a leading infectious cause of birth defects. Previous studies have reported birth defects with multiple organ maldevelopment in congenital HCMV-infected neonates. Multipotent mesenchymal stromal cells (MSCs) are a group of stem/progenitor cells that are multi-potent and can self-renew, and they play a vital role in multi-organ formation. Whether MSCs are susceptible to HCMV infection is unclear. In this study, MSCs were isolated from Wharton's jelly of the human umbilical cord and identified by their plastic adherence, surface marker pattern, and differentiation capacity. Then, the MSCs were infected with the HCMV Towne strain, and infection status was assessed via determination of viral entry, replication initiation, viral protein expression, and infectious virion release using western blotting, immunofluorescence assays, and plaque forming assays. The results indicate that the isolated MSCs were fully permissive for HCMV infection and provide a preliminary basis for understanding the pathogenesis of HCMV infection in non-nervous system diseases, including multi-organ malformation during fetal development.

  20. Roles of host and viral microRNAs in human cytomegalovirus biology

    PubMed Central

    Dhuruvasan, Kavitha; Sivasubramanian, Geetha; Pellett, Philip E.

    2011-01-01

    Human cytomegalovirus (HCMV) has a relatively large and complex genome, a protracted lytic replication cycle, and employs a strategy of replicational latency as part of its lifelong persistence in the infected host. An important form of gene regulation in plants and animals revolves around a type of small RNA known as microRNA (miRNA). miRNAs can serve as major regulators of key developmental pathways, as well as provide subtle forms of regulatory control. The human genome encodes over 900 miRNAs, and miRNAs are also encoded by some viruses, including HCMV, which encodes at least 14 miRNAs. Some of the HCMV miRNAs are known to target both viral and cellular genes, including important immunomodulators. In addition to expressing their own miRNAs, infections with some viruses, including HCMV, can result in changes in the expression of cellular miRNAs that benefit virus replication. In this review, we summarize the connections between miRNAs and HCMV biology. We describe the nature of miRNA genes, miRNA biogenesis and modes of action, methods for studying miRNAs, HCMV-encoded miRNAs, effects of HCMV infection on cellular miRNA expression, roles of miRNAs in HCMV biology, and possible HCMV-related diagnostic and therapeutic applications of miRNAs. PMID:20969901

  1. Occupational trichloroethylene hypersensitivity syndrome with human herpesvirus-6 and cytomegalovirus reactivation.

    PubMed

    Watanabe, Hideaki; Tohyama, Mikiko; Kamijima, Michihiro; Nakajima, Tamie; Yoshida, Takemi; Hashimoto, Koji; Iijima, Masafumi

    2010-08-01

    Patients having a generalised rash with severe liver dysfunction associated with exposure to trichloroethylene (TCE) have been reported mainly in Asian countries. However, no case has been reported in Japan since the 1990s. Here, we describe a case of hypersensitivity syndrome (HS) caused by TCE in a 30-year-old Japanese man. The patient developed a rash, fever and liver dysfunction 21 days after he had been exposed to TCE at his workplace. Serum human herpesvirus (HHV)-6 and cytomegalovirus (CMV) DNA were detected 4 and 7 weeks, respectively, after the onset; the IgG antibody titres to HHV-6 and CMV were significantly elevated 6 and 9 weeks, respectively, after the onset. Patch testing was positive for the metabolites of TCE (i.e. trichloroethanol, trichloroacetic acid and chloral hydrate) but not for TCE itself; these results suggest that the TCE metabolites induced this disease. Human leucocyte antigen-B*1301, which has been reported to be strongly associated with TCE-induced HS, was identified in this patient. In addition, the clinical findings, laboratory data and period of virus reactivation after onset were quite similar to those of drug-induced hypersensitivity syndrome (DIHS). We also review TCE-induced HS from the viewpoint of the similarity to DIHS in this article. Copyright 2010 S. Karger AG, Basel.

  2. Kissing as an evolutionary adaptation to protect against Human Cytomegalovirus-like teratogenesis.

    PubMed

    Hendrie, C A; Brewer, G

    2010-02-01

    Mouth to mouth sexual kissing is seen in more than 90% of human cultures. Various theories have been put forward to account for this but none offer a full explanation within an evolutionary framework. As mouth to mouth sexual kissing exposes each participant to the diseases of the other, it must confer significant benefit. Human Cytomegalovirus (HCMV) is a ubiquitous infection that carries a severe teratogenic risk if primary infection is acquired during certain critical periods. As HCMV is present in salivary gland epithelial cells and sheds from periodontitis induced lesions, female inoculation with a specific male's HCMV is most efficiently achieved through mouth to mouth contact and saliva exchange, particularly where the flow of saliva is from the male to the typically shorter female. The current hypothesis proposes that mouth to mouth sexual kissing enables females to control when they become infected with a particular male's HCMV and so protect their offspring from the threat of teratogenesis from primary infection during vulnerable times in their development. Females only gain this benefit if they also avoid becoming infected by other males. Hence HCMV induced teratogenesis is a strong viral pressure towards the development of monogamy as well as kissing as a behavioural strategy to protect against it.

  3. In vitro generation of human cytomegalovirus pp65 antigenemia, viremia, and leukoDNAemia.

    PubMed Central

    Revello, M G; Percivalle, E; Arbustini, E; Pardi, R; Sozzani, S; Gerna, G

    1998-01-01

    Immunocompromised patients with disseminated human cytomegalovirus (HCMV) infection have circulating PMN carrying HCMV pp65 (antigenemia), infectious virus (viremia), and viral DNA (leukoDNAemia). Because HCMV does not fully replicate in PMN, it is generally hypothesized that virions and viral materials are taken up by phagocytosis from fully permissive HCMV-infected endothelial cells. However, no experimental evidence has ever been provided for these PMN-endothelium interactions. PMN from 11 donors were cocultured with endothelial cells infected with an endothelium-adapted HCMV strain and with human fibroblasts infected with low-passaged clinical and laboratory-adapted HCMV strains. pp65-positive PMN were detected after coculture with both HCMV-infected endothelial and fibroblast cells, provided that wild and not laboratory-adapted strains were used. In addition, cocultured PMN carried infectious virus as demonstrated by virus isolation and presence of complete virus particles by electron microscopy. Moreover, high levels of viral DNA were consistently detected by quantitative PCR in cocultured PMN. Thus, we have generated in vitro the three most important viral parameters detected in patients with disseminated HCMV infection (antigenemia, viremia, and leukoDNAemia). The failure of laboratory-adapted HCMV strain to induce this phenomenon demonstrates that important modifications have occurred in attenuated viral strains affecting basic biological functions. PMID:9637702

  4. Human cytomegalovirus inhibits a DNA damage response by mislocalizing checkpoint proteins

    NASA Astrophysics Data System (ADS)

    Gaspar, Miguel; Shenk, Thomas

    2006-02-01

    The DNA damage checkpoint pathway responds to DNA damage and induces a cell cycle arrest to allow time for DNA repair. Several viruses are known to activate or modulate this cellular response. Here we show that the ataxia-telangiectasia mutated checkpoint pathway, which responds to double-strand breaks in DNA, is activated in response to human cytomegalovirus DNA replication. However, this activation does not propagate through the pathway; it is blocked at the level of the effector kinase, checkpoint kinase 2 (Chk2). Late after infection, several checkpoint proteins, including ataxia-telangiectasia mutated and Chk2, are mislocalized to a cytoplasmic virus assembly zone, where they are colocalized with virion structural proteins. This colocalization was confirmed by immunoprecipitation of virion proteins with an antibody that recognizes Chk2. Virus replication was resistant to ionizing radiation, which causes double-strand breaks in DNA. We propose that human CMV DNA replication activates the checkpoint response to DNA double-strand breaks, and the virus responds by altering the localization of checkpoint proteins to the cytoplasm and thereby inhibiting the signaling pathway. ionizing radiation | ataxia-telangiectasia mutated pathway

  5. Induction of Pluripotent Protective Immunity Following Immunisation with a Chimeric Vaccine against Human Cytomegalovirus

    PubMed Central

    Zhong, Jie; Rist, Michael; Cooper, Leanne; Smith, Corey; Khanna, Rajiv

    2008-01-01

    Based on the life-time cost to the health care system, the Institute of Medicine has assigned the highest priority for a vaccine to control human cytomegalovirus (HCMV) disease in transplant patients and new born babies. In spite of numerous attempts successful licensure of a HCMV vaccine formulation remains elusive. Here we have developed a novel chimeric vaccine strategy based on a replication-deficient adenovirus which encodes the extracellular domain of gB protein and multiple HLA class I & II-restricted CTL epitopes from HCMV as a contiguous polypeptide. Immunisation with this chimeric vaccine consistently generated strong HCMV-specific CD8+ and CD4+ T-cells which co-expressed IFN-γ and TNF-α, while the humoral response induced by this vaccine showed strong virus neutralizing capacity. More importantly, immunization with adenoviral chimeric vaccine also afforded protection against challenge with recombinant vaccinia virus encoding HCMV antigens and this protection was associated with the induction of a pluripotent antigen-specific cellular and antibody response. Furthermore, in vitro stimulation with this adenoviral chimeric vaccine rapidly expanded multiple antigen-specific human CD8+ and CD4+ T-cells from healthy virus carriers. These studies demonstrate that the adenovirus chimeric HCMV vaccine provides an excellent platform for reconstituting protective immunity to prevent HCMV diseases in different clinical settings. PMID:18806877

  6. Germline V-genes sculpt the binding site of a family of antibodies neutralizing human cytomegalovirus

    SciTech Connect

    Thomson, Christy A.; Bryson, Steve; McLean, Gary R.; Creagh, A. Louise; Pai, Emil F.; Schrader, John W.

    2008-10-17

    Immunoglobulin genes are generated somatically through specialized mechanisms resulting in a vast repertoire of antigen-binding sites. Despite the stochastic nature of these processes, the V-genes that encode most of the antigen-combining site are under positive evolutionary selection, raising the possibility that V-genes have been selected to encode key structural features of binding sites of protective antibodies against certain pathogens. Human, neutralizing antibodies to human cytomegalovirus that bind the AD-2S1 epitope on its gB envelope protein repeatedly use a pair of well-conserved, germline V-genes IGHV3-30 and IGKV3-11. Here, we present crystallographic, kinetic and thermodynamic analyses of the binding site of such an antibody and that of its primary immunoglobulin ancestor. These show that these germline V-genes encode key side chain contacts with the viral antigen and thereby dictate key structural features of the hypermutated, high-affinity neutralizing antibody. V-genes may thus encode an innate, protective immunological memory that targets vulnerable, invariant sites on multiple pathogens.

  7. Bacterial Muramyl Dipeptide (MDP) Restricts Human Cytomegalovirus Replication via an IFN-β-Dependent Pathway

    PubMed Central

    Kapoor, Arun; Fan, Yi-Hsin; Arav-Boger, Ravit

    2016-01-01

    We recently reported that induction of NOD2 by human Cytomegalovirus (HCMV) resulted in virus inhibition and upregulation of antiviral and inflammatory cytokines. Here we investigated the effects of muramyl dipeptide (MDP), a bacterial cell wall component that activates NOD2, on HCMV replication and antiviral responses. HCMV infection of human foreskin fibroblasts induced NOD2, the downstream receptor-interacting serine/threonine-protein kinase 2 (RIPK2), resulting in phosphorylation of TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3). MDP treatment following infection at low multiplicity (MOI = 0.1 PFU/cell) inhibited HCMV in a dose-dependent manner and further induced phosphorylation of TBK1, IRF3 and expression of IFN-β. None of these effects of MDP were observed following infection at multiplicity of 1. In infected NOD2 knocked-down cells MDP did not induce IFN-β, irrespective of MOI. Treatment with MDP before infection also inhibited HCMV, an effect augmented with treatment duration. Treatment with an IFN-β receptor blocking antibody or knockdown of IFN-β significantly attenuated the inhibitory effect of MDP on HCMV. MDP treatment before or after infection with herpesvirus 1 did not inhibit its replication. Summarized, NOD2 activation exerts anti-HCMV activities predominantly via IFN-β. Since MDP is a bacterial cell wall component, ongoing microbial exposure may influence HCMV replication. PMID:26830977

  8. Inhibition of UL54 and UL97 genes of human cytomegalovirus by RNA interference.

    PubMed

    Shin, M-C; Hong, S-K; Yoon, J-S; Park, S-S; Lee, S-G; Lee, D-G; Min, W-S; Shin, W-S; Paik, S-Y

    2006-01-01

    Short interfering RNAs (siRNAs), namely siUL54-1 and siU54-2 targeting UL54 (DNA polymerase) gene, and siUL97-1 and siUL97-2 targeting UL97 (phosphotransferase) gene, were used to inhibit respective genes of Human cytomegalovirus (HCMV) and consequently the virus infection process in human foreskin fibroblast (HFF) cultures. The virus infection was monitored by cell morphology (CPE), levels of UL83 and IE86 mRNAs, and virus antigen. The results showed that siUL97-2 remarkably inhibited viral CPE while other siRNAs were less inhibitory. The siRNAs reduced the levels of UL83 mRNA but not that of IE86 mRNA; again, siUL97-2 was most inhibitory. Particularly, siUL97-2 reduced the UL83 mRNA level 14, 19, 203, and 37 times at 24, 48, 72, and 96 hrs post infection (p.i.), respectively. When tested for the effect on viral antigen by immunofluorescent assay (IFA), UL97-2 exerted a marked inhibition. These results demonstrate the effectiveness of siRNAs against experimental HCMV infection and indicate their therapeutic potential.

  9. Upregulation of functionally active vascular endothelial growth factor by human cytomegalovirus.

    PubMed

    Reinhardt, Barbara; Schaarschmidt, Peter; Bossert, Andrea; Lüske, Anke; Finkenzeller, Günter; Mertens, Thomas; Michel, Detlef

    2005-01-01

    Human cytomegalovirus (HCMV) infection is known to modulate host gene expression and has been linked to the pathogenesis of vasculopathies; however, relevant pathomechanisms are still unclear. It was shown that HCMV infection leads to upregulation of vascular endothelial growth factor (VEGF) expression in human foreskin fibroblasts and coronary artery smooth muscle cells (SMC). Activation of VEGF transcription by HCMV infection was confirmed by transient-expression experiments, which revealed that a short promoter fragment, pLuc135 (-85 to +50), is sufficient for activation. Site-directed mutagenesis of Sp1-recognition sites within this fragment abolished the upregulation of transcription. Functional VEGF protein is released into the culture supernatant of infected SMC. Incubation of endothelial cells with supernatants from HCMV-infected SMC cultures induced upregulation of VEGF receptor-2 expression on endothelial cells, as well as a significant upregulation of DNA synthesis, implicating cell proliferation. The mean incline of DNA synthesis at 48 and 72 h post-infection was 148 and 197 %, respectively. Addition of neutralizing antibodies against VEGF completely abolished this effect. Supernatants from SMC cultures incubated with UV-inactivated virus induced a comparable effect. This virus-induced paracrine effect may represent a molecular mechanism for HCMV-induced pathogenesis, such as inflammatory vasculopathies, by inducing a proatherogenic phenotype in SMC.

  10. Human Cytomegalovirus DNA Quantification and Gene Expression in Gliomas of Different Grades

    PubMed Central

    Medeiros, Raphael Salles Scortegagna; Guerra, Juliana Mariotti; Kimura, Lidia Midori; Shirata, Neuza Kazumi; Nonogaki, Suely; dos Santos, Claudia Januário; Carlan Silva, Maria Cristina

    2016-01-01

    Gliomas are the most common type of primary brain tumors. The most aggressive type, Glioblastoma multiforme (GBM), is one of the deadliest human diseases, with an average survival at diagnosis of about 1 year. Previous evidence suggests a link between human cytomegalovirus (HCMV) and gliomas. HCMV has been shown to be present in these tumors and several viral proteins can have oncogenic properties in glioma cells. Here we have investigated the presence of HCMV DNA, RNA and proteins in fifty-two gliomas of different grades of malignancy. The UL83 viral region, the early beta 2.7 RNA and viral protein were detected in 73%, 36% and 57% by qPCR, ISH and IHC, respectively. Positivity of the viral targets and viral load was independent of tumor type or grade suggesting no correlation between viral presence and tumor progression. Our results demonstrate high prevalence of the virus in gliomas from Brazilian patients, contributing to a better understanding of the association between HCMV infection and gliomas worldwide and supporting further investigations of the virus oncomodulatory properties. PMID:27458810

  11. Enhanced capacity of DNA repair in human cytomegalovirus-infected cells

    SciTech Connect

    Nishiyama, Y.; Rapp, F.

    1981-04-01

    Plaque formation in Vero cells by UV-irradiated herpes simplex virus was enhanced by infection with human cytomegalovirus (HCMV), UV irradiation, or treatment with methylmethanesulfonate. Preinfection of Vero cells with HCMV enhanced reactivation of UV-irradiated herpes simplex virus more significantly than did treatment with UV or methylmethanesulfonate alone. A similar enhancement by HCMV was observed in human embryonic fibroblasts, but not in xeroderma pigmentosum (XP12BE) cells. It was also found that HCMV infection enhanced hydroxyurea-resistant DNA synthesis induced by UV light or methylmethanesulfonate. Alkaline sucrose gradient sedimentation analysis revealed an enhanced rate of synthesis of all size classes of DNA in UV-irradiated HCMV-infected Vero cells. However, HCMV infection did not induce repairable lesions in cellular DNA and did not significantly inhibit host cell DNA synthesis, unlike UV or methylmethanesulfonate. These results indicate that HCMV enhanced DNA repair capacity in the host cells without producing detectable lesions in cellular DNA and without inhibiting DNA synthesis. This repair appeared to be error proof for UV-damaged herpes simplex virus DNA when tested with herpes simplex virus thymidine kinase-negative mutants.

  12. Human cytomegalovirus renders cells non-permissive for replication of herpes simplex viruses

    SciTech Connect

    Cockley, K.D.

    1988-01-01

    The herpes simplex virus (HSV) genome during production infection in vitro may be subject to negative regulation which results in modification of the cascade of expression of herpes virus macromolecular synthesis leading to establishment of HSV latency. In the present study, human embryonic lung (HEL) cells infected with human cytomegalovirus (HCMV) restricted the replication of HSV type-1 (HSV-1). A delay in HSV replication of 15 hr as well as a consistent, almost 1000-fold inhibition of HSV replication in HCMV-infected cell cultures harvested 24 to 72 hr after superinfection were observed compared with controls infected with HSV alone. HSV type-2 (HSV-2) replication was similarly inhibited in HCMV-infected HEL cells. Prior ultraviolet-irradiation (UV) of HCMV removed the block to HSV replication, demonstrating the requirement for an active HCMV genome. HCMV deoxyribonucleic acid (DNA) negative temperature-sensitive (ts) mutants inhibited HSV replications as efficiently as wild-type (wt) HCMV at the non-permissive temperature. Evidence for penetration and replication of superinfecting HSV into HCMV-infected cells was provided by blot hybridization of HSV DNA synthesized in HSV-superinfected cell cultures and by cesium chloride density gradient analysis of ({sup 3}H)-labeled HSV-1-superinfected cells.

  13. Human cytomegalovirus infection downregulates vitamin-D receptor in mammalian cells.

    PubMed

    Rieder, Franz J J; Gröschel, Charlotte; Kastner, Marie-Theres; Kosulin, Karin; Laengle, Johannes; Zadnikar, Rene; Marculescu, Rodrig; Schneider, Martina; Lion, Thomas; Bergmann, Michael; Kallay, Enikö; Steininger, Christoph

    2017-01-01

    Vitamin D (VD) is essential for the human body and involved in a wide variety of critical physiological processes including bone, muscle, and cardiovascular health, as well as innate immunity and antimicrobial responses. Here, we elucidated the significance of the VD system in cytomegalovirus (CMV) infection, which is one of the most common opportunistic infections in immunocompromised or -suppressed patients. We found that expression of vitamin D receptor (VDR) was downregulated in CMV-infected cells within 12h [hrs] post infection [p.i.] to 12% relative to VDR expression in mock-infected fibroblasts and did not recover during the CMV replication cycle of 96h. None of the biologically active metabolites of VD, cholecalciferol, calcidiol, or calcitriol, inhibit CMV replication significantly in human fibroblasts. In a feedback loop, expression of CYP24A1 dropped to 3% by 12h p.i. and expression of CYP27B1 increased gradually during the replication cycle of CMV to 970% probably as a consequence of VDR inhibition. VDR expression was not downregulated during influenza virus or adenovirus replication. The potent synthetic vitamin D analog EB-1089 was not able to inhibit CMV replication or antagonize its effect on VDR expression. Only CMV replication, and none of the other viral pathogens evaluated, inhibited the vitamin D system in vitro. In view of the pleiotropism of VDR, CMV-mediated downregulation may have far-reaching virological, immunological, and clinical implications and thus warrant further evaluations in vitro and in vivo.

  14. Generation of potent neutralizing human monoclonal antibodies against cytomegalovirus infection from immune B cells

    PubMed Central

    Funaro, Ada; Gribaudo, Giorgio; Luganini, Anna; Ortolan, Erika; Lo Buono, Nicola; Vicenzi, Elisa; Cassetta, Luca; Landolfo, Santo; Buick, Richard; Falciola, Luca; Murphy, Marianne; Garotta, Gianni; Malavasi, Fabio

    2008-01-01

    Background Human monoclonal antibodies (mAbs) generated as a result of the immune response are likely to be the most effective therapeutic antibodies, particularly in the case of infectious diseases against which the immune response is protective. Human cytomegalovirus (HCMV) is an ubiquitous opportunistic virus that is the most serious pathogenic agent in transplant patients. The available therapeutic armamentarium (e.g. HCMV hyperimmune globulins or antivirals) is associated with severe side effects and the emergence of drug-resistant strains; therefore, neutralizing human mAb may be a decisive alternative in the prevention of primary and re-activated HCMV infections in these patients. Results The purpose of this study was to generate neutralizing mAb against HCMV from the immunological repertoire of immune donors. To this aim, we designed an efficient technology relying on two discrete and sequential steps: first, human B-lymphocytes are stimulated with TLR9-agonists and IL-2; second, after both additives are removed, the cells are infected with EBV. Using this strategy we obtained 29 clones secreting IgG neutralizing the HCMV infectivity; four among these were further characterized. All of the mAbs neutralize the infection in different combinations of HCMV strains and target cells, with a potency ~20 fold higher than that of the HCMV hyperimmune globulins, currently used in transplant recipients. Recombinant human monoclonal IgG1 suitable as a prophylactic or therapeutic tool in clinical applications has been generated. Conclusion The technology described has proven to be more reproducible, efficient and rapid than previously reported techniques, and can be adopted at low overall costs by any cell biology laboratory for the development of fully human mAbs for immunotherapeutic uses. PMID:19014469

  15. Evolution of the ability to modulate host chemokine networks via gene duplication in human cytomegalovirus (HCMV).

    PubMed

    Scarborough, Jessica A; Paul, John R; Spencer, Juliet V

    2017-03-14

    Human cytomegalovirus (HCMV) is a widespread pathogen that is particularly skillful at evading immune detection and defense mechanisms, largely due to extensive co-evolution with its host. One aspect of this co-evolution involves the acquisition of virally encoded G protein-coupled receptors (GPCRs) with homology to the chemokine receptor family. GPCRs are the largest family of cell surface proteins, found in organisms from yeast to humans, and they regulate a variety of cellular processes including development, sensory perception, and immune cell trafficking. The US27 and US28 genes are encoded by human and primate CMVs, but homologs are not found in the genomes of viruses infecting rodents or other species. Phylogenetic analysis was used to investigate the US27 and US28 genes, which are adjacent in the unique short (US) region of the HCMV genome, and their relationship to one another and to human chemokine receptor genes. The results indicate that both US27 and US28 share the same common ancestor with human chemokine receptor CX3CR1, suggesting that a single host gene was captured and a subsequent viral gene duplication event occurred. The US28 gene product (pUS28) has maintained the function of the ancestral gene and has the ability to bind and signal in response to CX3CL1/fractalkine, the natural ligand for CX3CR1. In contrast, pUS27 does not bind to any known chemokine ligand, and the sequence has diverged significantly, highlighted by the fact that pUS27 currently exhibits greater sequence similarity to human CCR1. While the evolutionary advantage of the gene duplication and neofunctionalization event remains unclear, the US27 and US28 genes are highly conserved among different HCMV strains and retained even in laboratory strains that have lost many virulence genes, suggesting that US27 and US28 have each evolved distinct, important functions during virus infection.

  16. Induction of chromosome aberrations and mitotic arrest by cytomegalovirus in human cells

    SciTech Connect

    AbuBakar, S.; Au, W.W.; Legator, M.S.; Albrecht, T.

    1988-01-01

    Human cytomegalovirus (CMV) is potentially an effective but often overlooked genotoxic agent in humans. We report here evidence that indicates that infection by CMV can induce chromosome alterations and mitotic inhibition. The frequency of chromosome aberrations induced was dependent on the input multiplicity of infection (m.o.i.) for human lung fibroblasts (LU), but not for human peripheral blood lymphocytes (PBLs) when both cell types were infected at the GO phase of the cell cycle. The aberrations induced by CMV were mostly chromatid breaks and chromosome pulverizations that resembled prematurely condensed S-phase chromatin. Pulverized chromosomes were not observed in LU cells infected with virus stocks that had been rendered nonlytic by UV-irradiation at 24,000 ergs/mm2 or from infection of human lymphocytes. In LU cells infected with UV-irradiated CMV, the frequency of aberrations induced was inversely dependent on the extent of the exposure of the CMV stock to the UV-light. In permissive CMV infection of proliferating LU cells at 24 hr after subculture, a high percentage (greater than 40%) of the metaphase cells were arrested at their first metaphase and displayed severely condensed chromosomes when harvested 48 hr later. A significant increase (p less than 0.05) in the chromosome aberration frequency was also observed. Our study shows that CMV infection is genotoxic to host cells. The types and extent of damage are dependent on the viral genome expression and on the cell cycle stage of the cells at the time of infection. The possible mechanisms for induction of chromosome damage by CMV are discussed.

  17. Human Cytomegalovirus miR-UL148D Facilitates Latent Viral Infection by Targeting Host Cell Immediate Early Response Gene 5

    PubMed Central

    Li, Limin; Li, Donghai; Liu, Fenyong; Zhang, Chen-Yu; Zen, Ke

    2016-01-01

    The mechanisms underlying human cytomegalovirus (HCMV) latency remain incompletely understood. Here, we showed that a HCMV-encoded miRNA, miR-UL148D, robustly accumulates during late stages of experimental latent HCMV infection in host cells and promotes HCMV latency by modulating the immediate early response gene 5 (IER5)-cell division cycle 25B (CDC25B) axis in host cells. miR-UL148D inhibited IER5 expression by directly targeting the three-prime untranslated region(3’UTR) of IER5 mRNA and thus rescued CDC25B expression during the establishment of viral latency. Infection with NR-1ΔmiR-UL148D, a derivative of the HCMV clinical strain NR-1 with a miR-UL148D knockout mutation, resulted in sustained induction of IER5 expression but decreased CDC25B expression in host cells. Mechanistically, we further showed that CDC25B plays an important role in suppressing HCMV IE1 and lytic gene transcription by activating cyclin-dependent kinase 1 (CDK-1). Both gain-of-function and lose-of-function assays demonstrated that miR-UL148D promotes HCMV latency by helping maintain CDC25B activity in host cells. These results provide a novel mechanism through which a HCMV miRNA regulates viral latency. PMID:27824944

  18. Proteomic analyses of human cytomegalovirus strain AD169 derivatives reveal highly conserved patterns of viral and cellular proteins in infected fibroblasts.

    PubMed

    Reyda, Sabine; Büscher, Nicole; Tenzer, Stefan; Plachter, Bodo

    2014-01-07

    Human cytomegalovirus (HCMV) particle morphogenesis in infected cells is an orchestrated process that eventually results in the release of enveloped virions. Proteomic analysis has been employed to reveal the complexity in the protein composition of these extracellular particles. Only limited information is however available regarding the proteome of infected cells preceding the release of HCMV virions. We used quantitative mass spectrometry to address the pattern of viral and cellular proteins in cells, infected with derivatives of the AD169 laboratory strain. Our analyses revealed a remarkable conservation in the patterns of viral and of abundant cellular proteins in cells, infected for 2 hours, 2 days, or 4 days. Most viral proteins increased in abundance as the infection progressed over time. Of the proteins that were reliably detectable by mass spectrometry, only IE1 (pUL123), pTRS1, and pIRS1 were downregulated at 4 days after infection. In addition, little variation of viral proteins in the virions of the different viruses was detectable, independent of the expression of the major tegument protein pp65. Taken together these data suggest that there is little variation in the expression program of viral and cellular proteins in cells infected with related HCMVs, resulting in a conserved pattern of viral proteins ultimately associated with extracellular virions.

  19. Human Cytomegalovirus (HCMV)-Specific CD4(+) T Cells Are Polyfunctional and Can Respond to HCMV-Infected Dendritic Cells In Vitro.

    PubMed

    Jackson, Sarah E; Sedikides, George X; Mason, Gavin M; Okecha, Georgina; Wills, Mark R

    2017-03-15

    Human cytomegalovirus (HCMV) infection and periodic reactivation are generally well controlled by the HCMV-specific T cell response in healthy people. While the CD8(+) T cell response to HCMV has been extensively studied, the HCMV-specific CD4(+) T cell effector response is not as well understood, especially in the context of direct interactions with HCMV-infected cells. We screened the gamma interferon (IFN-γ) and interleukin-10 (IL-10) responses to 6 HCMV peptide pools (pp65, pp71, IE1, IE2, gB, and US3, selected because they were the peptides most frequently responded to in our previous studies) in 84 donors aged 23 to 74 years. The HCMV-specific CD4(+) T cell response to pp65, IE1, IE2, and gB was predominantly Th1 biased, with neither the loss nor the accumulation of these responses occurring with increasing age. A larger proportion of donors produced an IL-10 response to pp71 and US3, but the IFN-γ response was still dominant. CD4(+) T cells specific to the HCMV proteins studied were predominantly effector memory cells and produced both cytotoxic (CD107a expression) and cytokine (macrophage inflammatory protein 1β secretion) effector responses. Importantly, when we measured the CD4(+) T cell response to cytomegalovirus (CMV)-infected dendritic cells in vitro, we observed that the CD4(+) T cells produced a range of cytotoxic and secretory effector functions, despite the presence of CMV-encoded immune evasion molecules. CD4(+) T cell responses to HCMV-infected dendritic cells were sufficient to control the dissemination of virus in an in vitro assay. Together, the results show that HCMV-specific CD4(+) T cell responses, even those from elderly individuals, are highly functional and are directly antiviral.IMPORTANCE Human cytomegalovirus (HCMV) infection is carried for a lifetime and in healthy people is kept under control by the immune system. HCMV has evolved many mechanisms to evade the immune response, possibly explaining why the virus is never eliminated

  20. Sequence homology between HLA-bound cytomegalovirus and human peptides: A potential trigger for alloreactivity

    PubMed Central

    Koparde, Vishal N.; Jameson-Lee, Maximilian; Elnasseh, Abdelrhman G.; Scalora, Allison F.; Kobulnicky, David J.; Serrano, Myrna G.; Roberts, Catherine H.; Buck, Gregory A.; Neale, Michael C.; Nixon, Daniel E.; Toor, Amir A.

    2017-01-01

    Human cytomegalovirus (hCMV) reactivation may often coincide with the development of graft-versus-host-disease (GVHD) in stem cell transplantation (SCT). Seventy seven SCT donor-recipient pairs (DRP) (HLA matched unrelated donor (MUD), n = 50; matched related donor (MRD), n = 27) underwent whole exome sequencing to identify single nucleotide polymorphisms (SNPs) generating alloreactive peptide libraries for each DRP (9-mer peptide-HLA complexes); Human CMV CROSS (Cross-Reactive Open Source Sequence) database was compiled from NCBI; HLA class I binding affinity for each DRPs HLA was calculated by NetMHCpan 2.8 and hCMV- derived 9-mers algorithmically compared to the alloreactive peptide-HLA complex libraries. Short consecutive (≥6) amino acid (AA) sequence homology matching hCMV to recipient peptides was considered for HLA-bound-peptide (IC50<500nM) cross reactivity. Of the 70,686 hCMV 9-mers contained within the hCMV CROSS database, an average of 29,658 matched the MRD DRP alloreactive peptides and 52,910 matched MUD DRP peptides (p<0.001). In silico analysis revealed multiple high affinity, immunogenic CMV-Human peptide matches (IC50<500 nM) expressed in GVHD-affected tissue-specific manner. hCMV+GVHD was found in 18 patients, 13 developing hCMV viremia before GVHD onset. Analysis of patients with GVHD identified potential cross reactive peptide expression within affected organs. We propose that hCMV peptide sequence homology with human alloreactive peptides may contribute to the pathophysiology of GVHD. PMID:28800601

  1. The Transcription and Translation Landscapes during Human Cytomegalovirus Infection Reveal Novel Host-Pathogen Interactions

    PubMed Central

    Shitrit, Alina; Shani, Odem; Le-Trilling, Vu Thuy Khanh; Trilling, Mirko; Friedlander, Gilgi; Tanenbaum, Marvin; Stern-Ginossar, Noam

    2015-01-01

    Viruses are by definition fully dependent on the cellular translation machinery, and develop diverse mechanisms to co-opt this machinery for their own benefit. Unlike many viruses, human cytomegalovirus (HCMV) does suppress the host translation machinery, and the extent to which translation machinery contributes to the overall pattern of viral replication and pathogenesis remains elusive. Here, we combine RNA sequencing and ribosomal profiling analyses to systematically address this question. By simultaneously examining the changes in transcription and translation along HCMV infection, we uncover extensive transcriptional control that dominates the response to infection, but also diverse and dynamic translational regulation for subsets of host genes. We were also able to show that, at late time points in infection, translation of viral mRNAs is higher than that of cellular mRNAs. Lastly, integration of our translation measurements with recent measurements of protein abundance enabled comprehensive identification of dozens of host proteins that are targeted for degradation during HCMV infection. Since targeted degradation indicates a strong biological importance, this approach should be applicable for discovering central host functions during viral infection. Our work provides a framework for studying the contribution of transcription, translation and degradation during infection with any virus. PMID:26599541

  2. Human Cytomegalovirus Secretome Contains Factors That Induce Angiogenesis and Wound Healing

    SciTech Connect

    Dumortier, Jerome; Streblow, Daniel N.; Moses, Ashlee V.; Jacobs, Jon M.; Kreklywich, Craig N.; Camp, David G.; Smith, Richard D.; Orloff, Susan L.; Nelson, Jay

    2008-07-01

    Human cytomegalovirus (HCMV) is implicated in the acceleration of a number of vascular diseases including transplant vascular sclerosis (TVS), the lesion associated with chronic rejection (CR) of solid organ transplants. Although the virus persists in the allograft throughout the course of disease, few cells are directly infected by CMV. This observation is in contrast to the global effects that CMV has on the acceleration of TVS/CR, suggesting that CMV infection indirectly promotes the vascular disease process. Recent transcriptome analysis of CMV-infected heart allografts indicates that the virus induces cytokines and growth factors associated with angiogenesis (AG) and wound healing (WH), suggesting that CMV may accelerate TVS/CR through the induction and secretion of AG/WH factors from infected cells. We analyzed virus-free supernatants from HCMV-infected cells (HCMV secretomes) for growth factors, by mass spectrometry and immunoassays, and found that the HCMV secretome contains over 1,000 cellular proteins, many of which are involved in AG/WH. Importantly, functional assays demonstrated that CMV but not herpes simplex virus secretomes not only induce AG/WH but also promote neovessel stabilization and endothelial cell survival for 2 weeks. These findings suggest that CMV acceleration of TVS occurs through virus-induced growth factors and cytokines in the CMV secretome.

  3. Characterizing human cytomegalovirus reinfection in congenitally infected infants: an evolutionary perspective.

    PubMed

    Pokalyuk, Cornelia; Renzette, Nicholas; Irwin, Kristen K; Pfeifer, Susanne P; Gibson, Laura; Britt, William J; Yamamoto, Aparecida Y; Mussi-Pinhata, Marisa M; Kowalik, Timothy F; Jensen, Jeffrey D

    2017-04-01

    Given the strong selective pressures often faced by populations when colonizing a novel habitat, the level of variation present on which selection may act is an important indicator of adaptive potential. While often discussed in an ecological context, this notion is also highly relevant in our clinical understanding of viral infection, in which the novel habitat is a new host. Thus, quantifying the factors determining levels of variation is of considerable importance for the design of improved treatment strategies. Here, we focus on such a quantification of human cytomegalovirus (HCMV) - a virus which can be transmitted across the placenta, resulting in foetal infection that can potentially cause severe disease in multiple organs. Recent studies using genomewide sequencing data have demonstrated that viral populations in some congenitally infected infants diverge rapidly over time and between tissue compartments within individuals, while in other infants, the populations remain highly stable. Here, we investigate the underlying causes of these extreme differences in observed intrahost levels of variation by estimating the underlying demographic histories of infection. Importantly, reinfection (i.e. population admixture) appears to be an important, and previously unappreciated, player. We highlight illustrative examples likely to represent a single-population transmission from a mother during pregnancy and multiple-population transmissions during pregnancy and after birth.

  4. Virological and Immunological Characteristics of Human Cytomegalovirus Infection Associated With Alzheimer Disease

    PubMed Central

    Lurain, Nell S.; Hanson, Barbara A.; Martinson, Jeffrey; Leurgans, Sue E.; Landay, Alan L.; Bennett, David A.; Schneider, Julie A.

    2013-01-01

    Serum, cerebrospinal fluid (CSF), and cryopreserved lymphocytes from subjects in the Rush Alzheimer's Disease Center Religious Orders Study were analyzed for associations between cytomegalovirus (CMV) infection and clinical and pathological markers of Alzheimer disease. CMV antibody levels were associated with neurofibrillary tangles (NFTs). CSF interferon γ was only detected in seropositive subjects and was significantly associated with NFTs. The percentage of senescent T cells (CD4+ or CD8+CD28−CD57+) was significantly higher for CMV-seropositive as compared to CMV-seronegative subjects and was marginally associated with the pathologic diagnosis of Alzheimer disease (CD4+) or amyloid-β (CD8+). Immunocytochemical analysis showed induction of amyloid-β in human foreskin fibroblasts (HFFs) infected with each of 3 clinical CMV strains. In the same subjects, there was no association of herpes simplex virus type 1 (HSV-1) antibody levels with CMV antibody levels or clinical or pathological markers of Alzheimer disease. HSV-1 infection of HFFs did not induce amyloid-β. These data support an association between CMV and the development of Alzheimer disease. PMID:23661800

  5. Virological and immunological characteristics of human cytomegalovirus infection associated with Alzheimer disease.

    PubMed

    Lurain, Nell S; Hanson, Barbara A; Martinson, Jeffrey; Leurgans, Sue E; Landay, Alan L; Bennett, David A; Schneider, Julie A

    2013-08-15

    Serum, cerebrospinal fluid (CSF), and cryopreserved lymphocytes from subjects in the Rush Alzheimer's Disease Center Religious Orders Study were analyzed for associations between cytomegalovirus (CMV) infection and clinical and pathological markers of Alzheimer disease. CMV antibody levels were associated with neurofibrillary tangles (NFTs). CSF interferon γ was only detected in seropositive subjects and was significantly associated with NFTs. The percentage of senescent T cells (CD4+ or CD8+CD28-CD57+) was significantly higher for CMV-seropositive as compared to CMV-seronegative subjects and was marginally associated with the pathologic diagnosis of Alzheimer disease (CD4+) or amyloid-β (CD8+). Immunocytochemical analysis showed induction of amyloid-β in human foreskin fibroblasts (HFFs) infected with each of 3 clinical CMV strains. In the same subjects, there was no association of herpes simplex virus type 1 (HSV-1) antibody levels with CMV antibody levels or clinical or pathological markers of Alzheimer disease. HSV-1 infection of HFFs did not induce amyloid-β. These data support an association between CMV and the development of Alzheimer disease.

  6. Nuclear body formation and PML body remodeling by the human cytomegalovirus protein UL35

    SciTech Connect

    Salsman, Jayme; Wang Xueqi; Frappier, Lori

    2011-06-05

    The human cytomegalovirus (HCMV) UL35 gene encodes two proteins, UL35 and UL35a. Expression of UL35 in transfected cells results in the formation of UL35 nuclear bodies that associate with promyelocytic leukemia (PML) protein. PML forms the basis for PML nuclear bodies that are important for suppressing viral lytic gene expression. Given the important relationship between PML and viral infection, we have further investigated the association of UL35 with PML bodies. We demonstrate that UL35 bodies form independently of PML and subsequently recruit PML, Sp100 and Daxx. In contrast, UL35a did not form bodies; however, it could bind UL35 and inhibit the formation of UL35 bodies. The HCMV tegument protein pp71 promoted the formation of UL35 bodies and the cytoplasmic localization of UL35a. Similarly, UL35a shifted pp71 to the cytoplasm. These results indicate that the interplay between UL35, UL35a and pp71 affects their subcellular localization and likely their functions throughout infection.

  7. High frequency of Human Cytomegalovirus DNA in the Liver of Infants with Extrahepatic Neonatal Cholestasis

    PubMed Central

    De Tommaso, Adriana MA; Andrade, Paula D; Costa, Sandra CB; Escanhoela, Cecília AF; Hessel, Gabriel

    2005-01-01

    Background Biliary atresia (BA) is the most severe hepatic disorder in newborns and its etiopathogenesis remains unknown. Viral involvement has been proposed, including the human cytomegalovirus (HCMV). The aims of the study were to use the polymerase chain reaction (PCR) to screen the liver tissue of infants with extrahepatic cholestasis for HCMV and to correlate the results with serological antibodies against HCMV and histological findings. Methods A retrospective study in a tertiary care setting included 35 patients (31 BA, 1 BA associated with a choledochal cyst, 2 congenital stenosis of the distal common bile duct and 1 hepatic cyst). HCMV serology was determined by ELISA. Liver and porta hepatis were examined histologically. Liver samples from infants and a control group were screened for HCMV DNA. Results Twelve patients had HCMV negative serology, 9 were positive for IgG antibodies and 14 were positive for IgG and IgM. Nine liver and seven porta hepatis samples were positive for HCMV DNA but none of the control group were positive (general frequency of positivity was 34.3% – 12/35). There was no correlation between HCMV positivity by PCR and the histological findings. The accuracy of serology for detecting HCMV antibodies was low. Conclusion These results indicate an elevated frequency of HCMV in pediatric patients with extrahepatic neonatal cholestasis. They also show the low accuracy of serological tests for detecting active HCMV infection and the lack of correlation between HCMV positivity by PCR and the histopathological changes. PMID:16321152

  8. Neutralizing antibodies are unable to inhibit direct viral cell-to-cell spread of human cytomegalovirus.

    PubMed

    Jacob, Christian L; Lamorte, Louie; Sepulveda, Eliud; Lorenz, Ivo C; Gauthier, Annick; Franti, Michael

    2013-09-01

    Infection with human cytomegalovirus (CMV) during pregnancy is the most common cause of congenital disorders, and can lead to severe life-long disabilities with associated high cost of care. Since there is no vaccine or effective treatment, current efforts are focused on identifying potent neutralizing antibodies. A panel of CMV monoclonal antibodies identified from patent applications, was synthesized and expressed in order to reproduce data from the literature showing that anti-glycoprotein B antibodies neutralized virus entry into all cell types and that anti-pentameric complex antibodies are highly potent in preventing virus entry into epithelial cells. It had not been established whether antibodies could prevent subsequent rounds of infection that are mediated primarily by direct cell-to-cell transmission. A thorough validation of a plaque reduction assay to monitor cell-to-cell spread led to the conclusion that neutralizing antibodies do not significantly inhibit plaque formation or reduce plaque size when they are added post-infection. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. Commutability of the First World Health Organization International Standard for Human Cytomegalovirus

    PubMed Central

    Preiksaitis, J.; Tong, Y.; Pang, X.; Sun, Y.; Tang, L.; Cook, L.; Pounds, S.; Fryer, J.; Caliendo, A. M.

    2015-01-01

    Quantitative detection of cytomegalovirus (CMV) DNA has become a standard part of care for many groups of immunocompromised patients; recent development of the first WHO international standard for human CMV DNA has raised hopes of reducing interlaboratory variability of results. Commutability of reference material has been shown to be necessary if such material is to reduce variability among laboratories. Here we evaluated the commutability of the WHO standard using 10 different real-time quantitative CMV PCR assays run by eight different laboratories. Test panels, including aliquots of 50 patient samples (40 positive samples and 10 negative samples) and lyophilized CMV standard, were run, with each testing center using its own quantitative calibrators, reagents, and nucleic acid extraction methods. Commutability was assessed both on a pairwise basis and over the entire group of assays, using linear regression and correspondence analyses. Commutability of the WHO material differed among the tests that were evaluated, and these differences appeared to vary depending on the method of statistical analysis used and the cohort of assays included in the analysis. Depending on the methodology used, the WHO material showed poor or absent commutability with up to 50% of assays. Determination of commutability may require a multifaceted approach; the lack of commutability seen when using the WHO standard with several of the assays here suggests that further work is needed to bring us toward true consensus. PMID:26269622

  10. Sites and roles of phosphorylation of the human cytomegalovirus DNA polymerase subunit UL44

    SciTech Connect

    Silva, Laurie A.; Strang, Blair L.; Lin, Eric W.; Kamil, Jeremy P.; Coen, Donald M.

    2011-09-01

    The human cytomegalovirus DNA polymerase subunit UL44 is a phosphoprotein, but its sites and roles of phosphorylation have not been investigated. We compared sites of phosphorylation of UL44 in vitro by the viral protein kinase UL97 and cyclin-dependent kinase 1 with those in infected cells. Transient treatment of infected cells with a UL97 inhibitor greatly reduced labeling of two minor UL44 phosphopeptides. Viruses containing alanine substitutions of most UL44 residues that are phosphorylated in infected cells exhibited at most modest effects on viral DNA synthesis and yield. However, substitution of highly phosphorylated sites adjacent to the nuclear localization signal abolished viral replication. The results taken together are consistent with UL44 being phosphorylated directly by UL97 during infection, and a crucial role for phosphorylation-mediated nuclear localization of UL44 for viral replication, but lend little support to the widely held hypothesis that UL97-mediated phosphorylation of UL44 is crucial for viral DNA synthesis.

  11. Superresolution Imaging of Human Cytomegalovirus vMIA Localization in Sub-Mitochondrial Compartments

    PubMed Central

    Bhuvanendran, Shivaprasad; Salka, Kyle; Rainey, Kristin; Sreetama, Sen Chandra; Williams, Elizabeth; Leeker, Margretha; Prasad, Vidhya; Boyd, Jonathan; Patterson, George H.; Jaiswal, Jyoti K.; Colberg-Poley, Anamaris M.

    2014-01-01

    The human cytomegalovirus (HCMV) viral mitochondria-localized inhibitor of apoptosis (vMIA) protein, traffics to mitochondria-associated membranes (MAM), where the endoplasmic reticulum (ER) contacts the outer mitochondrial membrane (OMM). vMIA association with the MAM has not been visualized by imaging. Here, we have visualized this by using a combination of confocal and superresolution imaging. Deconvolution of confocal microscopy images shows vMIA localizes away from mitochondrial matrix at the Mitochondria-ER interface. By gated stimulated emission depletion (GSTED) imaging, we show that along this interface vMIA is distributed in clusters. Through multicolor, multifocal structured illumination microscopy (MSIM), we find vMIA clusters localize away from MitoTracker Red, indicating its OMM localization. GSTED and MSIM imaging show vMIA exists in clusters of ~100–150 nm, which is consistent with the cluster size determined by Photoactivated Localization Microscopy (PALM). With these diverse superresolution approaches, we have imaged the clustered distribution of vMIA at the OMM adjacent to the ER. Our findings directly compare the relative advantages of each of these superresolution imaging modalities for imaging components of the MAM and sub-mitochondrial compartments. These studies establish the ability of superresolution imaging to provide valuable insight into viral protein location, particularly in the sub-mitochondrial compartments, and into their clustered organization. PMID:24721787

  12. The eIF4AIII RNA helicase is a critical determinant of human cytomegalovirus replication

    PubMed Central

    Ziehr, Ben; Lenarcic, Erik; Cecil, Chad; Moorman, Nathaniel J.

    2016-01-01

    Human cytomegalovirus (HCMV) was recently shown to encode a large number of spliced mRNAs. While the nuclear export of unspliced viral transcripts has been extensively studied, the role of host mRNA export factors in HCMV mRNA trafficking remains poorly defined. We found that the eIF4AIII RNA helicase, a component of the exon junction complex, was necessary for efficient virus replication. Depletion of eIF4AIII limited viral DNA accumulation, export of viral mRNAs from the nucleus, and the production of progeny virus. However eIF4AIII was dispensable for the association of viral transcripts with ribosomes. We found that pateamine A, a natural compound that inhibits both eIF4AI/II and eIF4AIII, has potent antiviral activity and inhibits HCMV replication throughout the virus lytic cycle. Our results demonstrate that eIF4AIII is required for efficient HCMV replication, and suggest that eIF4A family helicases may be a new class of targets for the development of host-directed antiviral therapeutics. PMID:26773380

  13. Structural changes in human cytomegalovirus cytoplasmic assembly sites in the absence of UL97 kinase activity

    SciTech Connect

    Azzeh, Maysa; Honigman, Alik; Taraboulos, Albert; Rouvinski, Alexander; Wolf, Dana G. . E-mail: wolfd@md.huji.ac.il

    2006-10-10

    Studies of human cytomegalovirus (HCMV) UL97 kinase deletion mutant ({delta}UL97) indicated a multi-step role for this kinase in early and late phases of the viral life cycle, namely, in DNA replication, capsid maturation and nuclear egress. Here, we addressed its possible involvement in cytoplasmic steps of HCMV assembly. Using the {delta}UL97 and the UL97 kinase inhibitor NGIC-I, we demonstrate that the absence of UL97 kinase activity results in a modified subcellular distribution of the viral structural protein assembly sites, from compact structures impacting upon the nucleus to diffuse perinuclear structures punctuated by large vacuoles. Infection by either wild type or {delta}UL97 viruses induced a profound reorganization of wheat germ agglutinin (WGA)-positive Golgi-related structures. Importantly, the viral-induced Golgi remodeling along with the reorganization of the nuclear architecture was substantially altered in the absence of UL97 kinase activity. These findings suggest that UL97 kinase activity might contribute to organization of the viral cytoplasmic assembly sites.

  14. Four phosphoproteins with common amino termini are encoded by human cytomegalovirus AD169

    SciTech Connect

    Wright, D.A.; Staprans, S.I.; Spector, D.H.

    1988-01-01

    In this report, the authors identify the proteins encoded by the 2.2-kilobase class of early transcripts arising from a region of the strain AD169 human cytomegalovirus genome (map units 0.682 to 0.713) which contains cell-related sequences. These transcripts, encoded by adjacent EcoRI fragments R and d, have a complex spliced structure with 5' and 3' coterminal ends. Antiserum directed against a synthetic 11-amino-acid peptide corresponding to the predicted amino terminus of the proteins was generated and found to immunoprecipitate four-infected-cell proteins of 84, 50, 43, and 34 kilodaltons. These proteins were phosphorylated and were associated predominantly with the nuclei of infected cells. The 43-kilodalton protein was the most abundant of the four proteins, and its level of expression remained relatively constant throughout the infection. Expression of the other proteins increased as the infection progressed. Pulse-chase analysis failed to show a precursor-product relationship between any of the proteins. A comparison of the (/sup 35/S)methionine-labeled tryptic peptide maps of the four proteins from infected cells and an in vitro-generated polypeptide derived from the putative first exon showed that all four infected-cell proteins were of viral origin and contained a common amino-terminal region.

  15. Expression of a human cytomegalovirus receptor correlates with infectibility of cells.

    PubMed Central

    Nowlin, D M; Cooper, N R; Compton, T

    1991-01-01

    Previous studies have demonstrated that human cytomegalovirus (HCMV) specifically binds to a fibroblast membrane glycoprotein(s) with a molecular mass from 30 to 34 kDa. In this study, the distribution of the putative receptor proteins was analyzed in a variety of cell types, including cell types representative of those that are infected in vivo. Using a sensitive microbinding assay (to score virus attachment) and an indirect detection method (to score HCMV-binding proteins), we found that the 34- and 32-kDa HCMV binding proteins are ubiquitous molecules, broadly distributed among diverse cell types. In addition, the level of virus attachment was found to correlate with the abundance of the 34- and 32-kDa cellular proteins, while the ability of the virus to penetrate cells and initiate infection did not. The results support the hypothesis that the 34- and 32-kDa cellular proteins represent the HCMV (attachment) receptor. The data also support the notion that additional cellular components are required for virus entry and fusion. Images PMID:1851872

  16. Human cytomegalovirus secretome contains factors that induce angiogenesis and wound healing.

    PubMed

    Dumortier, Jerome; Streblow, Daniel N; Moses, Ashlee V; Jacobs, Jon M; Kreklywich, Craig N; Camp, David; Smith, Richard D; Orloff, Susan L; Nelson, Jay A

    2008-07-01

    Human cytomegalovirus (HCMV) is implicated in the acceleration of a number of vascular diseases including transplant vascular sclerosis (TVS), the lesion associated with chronic rejection (CR) of solid organ transplants. Although the virus persists in the allograft throughout the course of disease, few cells are directly infected by CMV. This observation is in contrast to the global effects that CMV has on the acceleration of TVS/CR, suggesting that CMV infection indirectly promotes the vascular disease process. Recent transcriptome analysis of CMV-infected heart allografts indicates that the virus induces cytokines and growth factors associated with angiogenesis (AG) and wound healing (WH), suggesting that CMV may accelerate TVS/CR through the induction and secretion of AG/WH factors from infected cells. We analyzed virus-free supernatants from HCMV-infected cells (HCMV secretomes) for growth factors, by mass spectrometry and immunoassays, and found that the HCMV secretome contains over 1,000 cellular proteins, many of which are involved in AG/WH. Importantly, functional assays demonstrated that CMV but not herpes simplex virus secretomes not only induce AG/WH but also promote neovessel stabilization and endothelial cell survival for 2 weeks. These findings suggest that CMV acceleration of TVS occurs through virus-induced growth factors and cytokines in the CMV secretome.

  17. Differential Ligand Binding to a Human Cytomegalovirus Chemokine Receptor Determines Cell Type–Specific Motility

    PubMed Central

    Vomaske, Jennifer; Melnychuk, Ryan M.; Smith, Patricia P.; Powell, Joshua; Hall, Laurel; DeFilippis, Victor; Früh, Klaus; Smit, Martine; Schlaepfer, David D.; Nelson, Jay A.; Streblow, Daniel N.

    2009-01-01

    While most chemokine receptors fail to cross the chemokine class boundary with respect to the ligands that they bind, the human cytomegalovirus (HCMV)-encoded chemokine receptor US28 binds multiple CC-chemokines and the CX3C-chemokine Fractalkine. US28 binding to CC-chemokines is both necessary and sufficient to induce vascular smooth muscle cell (SMC) migration in response to HCMV infection. However, the function of Fractalkine binding to US28 is unknown. In this report, we demonstrate that Fractalkine binding to US28 not only induces migration of macrophages but also acts to inhibit RANTES-mediated SMC migration. Similarly, RANTES inhibits Fractalkine-mediated US28 migration in macrophages. While US28 binding of both RANTES and Fractalkine activate FAK and ERK-1/2, RANTES signals through Gα12 and Fractalkine through Gαq. These findings represent the first example of differential chemotactic signaling via a multiple chemokine family binding receptor that results in migration of two different cell types. Additionally, the demonstration that US28-mediated chemotaxis is both ligand-specific and cell type–specific has important implications in the role of US28 in HCMV pathogenesis. PMID:19229316

  18. Human Cytomegalovirus Secretome Contains Factors That Induce Angiogenesis and Wound Healing▿

    PubMed Central

    Dumortier, Jerome; Streblow, Daniel N.; Moses, Ashlee V.; Jacobs, Jon M.; Kreklywich, Craig N.; Camp, David; Smith, Richard D.; Orloff, Susan L.; Nelson, Jay A.

    2008-01-01

    Human cytomegalovirus (HCMV) is implicated in the acceleration of a number of vascular diseases including transplant vascular sclerosis (TVS), the lesion associated with chronic rejection (CR) of solid organ transplants. Although the virus persists in the allograft throughout the course of disease, few cells are directly infected by CMV. This observation is in contrast to the global effects that CMV has on the acceleration of TVS/CR, suggesting that CMV infection indirectly promotes the vascular disease process. Recent transcriptome analysis of CMV-infected heart allografts indicates that the virus induces cytokines and growth factors associated with angiogenesis (AG) and wound healing (WH), suggesting that CMV may accelerate TVS/CR through the induction and secretion of AG/WH factors from infected cells. We analyzed virus-free supernatants from HCMV-infected cells (HCMV secretomes) for growth factors, by mass spectrometry and immunoassays, and found that the HCMV secretome contains over 1,000 cellular proteins, many of which are involved in AG/WH. Importantly, functional assays demonstrated that CMV but not herpes simplex virus secretomes not only induce AG/WH but also promote neovessel stabilization and endothelial cell survival for 2 weeks. These findings suggest that CMV acceleration of TVS occurs through virus-induced growth factors and cytokines in the CMV secretome. PMID:18448536

  19. Lymphoproliferative response in primary human cytomegalovirus (HCMV) infection is delayed in HCMV transmitter mothers.

    PubMed

    Revello, Maria Grazia; Lilleri, Daniele; Zavattoni, Maurizio; Furione, Milena; Genini, Emilia; Comolli, Giuditta; Gerna, Giuseppe

    2006-01-15

    The T cell-mediated immune response to human cytomegalovirus (HCMV) after primary infection, as well as the determinants of intrauterine transmission, are poorly understood. Sequential peripheral blood leukocyte samples from 74 pregnant women and 29 nonpregnant individuals with primary infection were examined for HCMV-specific CD4+ T cells by cytokine flow cytometry (CFC) and lymphoproliferative response (LPR) analysis. Immunological results for 19 transmitter and 21 nontransmitter mothers were compared. Comparison of CFC and LPR analysis results showed that (1) there was no difference between pregnant and nonpregnant individuals; (2) HCMV-specific CD4+ T cells were detected by CFC, in the absence of an LPR to HCMV, in the great majority or the totality (according to different intervals) of samples collected from both pregnant and nonpregnant individuals during follow-up; and (3) LPR to HCMV was significantly (P<.001) lowered or delayed in transmitter mothers, compared with that in nontransmitter mothers. Pregnancy does not influence the HCMV-specific immune response. A dissociation between CFC response and LPR is commonly observed in patients with primary infections, and ad hoc studies aimed at understanding the mechanism(s) of the reduced LPR in transmitter mothers are warranted.

  20. The immunology of human cytomegalovirus latency: could latent infection be cleared by novel immunotherapeutic strategies?

    PubMed

    Wills, Mark R; Poole, Emma; Lau, Betty; Krishna, Ben; Sinclair, John H

    2015-03-01

    While the host immune response following primary human cytomegalovirus (HCMV) infection is generally effective at stopping virus replication and dissemination, virus is never cleared by the host and like all herpesviruses, persists for life. At least in part, this persistence is known to be facilitated by the ability of HCMV to establish latency in myeloid cells in which infection is essentially silent with, importantly, a total lack of new virus production. However, although the viral transcription programme during latency is much suppressed, a number of viral genes are expressed during latent infection at the protein level and many of these have been shown to have profound effects on the latent cell and its environment. Intriguingly, many of these latency-associated genes are also expressed during lytic infection. Therefore, why the same potent host immune responses generated during lytic infection to these viral gene products are not recognized during latency, thereby allowing clearance of latently infected cells, is far from clear. Reactivation from latency is also a major cause of HCMV-mediated disease, particularly in the immune compromised and immune naive, and is also likely to be a major source of virus in chronic subclinical HCMV infection which has been suggested to be associated with long-term diseases such as atherosclerosis and some neoplasias. Consequently, understanding latency and why latently infected cells appear to be immunoprivileged is crucial for an understanding of the pathogenesis of HCMV and may help to design strategies to eliminate latent virus reservoirs, at least in certain clinical settings.

  1. The effect of human cytomegalovirus on the formation of CFU-MK in vitro.

    PubMed

    Yao, Junxia; Song, Sanjun; Hu, Lihua

    2004-01-01

    To investigate the mechanism and the suppressive effect of human cytomegalovirus (HCMV) on colony forming unit-megakaryocyte (CFU-MK), semi-solid culture system was used to observe the effect of HCMV AD169 strain on CFU-MK's growth of 18 cord blood samples. HCMV DNA and immediate early (IE) protein mRNA in CFU-MK was detected by PCR and reverse transcription-polymerase chain reaction (RT-PCR). Our results showed that HCMV AD169 significantly suppressed the formation of CFU-MK in vitro. Compared with the mock group, the CFU-MK colonies decreased by 21.6%, 33.8% and 46.3%, respectively, in all the 3 infected groups (P<0.05), suggesting the suppression and the titer of the virus was dose-dependent. Both HCMV DNA and the expression of HCMV IE protein mRNA were positively detected in the colony cells of viral infected group. It is concluded that HCMV AD169 strain could inhibit the differentiation and proliferation of CFU-MK by directly infecting their progenitors. There was early transcription of HCMV IE protein in CFU-MK infected by virus.

  2. Visualization of the dynamic multimerization of human Cytomegalovirus pp65 in punctuate nuclear foci

    SciTech Connect

    Cui Zongqiang; Zhang Ke; Zhang Zhiping; Liu Yalan; Zhou Yafeng; Wei Hongping; Zhang Xian-En

    2009-09-30

    The phosphorylated protein pp65 of human Cytomegalovirus (HCMV) is the predominant virion protein and the major tegument constituent. It plays important roles in HCMV infection and virion assembly. Live cell imaging and fluorescence recovery after photobleaching (FRAP) analysis showed that HCMV pp65 accumulated dynamically in punctuate nuclear foci when transiently expressed in mammalian cells. Fluorescence resonance energy transfer (FRET) imaging disclosed that pp65 can self-interact in its localization foci. Yeast two-hybrid assay verified that pp65 is a self-associating protein, and the N-terminal amino acids 14-22 were determined to be essential for pp65 self-association. However, these amino acids were not related to pp65 localization in the specific nuclear foci. The interaction of pp65 and ppUL97 was also studied by FRET microscopy, and the result suggested that there is another signal sequence in pp65, being the ppUL97 phosphorylation site, that is responsible for localization of pp65 in nuclear foci. These results help to understand the function of pp65 in HCMV infection and virion morphogenesis.

  3. Sequence and transcription analysis of the human cytomegalovirus DNA polymerase gene

    SciTech Connect

    Kouzarides, T.; Bankier, A.T.; Satchwell, S.C.; Weston, K.; Tomlinson, P.; Barrell, B.G.

    1987-01-01

    DNA sequence analysis has revealed that the gene coding for the human cytomegalovirus (HCMV) DNA polymerase is present within the long unique region of the virus genome. Identification is based on extensive amino acid homology between the predicted HCMV open reading frame HFLF2 and the DNA polymerase of herpes simplex virus type 1. The authors present here a 5280 base-pair DNA sequence containing the HCMV pol gene, along with the analysis of transcripts encoded within this region. Since HCMV pol also shows homology to the predicted Epstein-Barr virus pol, they were able to analyze the extent of homology between the DNA polymerases of three distantly related herpes viruses, HCMV, Epstein-Barr virus, and herpes simplex virus. The comparison shows that these DNA polymerases exhibit considerable amino acid homology and highlights a number of highly conserved regions; two such regions show homology to sequences within the adenovirus type 2 DNA polymerase. The HCMV pol gene is flanked by open reading frames with homology to those of other herpes viruses; upstream, there is a reading frame homologous to the glycoprotein B gene of herpes simplex virus type I and Epstein-Barr virus, and downstream there is a reading frame homologous to BFLF2 of Epstein-Barr virus.

  4. The Microtubule Inhibitor Podofilox Inhibits an Early Entry Step of Human Cytomegalovirus

    PubMed Central

    Cohen, Tobias; Schwarz, Toni M.; Vigant, Frederic; Gardner, Thomas J.; Hernandez, Rosmel E.; Lee, Benhur; Tortorella, Domenico

    2016-01-01

    Human cytomegalovirus is a ubiquitous β-herpesvirus that infects many different cell types through an initial binding to cell surface receptors followed by a fusion event at the cell membrane or endocytic vesicle. A recent high-throughput screen to identify compounds that block a step prior to viral gene expression identified podofilox as a potent and nontoxic inhibitor. Time-of-addition studies in combination with quantitative-PCR analysis demonstrated that podofilox limits an early step of virus entry at the cell surface. Podofilox was also able to drastically reduce infection by herpes simplex 1, an α-herpesvirus with a very similar entry process to CMV. Podofilox caused a reduced maximal plateau inhibition of infection by viruses with single step binding processes prior to fusion-like Newcastle disease virus, Sendai virus, and influenza A virus or viruses that enter via endocytosis like vesicular stomatitis virus and a clinical-like strain of CMV. These results indicate that microtubules appear to be participating in the post-binding step of virus entry including the pre- and post-penetration events. Modulation of the plasma membrane is required to promote virus entry for herpesviruses, and that podofilox, unlike colchicine or nocodazole, is able to preferentially target microtubule networks at the plasma membrane. PMID:27783035

  5. Genomic localization, sequence analysis, and transcription of the putative human cytomegalovirus DNA polymerase gene.

    PubMed Central

    Heilbronn, R; Jahn, G; Bürkle, A; Freese, U K; Fleckenstein, B; zur Hausen, H

    1987-01-01

    The human cytomegalovirus (HCMV)-induced DNA polymerase has been well characterized biochemically and functionally, but its genomic location has not yet been assigned. To identify the coding sequence, cross-hybridization with the herpes simplex virus type 1 (HSV-1) polymerase gene was used, as suggested by the close similarity of the herpes group virus-induced DNA polymerases to the HCMV DNA polymerase. A cosmid and plasmid library of the entire HCMV genome was screened with the BamHI Q fragment of HSV-1 at different stringency conditions. One PstI-HincII restriction fragment of 850 base pairs mapping within the EcoRI M fragment of HCMV cross-hybridized at Tm - 25 degrees C. Sequence analysis revealed one open reading frame spanning the entire sequence. The amino acid sequence showed a highly conserved domain of 133 amino acids shared with the HSV and putative Epstein-Barr virus polymerase sequences. This domain maps within the C-terminal part of the HSV polymerase gene, which has been suggested to contain part of the catalytic center of the enzyme. Transcription analysis revealed one 5.4-kilobase early transcript in the sense orientation with respect to the open reading frame identified. This transcript appears to code for the 140-kilodalton HCMV polymerase protein. Images PMID:3023689

  6. Human cytomegalovirus US28 facilitates cell-to-cell viral dissemination.

    PubMed

    Noriega, Vanessa M; Gardner, Thomas J; Redmann, Veronika; Bongers, Gerold; Lira, Sergio A; Tortorella, Domenico

    2014-03-12

    Human cytomegalovirus (HCMV) encodes a number of viral proteins with homology to cellular G protein-coupled receptors (GPCRs). These viral GPCRs, including US27, US28, UL33, and UL78, have been ascribed numerous functions during infection, including activating diverse cellular pathways, binding to immunomodulatory chemokines, and impacting virus dissemination. To investigate the role of US28 during virus infection, two variants of the clinical isolate TB40/E were generated: TB40/E-US28(YFP) expressing a C-terminal yellow fluorescent protein tag, and TB40/E-FLAG(YFP) in which a FLAG-YFP cassette replaces the US28 coding region. The TB40/E-US28(YFP) protein localized as large perinuclear fluorescent structures at late times post-infection in fibroblasts, endothelial, and epithelial cells. Interestingly, US28(YFP) is a non-glycosylated membrane protein throughout the course of infection. US28 appears to impact cell-to-cell spread of virus, as the DUS28 virus (TB40/E-FLAG(YFP)) generated a log-greater yield of extracellular progeny whose spread could be significantly neutralized in fibroblasts. Most strikingly, in epithelial cells, where dissemination of virus occurs exclusively by the cell-to-cell route, TB40/E-FLAG(YFP) (DUS28) displayed a significant growth defect. The data demonstrates that HCMV US28 may contribute at a late stage of the viral life cycle to cell-to-cell dissemination of virus.

  7. Detection of human cytomegalovirus in plasma of AIDS patients during acute visceral disease by DNA amplification.

    PubMed Central

    Spector, S A; Merrill, R; Wolf, D; Dankner, W M

    1992-01-01

    By using the polymerase chain reaction (PCR) amplification procedure, 19 (83%) of 23 plasma specimens obtained from individuals with AIDS and human cytomegalovirus (HCMV) visceral disease were found to be positive for plasma viremia as detected by PCR (PV-PCR), whereas 78% of cultures of peripheral blood leukocytes from the same samples were found to be positive. All 11 specimens prospectively obtained from individuals with acute HCMV disease were positive by PV-PCR. Plasma specimens from patients who received ganciclovir therapy rapidly became both culture and PV-PCR negative, and there was an excellent correlation between the two procedures. DNA detected by PV-PCR was unaffected by filtering plasma through a 0.2-microns-pore-size filter, although a conserved cellular gene, HLA-DQ alpha, was undetectable by PCR following filtration. HCMV DNA in plasma could be quantitated by PV-PCR by using endpoint serial dilutions, with detectable virus being present in 10(1) to 10(-2) microliters of plasma. A low titer of infectious virus could be detected in 2 of 11 plasma samples. The detection of HCMV DNA in plasma by PV-PCR promises to be a useful procedure for monitoring patients with AIDS suspected of having impending, acute, or recurrent HCMV visceral disease and suggests an additional route by which virus may disseminate in the immunocompromised host. Images PMID:1328287

  8. Infected T98G glioblastoma cells support human cytomegalovirus reactivation from latency.

    PubMed

    Cheng, Shuang; Jiang, Xuan; Yang, Bo; Wen, Le; Zhao, Fei; Zeng, Wen-Bo; Liu, Xi-Juan; Dong, Xiao; Sun, Jin-Yan; Ming, Ying-Zi; Zhu, Hua; Rayner, Simon; Tang, Qiyi; Fortunato, Elizabeth; Luo, Min-Hua

    2017-10-01

    T98G cells have been shown to support long-term human cytomegalovirus (HCMV) genome maintenance without infectious virus release. However, it remains unclear whether these viral genomes could be reactivated. To address this question, a recombinant HCMV (rHCMV) containing a GFP gene was used to infect T98G cells, and the infected cells absent of infectious virus production were designated T98G-LrV. Upon dibutyryl cAMP plus IBMX (cAMP/IBMX) treatment, a serial of phenomena were observed, including GFP signal increase, viral genome replication, lytic genes expression and infectious viruses release, indicating the reactivation of HCMV in T98G-LrV cells from a latent status. Mechanistically, HCMV reactivation in the T98G-LrV cells induced by cAMP/IBMX was associated with the PKA-CREB signaling pathway. These results demonstrate that HCMV was latent in T98G-LrV cells and could be reactivated. The T98G-LrV cells represent an effective model for investigating the mechanisms of HCMV reactivation from latency in the context of neural cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Nuclear trafficking of the human cytomegalovirus pp71 (ppUL82) tegument protein

    SciTech Connect

    Shen Weiping; Westgard, Elizabeth; Huang Liqun; Ward, Michael D.; Osborn, Jodi L.; Chau, Nha H.; Collins, Lindsay; Marcum, Benjamin; Koach, Margaret A.; Bibbs, Jennifer; Semmes, O. John; Kerry, Julie A.

    2008-06-20

    The human cytomegalovirus tegument protein pp71 localizes to the nucleus immediately upon infection, and functions to initiate viral gene expression. Analysis of a series of random insertion mutations revealed that sequences within the mid region (MR) of pp71 are important for localization to the nucleus. Fusion of MR sequences with eGFP revealed that amino acids 94 to 300 were sufficient to target proteins to the nucleus. Random substitution mutagenesis within this domain resulted in two double substitution mutants, pp71P203T/T223M and pp71T228M/L275Q, with a predominantly cytoplasmic localization. Disruption of nuclear targeting resulted in relocalization of the fusion proteins to a distinct perinuclear region. Using tandem mass spectrometry, we determined that threonine 223 can be phosphorylated. Mutation of this residue to a phosphomimetic amino acid resulted in abrogation of nuclear targeting. These results strongly suggest that the intracellular trafficking of pp71 is regulated by phosphorylation.

  10. Salivary glands and human congenital cytomegalovirus infection: What happens in early fetal life?

    PubMed

    Gabrielli, Liliana; Bonasoni, Maria Paola; Chiereghin, Angela; Piccirilli, Giulia; Santini, Donatella; Pavia, Claudia; Turello, Gabriele; Squarzoni, Diego; Lazzarotto, Tiziana

    2017-02-01

    Salivary glands are a site of human cytomegalovirus (CMV) replication, latency, and persistence. Prolonged secretion of virus in saliva for months following a primary infection contribute to horizontal transmission. In order to better understand the early effects of CMV on salivary glands and the mechanisms of viral persistent replication, submandibular glands of six CMV congenitally infected fetuses at 21 weeks gestation were studied. Three fetuses at the same gestational age from CMV-seronegative women were compared as negative controls. Tissue viral load and the type of inflammatory infiltrate were evaluated. Moreover, development and branching of salivary glands, the number of myoepithelial cells, cellular proliferation, and expression of secretory proteins of the saliva (Gross Cystic Disease Fluid Protein-15 and lysozyme) were studied. A low viral load and rare CMV-positive cells associated with T CD8 cytotoxic lymphocytes were observed. Branching was impaired with a decrease in terminal acinar structures, the number of myoepithelial cells, and cellular proliferation were reduced. In addition, a compromised secretion of defense proteins involved in the oral humoral immunity was observed. These findings suggest that CMV may affect salivary glands, impairing structure development and secretion of defense proteins, probably responsible for the prolonged viral shedding in saliva. J. Med. Virol. 89:318-323, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  11. RNase P Ribozymes Inhibit the Replication of Human Cytomegalovirus by Targeting Essential Viral Capsid Proteins.

    PubMed

    Yang, Zhu; Reeves, Michael; Ye, Jun; Trang, Phong; Zhu, Li; Sheng, Jingxue; Wang, Yu; Zen, Ke; Wu, Jianguo; Liu, Fenyong

    2015-06-24

    An engineered RNase P-based ribozyme variant, which was generated using the in vitro selection procedure, was used to target the overlapping mRNA region of two proteins essential for human cytomegalovirus (HCMV) replication: capsid assembly protein (AP) and protease (PR). In vitro studies showed that the generated variant, V718-A, cleaved the target AP mRNA sequence efficiently and its activity was about 60-fold higher than that of wild type ribozyme M1-A. Furthermore, we observed a reduction of 98%-99% in AP/PR expression and an inhibition of 50,000 fold in viral growth in cells with V718-A, while a 75% reduction in AP/PR expression and a 500-fold inhibition in viral growth was found in cells with M1-A. Examination of the antiviral effects of the generated ribozyme on the HCMV replication cycle suggested that viral DNA encapsidation was inhibited and as a consequence, viral capsid assembly was blocked when the expression of AP and PR was inhibited by the ribozyme. Thus, our study indicates that the generated ribozyme variant is highly effective in inhibiting HCMV gene expression and blocking viral replication, and suggests that engineered RNase P ribozyme can be potentially developed as a promising gene-targeting agent for anti-HCMV therapy.

  12. Synthetic lethal mutations in the cyclin A interface of human cytomegalovirus

    PubMed Central

    Vetter, Barbara

    2017-01-01

    Generally, the antagonism between host restriction factors and viral countermeasures decides on cellular permissiveness or resistance to virus infection. Human cytomegalovirus (HCMV) has evolved an additional level of self-imposed restriction by the viral tegument protein pp150. Depending on a cyclin A-binding motif, pp150 prevents the onset of viral gene expression in the S/G2 cell cycle phase of otherwise fully permissive cells. Here we address the physiological relevance of this restriction during productive HCMV infection by employing a cyclin A-binding deficient pp150 mutant virus. One consequence of unrestricted viral gene expression in S/G2 was the induction of a G2/M arrest. G2-arrested but not mitotic cells supported viral replication. Cyclin A destabilization by the viral gene product pUL21a was required to maintain the virus-permissive G2-arrest. An HCMV double-point mutant where both pp150 and pUL21a are disabled in cyclin A interaction forced mitotic entry of the majority of infected cells, with a severe negative impact on cell viability and virus growth. Thus, pp150 and pUL21a functionally cooperate, together building a cell cycle synchronization strategy of cyclin A targeting and avoidance that is essential for productive HCMV infection. PMID:28129404

  13. The multi-targeted kinase inhibitor sorafenib inhibits human cytomegalovirus replication.

    PubMed

    Michaelis, Martin; Paulus, Christina; Löschmann, Nadine; Dauth, Stephanie; Stange, Elisabeth; Doerr, Hans Wilhelm; Nevels, Michael; Cinatl, Jindrich

    2011-03-01

    Human cytomegalovirus (HCMV) is a major pathogen in immunocompromised individuals. Here, non-toxic concentrations of the anti-cancer kinase inhibitor sorafenib were shown to inhibit replication of different HCMV strains (including a ganciclovir-resistant strain) in different cell types. In contrast to established anti-HCMV drugs, sorafenib inhibited HCMV major immediate early promoter activity and HCMV immediate early antigen (IEA) expression. Sorafenib is known to inhibit Raf. Comparison of sorafenib with the MEK inhibitor U0126 suggested that sorafenib inhibits HCMV IEA expression through inhibition of Raf but independently of signaling through the Raf downstream kinase MEK 1/2. In concordance, siRNA-mediated depletion of Raf but not of MEK-reduced IEA expression. In conclusion, sorafenib diminished HCMV replication in clinically relevant concentrations and inhibited HCMV IEA expression, a pathophysiologically relevant event that is not affected by established anti-HCMV drugs. Moreover, we demonstrated for the first time that Raf activation is involved in HCMV IEA expression.

  14. US28, a Virally-Encoded GPCR as an Antiviral Target for Human Cytomegalovirus Infection

    PubMed Central

    Lee, Sungjin; Chung, Yoon Hee; Lee, Choongho

    2017-01-01

    Viruses continue to evolve a new strategy to take advantage of every aspect of host cells in order to maximize their survival. Due to their central roles in transducing a variety of transmembrane signals, GPCRs seem to be a prime target for viruses to pirate for their own use. Incorporation of GPCR functionality into the genome of herpesviruses has been demonstrated to be essential for pathogenesis of many herpesviruses-induced diseases. Here, we introduce US28 of human cytomegalovirus (HCMV) as the best-studied example of virally-encoded GPCRs to manipulate host GPCR signaling. In this review, we wish to summarize a number of US28-related topics including its regulation of host signaling pathways, its constitutive internalization, its structural and functional analysis, its roles in HCMV biology and pathogenesis, its proliferative activities and role in oncogenesis, and pharmacological modulation of its biological activities. This review will aid in our understanding of how pathogenic viruses usurp the host GPCR signaling for successful viral infection. This kind of knowledge will enable us to build a better strategy to control viral infection by normalizing the virally-dysregulated host GPCR signaling. PMID:28035083

  15. Significance of human cytomegalovirus and Epstein-Barr virus in inducing cytokine expression in periapical lesions.

    PubMed

    Sabeti, Mohammad; Kermani, Vali; Sabeti, Sara; Simon, James H

    2012-01-01

    Because herpesviruses might be etiologically involved in periapical pathosis of endodontic origin, this study aimed to determine the occurrence of human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), and the expression of mRNA transcripts of tumor necrosis factor (TNF)-α, γ-interferon (IFN), interleukin (IL)-1β, IL-6, IL-12, and IL-10 in periapical granulomatous lesions collected in conjunction with apicoectomy. A total of 9 symptomatic and 6 asymptomatic teeth with periapical lesions were studied. Periapical samples were collected in conjunction with apicoectomy, which was being performed because of radiographic evidence of incomplete periapical healing after conventional root canal therapy. By using established polymerase chain reaction primers and procedures, polymerase chain reaction assays were used to identify herpesvirus and cytokine gene expression. The difference in occurrence of HCMV, EBV, and cytokines between symptomatic and asymptomatic periapical lesions was statistically significant: HCMV (P = .048), EBV (P = .002), IFN (P = .001), IL-1 (P = .012), IL-6 (P = .026), IL-10 (P = .026), IL-12 (P = .012), and TNF (P < .001) (Mann-Whitney U test). There was a significant correlation between EBV, HCMV, and TNF, γ-IFN, IL-1, and IL-12 in symptomatic periapical lesions (Spearman test). The present findings provide evidence of a putative role of HCMV and EBV in the pathogenesis of symptomatic periapical pathosis. The release of tissue-destructive cytokines might be of pathogenetic significance. Copyright © 2012 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  16. No evidence of association between human cytomegalovirus infection and papillary thyroid cancer.

    PubMed

    Huang, Tung-Sun; Lee, Jie-Jen; Cheng, Shih-Ping

    2014-02-21

    Human cytomegalovirus (CMV) has been detected in the thyroid gland and thyroid tumors. CMV infection may activate the mitogen-activated protein kinase pathway, of which aberrant activation is frequently associated with BRAF mutation in papillary thyroid cancer. A total of 45 paired tumorous and adjacent non-neoplastic tissue samples, including 5 follicular adenoma and 40 papillary thyroid cancer, were obtained during thyroidectomy. BRAF mutational status was determined using direct sequencing. The presence of CMV DNA was determined using conventional PCR and quantitative real-time PCR. CMV protein in the tissue samples were evaluated with Western blot analysis. BRAF mutation was identified in the cancerous part of 31 (78%) papillary thyroid cancers. Papillary cancer with BRAF mutation was significantly associated with a larger tumor size (P = 0.045), extrathyroidal invasion (P = 0.012), lymph node metastasis (P = 0.008), and a higher TNM stage (P = 0.044). CMV DNA and protein were not detected in any studied samples. Our results suggest no association between CMV infection and papillary thyroid cancer.

  17. Divergent effects of human cytomegalovirus and herpes simplex virus-1 on cellular metabolism.

    PubMed

    Vastag, Livia; Koyuncu, Emre; Grady, Sarah L; Shenk, Thomas E; Rabinowitz, Joshua D

    2011-07-01

    Viruses rely on the metabolic network of the host cell to provide energy and macromolecular precursors to fuel viral replication. Here we used mass spectrometry to examine the impact of two related herpesviruses, human cytomegalovirus (HCMV) and herpes simplex virus type-1 (HSV-1), on the metabolism of fibroblast and epithelial host cells. Each virus triggered strong metabolic changes that were conserved across different host cell types. The metabolic effects of the two viruses were, however, largely distinct. HCMV but not HSV-1 increased glycolytic flux. HCMV profoundly increased TCA compound levels and flow of two carbon units required for TCA cycle turning and fatty acid synthesis. HSV-1 increased anapleurotic influx to the TCA cycle through pyruvate carboxylase, feeding pyrimidine biosynthesis. Thus, these two related herpesviruses drive diverse host cells to execute distinct, virus-specific metabolic programs. Current drugs target nucleotide metabolism for treatment of both viruses. Although our results confirm that this is a robust target for HSV-1, therapeutic interventions at other points in metabolism might prove more effective for treatment of HCMV.

  18. Human cytomegalovirus infection is detected frequently in stillbirths and is associated with fetal thrombotic vasculopathy.

    PubMed

    Iwasenko, Jenna M; Howard, Jonathan; Arbuckle, Susan; Graf, Nicole; Hall, Beverley; Craig, Maria E; Rawlinson, William D

    2011-06-01

    Human cytomegalovirus (CMV) is the most common congenital infection in developed countries and is a known cause of intrauterine fetal death. We examined CMV infection in stillbirths and the relationship with histopathological findings at autopsy. We collected liver, kidney, and placenta specimens from 130 stillbirths. CMV DNA and protein were detected using polymerase chain reaction and immunohistochemistry, along with routine autopsy of stillborn infants. Overall, CMV DNA was detected in 15% of singleton, >20-week stillborn infants. CMV DNA was detected in kidney (9%), liver (11%), and placenta (5%) specimens, with 75% of infections confirmed by immunohistochemistry. Fetal thrombotic vasculopathy was the only histopathological abnormality associated with CMV infection (in 60% CMV-infected vs 28% uninfected stillbirths P = .010). Stillbirth has multiple etiologies. However, the detection of CMV DNA in 15% of fetal tissues or placentae suggests a strong association between CMV infection in pregnancy and stillbirth. Molecular testing during postmortem investigation has an important role to determine the contribution of CMV infection.

  19. Human cytomegalovirus (HCMV) replication dynamics in HCMV-naive and -experienced immunocompromised hosts.

    PubMed

    Emery, Vincent C; Hassan-Walker, Aycan F; Burroughs, Andrew K; Griffiths, Paul D

    2002-06-15

    Human cytomegalovirus (HCMV) can infect both HCMV-naive and -experienced transplant patients. In this study, the growth rate of HCMV in HCMV-naive hosts (1.82 units/day; 95% confidence interval [CI], 1.44-2.56 units/day) was shown to be significantly faster than the growth rate of virus in HCMV-experienced hosts undergoing recurrent infection (0.61 units/day; 95% CI, 0.55-0.7 units/day; P<.0001). The basic reproductive number (R(0)) for HCMV-naive liver transplant patients was 15.1 (95% CI, 8.9-44) but was only 2.4 (95% CI, 2.35-2.8) for HCMV-experienced transplant recipients, corresponding to an anti-HCMV immune efficacy of approximately 84%, despite immunosuppressive therapy. The R(0) values suggest that an anti-HCMV drug or vaccine with an efficacy of >93% (95% CI, 89%-98%) is required to eliminate viral growth during infection of HCMV-naive liver transplant recipients, whereas lower efficacy levels are sufficient to reduce the R(0) value to <1 in hosts with prior HCMV immunity.

  20. Genomic localization, sequence analysis, and transcription of the putative human cytomegalovirus DNA polymerase gene

    SciTech Connect

    Heilbronn, T.; Jahn, G.; Buerkle, A.; Freese, U.K.; Fleckenstein, B.; Zur Hausen, H.

    1987-01-01

    The human cytomegalovirus (HCMV)-induced DNA polymerase has been well characterized biochemically and functionally, but its genomic location has not yet been assigned. To identify the coding sequence, cross-hybridization with the herpes simplex virus type 1 (HSV-1) polymerase gene was used, as suggested by the close similarity of the herpes group virus-induced DNA polymerases to the HCMV DNA polymerase. A cosmid and plasmid library of the entire HCMV genome was screened with the BamHI Q fragment of HSF-1 at different stringency conditions. One PstI-HincII restriction fragment of 850 base pairs mapping within the EcoRI M fragment of HCMV cross-hybridized at T/sub m/ - 25/degrees/C. Sequence analysis revealed one open reading frame spanning the entire sequence. The amino acid sequence showed a highly conserved domain of 133 amino acids shared with the HSV and putative Esptein-Barr virus polymerase sequences. This domain maps within the C-terminal part of the HSV polymerase gene, which has been suggested to contain part of the catalytic center of the enzyme. Transcription analysis revealed one 5.4-kilobase early transcript in the sense orientation with respect to the open reading frame identified. This transcript appears to code for the 140-kilodalton HCMV polymerase protein.

  1. Structural basis for translational stalling by human cytomegalovirus and fungal arginine attenuator peptide.

    PubMed

    Bhushan, Shashi; Meyer, Helge; Starosta, Agata L; Becker, Thomas; Mielke, Thorsten; Berninghausen, Otto; Sattler, Michael; Wilson, Daniel N; Beckmann, Roland

    2010-10-08

    Specific regulatory nascent chains establish direct interactions with the ribosomal tunnel, leading to translational stalling. Despite a wealth of biochemical data, structural insight into the mechanism of translational stalling in eukaryotes is still lacking. Here we use cryo-electron microscopy to visualize eukaryotic ribosomes stalled during the translation of two diverse regulatory peptides: the fungal arginine attenuator peptide (AAP) and the human cytomegalovirus (hCMV) gp48 upstream open reading frame 2 (uORF2). The C terminus of the AAP appears to be compacted adjacent to the peptidyl transferase center (PTC). Both nascent chains interact with ribosomal proteins L4 and L17 at tunnel constriction in a distinct fashion. Significant changes at the PTC were observed: the eukaryotic-specific loop of ribosomal protein L10e establishes direct contact with the CCA end of the peptidyl-tRNA (P-tRNA), which may be critical for silencing of the PTC during translational stalling. Our findings provide direct structural insight into two distinct eukaryotic stalling processes. Copyright © 2010 Elsevier Inc. All rights reserved.

  2. Cytomegalovirus iritis.

    PubMed

    Cheng, L; Rao, N A; Keefe, K S; Avila, C P; Macdonald, J C; Freeman, W R

    1998-11-01

    We describe a case of focal cytomegalovirus iritis in a patient with acquired immunodeficiency syndrome (AIDS) who had CMV retinitis. The autopsy showed histologic evidence of focal iritis in the left eye. This iritis was characterized by infiltration of acute inflammatory cells mixed with cytomegalic cells, which was confirmed by CMV-specific immunohistochemical staining. The case suggested that cytomegalovirus could be a direct causative agent of infectious iritis in AIDS patients.

  3. Human cytomegalovirus infection contributes to glioma disease progression via upregulating endocan expression.

    PubMed

    Xing, Yan; Wang, Yisong; Wang, Shijie; Wang, Xin; Fan, Dongying; Zhou, Dabiao; An, Jing

    2016-11-01

    The etiology of malignant glioma remains unclear. To examine the association between glioma and human cytomegalovirus (HCMV) infection and the possible mechanism through which HCMV contributes to malignant glioma, we investigated the expression of HCMV components and an angiogenesis marker, endocan, in 79 glioma specimens and 8 control brain samples. HCMV pp65 protein and DNA were detected in 65.8% (52 of 79) and 54.4% (43 of 79) of glioma specimens, respectively. The positive rate and expression levels of pp65 were significantly correlated with the glioma grades. The endocan expression was detected in 78.5% (62 of 79) of glioma specimens, and elevated endocan immunoreactivity was also significantly associated with high-grade glioma. The pp65 was predominantly detected and colocalized with endocan in the cytoplasm of tumor cells. Importantly, there was a significant positive correlation in detection rates between those 2 proteins. In control samples, neither HCMV pp65 nor endocan expression was detected. Moreover, the serum endocan levels in glioma patients were markedly higher than that in healthy subjects. In in vitro study, HCMV infection induced the expression of interleukin 6 and tumor necrosis factor-α in human glioblastoma U87 MG (U87) cells and human umbilical vein endothelial cells (HUVECs). Furthermore, elevated endocan levels were also observed in HCMV-infected U87 cells and HUVECs and antiviral treatment with ganciclovir reduced the endocan expression. These results suggest HCMV infection leads to glioma progression through an upregulation of endocan and the secretion of inflammatory cytokines. Thus, anti-HCMV treatment may represent a potentially novel therapeutic strategy for glioma. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  4. Effects of cytomegalovirus infection in human neural precursor cells depend on their differentiation state.

    PubMed

    González-Sánchez, H M; Monsiváis-Urenda, A; Salazar-Aldrete, C A; Hernández-Salinas, A; Noyola, D E; Jiménez-Capdeville, M E; Martínez-Serrano, A; Castillo, C G

    2015-08-01

    Cytomegalovirus (CMV) is the most common cause of congenital infection in developed countries and a major cause of neurological disability in children. Although CMV can affect multiple organs, the most important sequelae of intrauterine infection are related to lesions of the central nervous system. However, little is known about the pathogenesis and the cellular events responsible for neuronal damage in infants with congenital infection. Some studies have demonstrated that neural precursor cells (NPCs) show the greatest susceptibility to CMV infection in the developing brain. We sought to establish an in vitro model of CMV infection of the developing brain in order to analyze the cellular events associated with invasion by this virus. To this end, we employed two cell lines as a permanent source of NPC, avoiding the continuous use of human fetal tissue, the human SK-N-MC neuroblastoma cell line, and an immortalized cell line of human fetal neural origin, hNS-1. We also investigated the effect of the differentiation stage in relation to the susceptibility of these cell lines by comparing the neuroblastoma cell line with the multipotent cell line hNS-1. We found that the effects of the virus were more severe in the neuroblastoma cell line. Additionally, we induced hNS-1 to differentiate and evaluated the effect of CMV in these differentiated cells. Like SK-N-MC cells, hNS-1-differentiated cells were also susceptible to infection. Viability of differentiated hNS-1 cells decreased after CMV infection in contrast to undifferentiated cells. In addition, differentiated hNS-1 cells showed an extensive cytopathic effect whereas the effect was scarce in undifferentiated cells. We describe some of the effects of CMV in neural stem cells, and our observations suggest that the degree of differentiation is important in the acquisition of susceptibility.

  5. Analysis of the CoIE1 stability determinant Rcd.

    PubMed

    Sharpe, M E; Chatwin, H M; Macpherson, C; Withers, H L; Summers, D K

    1999-08-01

    Multimer formation is an important cause of instability for many multicopy plasmids. Plasmid CoIE1 is maintained stably because multimers are converted to monomers by Xer-mediated site-specific recombination at the cer site. However, multimer resolution is not the whole story; inactivation of a promoter (Pcer) within cer causes plasmid instability even though recombination is unaffected. The promoter directs the synthesis of a short transcript (Rcd) which is proposed to delay the division of multimer-containing cells. Mapping of the 5' terminus of Rcd confirms that transcription initiates from Pcer. The 3' terminus shows considerable heterogeneity, consistent with a primary transcript of 95 nt being degraded via intermediates of 79 and 70 nt. Secondary structure predictions for Rcd are presented. Of four mutations which abolish Rcd-mediated growth inhibition, one reduces the activity of Pcer while the other three map to the rcd coding sequence and reduce the steady-state level of the transcript. RNA folding analysis suggests that these three mutant transcripts adopt a common secondary structure in which the major stem-loop differs from that of wild-type Rcd. A survey of 24 cer-like multimer resolution sites revealed six which contain Pcer-like sequences. The putative transcripts from these sites have similar predicted secondary structures to Rcd and contain a highly conserved 15 base sequence. To test the hypothesis that Rcd acts as an anti-sense RNA, interacting with its target gene(s) through the 15 nt sequence, we used DNA hybridization and sequence analysis to find matches to this sequence in the Escherichia coli chromosome. Our failure to find plausible anti-sense targets has led to the suggestion that Rcd may interact directly with a protein target.

  6. Rapid intrahost evolution of human cytomegalovirus is shaped by demography and positive selection.

    PubMed

    Renzette, Nicholas; Gibson, Laura; Bhattacharjee, Bornali; Fisher, Donna; Schleiss, Mark R; Jensen, Jeffrey D; Kowalik, Timothy F

    2013-01-01

    Populations of human cytomegalovirus (HCMV), a large DNA virus, are highly polymorphic in patient samples, which may allow for rapid evolution within human hosts. To understand HCMV evolution, longitudinally sampled genomic populations from the urine and plasma of 5 infants with symptomatic congenital HCMV infection were analyzed. Temporal and compartmental variability of viral populations were quantified using high throughput sequencing and population genetics approaches. HCMV populations were generally stable over time, with ~88% of SNPs displaying similar frequencies. However, samples collected from plasma and urine of the same patient at the same time were highly differentiated with approximately 1700 consensus sequence SNPs (1.2% of the genome) identified between compartments. This inter-compartment differentiation was comparable to the differentiation observed in unrelated hosts. Models of demography (i.e., changes in population size and structure) and positive selection were evaluated to explain the observed patterns of variation. Evidence for strong bottlenecks (>90% reduction in viral population size) was consistent among all patients. From the timing of the bottlenecks, we conclude that fetal infection occurred between 13-18 weeks gestational age in patients analyzed, while colonization of the urine compartment followed roughly 2 months later. The timing of these bottlenecks is consistent with the clinical histories of congenital HCMV infections. We next inferred that positive selection plays a small but measurable role in viral evolution within a single compartment. However, positive selection appears to be a strong and pervasive driver of evolution associated with compartmentalization, affecting ≥ 34 of the 167 open reading frames (~20%) of the genome. This work offers the most detailed map of HCMV in vivo evolution to date and provides evidence that viral populations can be stable or rapidly differentiate, depending on host environment. The

  7. Role of human cytomegalovirus in the proliferation and invasion of extravillous cytotrophoblasts isolated from early placentae

    PubMed Central

    Liu, Tao; Zheng, Xiaofei; Li, Qin; Chen, Juanjuan; Yin, Zongzhi; Xiao, Juan; Zhang, Dandan; Li, Wei; Qiao, Yuan; Chen, Suhua

    2015-01-01

    Aim: We investigated the role of human cytomegalovirus (HCMV) and its mechanism in extravillous cytotrophoblast (EVT) proliferation and invasion in vitro. Methods: Differential enzymatic digestion combined with gradient centrifugation, was used to isolate primary EVT from human chorionic villi collected from early placentae of healthy pregnant women. HCMV infection was determined by immunofluorescence staining of HCMVpp65 antigen expression. An MTT assay was used to examine the role of HCMV in the proliferation of EVT. Quantitative real-time polymerase chain reaction (qRT-PCR), immunocytochemical staining and Western blots were carried out in a control group (EVT) and a virus group (EVT+HCMV) to examine the expression of major genes and protein in TGF-β/Smad signaling pathways in EVT 48 h after inoculation with HCMV. An in vitro cell invasion assay was performed to analyze the influence of HCMV on EVT invasion. Results: HCMV significantly inhibited the proliferation of EVT 48 h after viral infection (P < 0.05). The expression of TGF-β1, Smad1, Smad2, Smad3, Smad4, and Smad5 genes was significantly increased (P < 0.05), but that of TGF-β2, TGF-β3, TGFβRI, TGFβRII, Smad7, MMP2, and MMP9 was significantly decreased in the virus group 48 h after HCMV infection (P < 0.05). Smad7, MMP-2 and MMP-9 protein levels were significantly decreased and the TGF-β1 protein level was significantly increased in infected EVT (all P < 0.05). Conclusions: HCMV may act on multiple steps of the TGF-β/Smad signaling pathway to impede EVT proliferation and invasion. PMID:26770317

  8. In vivo expression of human cytomegalovirus (HCMV) microRNAs during latency.

    PubMed

    Meshesha, Mesfin K; Bentwich, Zvi; Solomon, Semaria A; Avni, Yonat Shemer

    2016-01-01

    Viral encoded microRNAs play key roles in regulating gene expression and the life cycle of human herpes viruses. Latency is one of the hallmarks of the human cytomegalovirus (HCMV or HHV5) life cycle, and its control may have immense practical applications. The present study aims to identify HCMV encoded microRNAs during the latency phase of the virus. We used a highly sensitive real time PCR (RTPCR) assay that involves a pre-amplification step before RTPCR. It can detect HCMV encoded microRNAs (miRNAs) during latency in purified monocytes and PBMCs from HCMV IgG positive donors and in latently infected monocytic THP-1 cell lines. During the latency phase, only eight HCMV encoded microRNAs were detected in PBMCs, monocytes and in the THP-1 cells. Five originated from the UL region of the virus genome and three from the US region. Reactivation of the virus from latency, in monocytes obtained from the same donor, using dexamethasone restored the expression of all known HCMV encoded miRNAs including those that were absent during latency. We observed a shift in the abundance of the two arms of mir-US29 between the productive and latency stages of the viral life cycle, suggesting that the star "passenger" form of this microRNA is preferentially expressed during latency. As a whole, our study demonstrates that HCMV expresses during the latency phase, both in vivo and in vitro, only a subset of its microRNAs, which may indicate that they play an important role in maintenance and reactivation of latency.

  9. Electrophoretic analysis of polypeptides immune precipitated from cytomegalovirus-infected cell extracts by human sera.

    PubMed Central

    Pereira, L; Hoffman, M; Cremer, N

    1982-01-01

    Serodiagnosis of cytomegalovirus (CMV) infection by complement fixation tests depends on showing a fourfold rise in antibody titer from acute- to convalescent-phase sera. Freeze-thaw and glycine-extracted, infected cell culture antigens used for these tests give markedly different titers in reactions with the same sera. In this study, we characterized the CMV-infected cell polypeptides contained in freeze-thaw and glycine-extracted antigens and identified the proteins precipitated by 23 pairs of human acute and convalescent sera. Our results were as follows. First, freeze-thaw and glycine-extracted antigens prepared from infected cells radiolabeled with [35S]methionine and subjected to electrophoresis in sodium dodecyl sulfate-polyacrylamide gels yielded similar patterns, and the bulk of the label was contained in late structural proteins and glycoproteins. Glycine-extracted preparations contained a greater proportion of soluble 66,000- and 50,000-molecular-weight proteins than did freeze-thaw antigens. Second, convalescent sera precipitated proteins migrating with apparent molecular weights of 150,000, 130,000, 110,000, 96,000, 74,000, 66,000, 50,000, 34,000, 32,000, and 25,000. Of these the 130,000-, 110,000-, 96,000-, 66,000-, 50,000-, and 25,000-molecular-weight proteins comigrated with glucosamine-labeled polypeptides. Both immunoglobulin G and M antibodies in human sera precipitated these proteins from CMV-infected cell preparations. Implications of the results for serodiagnosis of CMV infections are discussed. Images FIG. 1 FIG. 2 FIG. 3 FIG. 4 FIG. 5 FIG. 6 FIG. 7 FIG. 8 FIG. 9 FIG. 10 PMID:6284646

  10. The pentameric complex of human Cytomegalovirus: cell tropism, virus dissemination, immune response and vaccine development.

    PubMed

    Gerna, Giuseppe; Revello, Maria Grazia; Baldanti, Fausto; Percivalle, Elena; Lilleri, Daniele

    2017-09-01

    Between the 1980s and 1990s, three assays were developed for diagnosis of human cytomegalovirus (HCMV) infections: leuko (L)-antigenemia, l-viremia and l-DNAemia, detecting viral protein pp65, infectious virus and viral DNA, respectively, in circulating leukocytes Repeated initial attempts to reproduce the three assays in vitro using laboratory-adapted strains and infected cell cultures were consistently unsuccessful. Results were totally reversed when wild-type HCMV strains were used to infect either fibroblasts or endothelial cells. Careful analysis and sequencing of plaque-purified viruses from recent clinical isolates drew attention to the ULb' region of the HCMV genome. Using bacterial artificial chromosome technology, it was shown by both gain-of-function and loss-of-function experiments that UL131-128 genes are indispensable for virus growth in endothelial cells and virus transfer to leukocytes. In addition, a number of clinical isolates passaged in human fibroblasts had lost both properties (leuko-tropism and endothelial cell-tropism) when displaying a mutation in the UL131-128 locus (referred to as UL128L). In the following years, it was shown that pUL128L was complexed with gH and gL to form the pentameric complex (PC), which is required to infect endothelial, epithelial and myeloid cells. The immune response to PC was studied extensively, particularly its humoral component, showing that the great majority of the neutralizing antibody response is directed to PC. Although anti-HCMV antibodies may act with other mechanisms than mere neutralizing activity, these findings definitely favour their protective activity, thus paving the way to the development of a potentially protective HCMV vaccine.

  11. Human cytomegalovirus detection in gastric cancer and its possible association with lymphatic metastasis.

    PubMed

    Zhang, Liang; Guo, Gangqiang; Xu, Jianfeng; Sun, Xiangwei; Chen, Wenjing; Jin, Jinji; Hu, Changyuan; Zhang, Peichen; Shen, Xian; Xue, Xiangyang

    2017-02-08

    Increasing evidence suggests that human cytomegalovirus (HCMV) is associated with many human malignancies. However, its prevalence in gastric cancer (GC) and clinical association remain unknown. HCMV IgG and IgM antibodies in the sera of 80 GC patients and 80 healthy controls were detected using a microparticle enzyme immunoassay. The prevalence of HCMV UL47, UL55, UL56, and UL77 genes among 102 GC tumor tissues and adjacent normal specimens was measured by polymerase chain reaction (PCR) or nested PCR. Quantitative real-time PCR (Q-PCR) was used to determine viral load. Virus localization in neoplastic tissues was determined by immunohistochemistry. No significant difference of HCMV IgG and IgM seropositivity was found between GC patients and the healthy group. However, the overall HCMV DNA positivity rate was significantly higher in GC cancerous tissue compared with in paired normal tissue (P<0.01). HCMV infection was mainly localized in the tumorous epithelium. Q-PCR in HCMV-positive specimens indicated that the viral copy number was notably higher in GC tissues than in adjacent normal specimens (P<0.001). Clinical statistical analysis indicated that HCMV load in GC tumor tissue was positively associated with lymphatic metastasis (P=0.043), the area under the receiver operating characteristic (ROC) curve was 0.6638. Our data clearly provide the prevalence of HCMV in GC patients. We conclude that HCMV infection in malignant tissues might be associated with carcinogenesis or progression of GC and possibly relates to lymphatic metastasis.

  12. High-Resolution Profiling and Analysis of Viral and Host Small RNAs during Human Cytomegalovirus Infection

    PubMed Central

    Stark, Thomas J.; Arnold, Justin D.; Spector, Deborah H.

    2012-01-01

    Human cytomegalovirus (HCMV) contributes its own set of microRNAs (miRNAs) during lytic infection of cells, likely fine-tuning conditions important for viral replication. To enhance our understanding of this component of the HCMV-host transcriptome, we have conducted deep-sequencing analysis of small RNAs (smRNA-seq) from infected human fibroblast cells. We found that HCMV-encoded miRNAs accumulate to ∼20% of the total smRNA population at late stages of infection, and our analysis led to improvements in viral miRNA annotations and identification of two novel HCMV miRNAs, miR-US22 and miR-US33as. Both of these miRNAs were capable of functionally repressing synthetic targets in transient transfection experiments. Additionally, through cross-linking and immunoprecipitation (CLIP) of Argonaute (Ago)-bound RNAs from infected cells, followed by high-throughput sequencing, we have obtained direct evidence for incorporation of all HCMV miRNAs into the endogenous host silencing machinery. Surprisingly, three HCMV miRNA precursors exhibited differential incorporation of their mature miRNA arms between Ago2 and Ago1 complexes. Host miRNA abundances were also affected by HCMV infection, with significant upregulation observed for an miRNA cluster containing miR-96, miR-182, and miR-183. In addition to miRNAs, we also identified novel forms of virus-derived smRNAs, revealing greater complexity within the smRNA population during HCMV infection. PMID:22013051

  13. A Role for Nuclear F-Actin Induction in Human Cytomegalovirus Nuclear Egress

    PubMed Central

    Wilkie, Adrian R.; Lawler, Jessica L.

    2016-01-01

    ABSTRACT Herpesviruses, which include important pathogens, remodel the host cell nucleus to facilitate infection. This remodeling includes the formation of structures called replication compartments (RCs) in which herpesviruses replicate their DNA. During infection with the betaherpesvirus, human cytomegalovirus (HCMV), viral DNA synthesis occurs at the periphery of RCs within the nuclear interior, after which assembled capsids must reach the inner nuclear membrane (INM) for translocation to the cytoplasm (nuclear egress). The processes that facilitate movement of HCMV capsids to the INM during nuclear egress are unknown. Although an actin-based mechanism of alphaherpesvirus capsid trafficking to the INM has been proposed, it is controversial. Here, using a fluorescently-tagged, nucleus-localized actin-binding peptide, we show that HCMV, but not herpes simplex virus 1, strongly induced nuclear actin filaments (F-actin) in human fibroblasts. Based on studies using UV inactivation and inhibitors, this induction depended on viral gene expression. Interestingly, by 24 h postinfection, nuclear F-actin formed thicker structures that appeared by super-resolution microscopy to be bundles of filaments. Later in infection, nuclear F-actin primarily localized along the RC periphery and between the RC periphery and the nuclear rim. Importantly, a drug that depolymerized nuclear F-actin caused defects in production of infectious virus, capsid accumulation in the cytoplasm, and capsid localization near the nuclear rim, without decreasing capsid accumulation in the nucleus. Thus, our results suggest that for at least one herpesvirus, nuclear F-actin promotes capsid movement to the nuclear periphery and nuclear egress. We discuss our results in terms of competing models for these processes. PMID:27555312

  14. Human cytomegalovirus tropism for endothelial/epithelial cells: scientific background and clinical implications.

    PubMed

    Revello, M Grazia; Gerna, Giuseppe

    2010-05-01

    Human cytomegalovirus (HCMV) has been routinely isolated from and propagated in vitro in human embryonic lung fibroblast (HELF) cell cultures, while in vivo it is known to infect predominantly endothelial and epithelial cells. In recent years, genetic determinants of the HCMV tropism for endothelial/epithelial cells were identified in the UL131A/UL130/UL128 locus of HCMV genome of wild-type strains. UL131A-UL128 gene products form a complex with glycoprotein H (gH) and L (gL) resulting in a gH/gL/UL131A-UL128 complex that is required for HCMV entry into endothelial/epithelial cells. In contrast, virus entry into fibroblasts has its genetic determinants in the complex gH/gL/gO (or gH/gL). During primary HCMV infection, the neutralising antibody response measured in endothelial cells (EC) is potent, occurs very early and is directed mostly against combinations of two or three gene products of the UL131A-128 locus. On the contrary, neutralising antibodies measured in fibroblasts appear late, are relatively weak in potency and are directed against gH and gB. The T-cell immune response to UL131A-UL128 gene products remains to be investigated. Recently, a role has been proposed for neutralising antibody in conferring prevention/protection against HCMV infection/disease in pregnant women with primary HCMV infection. However, the level of cooperation between humoral immunity and the well-established T-cell protection remains to be defined.

  15. Identification of Human Cytomegalovirus Genes Important for Biogenesis of the Cytoplasmic Virion Assembly Complex

    PubMed Central

    Das, Subhendu; Ortiz, Daniel A.; Gurczynski, Stephen J.; Khan, Fatin

    2014-01-01

    ABSTRACT Human cytomegalovirus (HCMV) has many effects on cells, including remodeling the cytoplasm to form the cytoplasmic virion assembly complex (cVAC), the site of final virion assembly. Viral tegument, envelope, and some nonstructural proteins localize to the cVAC, and cytoskeletal filaments radiate from a microtubule organizing center in the cVAC. The endoplasmic reticulum (ER)-to-Golgi intermediate compartment, Golgi apparatus, and trans-Golgi network form a ring that outlines the cVAC. The center of the cVAC ring is occupied by numerous vesicles that share properties with recycling endosomes. In prior studies, we described the three-dimensional structure and the extensive remodeling of the cytoplasm and shifts in organelle identity that occur during development of the cVAC. The objective of this work was to identify HCMV proteins that regulate cVAC biogenesis. Because the cVAC does not form in the absence of viral DNA synthesis, we employed HCMV-infected cells transfected with synthetic small interfering RNAs (siRNAs) that targeted 26 candidate early-late and late protein-coding genes required for efficient virus replication. We identified three HCMV genes (UL48, UL94, and UL103) whose silencing had major effects on cVAC development, including failure to form the Golgi ring and dispersal of markers of early and recycling endosomes. To confirm and extend the siRNA results, we constructed recombinant viruses in which pUL48 and pUL103 are fused with a regulatable protein destabilization domain (dd-FKBP). In the presence of a stabilizing ligand (Shield-1), the cVAC appeared to develop normally. In its absence, cVAC development was abrogated, verifying roles for pUL48 and pUL103 in cVAC biogenesis. IMPORTANCE Human cytomegalovirus (HCMV) is an important human pathogen that causes disease and disability in immunocompromised individuals and in children infected before birth. Few drugs are available for treatment of HCMV infections. HCMV remodels the interior of

  16. Restriction of Human Cytomegalovirus Replication by ISG15, a Host Effector Regulated by cGAS-STING Double-Stranded-DNA Sensing.

    PubMed

    Bianco, Christopher; Mohr, Ian

    2017-05-01

    Accumulation of the interferon-stimulated gene 15 (ISG15) protein product, which is reversibly conjugated to numerous polypeptide targets, impacts the proteome and physiology of uninfected and infected cells. While many viruses, including human cytomegalovirus (HCMV), blunt host antiviral defenses by limiting ISG expression, the overall abundance of ISG15 monomer and protein conjugates rises in HCMV-infected cells. However, the molecular signals underlying ISG15 accumulation and whether the ISG15 polypeptide itself influences HCMV infection biology remain unknown. Here, we establish that the ISG15 gene product itself directly regulates HCMV replication and that its accumulation restricts productive virus growth. Although ISG15 monomer and protein conjugate accumulation was induced in cells infected with UV-inactivated HCMV, it was subsequently reduced, but not eliminated, by an immediate-early (IE) or early (E) virus-encoded function(s). Instead, HCMV-induced ISG15 monomer and protein conjugate accumulation was dependent upon the double-stranded DNA (dsDNA) sensor cyclic GMP-AMP synthase (cGAS), the innate immune adaptor STING, and interferon signaling. Significantly, dsDNA itself was sufficient to induce cGAS-, STING-, and interferon signaling-dependent ISG15 monomer and conjugate protein accumulation in uninfected cells. Accumulation of ISGylated proteins in uninfected cells treated with dsDNA was prevented by expressing the HCMV multifunctional IE1 transactivator. This demonstrates that expression of a single host interferon-stimulated gene, ISG15, restricts HCMV replication, and that IE1 is sufficient to blunt ISGylation in response to dsDNA sensing in uninfected cells. Moreover, it establishes that ISGylation modifies the proteomes of virus-infected and uninfected normal cells in response to cell-intrinsic dsDNA sensing dependent upon cGAS-STING.IMPORTANCE By antagonizing type I interferon production and action, many viruses, including human cytomegalovirus

  17. In Vivo Replication of Recombinant Murine Cytomegalovirus Driven by the Paralogous Major Immediate-Early Promoter-Enhancer of Human Cytomegalovirus

    PubMed Central

    Grzimek, Natascha K. A.; Podlech, Jürgen; Steffens, Hans-Peter; Holtappels, Rafaela; Schmalz, Susanne; Reddehase, Matthias J.

    1999-01-01

    Transcription of the major immediate-early (MIE) genes of cytomegaloviruses (CMV) is driven by a strong promoter-enhancer (MIEPE) complex. Transactivator proteins encoded by these MIE genes are essential for productive infection. Accordingly, the MIEPE is a crucial control point, and its regulation by activators and repressors is pertinent to virus replication. Since the MIEPE contains multiple regulatory elements, it was reasonable to assume that specific sequence motifs are irreplaceable for specifying the cell-type tropism and replication pattern. Recent work on murine CMV infectivity (A. Angulo, M. Messerle, U. H. Koszinowski, and P. Ghazal, J. Virol. 72:8502–8509, 1998) has documented the proposed enhancing function of the enhancer in that its resection or its replacement by a nonregulatory stuffer sequence resulted in a significant reduction of infectivity, even though replication competence was maintained by a basal activity of the spared authentic MIE promoter. Notably, full capacity for productive in vitro infection of fibroblasts was restored in recombinant viruses by the human CMV enhancer. Using two-color in situ hybridization with MIEPE-specific polynucleotide probes, we demonstrated that a murine CMV recombinant in which the complete murine CMV MIEPE is replaced by the paralogous human CMV core promoter and enhancer (recombinant virus mCMVhMIEPE) retained the potential to replicate in vivo in all tissues relevant to CMV disease. Notably, mCMVhMIEPE was also found to replicate in the liver, a site at which transgenic hCMV MIEPE is silenced. We conclude that productive in vivo infection with murine CMV does not strictly depend on a MIEPE type-specific regulation. PMID:10233967

  18. Human Cytomegalovirus Requires Epidermal Growth Factor Receptor Signaling To Enter and Initiate the Early Steps in the Establishment of Latency in CD34(+) Human Progenitor Cells.

    PubMed

    Kim, Jung Heon; Collins-McMillen, Donna; Buehler, Jason C; Goodrum, Felicia D; Yurochko, Andrew D

    2017-03-01

    The establishment of human cytomegalovirus (HCMV) latency and persistence relies on the successful infection of hematopoietic cells, which serve as sites of viral persistence and contribute to viral spread. Here, using blocking antibodies and pharmacological inhibitors, we document that HCMV activation of the epidermal growth factor receptor (EGFR) and downstream phosphatidylinositol 3-kinase (PI3K) mediates viral entry into CD34(+) human progenitor cells (HPCs), resulting in distinct cellular trafficking and nuclear translocation of the virus compared to that in other immune cells, such as we have documented in monocytes. We argue that the EGFR allows HCMV to regulate the cellular functions of these replication-restricted cells via its signaling activity following viral binding. In addition to regulating HCMV entry/trafficking, EGFR signaling may also shape the early steps required for the successful establishment of viral latency in CD34(+) cells, as pharmacological inhibition of EGFR increases the transcription of lytic IE1/IE2 mRNA while curbing the expression of latency-associated UL138 mRNA. EGFR signaling following infection of CD34(+) HPCs may also contribute to changes in hematopoietic potential, as treatment with the EGFR kinase (EGFRK) inhibitor AG1478 alters the expression of the cellular hematopoietic cytokine interleukin 12 (IL-12) in HCMV-infected cells but not in mock-infected cells. These findings, along with our previous work with monocytes, suggest that EGFR likely serves as an important determinant of HCMV tropism for select subsets of hematopoietic cells. Moreover, our new data suggest that EGFR is a key receptor for efficient viral entry and that the ensuing signaling regulates important early events required for successful infection of CD34(+) HPCs by HCMV.IMPORTANCE HCMV establishes lifelong persistence within the majority of the human population without causing overt pathogenesis in healthy individuals. Despite this, reactivation of HCMV

  19. Human cytomegalovirus infection interferes with the maintenance and differentiation of trophoblast progenitor cells of the human placenta.

    PubMed

    Tabata, Takako; Petitt, Matthew; Zydek, Martin; Fang-Hoover, June; Larocque, Nicholas; Tsuge, Mitsuru; Gormley, Matthew; Kauvar, Lawrence M; Pereira, Lenore

    2015-05-01

    Human cytomegalovirus (HCMV) is a major cause of birth defects that include severe neurological deficits, hearing and vision loss, and intrauterine growth restriction. Viral infection of the placenta leads to development of avascular villi, edema, and hypoxia associated with symptomatic congenital infection. Studies of primary cytotrophoblasts (CTBs) revealed that HCMV infection impedes terminal stages of differentiation and invasion by various molecular mechanisms. We recently discovered that HCMV arrests earlier stages involving development of human trophoblast progenitor cells (TBPCs), which give rise to the mature cell types of chorionic villi-syncytiotrophoblasts on the surfaces of floating villi and invasive CTBs that remodel the uterine vasculature. Here, we show that viral proteins are present in TBPCs of the chorion in cases of symptomatic congenital infection. In vitro studies revealed that HCMV replicates in continuously self-renewing TBPC lines derived from the chorion and alters expression and subcellular localization of proteins required for cell cycle progression, pluripotency, and early differentiation. In addition, treatment with a human monoclonal antibody to HCMV glycoprotein B rescues differentiation capacity, and thus, TBPCs have potential utility for evaluation of the efficacies of novel antiviral antibodies in protecting and restoring placental development. Our results suggest that HCMV replicates in TBPCs in the chorion in vivo, interfering with the earliest steps in the growth of new villi, contributing to virus transmission and impairing compensatory development. In cases of congenital infection, reduced responsiveness of the placenta to hypoxia limits the transport of substances from maternal blood and contributes to fetal growth restriction. Human cytomegalovirus (HCMV) is a leading cause of birth defects in the United States. Congenital infection can result in permanent neurological defects, mental retardation, hearing loss, visual

  20. Human Cytomegalovirus Infection Interferes with the Maintenance and Differentiation of Trophoblast Progenitor Cells of the Human Placenta

    PubMed Central

    Tabata, Takako; Petitt, Matthew; Zydek, Martin; Fang-Hoover, June; Larocque, Nicholas; Tsuge, Mitsuru; Gormley, Matthew; Kauvar, Lawrence M.

    2015-01-01

    ABSTRACT Human cytomegalovirus (HCMV) is a major cause of birth defects that include severe neurological deficits, hearing and vision loss, and intrauterine growth restriction. Viral infection of the placenta leads to development of avascular villi, edema, and hypoxia associated with symptomatic congenital infection. Studies of primary cytotrophoblasts (CTBs) revealed that HCMV infection impedes terminal stages of differentiation and invasion by various molecular mechanisms. We recently discovered that HCMV arrests earlier stages involving development of human trophoblast progenitor cells (TBPCs), which give rise to the mature cell types of chorionic villi—syncytiotrophoblasts on the surfaces of floating villi and invasive CTBs that remodel the uterine vasculature. Here, we show that viral proteins are present in TBPCs of the chorion in cases of symptomatic congenital infection. In vitro studies revealed that HCMV replicates in continuously self-renewing TBPC lines derived from the chorion and alters expression and subcellular localization of proteins required for cell cycle progression, pluripotency, and early differentiation. In addition, treatment with a human monoclonal antibody to HCMV glycoprotein B rescues differentiation capacity, and thus, TBPCs have potential utility for evaluation of the efficacies of novel antiviral antibodies in protecting and restoring placental development. Our results suggest that HCMV replicates in TBPCs in the chorion in vivo, interfering with the earliest steps in the growth of new villi, contributing to virus transmission and impairing compensatory development. In cases of congenital infection, reduced responsiveness of the placenta to hypoxia limits the transport of substances from maternal blood and contributes to fetal growth restriction. IMPORTANCE Human cytomegalovirus (HCMV) is a leading cause of birth defects in the United States. Congenital infection can result in permanent neurological defects, mental retardation

  1. Quantification of Epstein-Barr Virus and Human Cytomegalovirus in Chronic Periodontal Patients

    PubMed Central

    Khosropanah, Hengameh; Karandish, Maryam; Ziaeyan, Mazyar; Jamalidoust, Marzieh

    2015-01-01

    Background: Although studies focused mainly on the identification of periopathogenic bacteria, recent reports have suggested that various herpes viruses may also be involved in the occurrence and progression of different forms of periodontal diseases. Objectives: This study aimed to compare the prevalence and load of Epstein-Barr Virus (EBV) and Human Cytomegalovirus (HCMV) in subgingival tissue specimens between chronic periodontitis and healthy sites. Patients and Methods: A total of 60 samples from the systematically healthy patients with chronic periodontitis participated in this study (mean age, 35 ± 7). Clinical periodontal evaluation included the plaque index (PI) (Loe and Silness), bleeding on probing (BOP) (O’Leary), bleeding index, periodontal pocket depth (PPD) and clinical attachment level measurement. Tissue specimens harvested from > 6 mm periodontal pockets and from ≤ 3 mm sulcus depth in a quadrant of the same patient using periodontal curettes. Moreover, the unstimulated whole saliva was gathered as a shedding medium. A Taq-man Real-Time Polymerase Chain Reaction assay was used to identify genomic copies of periodontal HCMV and EBV. Data were analyzed by the Wilcoxon-signed ranks and Friedman tests using the SPSS 16 software. Results: Out of 60 samples of subgingival tissues taken from the patients with chronic periodontitis, EBV count was the highest in saliva and the least in the tissue sample with PD < 3 mm (P < 0.05). The highest HCMV count was in saliva and tissue samples with PD > 6 mm (P < 0.05). Conclusions: According to the results of this study, quantification of HCMV and EBV observed in this study is high in periodontal tissue samples of severe chronic periodontitis. PMID:26322203

  2. The immunology of human cytomegalovirus latency: could latent infection be cleared by novel immunotherapeutic strategies?

    PubMed Central

    Wills, Mark R; Poole, Emma; Lau, Betty; Krishna, Ben; Sinclair, John H

    2015-01-01

    While the host immune response following primary human cytomegalovirus (HCMV) infection is generally effective at stopping virus replication and dissemination, virus is never cleared by the host and like all herpesviruses, persists for life. At least in part, this persistence is known to be facilitated by the ability of HCMV to establish latency in myeloid cells in which infection is essentially silent with, importantly, a total lack of new virus production. However, although the viral transcription programme during latency is much suppressed, a number of viral genes are expressed during latent infection at the protein level and many of these have been shown to have profound effects on the latent cell and its environment. Intriguingly, many of these latency-associated genes are also expressed during lytic infection. Therefore, why the same potent host immune responses generated during lytic infection to these viral gene products are not recognized during latency, thereby allowing clearance of latently infected cells, is far from clear. Reactivation from latency is also a major cause of HCMV-mediated disease, particularly in the immune compromised and immune naive, and is also likely to be a major source of virus in chronic subclinical HCMV infection which has been suggested to be associated with long-term diseases such as atherosclerosis and some neoplasias. Consequently, understanding latency and why latently infected cells appear to be immunoprivileged is crucial for an understanding of the pathogenesis of HCMV and may help to design strategies to eliminate latent virus reservoirs, at least in certain clinical settings. PMID:25132454

  3. Nuclear domain 10 components upregulated via interferon during human cytomegalovirus infection potently regulate viral infection.

    PubMed

    Ashley, Caroline L; Glass, Mandy S; Abendroth, Allison; McSharry, Brian P; Slobedman, Barry

    2017-07-01

    Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus that causes life-threatening disease in immunocompromised and immunonaïve individuals. Type I interferons (IFNs) are crucial molecules in the innate immune response to HCMV and are also known to upregulate several components of the interchromosomal multiprotein aggregates collectively referred to as nuclear domain 10 (ND10). In the context of herpesvirus infection, ND10 components are known to restrict gene expression. This raises the question as to whether key ND10 components (PML, Sp100 and hDaxx) act as anti-viral IFN-stimulated genes (ISGs) during HCMV infection. In this study, analysis of ND10 component transcription during HCMV infection demonstrated that PML and Sp100 were significantly upregulated whilst hDaxx expression remained unchanged. In cells engineered to block the production of, or response to, type I IFNs, upregulation of PML and Sp100 was not detected during HCMV infection. Furthermore, pre-treatment with an IFN-β neutralizing antibody inhibited upregulation of PML and Sp100 during both infection and treatment with HCMV-infected cell supernatant. The significance of ND10 components functioning as anti-viral ISGs during HCMV infection was determined through knockdown of PML, Sp100 and hDaxx. ND10 knockdown cells were significantly more permissive to HCMV infection, as previously described but, in contrast to control cells, could support HCMV plaque formation following IFN-β pre-treatment. This ability of HCMV to overcome the potently anti-viral effects of IFN-β in ND10 expression deficient cells provides evidence that ND10 component upregulation is a key mediator of the anti-viral activity of IFN-β.

  4. Limited Effector Memory B-Cell Response to Envelope Glycoprotein B During Primary Human Cytomegalovirus Infection.

    PubMed

    Dauby, Nicolas; Sartori, Delphine; Kummert, Caroline; Lecomte, Sandra; Haelterman, Edwige; Delforge, Marie-Luce; Donner, Catherine; Mach, Michael; Marchant, Arnaud

    2016-05-15

    Following primary human cytomegalovirus (HCMV) infection, the production of antibodies against envelope glycoprotein B (gB) is delayed, compared with production of antibodies against tegument proteins, and this likely reduces the control of HCMV dissemination. The frequency and the phenotype of gB-specific and tegument protein-specific B cells were studied in a cohort of pregnant women with primary HCMV infection. Healthy adults who had chronic HCMV infection or were recently immunized with tetanus toxoid (TT) were included as controls. Primary HCMV infection was associated with high and similar frequencies of gB-specific and tegument protein-specific B cells following primary HCMV infection. During primary infection, tegument protein-specific B cells expressed an activated (CD21(low)) memory B-cell (MBC) phenotype. Activated MBCs were also induced by TT booster immunization, indicating that the expansion of this subset is part of the physiological B-cell response to protein antigens. In contrast, gB-specific B cells had a predominant classical (CD21(+)) MBC phenotype during both primary and chronic infections. The delayed production of gB-specific immunoglobulin G (IgG) during primary HCMV infection is associated with a limited induction of MBCs with effector potential. This novel mechanism by which HCMV may interfere with the production of neutralizing antibodies could represent a target for therapeutic immunization. © The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  5. Diagnosis and Management of Human Cytomegalovirus Infection in the Mother, Fetus, and Newborn Infant

    PubMed Central

    Revello, Maria Grazia; Gerna, Giuseppe

    2002-01-01

    Human cytomegalovirus (HCMV) is the leading cause of congenital viral infection and mental retardation. HCMV infection, while causing asymptomatic infections in most immunocompetent subjects, can be transmitted during pregnancy from the mother with primary (and also recurrent) infection to the fetus. Hence, careful diagnosis of primary infection is required in the pregnant woman based on the most sensitive serologic assays (immunoglobulin M [IgM] and IgG avidity assays) and conventional virologic and molecular procedures for virus detection in blood. Maternal prognostic markers of fetal infection are still under investigation. If primary infection is diagnosed in a timely manner, prenatal diagnosis can be offered, including the search for virus and virus components in fetal blood and amniotic fluid, with fetal prognostic markers of HCMV disease still to be defined. However, the final step for definite diagnosis of congenital HCMV infection is detection of virus in the blood or urine in the first 1 to 2 weeks of life. To date, treatment of congenital infection with antiviral drugs is only palliative both prior to and after birth, whereas the only efficacious preventive measure seems to be the development of a safe and immunogenic vaccine, including recombinant, subunit, DNA, and peptide-based vaccines now under investigation. The following controversial issues are discussed in the light of the most recent advances in the field: the actual perception of the problem; universal serologic screening before pregnancy; the impact of correct counseling on decision making by the couple involved; the role of prenatal diagnosis in ascertaining transmission of virus to the fetus; the impact of preconceptional and periconceptional infections on the prevalence of congenital infection; and the prevalence of congenitally infected babies born to mothers who were immune prior to pregnancy compared to the number born to mothers undergoing primary infection during pregnancy. PMID

  6. Identification of persistent RNA-DNA hybrid structures within the origin of replication of human cytomegalovirus.

    PubMed

    Prichard, M N; Jairath, S; Penfold, M E; St Jeor, S; Bohlman, M C; Pari, G S

    1998-09-01

    Human cytomegalovirus (HCMV) lytic-phase DNA replication initiates at the cis-acting origin of replication, oriLyt. oriLyt is a structurally complex region containing repeat elements and transcription factor binding sites. We identified two site-specific alkali-labile regions within oriLyt which flank an alkali-resistant DNA segment. These alkali-sensitive regions were the result of the degradation of two RNA species embedded within oriLyt and covalently linked to viral DNA. The virus-associated RNA, vRNA, was identified by DNase I treatment of HCMV DNA obtained from sucrose gradient purified virus. This heterogeneous population of vRNA was end labeled and used as a hybridization probe to map the exact location of vRNAs within oriLyt. vRNA-1 is localized between restriction endonuclease sites XhoI at nucleotide (nt) 93799 and SacI at nt 94631 and is approximately 500 bases long. The second vRNA, vRNA-2, lies within a region which exhibits a heterogeneous restriction pattern located between the SphI (nt 92636) and BamHI (nt 93513) and is approximately 300 bases long. This region was previously shown to be required for oriLyt replication (D. G. Anders, M. A. Kacica, G. S. Pari, and S. M. Punturieri, J. Virol. 66:3373-3384, 1992). RNase H analysis determined that vRNA-2 forms a persistent RNA-DNA hybrid structure in the context of the viral genome and in an oriLyt-containing plasmid used in the transient-replication assay.

  7. Identification of proteins in human cytomegalovirus (HCMV) particles: the HCMV proteome.

    PubMed

    Varnum, Susan M; Streblow, Daniel N; Monroe, Matthew E; Smith, Patricia; Auberry, Kenneth J; Pasa-Tolic, Ljiljana; Wang, Dai; Camp, David G; Rodland, Karin; Wiley, Steven; Britt, William; Shenk, Thomas; Smith, Richard D; Nelson, Jay A

    2004-10-01

    Human cytomegalovirus (HCMV), a member of the herpesvirus family, is a large complex enveloped virus composed of both viral and cellular gene products. While the sequence of the HCMV genome has been known for over a decade, the full set of viral and cellular proteins that compose the HCMV virion are unknown. To approach this problem we have utilized gel-free two-dimensional capillary liquid chromatography-tandem mass spectrometry (MS/MS) and Fourier transform ion cyclotron resonance MS to identify and determine the relative abundances of viral and cellular proteins in purified HCMV AD169 virions and dense bodies. Analysis of the proteins from purified HCMV virion preparations has indicated that the particle contains significantly more viral proteins than previously known. In this study, we identified 71 HCMV-encoded proteins that included 12 proteins encoded by known viral open reading frames (ORFs) previously not associated with virions and 12 proteins from novel viral ORFs. Analysis of the relative abundance of HCMV proteins indicated that the predominant virion protein was the pp65 tegument protein and that gM rather than gB was the most abundant glycoprotein. We have also identified over 70 host cellular proteins in HCMV virions, which include cellular structural proteins, enzymes, and chaperones. In addition, analysis of HCMV dense bodies indicated that these viral particles are composed of 29 viral proteins with a reduced quantity of cellular proteins in comparison to HCMV virions. This study provides the first comprehensive quantitative analysis of the viral and cellular proteins that compose infectious particles of a large complex virus.

  8. Identification of Proteins in Human Cytomegalovirus (HCMV) Particles: the HCMV Proteome

    SciTech Connect

    Varnum, Susan M.; Streblow, Daniel N.; Monroe, Matthew E.; Smith, Patricia; Auberry, Kenneth J.; Pasa-Tolic, Liljiana; Wang, Dai; Camp, David G.; Rodland, Karin D.; Wiley, H S.; Britt, William; Shenk, Thomas; Smith, Richard D.; Nelson, Jay

    2004-10-15

    Human cytomegalovirus (HCMV), a member of the herpes virus family, is a large complex enveloped virus composed of both viral and cellular gene products. While the sequence of the HCMV genome has been known for over a decade, the full set of viral and cellular proteins that compose the HCMV virion are unknown. To approach this problem we have utilized gel-free two-dimensional capillary liquid chromatography-tandem mass spectrometry (MS/MS) and Fourier transform ion cyclotron resonance MS to identify and determine the relative abundances of viral and cellular proteins in purified HCMV AD169 virions and dense bodies. Analysis of the proteins from purified HCMV virion preparations has indicated that the particle contains significantly more viral proteins than previously known. In this study, we identified 71 HCMV-encoded proteins that included 12 proteins encoded by known viral open reading frames (ORFs) previously not associated with virions and 12 proteins from novel viral ORFs. Analysis of the relative abundance of HCMV proteins indicated that the predominant virion protein was the pp65 tegument protein and that gM rather than gB was the most abundant glycoprotein. We have also identified over 70 host cellular proteins in HCMV virions, which include cellular structural proteins, enzymes, and chaperones. In addition, analysis of HCMV dense bodies indicated that these viral particles are composed of 29 viral proteins with a reduced quantity of cellular proteins in comparison to HCMV virions. This study provides the first comprehensive quantitative analysis of the viral and cellular proteins that compose infectious particles of a large complex virus.

  9. Inter-laboratory assessment of different digital PCR platforms for quantification of human cytomegalovirus DNA.

    PubMed

    Pavšič, Jernej; Devonshire, Alison; Blejec, Andrej; Foy, Carole A; Van Heuverswyn, Fran; Jones, Gerwyn M; Schimmel, Heinz; Žel, Jana; Huggett, Jim F; Redshaw, Nicholas; Karczmarczyk, Maria; Mozioğlu, Erkan; Akyürek, Sema; Akgöz, Müslüm; Milavec, Mojca

    2017-04-01

    Quantitative PCR (qPCR) is an important tool in pathogen detection. However, the use of different qPCR components, calibration materials and DNA extraction methods reduces comparability between laboratories, which can result in false diagnosis and discrepancies in patient care. The wider establishment of a metrological framework for nucleic acid tests could improve the degree of standardisation of pathogen detection and the quantification methods applied in the clinical context. To achieve this, accurate methods need to be developed and implemented as reference measurement procedures, and to facilitate characterisation of suitable certified reference materials. Digital PCR (dPCR) has already been used for pathogen quantification by analysing nucleic acids. Although dPCR has the potential to provide robust and accurate quantification of nucleic acids, further assessment of its actual performance characteristics is needed before it can be implemented in a metrological framework, and to allow adequate estimation of measurement uncertainties. Here, four laboratories demonstrated reproducibility (expanded measurement uncertainties below 15%) of dPCR for quantification of DNA from human cytomegalovirus, with no calibration to a common reference material. Using whole-virus material and extracted DNA, an intermediate precision (coefficients of variation below 25%) between three consecutive experiments was noted. Furthermore, discrepancies in estimated mean DNA copy number concentrations between laboratories were less than twofold, with DNA extraction as the main source of variability. These data demonstrate that dPCR offers a repeatable and reproducible method for quantification of viral DNA, and due to its satisfactory performance should be considered as candidate for reference methods for implementation in a metrological framework.

  10. Toxic megacolon and human Cytomegalovirus in a series of severe ulcerative colitis patients.

    PubMed

    Criscuoli, Valeria; Rizzuto, Maria Rosa; Gallo, Elena; Orlando, Ambrogio; Cottone, Mario

    2015-05-01

    Human Cytomegalovirus (HCMV) infection has been reported to be a cause of refractory ulcerative colitis (UC). Toxic megacolon (TM) is a rare but severe complication of an acute attack of UC. Aim of this study is to evaluate in a case-control study the association between HCMV and TM. All patients who were admitted at Medicine Department of V. Cervello Hospital in Palermo (tertiary referral center) for a severe UC flare-up complicated by the onset of TM (diameter of the transverse colon>6 cm) between January 1990 and November 2011 were identified through the electronic database. A total of 24 consecutive patients (16 male/8 female) with TM were identified. Each case of TM were individually matched by sex, age, extent of the underlying disease to 24 severe UC controls who did not develop TM. A further non matched control population of 48 severe UC was included. Haematoxilin and eosin stain, immunohistochemical procedure and nested polymerase chain reaction were performed to detect HCMV genes and proteins on rectal biopsies or surgical specimens. Pp65 antigenemia was performed in order to diagnose any possible systemic infection. HCMV frequency was compared between patients with and without TM during follow-up, using Fisher's Exact test. HCMV was detected in histological specimens of 11 patients (46%) with TM compared to 2 (9%) severe UC matched controls (P = 0.0078) and 7 (14%) unmatched controls (p = 0,003). In severe colitis the presence of HCMV is more frequently associated with TM. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Human cytomegalovirus US3 modulates destruction of MHC class I molecules.

    PubMed

    Noriega, Vanessa M; Hesse, Julia; Gardner, Thomas J; Besold, Katrin; Plachter, Bodo; Tortorella, Domenico

    2012-06-01

    Human cytomegalovirus (HCMV), a member of the Herpesviridae family, is proficient at establishing lifelong persistence within the host in part due to immune modulating genes that limit immune recognition. HCMV encodes at least five glycoproteins within its unique short (US) genomic region that interfere with MHC class I antigen presentation, thus hindering viral clearance by cytotoxic T lymphocytes (CTL). Specifically, US3 retains class I within the endoplasmic reticulum (ER), while US2 and US11 induce class I heavy chain destruction. A cooperative effect on class I down-regulation during stable expression of HCMV US2 and US3 has been established. To address the impact of US3 on US11-mediated MHC class I down-regulation, the fate of class I molecules was examined in US3/US11-expressing cells and virus infection studies. Co-expression of US3 and US11 resulted in a decrease of surface expression of class I molecules. However, the class I molecules in US3/US11 cells were mostly retained in the ER with an attenuated rate of proteasome destruction. Analysis of class I levels from virus-infected cells using HCMV variants either expressing US3 or US11 revealed efficient surface class I down-regulation upon expression of both viral proteins. Cells infected with both US3 and US11 expressing viruses demonstrate enhanced retention of MHC class I complexes within the ER. Collectively, the data suggests a paradigm where HCMV-induced surface class I down-regulation occurs by diverse mechanisms dependent on the expression of specific US genes. These results validate the commitment of HCMV to limiting the surface expression of class I levels during infection.

  12. Cytomegalovirus and cancer after kidney transplantation: Role of the human leukocyte antigen system?

    PubMed

    Wong, Germaine; Chakera, Aron; Chapman, Jeremy R; Chadban, Steve C; Pilmore, Helen; Craig, Jonathan C; Lim, Wai H

    2017-02-01

    The role of cytomegalovirus (CMV) in cancer development after transplantation remains uncertain. We aimed to determine the association between donor and recipient CMV serological status and the risk of cancer development after kidney transplantation. Using data from the Australian and New Zealand Dialysis and Transplant (ANZDATA) Registry, we assessed the association between CMV donor/recipient (D/R) serological status and the risk of solid organ cancers in primary adult deceased-donor kidney transplant patients between 1990 and 2012. Of 8140 recipients, a total of 895 (11%) recipients developed incident cancers during a follow-up time of 51 555 person-years. Human leukocyte antigen (HLA) mismatches was an effect modifier between CMV serological status and cancer (P=.03 for interaction). In recipients who have received 0-2 HLA-ABDR mismatched kidneys, the adjusted hazard ratios for cancer incidence among those with CMV D-/R-, CMV D-/R+, and CMV D+/R- were 0.47 (95% confidence interval [CI]: 0.24-0.91), 1.42 (95% CI: 0.97-2.07), and 1.02 (95% CI: 0.67-1.57), respectively compared with the reference of CMV D+/R+. A similar association was not observed in those with >2 HLA-ABDR mismatches. CMV D-/R- status was associated with a reduced risk of cancer in kidney transplant recipients who have received well-matched renal allografts, suggesting a potential role of HLA matching in cancer development. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  13. The eIF4AIII RNA helicase is a critical determinant of human cytomegalovirus replication

    SciTech Connect

    Ziehr, Ben; Lenarcic, Erik; Cecil, Chad; Moorman, Nathaniel J.

    2016-02-15

    Human cytomegalovirus (HCMV) was recently shown to encode a large number of spliced mRNAs. While the nuclear export of unspliced viral transcripts has been extensively studied, the role of host mRNA export factors in HCMV mRNA trafficking remains poorly defined. We found that the eIF4AIII RNA helicase, a component of the exon junction complex, was necessary for efficient virus replication. Depletion of eIF4AIII limited viral DNA accumulation, export of viral mRNAs from the nucleus, and the production of progeny virus. However eIF4AIII was dispensable for the association of viral transcripts with ribosomes. We found that pateamine A, a natural compound that inhibits both eIF4AI/II and eIF4AIII, has potent antiviral activity and inhibits HCMV replication throughout the virus lytic cycle. Our results demonstrate that eIF4AIII is required for efficient HCMV replication, and suggest that eIF4A family helicases may be a new class of targets for the development of host-directed antiviral therapeutics. - Highlights: • The host eIF4AIII RNA helicase is required for efficient HCMV replication. • Depleting eIF4AIII inhibited the nuclear export of HCMV mRNAs. • HCMV mRNAs did not require eIF4AIII to associate with polyribosomes. • The eIF4A family helicases may be new targets for host-directed antiviral drugs.

  14. Human Cytomegalovirus-Infected Glioblastoma Cells Display Stem Cell-Like Phenotypes

    PubMed Central

    Liu, Che; Clark, Paul A.; Kuo, John S.

    2017-01-01

    ABSTRACT Glioblastoma multiforme (GBM) is the most common brain tumor in adults. Human cytomegalovirus (HCMV) genomes are present in GBM tumors, yielding hope that antiviral treatments could prove therapeutic and improve the poor prognosis of GBM patients. We discovered that GBM cells infected in vitro with HCMV display properties of cancer stem cells. HCMV-infected GBM cells grow more slowly than mock-infected controls, demonstrate a higher capacity for self-renewal determined by a sphere formation assay, and display resistance to the chemotherapeutic drug temozolomide. Our data suggest that HCMV, while present in only a minority of the cells within a tumor, could contribute to the pathogenesis of GBMs by promoting or prolonging stem cell-like phenotypes, thereby perpetuating tumors in the face of chemotherapy. Importantly, we show that temozolomide sensitivity is restored by the antiviral drug ganciclovir, indicating a potential mechanism underlying the positive effects observed in GBM patients treated with antiviral therapy. IMPORTANCE A role for HCMV in GBMs remains controversial for several reasons. Some studies find HCMV in GBM tumors, while others do not. Few cells within a GBM may harbor HCMV, making it unclear how the virus could be contributing to the tumor phenotype without infecting every cell. Finally, HCMV does not overtly transform cells in vitro. However, tumors induced by other viruses can be treated with antiviral remedies, and initial results indicate that this may be true for anti-HCMV therapies and GBMs. With such a poor prognosis for GBM patients, any potential new intervention deserves exploration. Our work here describes an evidence-based model for how HCMV could contribute to GBM biology while infecting very few cells and without transforming them. It also illuminates why anti-HCMV treatments may be beneficial to GBM patients. Our observations provide blueprints for future in vitro studies examining how HCMV manipulates stem cell

  15. Protein kinases responsible for the phosphorylation of the nuclear egress core complex of human cytomegalovirus.

    PubMed

    Sonntag, Eric; Milbradt, Jens; Svrlanska, Adriana; Strojan, Hanife; Häge, Sigrun; Kraut, Alexandra; Hesse, Anne-Marie; Amin, Bushra; Sonnewald, Uwe; Couté, Yohann; Marschall, Manfred

    2017-10-01

    Nuclear egress of herpesvirus capsids is mediated by a multi-component nuclear egress complex (NEC) assembled by a heterodimer of two essential viral core egress proteins. In the case of human cytomegalovirus (HCMV), this core NEC is defined by the interaction between the membrane-anchored pUL50 and its nuclear cofactor, pUL53. NEC protein phosphorylation is considered to be an important regulatory step, so this study focused on the respective role of viral and cellular protein kinases. Multiply phosphorylated pUL50 varieties were detected by Western blot and Phos-tag analyses as resulting from both viral and cellular kinase activities. In vitro kinase analyses demonstrated that pUL50 is a substrate of both PKCα and CDK1, while pUL53 can also be moderately phosphorylated by CDK1. The use of kinase inhibitors further illustrated the importance of distinct kinases for core NEC phosphorylation. Importantly, mass spectrometry-based proteomic analyses identified five major and nine minor sites of pUL50 phosphorylation. The functional relevance of core NEC phosphorylation was confirmed by various experimental settings, including kinase knock-down/knock-out and confocal imaging, in which it was found that (i) HCMV core NEC proteins are not phosphorylated solely by viral pUL97, but also by cellular kinases; (ii) both PKC and CDK1 phosphorylation are detectable for pUL50; (iii) no impact of PKC phosphorylation on NEC functionality has been identified so far; (iv) nonetheless, CDK1-specific phosphorylation appears to be required for functional core NEC interaction. In summary, our findings provide the first evidence that the HCMV core NEC is phosphorylated by cellular kinases, and that the complex pattern of NEC phosphorylation has functional relevance.

  16. A viral regulator of glycoprotein complexes contributes to human cytomegalovirus cell tropism.

    PubMed

    Li, Gang; Nguyen, Christopher C; Ryckman, Brent J; Britt, William J; Kamil, Jeremy P

    2015-04-07

    Viral glycoproteins mediate entry of enveloped viruses into cells and thus play crucial roles in infection. In herpesviruses, a complex of two viral glycoproteins, gH and gL (gH/gL), regulates membrane fusion events and influences virion cell tropism. Human cytomegalovirus (HCMV) gH/gL can be incorporated into two different protein complexes: a glycoprotein O (gO)-containing complex known as gH/gL/gO, and a complex containing UL128, UL130, and UL131 known as gH/gL/UL128-131. Variability in the relative abundance of the complexes in the virion envelope correlates with differences in cell tropism exhibited between strains of HCMV. Nonetheless, the mechanisms underlying such variability have remained unclear. We have identified a viral protein encoded by the UL148 ORF (UL148) that influences the ratio of gH/gL/gO to gH/gL/UL128-131 and the cell tropism of HCMV virions. A mutant disrupted for UL148 showed defects in gH/gL/gO maturation and enhanced infectivity for epithelial cells. Accordingly, reintroduction of UL148 into an HCMV strain that lacked the gene resulted in decreased levels of gH/gL/UL128-131 on virions and, correspondingly, decreased infectivity for epithelial cells. UL148 localized to the endoplasmic reticulum, but not to the cytoplasmic sites of virion envelopment. Coimmunoprecipitation results indicated that gH, gL, UL130, and UL131 associate with UL148, but that gO and UL128 do not. Taken together, the findings suggest that UL148 modulates HCMV tropism by regulating the composition of alternative gH/gL complexes.

  17. Sequence requirements for proteolytic processing of glycoprotein B of human cytomegalovirus strain Towne.

    PubMed Central

    Spaete, R R; Saxena, A; Scott, P I; Song, G J; Probert, W S; Britt, W J; Gibson, W; Rasmussen, L; Pachl, C

    1990-01-01

    Truncated versions of the human cytomegalovirus (CMV) strain Towne glycoprotein B (gB) gene were stably expressed in CHO cell lines. The calcium-specific ionophore A23187 inhibited proteolytic cleavage of C-terminal-truncated gB expressed by cell line 67.77. These inhibition studies also showed that the 93-kilodalton cleavage product most likely represents the N-terminal cleavage fragment of gB. The ionophore carboxyl cyanide m-chlorophenyl-hydrazone was used to show that proteolytic cleavage of gB did not occur in the endoplasmic reticulum. Two-dimensional polyacrylamide gel electrophoresis demonstrated that the N- and C-terminal cleavage products of gB remained associated by disulfide linkages after cleavage. Expression studies using constructs in which 80% or all of the N terminus was deleted demonstrated that the N terminus was required for secretion of the gB molecule. The amino acid sequence at the site of cleavage was shown to be critical for cleavage by a cellular protease. Our results indicate that an arginine-to-threonine change at either amino acid 457 or 460, a lysine-to-glutamine change at amino acid 459, or all three substitutions together block gB cleavage. The effect on proteolysis of the arginine-to-threonine amino acid change at residue 457 (position -4 relative to the cleavage site) demonstrated that a basic pair of amino acids at the endoproteolytic processing site is not the only requirement in cis for gB cleavage. Images PMID:2159553

  18. Modeling of Human Cytomegalovirus Maternal-Fetal Transmission in a Novel Decidual Organ Culture ▿

    PubMed Central

    Weisblum, Yiska; Panet, Amos; Zakay-Rones, Zichria; Haimov-Kochman, Ronit; Goldman-Wohl, Debra; Ariel, Ilana; Falk, Haya; Natanson-Yaron, Shira; Goldberg, Miri D.; Gilad, Ronit; Lurain, Nell S.; Greenfield, Caryn; Yagel, Simcha; Wolf, Dana G.

    2011-01-01

    Human cytomegalovirus (HCMV) is the leading cause of congenital infection, associated with severe birth defects and intrauterine growth retardation. The mechanism of HCMV transmission via the maternal-fetal interface is largely unknown, and there are no animal models for HCMV. The initial stages of infection are believed to occur in the maternal decidua. Here we employed a novel decidual organ culture, using both clinically derived and laboratory-derived viral strains, for the ex vivo modeling of HCMV transmission in the maternal-fetal interface. Viral spread in the tissue was demonstrated by the progression of infected-cell foci, with a 1.3- to 2-log increase in HCMV DNA and RNA levels between days 2 and 9 postinfection, the expression of immediate-early and late proteins, the appearance of typical histopathological features of natural infection, and dose-dependent inhibition of infection by ganciclovir and acyclovir. HCMV infected a wide range of cells in the decidua, including invasive cytotrophoblasts, macrophages, and endothelial, decidual, and dendritic cells. Cell-to-cell viral spread was revealed by focal extension of infected-cell clusters, inability to recover infectious extracellular virus, and high relative proportions (88 to 93%) of cell-associated viral DNA. Intriguingly, neutralizing HCMV hyperimmune globulins exhibited inhibitory activity against viral spread in the decidua even when added at 24 h postinfection—providing a mechanistic basis for their clinical use in prenatal prevention. The ex vivo-infected decidual cultures offer unique insight into patterns of viral tropism and spread, defining initial stages of congenital HCMV transmission, and can facilitate evaluation of the effects of new antiviral interventions within the maternal-fetal interface milieu. PMID:21976654

  19. Human Cytomegalovirus Exploits ESCRT Machinery in the Process of Virion Maturation ▿

    PubMed Central

    Tandon, Ritesh; AuCoin, David P.; Mocarski, Edward S.

    2009-01-01

    The endosomal sorting complex required for transport (ESCRT) machinery controls the incorporation of cargo into intraluminal vesicles of multivesicular bodies. This machinery is used during envelopment of many RNA viruses and some DNA viruses, including herpes simplex virus type 1. Other viruses mature independent of ESCRT components, instead relying on the intrinsic behavior of viral matrix and envelope proteins to drive envelopment. Human cytomegalovirus (HCMV) maturation has been reported to proceed independent of ESCRT components (A. Fraile-Ramos et al. Cell. Microbiol. 9:2955-2967, 2007). A virus complementation assay was used to evaluate the role of dominant-negative (DN) form of a key ESCRT ATPase, vacuolar protein sorting-4 (Vps4DN) in HCMV replication. Vps4DN specifically inhibited viral replication, whereas wild-type-Vps4 had no effect. In addition, a DN form of charged multivesicular body protein 1 (CHMP1DN) was found to inhibit HCMV. In contrast, DN tumor susceptibility gene-101 (Tsg101DN) did not impact viral replication despite the presence of a PTAP motif within pp150/ppUL32, an essential tegument protein involved in the last steps of viral maturation and release. Either Vps4DN or CHMP1DN blocked viral replication at a step after the accumulation of late viral proteins, suggesting that both are involved in maturation. Both Vps4A and CHMP1A localized in the vicinity of viral cytoplasmic assembly compartments, sites of viral maturation that develop in CMV-infected cells. Thus, ESCRT machinery is involved in the final steps of HCMV replication. PMID:19640981

  20. Human cytomegalovirus exploits ESCRT machinery in the process of virion maturation.

    PubMed

    Tandon, Ritesh; AuCoin, David P; Mocarski, Edward S

    2009-10-01

    The endosomal sorting complex required for transport (ESCRT) machinery controls the incorporation of cargo into intraluminal vesicles of multivesicular bodies. This machinery is used during envelopment of many RNA viruses and some DNA viruses, including herpes simplex virus type 1. Other viruses mature independent of ESCRT components, instead relying on the intrinsic behavior of viral matrix and envelope proteins to drive envelopment. Human cytomegalovirus (HCMV) maturation has been reported to proceed independent of ESCRT components (A. Fraile-Ramos et al. Cell. Microbiol. 9:2955-2967, 2007). A virus complementation assay was used to evaluate the role of dominant-negative (DN) form of a key ESCRT ATPase, vacuolar protein sorting-4 (Vps4DN) in HCMV replication. Vps4DN specifically inhibited viral replication, whereas wild-type-Vps4 had no effect. In addition, a DN form of charged multivesicular body protein 1 (CHMP1DN) was found to inhibit HCMV. In contrast, DN tumor susceptibility gene-101 (Tsg101DN) did not impact viral replication despite the presence of a PTAP motif within pp150/ppUL32, an essential tegument protein involved in the last steps of viral maturation and release. Either Vps4DN or CHMP1DN blocked viral replication at a step after the accumulation of late viral proteins, suggesting that both are involved in maturation. Both Vps4A and CHMP1A localized in the vicinity of viral cytoplasmic assembly compartments, sites of viral maturation that develop in CMV-infected cells. Thus, ESCRT machinery is involved in the final steps of HCMV replication.

  1. Identification of novel allosteric modulators for the G-protein coupled US28 receptor of human cytomegalovirus.

    PubMed

    Kralj, Ana; Wetzel, Alexander; Mahmoudian, Shohreh; Stamminger, Thomas; Tschammer, Nuska; Heinrich, Markus R

    2011-09-15

    The highly constitutively active G-protein coupled receptor US28 of human cytomegalovirus (HCMV) is an interesting pharmacological target because of its implication on viral dissemination, cardiovascular diseases and tumorigenesis. We found that dihydroisoquinolinone and tetrahydroisoquinoline scaffolds may be promising lead structures for novel US28 allosteric inverse agonists. These scaffolds were rapidly synthesized by radical carboamination reactions followed by non-radical transformations. Our novel US28 allosteric modulators provide valuable scaffolds for further ligand optimization and may be helpful chemical tools to investigate molecular mechanisms of US28 constitutive signaling and its role in pathogenesis.

  2. Human cytomegalovirus latent infection alters the expression of cellular and viral microRNA.

    PubMed

    Fu, Miao; Gao, Yan; Zhou, Qiuju; Zhang, Qi; Peng, Ying; Tian, Kegang; Wang, Jinhua; Zheng, Xiaoqun

    2014-02-25

    MicroRNAs (miRNAs) play important roles in regulating gene expression of plants, animals and viruses. Comprehensive characterization of host and viral miRNA will help uncover the molecular mechanisms that underlie the progression of human cytomegalovirus (HCMV) latent infection. To investigate the miRNA expression profile of HCMV and host cells during latent infection, we performed deep-sequencing analysis of the small RNAs isolated from HCMV-infected and mock-infected human monocytic leukemia cell line, THP-1. We established a HCMV latent infection cell model using the THP-1 cells. High-throughput sequencing technology was used to sequence small RNA libraries of the HCMV-infected and mock-infected THP-1 and to investigate their small RNA transcriptomes. We found eight miRNAs including miR-US25-1, miR-US25-2-5p and miR-UL112 that were expressed by HCMV during latent infection. The expressions of the host miRNAs were also affected by HCMV latent infection. At least 49 cellular miRNAs were differentially expressed: 39 were up-regulated and 10 were down-regulated upon HCMV latent infection. The expression of the human miRNA hsa-miR-124-3p was significantly up-regulated in the HCMV latent infection library. In addition, we found 14 cellular novel miRNAs in the HCMV-infected and mock-infected THP-1 libraries. Functional annotation of the target genes of the differentially expressed miRNAs suggested that the majority of the genes are involved in melanogenesis, pathways in cancer, endocytosis and wnt signaling pathway. The small RNA transcriptomes obtained in this study demonstrate the usefulness of the deep-sequencing combined with bioinformatics approach in understanding of the expression and function of host and viral small RNAs in HCMV latent infection. This approach can also be applied to the study of other kinds of viruses. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. Transient Oral Human Cytomegalovirus Infections Indicate Inefficient Viral Spread from Very Few Initially Infected Cells.

    PubMed

    Mayer, Bryan T; Krantz, Elizabeth M; Swan, David; Ferrenberg, James; Simmons, Karen; Selke, Stacy; Huang, Meei-Li; Casper, Corey; Corey, Lawrence; Wald, Anna; Schiffer, Joshua T; Gantt, Soren

    2017-06-15

    Cytomegalovirus (CMV) is acquired by the oral route in children, and primary infection is associated with abundant mucosal replication, as well as the establishment of latency in myeloid cells that results in lifelong infection. The efficiency of primary CMV infection in humans following oral exposure, however, is unknown. We consistently detected self-limited, low-level oral CMV shedding events, which we termed transient CMV infections, in a prospective birth cohort of 30 highly exposed CMV-uninfected infants. We estimated the likelihood of transient oral CMV infections by comparing their observed frequency to that of established primary infections, characterized by persistent high-level shedding, viremia, and seroconversion. We developed mathematical models of viral dynamics upon initial oral CMV infection and validated them using clinical shedding data. Transient infections comprised 76 to 88% of oral CMV shedding events. For this high percentage of transient infections to occur, we identified two mathematical prerequisites: a very small number of initially infected oral cells (1 to 4) and low viral infectivity (<1.5 new cells infected/cell). These observations indicate that oral CMV infection in infants typically begins with a single virus that spreads inefficiently to neighboring cells. Thus, although the incidence of CMV infection is high during infancy, our data provide a mechanistic framework to explain why multiple CMV exposures are typically required before infection is successfully established. These findings imply that a sufficiently primed immune response could prevent CMV from establishing latent infection in humans and support the achievability of a prophylactic CMV vaccine.IMPORTANCE CMV infects the majority of the world's population and is a major cause of birth defects. Developing a vaccine to prevent CMV infection would be extremely valuable but would be facilitated by a better understanding of how natural human CMV infection is acquired. We

  4. Immediate-early gene region of human cytomegalovirus trans-activates the promoter of human immunodeficiency virus

    SciTech Connect

    Davis, M.G.; Kenney, S.C.; Kamine, J.; Pagano, J.S.; Huang, E.S.

    1987-12-01

    Almost all homosexual patients with acquired immunodeficiency syndrome are also actively infected with human cytomegalovirus (HCMV). The authors have hypothesized that an interaction between HCMV and human immunodeficiency virus (HIV), the agent that causes acquired immunodeficiency syndrome, may exist at a molecular level and contribute to the manifestations of HIV infection. In this report, they demonstrate that the immediate-early gene region of HCMV, in particular immediate-early region 2, trans-activates the expression of the bacterial gene chloramphenicol acetyltransferase that is fused to the HIV long terminal repeat and carried by plasmid pHIV-CAT. The HCMV immediate-early trans-activator increases the level of mRNA from the plamid pHIV-CAT. The sequences of HIV that are responsive to trans-activation by the HDMV immediate-early region are distinct from HIV sequences that are required for response to the HIV tat. The stimulation of HIV gene expression by HDMV gene functions could enhance the consequences of HIV infection in persons with previous or concurrent HCMV infection.

  5. Diagnostic value of amplification of human cytomegalovirus DNA from gastrointestinal biopsies from human immunodeficiency virus-infected patients.

    PubMed Central

    Cotte, L; Drouet, E; Bissuel, F; Denoyel, G A; Trepo, C

    1993-01-01

    In order to assess the value of human cytomegalovirus (HCMV) DNA amplification of gastrointestinal biopsies, we studied 57 human immunodeficiency virus-infected patients with and without gastrointestinal HCMV diseases. After DNA extraction, a 406-bp fragment from the unique short region of the HCMV genome was amplified by 35 cycles of polymerase chain reaction (PCR) and semiquantified from 80 to 80,000 HCMV genomic copies. Among 12 non-AIDS patients, the PCR assay was negative for 11 of 12 duodenal and 8 of 8 colorectal samples. It was also negative for 28 of 31 duodenal and 12 of 15 colorectal samples from 31 AIDS patients without gastrointestinal HCMV diseases. Among 14 AIDS patients with gastrointestinal HCMV diseases, the PCR assay was positive for 12 of 12 patients with HCMV duodenitis and for 13 of 13 patients with HCMV colitis. Results were dichotomized between high and low HCMV-DNA copy numbers. For duodenitis, sensitivity was 92% and specificity was 100%. For colitis, sensitivity was 92% and specificity was 93%. Specificity and sensitivity were not influenced by shedding status for HCMV or by other gastrointestinal infections. HCMV DNA amplification of gastrointestinal biopsies is a sensitive and specific tool for the diagnosis of gastrointestinal HCMV diseases in AIDS patients. Images PMID:8396587

  6. Human cytomegalovirus infection leads to elevated levels of transplant arteriosclerosis in a humanized mouse aortic xenograft model.

    PubMed

    Abele-Ohl, S; Leis, M; Wollin, M; Mahmoudian, S; Hoffmann, J; Müller, R; Heim, C; Spriewald, B M; Weyand, M; Stamminger, T; Ensminger, S M

    2012-07-01

    Recent findings emphasized an important role of human cytomegalovirus (HCMV) infection in the development of transplant arteriosclerosis. Therefore, the aim of this study was to develop a human peripheral blood lymphocyte (hu-PBL)/Rag-2(-/-) γc(-/-) mouse-xenograft-model to investigate both immunological as well as viral effector mechanisms in the progression of transplant arteriosclerosis. For this, sidebranches from the internal mammary artery were recovered during coronary artery bypass graft surgery, tissue-typed and infected with HCMV. Then, size-matched sidebranches were implanted into the infrarenal aorta of Rag-2(-/-) γc(-/-) mice. The animals were reconstituted with human peripheral blood mononuclear cells (PBMCs) 7 days after transplantation. HCMV-infection was confirmed by Taqman-PCR and immunofluorescence analyses. Arterial grafts were analyzed by histology on day 40 after transplantation. PBMC-reconstituted Rag-2(-/-) γc(-/-) animals showed splenic chimerism levels ranging from 1-16% human cells. After reconstitution, Rag-2(-/-) γc(-/-) mice developed human leukocyte infiltrates in their grafts and vascular lesions that were significantly elevated after infection. Cellular infiltration revealed significantly increased ICAM-1 and PDGF-R-β expression after HCMV-infection of the graft. Arterial grafts from unreconstituted Rag-2(-/-) γc(-/-) recipients showed no vascular lesions. These data demonstrate a causative relationship between HCMV-infection as an isolated risk factor and the development of transplant-arteriosclerosis in a humanized mouse arterial-transplant-model possibly by elevated ICAM-1 and PDGF-R-β expression.

  7. Human Cytomegalovirus Vaccine Based on the Envelope gH/gL Pentamer Complex

    PubMed Central

    Martinez, Joy; Campo, John; Johnson, Erica; Flechsig, Christin; Newell, Maegan; Tran, Elaine; Ortiz, Jose; La Rosa, Corinna; Herrmann, Andreas; Longmate, Jeff; Chakraborty, Rana; Barry, Peter A.; Diamond, Don J.

    2014-01-01

    Human Cytomegalovirus (HCMV) utilizes two different pathways for host cell entry. HCMV entry into fibroblasts requires glycoproteins gB and gH/gL, whereas HCMV entry into epithelial and endothelial cells (EC) requires an additional complex composed of gH, gL, UL128, UL130, and UL131A, referred to as the gH/gL-pentamer complex (gH/gL-PC). While there are no established correlates of protection against HCMV, antibodies are thought to be important in controlling infection. Neutralizing antibodies (NAb) that prevent gH/gL-PC mediated entry into EC are candidates to be assessed for in vivo protective function. However, these potent NAb are predominantly directed against conformational epitopes derived from the assembled gH/gL-PC. To address these concerns, we constructed Modified Vaccinia Ankara (MVA) viruses co-expressing all five gH/gL-PC subunits (MVA-gH/gL-PC), subsets of gH/gL-PC subunits (gH/gL or UL128/UL130/UL131A), or the gB subunit from HCMV strain TB40/E. We provide evidence for cell surface expression and assembly of complexes expressing full-length gH or gB, or their secretion when the corresponding transmembrane domains are deleted. Mice or rhesus macaques (RM) were vaccinated three times with MVA recombinants and serum NAb titers that prevented 50% infection of human EC or fibroblasts by HCMV TB40/E were determined. NAb responses induced by MVA-gH/gL-PC blocked HCMV infection of EC with potencies that were two orders of magnitude greater than those induced by MVA expressing gH/gL, UL128-UL131A, or gB. In addition, MVA-gH/gL-PC induced NAb responses that were durable and efficacious to prevent HCMV infection of Hofbauer macrophages, a fetal-derived cell localized within the placenta. NAb were also detectable in saliva of vaccinated RM and reached serum peak levels comparable to NAb titers found in HCMV hyperimmune globulins. This vaccine based on a translational poxvirus platform co-delivers all five HCMV gH/gL-PC subunits to achieve robust humoral

  8. Xenotransplantation and porcine cytomegalovirus.

    PubMed

    Denner, Joachim

    2015-01-01

    Porcine microorganisms may be transmitted to the human recipient when xenotransplantation with pig cells, tissues, and organs will be performed. Most of such microorganisms can be eliminated from the donor pig by specified or designated pathogen-free production of the animals. As human cytomegalovirus causes severe transplant rejection in allotransplantation, considerable concern is warranted on the potential pathogenicity of porcine cytomegalovirus (PCMV) in the setting of xenotransplantation. On the other hand, despite having a similar name, PCMV is different from HCMV. The impact of PCMV infection on pigs is known; however, the influence of PCMV on the human transplant recipient is unclear. However, first transplantations of pig organs infected with PCMV into non-human primates were associated with a significant reduction of the survival time of the transplants. Sensitive detection methods and strategies for elimination of PCMV from donor herds are required.

  9. Cytomegalovirus Infections

    MedlinePlus

    Cytomegalovirus (CMV) is a virus found around the world. It is related to the viruses that cause chickenpox and infectious mononucleosis (mono). Between 50 percent ... in the United States have had a CMV infection by age 40. Once CMV is in a ...

  10. Detection of human cytomegalovirus in dental plaque from individual periodontal sites by real-time polymerase chain reaction.

    PubMed

    Combs, Darrin R; Reilly, Elizabeth A; Dawson, Dolphus R; Avdiushko, Sergei A; Danaher, Robert J; Miller, Craig S

    2008-12-01

    The aim was to evaluate three primer-probe sets and real-time polymerase chain reaction (PCR) for the detection of human cytomegalovirus (HCMV) in dental plaque from individual periodontal sites. Fifty subgingival plaque specimens from 13 healthy subjects (on average at least 2 healthy and 2 periodontal disease sites per subject) and 50 saliva specimens from 24 subjects, including 16 controls, were assessed using 3 primer-probe sets (polymerase [POL], glycoprotein B [gB], and US14) and real-time PCR. Kappa statistics were performed to measure agreement between the primer-probe sets. There was excellent agreement between the gB and POL primers in the detection of HCMV (kappa statistic = 0.85 [95% confidence interval 0.71-0.99]), yielding a prevalence of 4% (2 out of 50) at individual periodontal disease sites and a similar rate of 8.8% (3 out of 34) in saliva. Human cytomegalovirus was infrequently detected in dental plaque. Of 3 primer-probe sets evaluated, those targeting the POL and gB genes were more accurate in the detection of HCMV than that targeting US14.

  11. Modulation of Homology-Directed Repair in T98G Glioblastoma Cells Due to Interactions between Wildtype p53, Rad51 and HCMV IE1-72

    PubMed Central

    Kulkarni, Amit S.; Fortunato, Elizabeth A.

    2014-01-01

    Human cytomegalovirus (HCMV) is a ubiquitous pathogen capable of causing life threatening consequences in neonates and immune-compromised individuals. HCMV inflicts site-specific double strand breaks (DSBs) in the cellular genome. DNA damage infliction raises the corollary question of virus modulation of DNA repair. We recently reported HDR was stimulated in wt human foreskin fibroblasts (HFFs) during fully permissive infection or expression of the HCMV protein IE1-72 (IE72). These studies have been extended into semi-permissive T98G glioblastoma cells. T98Gs encode a mutant p53, which may contribute to their high baseline rate of HDR. We fully expected HCMV infection to increase HDR in T98Gs, similar to its effects in HFFs. Surprisingly in T98Gs HCMV infection, or sole expression of IE72, decreased HDR by two-fold. Transient expression of wt p53 in T98Gs also reduced HDR by two-fold. Dual transient expression of wt p53 and IE72 restored high baseline HDR levels. GST pulldown experiments revealed that both IE72 and wt p53 bound the important HDR protein, Rad51. We conclude that the expression of certain HCMV proteins can modulate HDR in an infected cell, dependent upon p53 status. We propose a model of the protein interactions explaining this behavior. PMID:24576846

  12. Human cytomegalovirus UL49 encodes an early, virion-associated protein essential for virus growth in human foreskin fibroblasts.

    PubMed

    Zhu, Feng; Yuan, Jian; Li, Hong-Jian; Zeng, Zhi-Feng; Luo, Zhi-Wen; Li, Shi-Qian; He, Chi-Qiang; Jia, Xue-Fang; Zhang, Xin; Zuo, Hui; Liu, Yi-Min; Chang, Martin; Li, Yue-Qin; Zhou, Tian-Hong

    2016-05-01

    Despite recent results of deletion experiments showing that open reading frame (ORF) UL49 of human cytomegalovirus (HCMV) is essential, the expression, function and functional location of its encoded protein remain unknown. We generated an antibody specific for pUL49 to investigate the protein product encoded by the UL49 ORF and identified its function in HCMV-infected host foreskin fibroblasts. A bacterial artificial chromosome (BAC) of HCMV strain Towne (pRV-Towne) and the UL49-deleted mutant pRV-delUL49Towne were used to observe virus growth by plaque assay. Using a UL49-protein-binding antibody, we located pUL49 in the fibroblast cytoplasm. pUL49 exhibited expression kinetics resembling those of the class β-2 proteins and was detected in the virion tegument. Following deletion of UL49 ORF, the virus failed to replicate, but it could be recovered by addition of pUL49 from pCDNA3.1 (+)-UL49. Our findings indicate that UL49 ORF is essential for HCMV replication in host foreskin fibroblasts.

  13. Human Cytomegalovirus Can Procure Deoxyribonucleotides for Viral DNA Replication in the Absence of Retinoblastoma Protein Phosphorylation

    PubMed Central

    Kuny, Chad V.

    2016-01-01

    ABSTRACT Viral DNA replication requires deoxyribonucleotide triphosphates (dNTPs). These molecules, which are found at low levels in noncycling cells, are generated either by salvage pathways or through de novo synthesis. Nucleotide synthesis utilizes the activity of a series of nucleotide-biosynthetic enzymes (NBEs) whose expression is repressed in noncycling cells by complexes between the E2F transcription factors and the retinoblastoma (Rb) tumor suppressor. Rb-E2F complexes are dissociated and NBE expression is activated during cell cycle transit by cyclin-dependent kinase (Cdk)-mediated Rb phosphorylation. The DNA virus human cytomegalovirus (HCMV) encodes a viral Cdk (v-Cdk) (the UL97 protein) that phosphorylates Rb, induces the expression of cellular NBEs, and is required for efficient viral DNA synthesis. A long-held hypothesis proposed that viral proteins with Rb-inactivating activities functionally similar to those of UL97 facilitated viral DNA replication in part by inducing the de novo production of dNTPs. However, we found that dNTPs were limiting even in cells infected with wild-type HCMV in which UL97 is expressed and Rb is phosphorylated. Furthermore, we revealed that both de novo and salvage pathway enzymes contribute to viral DNA replication during HCMV infection and that Rb phosphorylation by cellular Cdks does not correct the viral DNA replication defect observed in cells infected with a UL97-deficient virus. We conclude that HCMV can obtain dNTPs in the absence of Rb phosphorylation and that UL97 can contribute to the efficiency of DNA replication in an Rb phosphorylation-independent manner. IMPORTANCE Transforming viral oncoproteins, such as adenovirus E1A and papillomavirus E7, inactivate Rb. The standard hypothesis for how Rb inactivation facilitates infection with these viruses is that it is through an increase in the enzymes required for DNA synthesis, which include nucleotide-biosynthetic enzymes. However, HCMV UL97, which functionally

  14. Severe Symptomatic Primary Human Cytomegalovirus Infection despite Effective Innate and Adaptive Immune Responses.

    PubMed

    Riou, Raphaëlle; Bressollette-Bodin, Céline; Boutoille, David; Gagne, Katia; Rodallec, Audrey; Lefebvre, Maeva; Raffi, François; Senitzer, David; Imbert-Marcille, Berthe-Marie; Retière, Christelle

    2017-03-01

    Primary human cytomegalovirus (HCMV) infection usually goes unnoticed, causing mild or no symptoms in immunocompetent individuals. However, some rare severe clinical cases have been reported without investigation of host immune responses or viral virulence. In the present study, we investigate for the first time phenotypic and functional features, together with gene expression profiles in immunocompetent adults experiencing a severe primary HCMV infection. Twenty primary HCMV-infected patients (PHIP) were enrolled, as well as 26 HCMV-seronegative and 39 HCMV-seropositive healthy controls. PHIP had extensive lymphocytosis marked by massive expansion of natural killer (NK) and T cell compartments. Interestingly, PHIP mounted efficient innate and adaptive immune responses with a deep HCMV imprint, revealed mainly by the expansion of NKG2C(+) NK cells, CD16(+) Vδ2(-) γδ T cells, and conventional HCMV-specific CD8(+) T cells. The main effector lymphocytes were activated and displayed an early immune phenotype that developed toward a more mature differentiated status. We suggest that both massive lymphocytosis and excessive lymphocyte activation could contribute to massive cytokine production, known to mediate tissue damage observed in PHIP. Taken together, these findings bring new insights into the comprehensive understanding of immune mechanisms involved during primary HCMV infection in immunocompetent individuals.IMPORTANCE HCMV-specific immune responses have been extensively documented in immunocompromised patients and during in utero acquisition. While it usually goes unnoticed, some rare severe clinical cases of primary HCMV infection have been reported in immunocompetent patients. However, host immune responses or HCMV virulence in these patients has not so far been investigated. In the present study, we show massive expansion of NK and T cell compartments during the symptomatic stage of acute HCMV infection. The patients mounted efficient innate and adaptive

  15. Differences in Growth Properties among Two Human Cytomegalovirus Glycoprotein O Genotypes.

    PubMed

    Kalser, Julia; Adler, Barbara; Mach, Michael; Kropff, Barbara; Puchhammer-Stöckl, Elisabeth; Görzer, Irene

    2017-01-01

    Glycoprotein O (gO) of the human cytomegalovirus (HCMV) is the critical subunit of the envelope trimer gH/gL/gO as it interacts with platelet-derived growth factor alpha receptor upon fibroblast entry, and triggers gB-mediated fusion for fibroblast and epithelial cell infection. Eight genotypes (GT) of the highly polymorphic gO gene are described, yet it is unclear whether the distinct GTs differ in their function. Thus, we aimed to elucidate potential functional differences between two highly diverse gO GTs in an otherwise genomically identical HCMV strain. Therefore, resident gO GT1c sequence of strain TB40-BAC4-luc was entirely replaced by gO GT4 of strain Towne and both, GT1c and GT4 viruses, were investigated for their growth properties in fibroblasts and epithelial cells. In addition, two conserved gO cysteines involved in gH/gL/gO stabilization were mutated to serine either in GT1c (C218S and C343S) or GT4 (C216S and C336S) and their effects on cell-free infectivity were assessed. GT4 viruses displayed a significantly enhanced epithelial cell tropism and this resulted in higher virus release upon replication in epithelial cells when compared to GT1c viruses. Further, when the two cysteines were individually mutated in gO GT1c no impairment in cell-free infectivity was observed. This, however, was in sharp contrast to gO GT4, in which both of the corresponding cysteine mutations led to a substantial reduction in cell-free infectivity which was even more pronounced upon mutation of GT4-C336 than of GT4-C216. In conclusion, these findings provide evidence that the two highly diverse gO genotypes, GT1c and GT4, differ in their functional properties as revealed by their different infection capacities for epithelial cells and by their different responsiveness to mutation of strictly conserved cysteine residues. Thus, it is likely that the gO heterogeneity influences cell-free infectivity of HCMV also in vivo which may have important implications for virus host

  16. [Viral infection of herpes simplex, Epstein-Barr, varicela zoster, human papilloma, cytomegalovirus, or adenovirus are not related to sinonasal adenocarcinomas].

    PubMed

    Pérez Escuredo, Jhudit; Llorente, José Luis; Melón, Santiago; de Oña, María; García Martínez, Jorge; Alvarez Marcos, César; Hermsen, Mario

    2007-01-01

    Several types of virus have been implicated in the development of head and neck tumors. However, until now sinonasal adenocarcinomas (ACN) have not been studied. The aim of this study is to screen a series of ACN for the presence of a number of viruses known to play a role in cancer. Viral DNA sequences of herpes simplex virus, Epstein-Barr, varicela zoster, human papilloma, cytomegalovirus, and adenovirus were analysed by PCR in 37 primary ACN. Three tumors (8.1%) were positive for Epstein-Barr virus and 1 case (2.7%) for cytomegalovirus. Viral infections do not seem to play a role in the etiology of ACN.

  17. Human Cytomegalovirus UL97 Kinase Activity Is Required for the Hyperphosphorylation of Retinoblastoma Protein and Inhibits the Formation of Nuclear Aggresomes

    SciTech Connect

    Prichard, Mark N.; Sztul, Elizabeth; Daily, Shannon L.; Perry, Amie L.; Frederick, Samuel L.; Gill, Rachel B.; Hartline, Caroll B.; Streblow, Daniel N.; Varnum, Susan M.; Smith, Richard D.; Kern, Earl R.

    2008-05-01

    Cells infected with human cytomegalovirus in the absence of UL97 kinase activity produce large nuclear aggregates that sequester considerable quantities of viral proteins. A transient expression assay suggested that pp71 and IE1 were also involved in this process, and this suggestion was significant, since both proteins have been reported to interact with components of promyelocytic leukemia (PML) bodies (ND10) and also interact functionally with retinoblastoma pocket proteins (RB). PML bodies have been linked to the formation of nuclear aggresomes, and colocalization studies suggested that viral proteins were recruited to these structures and that UL97 kinase activity inhibited their formation. Proteins associated with PML bodies were examined by Western blot analysis, and pUL97 appeared to specifically affect the phosphorylation of RB in a kinasedependent manner. Three consensus RB binding motifs were identified in the UL97 kinase, and recombinant viruses were constructed in which each was mutated to assess a potential role in the phosphorylation of RB and the inhibition of nuclear aggresome formation. The mutation of either the conserved LxCxE RB binding moti for the lysine required for kinase activity impaired the ability of the virus to stabilize and phosphorylate RB. We concluded from these studies that both UL97 kinase activity and the LxCxE RB binding motif are required for the phosphorylation and stabilization of RB in infected cells and that this effect can be antagonized by the antiviral drug maribavir. These data also suggest a potential link between RB function and the formation of aggresomes.

  18. RNA interference-mediated targeting of human cytomegalovirus immediate-early or early gene products inhibits viral replication with differential effects on cellular functions.

    PubMed

    Xiaofei, E; Stadler, Bradford M; Debatis, Michelle; Wang, Shixia; Lu, Shan; Kowalik, Timothy F

    2012-05-01

    Viral drug toxicity, resistance, and an increasing immunosuppressed population warrant continued research into new avenues for limiting diseases associated with human cytomegalovirus (HCMV). In this study, a small interfering RNA (siRNA), siX3, was designed to target coding sequences within shared exon 3 of UL123 and UL122 transcripts encoding IE1 and IE2 immediate-early proteins of HCMV. Pretreatment of cells with siX3 reduced the levels of viral protein expression, DNA replication, and progeny virus production compared to control siRNA. Two siRNAs against UL54 and overlapping transcripts (UL55-57) were compared to siX3 in HCMV infection and were also found to be effective at inhibiting HCMV replication. Further investigation into the effects of the siRNAs on viral replication showed that pretreatment with each of the siRNAs resulted in an inhibition in the formation of mature replication compartments. The ability of these siRNAs to prevent or reduce certain cytopathic effects associated with HCMV infection was also examined. Infected cells pretreated with siX3, but not siUL54, retained promyelocytic leukemia (PML) protein in cellular PML bodies, an essential component of this host intrinsic antiviral defense. DNA damage response proteins, which are localized in nuclear viral replication compartments, were reduced in the siX3- and siUL54-treated cells. siX3, but not siUL54, prevented DNA damage response signaling early after infection. Therapeutic efficacy was demonstrated by treating cells with siRNAs after HCMV replication had commenced. Together, these findings suggest that siRNAs targeting exon 3 of the major IE genes or the UL54-57 transcripts be further studied for their potential development into anti-HCMV therapeutics.

  19. cGAS Senses Human Cytomegalovirus and Induces Type I Interferon Responses in Human Monocyte-Derived Cells

    PubMed Central

    Paijo, Jennifer; Döring, Marius; Spanier, Julia; Grabski, Elena; Nooruzzaman, Mohammed; Schmidt, Tobias; Witte, Gregor; Messerle, Martin; Hornung, Veit; Kaever, Volkhard; Kalinke, Ulrich

    2016-01-01

    Human cytomegalovirus (HCMV) infections of healthy individuals are mostly unnoticed and result in viral latency. However, HCMV can also cause devastating disease, e.g., upon reactivation in immunocompromised patients. Yet, little is known about human immune cell sensing of DNA-encoded HCMV. Recent studies indicated that during viral infection the cyclic GMP/AMP synthase (cGAS) senses cytosolic DNA and catalyzes formation of the cyclic di-nucleotide cGAMP, which triggers stimulator of interferon genes (STING) and thus induces antiviral type I interferon (IFN-I) responses. We found that plasmacytoid dendritic cells (pDC) as well as monocyte-derived DC and macrophages constitutively expressed cGAS and STING. HCMV infection further induced cGAS, whereas STING expression was only moderately affected. Although pDC expressed particularly high levels of cGAS, and the cGAS/STING axis was functional down-stream of STING, as indicated by IFN-I induction upon synthetic cGAMP treatment, pDC were not susceptible to HCMV infection and mounted IFN-I responses in a TLR9-dependent manner. Conversely, HCMV infected monocyte-derived cells synthesized abundant cGAMP levels that preceded IFN-I production and that correlated with the extent of infection. CRISPR/Cas9- or siRNA-mediated cGAS ablation in monocytic THP-1 cells and primary monocyte-derived cells, respectively, impeded induction of IFN-I responses following HCMV infection. Thus, cGAS is a key sensor of HCMV for IFN-I induction in primary human monocyte-derived DC and macrophages. PMID:27058035

  20. High-Throughput Analysis of Human Cytomegalovirus Genome Diversity Highlights the Widespread Occurrence of Gene-Disrupting Mutations and Pervasive Recombination

    PubMed Central

    Thys, Kim; Mbong Ngwese, Mirabeau; Van Damme, Ellen; Dvorak, Jan; Van Loock, Marnix; Li, Guangdi; Tachezy, Ruth; Busson, Laurent; Aerssens, Jeroen; Van Ranst, Marc

    2015-01-01

    ABSTRACT Human cytomegalovirus is a widespread pathogen of major medical importance. It causes significant morbidity and mortality in immunocompromised individuals, and congenital infections can result in severe disabilities or stillbirth. Development of a vaccine is prioritized, but no candidate is close to release. Although correlations of viral genetic variability with pathogenicity are suspected, knowledge about the strain diversity of the 235-kb genome is still limited. In this study, 96 full-length human cytomegalovirus genomes from clinical isolates were characterized, quadrupling the amount of information available for full-genome analysis. These data provide the first high-resolution map of human cytomegalovirus interhost diversity and evolution. We show that cytomegalovirus is significantly more divergent than all other human herpesviruses and highlight hot spots of diversity in the genome. Importantly, 75% of strains are not genetically intact but contain disruptive mutations in a diverse set of 26 genes, including the immunomodulatory genes UL40 and UL111A. These mutants are independent of culture passage artifacts and circulate in natural populations. Pervasive recombination, which is linked to the widespread occurrence of multiple infections, was found throughout the genome. The recombination density was significantly higher than those of other human herpesviruses and correlated with strain diversity. While the overall effects of strong purifying selection on virus evolution are apparent, evidence of diversifying selection was found in several genes encoding proteins that interact with the host immune system, including UL18, UL40, UL142, and UL147. These residues may present phylogenetic signatures of past and ongoing virus-host interactions. IMPORTANCE Human cytomegalovirus has the largest genome of all viruses that infect humans. Currently, there is a great interest in establishing associations between genetic variants and strain pathogenicity of

  1. High-throughput analysis of human cytomegalovirus genome diversity highlights the widespread occurrence of gene-disrupting mutations and pervasive recombination.

    PubMed

    Sijmons, Steven; Thys, Kim; Mbong Ngwese, Mirabeau; Van Damme, Ellen; Dvorak, Jan; Van Loock, Marnix; Li, Guangdi; Tachezy, Ruth; Busson, Laurent; Aerssens, Jeroen; Van Ranst, Marc; Maes, Piet

    2015-05-13

    Human cytomegalovirus is a widespread pathogen of major medical importance. It causes significant morbidity and mortality in the immunocompromised and congenital infections can result in severe disabilities or stillbirth. Development of a vaccine is prioritized, but no candidate is close to release. Although correlations of viral genetic variability with pathogenicity are suspected, knowledge about strain diversity of the 235kb genome is still limited. In this study, 96 full-length human cytomegalovirus genomes from clinical isolates were characterized, quadrupling the available information for full-genome analysis. These data provide the first high-resolution map of human cytomegalovirus interhost diversity and evolution. We show that cytomegalovirus is significantly more divergent than all other human herpesviruses and highlight hotspots of diversity in the genome. Importantly, 75% of strains are not genetically intact, but contain disruptive mutations in a diverse set of 26 genes, including immunomodulative genes UL40 and UL111A. These mutants are independent from culture passaging artifacts and circulate in natural populations. Pervasive recombination, which is linked to the widespread occurrence of multiple infections, was found throughout the genome. Recombination density was significantly higher than in other human herpesviruses and correlated with strain diversity. While the overall effects of strong purifying selection on virus evolution are apparent, evidence of diversifying selection was found in several genes encoding proteins that interact with the host immune system, including UL18, UL40, UL142 and UL147. These residues may present phylogenetic signatures of past and ongoing virus-host interactions. Human cytomegalovirus has the largest genome of all viruses that infect humans. Currently, there is a great interest in establishing associations between genetic variants and strain pathogenicity of this herpesvirus. Since the number of publicly available

  2. Inhibition of human cytomegalovirus IE gene expression by dihydro-beta-agarofuran sesquiterpenes isolated from Euonymus species.

    PubMed

    Pusztai, Rozália; Hohmann, Judit; Rédei, Dora; Engi, Helga; Molnár, Joseph

    2008-01-01

    The development of strategies intended to inhibit human cytomegalovirus (HCMV) immediate-early (IE) antigen expression is an important goal in research designed to prevent and treat certain forms of cancer. The aim of this study was to identify potent IE antigen-targeting natural compounds as antitumor promoters in an in vitro model of tumor promotion. Nineteen dihydro-beta-agarofuran sesquiterpenes isolated from Euonymus species were evaluated for their ability to inhibit HCMV IE antigen expression in human lung adenocarcinoma (A549) cells. Five esters of penta- and hexahydroxydihydro-beta-agarofuran proved to be active components in these Euonymus species, inhibiting the IE antigen expression of HCMV. The highest activity was displayed by 2beta,6alpha,15-triacetoxy1beta-benzoyloxy-9alpha-nicotinoyloxydihydro-beta-agarofuran. These effective compounds may be regarded as prototypes of antitumor promoters, as secondary chemopreventive agents which can modify or halt tumor promotion in general.

  3. Quantitative analysis of human herpesvirus-6 and human cytomegalovirus in blood and saliva from patients with acute leukemia.

    PubMed

    Nefzi, Faten; Ben Salem, Nabil Abid; Khelif, Abderrahim; Feki, Salma; Aouni, Mahjoub; Gautheret-Dejean, Agnès

    2015-03-01

    Human herpesvirus-6 (HHV-6) and human cytomegalovirus (HCMV) DNAs were quantified by real-time PCR assays in blood and saliva obtained from 50 patients with acute leukemia at the time of diagnosis (50 of each matrix), aplasia (65 of each matrix), remission (55 of each matrix), and relapse (20 of each matrix) to evaluate which biological matrix was more suitable to identify a viral reactivation, search for a possible link between HHV-6 and HCMV reactivations, and evaluate the relations between viral loads and count of different leukocyte types in blood. The median HHV-6 loads were 136; 219; 226, and 75 copies/million cells in blood at diagnosis, aplasia, remission and relapse, respectively. The HCMV loads were 193 and 317 copies/million cells in blood at diagnosis and remission. In the saliva samples, the HHV-6 loads were 22,165; 15,238; 30,214, and 17,454 copies/million cells at diagnosis, aplasia, remission, and relapse, respectively. The HCMV loads were 8,991; 1,461; 2,980, and 4,283 copies/million cells at diagnosis, aplasia, remission, and relapse, respectively. The HHV-6 load in the blood was correlated to the counts of polymorphonuclear leukocytes (R(2)  = 0.5; P < 0.0001) and lymphocytes (R(2)  = 0.4; P = 0.001) and was not correlated to the monocyte counts (R(2)  = 0.07; P = 0.7). Saliva appears to be a more sensitive biological matrix than whole blood in the detection of HHV-6 or HCMV reactivations. The HHV-6 and HCMV reactivations were linked only in saliva.

  4. Genetic Stability of Bacterial Artificial Chromosome-Derived Human Cytomegalovirus during Culture In Vitro.

    PubMed

    Murrell, Isa; Wilkie, Gavin S; Davison, Andrew J; Statkute, Evelina; Fielding, Ceri A; Tomasec, Peter; Wilkinson, Gavin W G; Stanton, Richard J

    2016-04-01

    Clinical human cytomegalovirus (HCMV) strains invariably mutate when propagatedin vitro Mutations in gene RL13 are selected in all cell types, whereas in fibroblasts mutants in the UL128 locus (UL128L; genes UL128, UL130, and UL131A) are also selected. In addition, sporadic mutations are selected elsewhere in the genome in all cell types. We sought to investigate conditions under which HCMV can be propagated without incurring genetic defects. Bacterial artificial chromosomes (BACs) provide a stable, genetically defined source of viral genome. Viruses were generated from BACs containing the genomes of strains TR, TB40, FIX, and Merlin, as well as from Merlin-BAC recombinants containing variant nucleotides in UL128L from TB40-BAC4 or FIX-BAC. Propagation of viruses derived from TR-BAC, TB40-BAC4, and FIX-BAC in either fibroblast or epithelial cells was associated with the generation of defects around the prokaryotic vector, which is retained in the unique short (US) region of viruses. This was not observed for Merlin-BAC, from which the vector is excised in derived viruses; however, propagation in epithelial cells was consistently associated with mutations in the unique longb' (UL/b') region, all impacting on gene UL141. Viruses derived from Merlin-BAC in fibroblasts had mutations in UL128L, but mutations occurred less frequently with recombinants containing UL128L nucleotides from TB40-BAC4 or FIX-BAC. Viruses derived from a Merlin-BAC derivative in which RL13 and UL128L were either mutated or repressed were remarkably stable in fibroblasts. Thus, HCMV containing a wild-type gene complement can be generatedin vitroby deriving virus from a self-excising BAC in fibroblasts and repressing RL13 and UL128L. Researchers should aim to study viruses that accurately represent the causative agents of disease. This is problematic for HCMV because clinical strains mutate rapidly when propagatedin vitro, becoming less cell associated, altered in tropism, more susceptible to

  5. Use of recombination-mediated genetic engineering for construction of rescue human cytomegalovirus bacterial artificial chromosome clones.

    PubMed

    Dulal, Kalpana; Silver, Benjamin; Zhu, Hua

    2012-01-01

    Bacterial artificial chromosome (BAC) technology has contributed immensely to manipulation of larger genomes in many organisms including large DNA viruses like human cytomegalovirus (HCMV). The HCMV BAC clone propagated and maintained inside E. coli allows for accurate recombinant virus generation. Using this system, we have generated a panel of HCMV deletion mutants and their rescue clones. In this paper, we describe the construction of HCMV BAC mutants using a homologous recombination system. A gene capture method, or gap repair cloning, to seize large fragments of DNA from the virus BAC in order to generate rescue viruses, is described in detail. Construction of rescue clones using gap repair cloning is highly efficient and provides a novel use of the homologous recombination-based method in E. coli for molecular cloning, known colloquially as recombineering, when rescuing large BAC deletions. This method of excising large fragments of DNA provides important prospects for in vitro homologous recombination for genetic cloning.

  6. Detection of human cytomegalovirus by slot-blot hybridization assay employing oligo-primed /sup 32/P-labelled probe

    SciTech Connect

    Agha, S.A.; Coleman, J.C.; Selwyn, S.; Mahmound, L.A.; Abd-Elaal, A.M.; Archard, L.C.

    1988-12-01

    A /sup 32/P-labelled Hind III-0 DNA fragment (nine Kilobases; Kb) from human cytomegalovirus AD-169 (HCMV) was used in slot-blot hybridization assay for the detection of HCMV in clinical samples. The results obtained with DNA hybridization assay (DNA HA) were compared with virus isolation using conventional tube cell culture (CTC) and centrifugation vial culture (CVC), immunofluorescence (IF), and complement fixation test (CFT). Of 15 CTC-positive samples, 13 were positive with DNA HA (sensitivity 86.7%). Also, 14 additional samples were DNA HA-positive but CTC-negative. CVC and/or IF confirmed the diagnosis in nine of 14; the remaining five samples were from three patients who showed fourfold rising antibody titre by CFT. Although DNA HA using /sup 32/P-labelled probes is relatively cumbersome and expensive, it is a valuable test for quantitation of viral shedding in patients with HCMV infections who may benefit from antiviral therapy.

  7. Expression of human cytomegalovirus pp150 gene in transgenic Vicia faba L. and immunogenicity of pp150 protein in mice.

    PubMed

    Yan, Hua; Yan, Huishen; Li, Guocai; Gong, Weijuan; Jiao, Hongmei; Chen, Hongju; Ji, Mingchun

    2010-03-01

    The pp150 gene of human cytomegalovirus (HCMV) was transferred into Vicia faba plants by Agrobacterium tumefaciens-mediated transformation. Three of five hygromycin resistant V. faba plants were identified as positive by PCR and dot-blot hybridization. The ELISA results indicated that pp150 protein from three plants of transformed V. faba leaves and seeds made up 0.005-0.015% of the total soluble protein. The results of detection by immunoblot and inhibition of immunofluorescent assay (IFA) showed that pp150 soluble protein had immunity activity. HCMV pp150-specific antibody (IgG, IgA) and IFN-gamma producing T cells were detected in 100% of the mice immunized with pp150 transgenic V. faba seeds by ELISA and intracellular staining and flow cytometry analysis, respectively. The transgenic V. faba plants will provide new material for the development of edible vaccination against HCMV infection.

  8. Increased expression of LDL receptor-related protein 1 during human cytomegalovirus infection reduces virion cholesterol and infectivity

    PubMed Central

    Gudleski-O’Regan, Nicole; Greco, Todd M.; Cristea, Ileana M.; Shenk, Thomas

    2012-01-01

    In response to virus infection, cells can alter protein expression to modify cellular functions and limit viral replication. To examine host protein expression during infection with human cytomegalovirus (HCMV), an enveloped DNA virus, we performed a semi-quantitative, temporal analysis of the cell surface proteome in infected fibroblasts. We determined that resident low density lipoprotein related receptor 1 (LRP1), a plasma membrane receptor that regulates lipid metabolism, is elevated early after HCMV infection, resulting in decreased intracellular cholesterol. siRNA knockdown or antibody-mediated inhibition of LRP1 increased intracellular cholesterol, and concomitantly increased the infectious virus yield. Virions produced under these conditions contained elevated cholesterol, resulting in increased infectivity. Depleting cholesterol from virions reduced their infectivity by blocking fusion of the virion envelope with the cell membrane. Thus, LRP1 restricts HCMV infectivity by controlling the availability of cholesterol for the virion envelope and increased LRP1 expression is likely a defense response to infection. PMID:22817990

  9. Interleukin-2 from Adaptive T Cells Enhances Natural Killer Cell Activity against Human Cytomegalovirus-Infected Macrophages.

    PubMed

    Wu, Zeguang; Frascaroli, Giada; Bayer, Carina; Schmal, Tatjana; Mertens, Thomas

    2015-06-01

    Control of human cytomegalovirus (HCMV) requires a continuous immune surveillance, thus HCMV is the most important viral pathogen in severely immunocompromised individuals. Both innate and adaptive immunity contribute to the control of HCMV. Here, we report that peripheral blood natural killer cells (PBNKs) from HCMV-seropositive donors showed an enhanced activity toward HCMV-infected autologous macrophages. However, this enhanced response was abolished when purified NK cells were applied as effectors. We demonstrate that this enhanced PBNK activity was dependent on the interleukin-2 (IL-2) secretion of CD4(+) T cells when reexposed to the virus. Purified T cells enhanced the activity of purified NK cells in response to HCMV-infected macrophages. This effect could be suppressed by IL-2 blocking. Our findings not only extend the knowledge on the immune surveillance in HCMV-namely, that NK cell-mediated innate immunity can be enhanced by a preexisting T cell antiviral immunity-but also indicate a potential clinical implication for patients at risk for severe HCMV manifestations due to immunosuppressive drugs, which mainly suppress IL-2 production and T cell responsiveness. Human cytomegalovirus (HCMV) is never cleared by the host after primary infection but instead establishes a lifelong latent infection with possible reactivations when the host's immunity becomes suppressed. Both innate immunity and adaptive immunity are important for the control of viral infections. Natural killer (NK) cells are main innate effectors providing a rapid response to virus-infected cells. Virus-specific T cells are the main adaptive effectors that are critical for the control of the latent infection and limitation of reinfection. In this study, we found that IL-2 secreted by adaptive CD4(+) T cells after reexposure to HCMV enhances the activity of NK cells in response to HCMV-infected target cells. This is the first direct evidence that the adaptive T cells can help NK cells to act

  10. Structure of Human Cytomegalovirus UL141 Binding to TRAIL-R2 Reveals Novel, Non-canonical Death Receptor Interactions

    PubMed Central

    Nemčovičová, Ivana; Benedict, Chris A.; Zajonc, Dirk M.

    2013-01-01

    The TRAIL (TNF-related apoptosis inducing ligand) death receptors (DRs) of the tumor necrosis factor receptor superfamily (TNFRSF) can promote apoptosis and regulate antiviral immunity by maintaining immune homeostasis during infection. In turn, human cytomegalovirus (HCMV) expresses immunomodulatory proteins that down-regulate cell surface expression of TNFRSF members as well as poliovirus receptor-related proteins in an effort to inhibit host immune effector pathways that would lead to viral clearance. The UL141 glycoprotein of human cytomegalovirus inhibits host defenses by blocking cell surface expression of TRAIL DRs (by retention in ER) and poliovirus receptor CD155, a nectin-like Ig-fold molecule. Here we show that the immunomodulatory function of HCMV UL141 is associated with its ability to bind diverse proteins, while utilizing at least two distinct binding sites to selectively engage TRAIL DRs or CD155. Binding studies revealed high affinity interaction of UL141 with both TRAIL-R2 and CD155 and low affinity binding to TRAIL-R1. We determined the crystal structure of UL141 bound to TRAIL-R2 at 2.1 Å resolution, which revealed that UL141 forms a homodimer that engages two TRAIL-R2 monomers 90° apart to form a heterotetrameric complex. Our structural and biochemical data reveal that UL141 utilizes its Ig-domain to facilitate non-canonical death receptor interactions while UL141 partially mimics the binding site of TRAIL on TRAIL-R2, which we found to be distinct from that of CD155. Moreover, UL141 also binds to an additional surface patch on TRAIL-R2 that is distinct from the TRAIL binding site. Therefore, the breadth of UL141-mediated effects indicates that HCMV has evolved sophisticated strategies to evade the immune system by modulating multiple effector pathways. PMID:23555243

  11. Structure of human cytomegalovirus UL141 binding to TRAIL-R2 reveals novel, non-canonical death receptor interactions.

    PubMed

    Nemčovičová, Ivana; Benedict, Chris A; Zajonc, Dirk M

    2013-03-01

    The TRAIL (TNF-related apoptosis inducing ligand) death receptors (DRs) of the tumor necrosis factor receptor superfamily (TNFRSF) can promote apoptosis and regulate antiviral immunity by maintaining immune homeostasis during infection. In turn, human cytomegalovirus (HCMV) expresses immunomodulatory proteins that down-regulate cell surface expression of TNFRSF members as well as poliovirus receptor-related proteins in an effort to inhibit host immune effector pathways that would lead to viral clearance. The UL141 glycoprotein of human cytomegalovirus inhibits host defenses by blocking cell surface expression of TRAIL DRs (by retention in ER) and poliovirus receptor CD155, a nectin-like Ig-fold molecule. Here we show that the immunomodulatory function of HCMV UL141 is associated with its ability to bind diverse proteins, while utilizing at least two distinct binding sites to selectively engage TRAIL DRs or CD155. Binding studies revealed high affinity interaction of UL141 with both TRAIL-R2 and CD155 and low affinity binding to TRAIL-R1. We determined the crystal structure of UL141 bound to TRAIL-R2 at 2.1 Å resolution, which revealed that UL141 forms a homodimer that engages two TRAIL-R2 monomers 90° apart to form a heterotetrameric complex. Our structural and biochemical data reveal that UL141 utilizes its Ig-domain to facilitate non-canonical death receptor interactions while UL141 partially mimics the binding site of TRAIL on TRAIL-R2, which we found to be distinct from that of CD155. Moreover, UL141 also binds to an additional surface patch on TRAIL-R2 that is distinct from the TRAIL binding site. Therefore, the breadth of UL141-mediated effects indicates that HCMV has evolved sophisticated strategies to evade the immune system by modulating multiple effector pathways.

  12. Human cytomegalovirus gene UL21a encodes a short-lived cytoplasmic protein and facilitates virus replication in fibroblasts.

    PubMed

    Fehr, Anthony R; Yu, Dong

    2010-01-01

    The human cytomegalovirus (HCMV) gene UL21a was recently annotated by its conservation in chimpanzee cytomegalovirus. Two large-scale mutagenic analyses showed that mutations in overlapping UL21a/UL21 resulted in a severe defect of virus growth in fibroblasts. Here, we characterized UL21a and demonstrated its role in HCMV infection. We mapped a UL21a-specific transcript of approximately 600 bp that was expressed with early kinetics. UL21a encoded pUL21a, a protein of approximately 15 kDa, which was unstable and localized predominantly to the cytoplasm during HCMV infection or when expressed alone. Interestingly, pUL21a was drastically stabilized in the presence of proteasome inhibitor MG132, but its instability was independent of a functional ubiquitin-mediated pathway, suggesting that pUL21a underwent proteasome-dependent, ubiquitin-independent degradation. A UL21a deletion virus was attenuated in primary human newborn foreskin fibroblasts (HFFs) and embryonic lung fibroblasts (MRC-5), whereas a marker-rescued virus and mutant viruses lacking the neighboring or overlapping genes UL20, UL21, or UL21.5-UL23 replicated at wild-type levels. The growth defect of UL21a-deficient virus in MRC-5 cells was more pronounced than that in HFFs. At a high multiplicity of infection, the UL21a deletion virus synthesized viral proteins with wild-type kinetics but had a two- to threefold defect in viral DNA replication. More importantly, although pUL21a was not detected in the virion, progeny virions produced by the mutant virus were approximately 10 times less infectious than wild-type virus, suggesting that UL21a is required for HCMV to establish efficient productive infection. We conclude that UL21a encodes a short-lived cytoplasmic protein and facilitates HCMV replication in fibroblasts.

  13. SPAM-8, a mouse-human heteromyeloma fusion partner in the production of human monoclonal antibodies. Establishment of a human monoclonal antibody against cytomegalovirus.

    PubMed

    Gustafsson, B; Jondal, M; Sundqvist, V A

    1991-01-01

    A heteromyeloma (mouse x human) cell line (SPAM-8) was produced by fusing mouse myeloma cells (SP2/0) with human peripheral blood lymphocytes. The cells were sensitive to aminopterin and resistant to ouabain. The cells showed a doubling time of about 19 hours and a cloning efficiency of 0.8 cells/well (to obtain growth in 50% of wells seeded) using mouse thymocytes as feeder cells. The number of chromosomes was about 86 and 1% of the total DNA was of human origin. Fusion of SPAM-8 cells with lymphocytes prepared from human spleens resulted in approximately one hybridoma per 10(5) seeded lymphocytes. A trioma (human x [mouse x human]) cell line was established by fusing cells of an Epstein-Barr virus-transformed B cell line with SPAM-8 cells. The trioma cells produced antibodies (IgG1, K) against cytomegalovirus, in a concentration of 7 micrograms/ml in spent medium, over a period of six months of continuous culture. The results obtained indicate that the heteromyeloma SPAM-8 may be used as a fusion partner in the production of human monoclonal antibodies.

  14. Human cytomegalovirus (HCMV) immediate-early enhancer/promoter specificity during embryogenesis defines target tissues of congenital HCMV infection.

    PubMed Central

    Koedood, M; Fichtel, A; Meier, P; Mitchell, P J

    1995-01-01

    Congenital human cytomegalovirus (HCMV) infection is a common cause of deafness and neurological disabilities. Many aspects of this prenatal infection, including which cell types are infected and how infection proceeds, are poorly understood. Transcription of HCMV immediate-early (IE) genes is required for expression of all other HCMV genes and is dependent on host cell transcription factors. Cell type-specific differences in levels of IE transcription are believed to underlie differences in infection permissivity. However, DNA transfection experiments have paradoxically suggested that the HCMV major IE enhancer/promoter is a broadly active transcriptional element with little cell type specificity. In contrast, we show here that expression of a lacZ gene driven by the HCMV major IE enhancer/promoter -524 to +13 segment is restricted in transgenic mouse embryos to sites that correlate with known sites of congenital HCMV infection in human fetuses. This finding suggests that the IE enhancer/promoter is a major determinant of HCMV infection sites in humans and that transcription factors responsible for its regulation are cell type-specifically conserved between humans and mice. The lacZ expression patterns of these transgenic embryos yield insight into congenital HCMV pathogenesis by providing a spatiotemporal map of the sets of vascular, neural, and epithelial cells that are likely targets of infection. These transgenic mice may constitute a useful model system for investigating IE enhancer/promoter regulation in vivo and for identifying factors that modulate active and latent HCMV infections in humans. PMID:7884867

  15. Cervical shedding of cytomegalovirus in human immunodeficiency virus type 1-infected women.

    PubMed

    Mostad, S B; Kreiss, J K; Ryncarz, A J; Overbaugh, J; Mandaliya, K; Chohan, B; Ndinya-Achola, J; Bwayo, J J; Corey, L

    1999-12-01

    Cervical shedding of cytomegalovirus (CMV) is important in transmission of CMV to exposed sexual partners and neonates. We evaluated prevalence and correlates of CMV DNA shedding in cervical secretions from a large cohort of HIV-1-seropositive women. Using polymerase chain reaction (PCR) assays, CMV DNA was detected in 183 (59%) cervical swab samples from 311 women. Cervical shedding of CMV DNA was significantly associated with shedding of HIV-1 DNA (odds ratio 1.8; 95% confidence interval 1.1-2.8). CMV shedding was also more frequent in women with Neisseria gonorrhoeae and Trichomonas vaginalis infections, but these associations were not statistically significant. Cervical shedding of CMV in HIV-1-infected women is very frequent and may reflect higher risk of transmission to sexual partners and neonates than previously appreciated.

  16. Human cytomegalovirus gH stability and trafficking are regulated by ER-associated degradation and transmembrane architecture.

    PubMed

    Gardner, Thomas J; Hernandez, Rosmel E; Noriega, Vanessa M; Tortorella, Domenico

    2016-03-30

    The prototypic betaherpesvirus human cytomegalovirus (CMV) establishes life-long persistence within its human host. While benign in healthy individuals, CMV poses a significant threat to the immune compromised, including transplant recipients and neonates. The CMV glycoprotein complex gH/gL/gO mediates infection of fibroblasts, and together with the gH/gL/UL128/130/131 a pentameric complex permits infection of epithelial, endothethial, and myeloid cells. Given the central role of the gH/gL complex during infection, we were interested in studying cellular trafficking of the gH/gL complex through generation of human cells that stably express gH and gL. When expressed alone, CMV gH and gL were degraded through the ER-associated degradation (ERAD) pathway. However, co-expression of these proteins stabilized the polypeptides and enhanced their cell-surface expression. To further define regulatory factors involved in gH/gL trafficking, a CMV gH chimera in which the gH transmembrane and cytoplasmic tail were replaced with that of human CD4 protein permitted cell surface gH expression in absence of gL. We thus demonstrate the ability of distinct cellular processes to regulate the trafficking of viral glycoproteins. Collectively, the data provide insight into the processing and trafficking requirements of CMV envelope protein complexes and provide an example of the co-opting of cellular processes by CMV.

  17. Human cytomegalovirus gH stability and trafficking are regulated by ER-associated degradation and transmembrane architecture

    PubMed Central

    Gardner, Thomas J.; Hernandez, Rosmel E.; Noriega, Vanessa M.; Tortorella, Domenico

    2016-01-01

    The prototypic betaherpesvirus human cytomegalovirus (CMV) establishes life-long persistence within its human host. While benign in healthy individuals, CMV poses a significant threat to the immune compromised, including transplant recipients and neonates. The CMV glycoprotein complex gH/gL/gO mediates infection of fibroblasts, and together with the gH/gL/UL128/130/131 a pentameric complex permits infection of epithelial, endothethial, and myeloid cells. Given the central role of the gH/gL complex during infection, we were interested in studying cellular trafficking of the gH/gL complex through generation of human cells that stably express gH and gL. When expressed alone, CMV gH and gL were degraded through the ER-associated degradation (ERAD) pathway. However, co-expression of these proteins stabilized the polypeptides and enhanced their cell-surface expression. To further define regulatory factors involved in gH/gL trafficking, a CMV gH chimera in which the gH transmembrane and cytoplasmic tail were replaced with that of human CD4 protein permitted cell surface gH expression in absence of gL. We thus demonstrate the ability of distinct cellular processes to regulate the trafficking of viral glycoproteins. Collectively, the data provide insight into the processing and trafficking requirements of CMV envelope protein complexes and provide an example of the co-opting of cellular processes by CMV. PMID:27026399

  18. Letermovir and inhibitors of the terminase complex: a promising new class of investigational antiviral drugs against human cytomegalovirus

    PubMed Central

    Melendez, Dante P; Razonable, Raymund R

    2015-01-01

    Infection with cytomegalovirus is prevalent in immunosuppressed patients. In solid organ transplant and hematopoietic stem cell transplant recipients, cytomegalovirus infection is associated with high morbidity and preventable mortality. Prevention and treatment of cytomegalovirus with currently approved antiviral drugs is often associated with side effects that sometimes preclude their use. Moreover, cytomegalovirus has developed mutations that confer resistance to standard antiviral drugs. During the last decade, there have been calls to develop novel antiviral drugs that could provide better options for prevention and treatment of cytomegalovirus. Letermovir (AIC246) is a highly specific antiviral drug that is currently undergoing clinical development for the management of cytomegalovirus infection. It acts by inhibiting the viral terminase complex. Letermovir is highly potent in vitro and in vivo against cytomegalovirus. Because of a distinct mechanism of action, it does not exhibit cross-resistance with other antiviral drugs. It is predicted to be active against strains that are resistant to ganciclovir, foscarnet, and cidofovir. To date, early-phase clinical trials suggest a very low incidence of adverse effects. Herein, we present a comprehensive review on letermovir, from its postulated novel mechanism of action to the results of most recent clinical studies. PMID:26345608

  19. The sequence and antiapoptotic functional domains of the human cytomegalovirus UL37 exon 1 immediate early protein are conserved in multiple primary strains.

    PubMed

    Hayajneh, W A; Colberg-Poley, A M; Skaletskaya, A; Bartle, L M; Lesperance, M M; Contopoulos-Ioannidis, D G; Kedersha, N L; Goldmacher, V S

    2001-01-05

    The human cytomegalovirus UL37 exon 1 gene encodes the immediate early protein pUL37x1 that has antiapoptotic and regulatory activities. Deletion mutagenesis analysis of the open reading frame of UL37x1 identified two domains that are necessary and sufficient for its antiapoptotic activity. These domains are confined within the segments between amino acids 5 to 34, and 118 to 147, respectively. The first domain provides the targeting of the protein to mitochondria. Direct PCR sequencing of UL37 exon 1 amplified from 26 primary strains of human cytomegalovirus demonstrated that the promoter, polyadenylation signal, and the two segments of pUL37x1 required for its antiapoptotic function were invariant in all sequenced strains and identical to those in AD169 pUL37x1. In total, UL37 exon 1 varies between 0.0 and 1.6% at the nucleotide level from strain AD169. Only 11 amino acids were found to vary in one or more viral strains, and these variations occurred only in the domains of pUL37x1 dispensable for its antiapoptotic function. We infer from this remarkable conservation of pUL37x1 in primary strains that this protein and, probably, its antiapoptotic function are required for productive replication of human cytomegalovirus in humans.

  20. Human cytomegalovirus immediate early gene expression in the osteosarcoma line U2OS is repressed by the cell protein ATRX.

    PubMed

    McFarlane, Steven; Preston, Chris M

    2011-04-01

    The control of human cytomegalovirus (HCMV) immediate early (IE) gene expression in infected human fibroblasts was compared with that in the U2OS human osteosarcoma cells. Viral IE expression was stimulated by the virion protein pp71 and repressed by the cell protein hDaxx in fibroblasts, as expected from published data. Neither of these events occurred in infected U2OS cells, suggesting that this cell line lacks one or more factors that repress HCMV IE expression. The chromatin remodeling factor ATRX is absent from U2OS cells, therefore the effect of introducing this protein by electroporation of plasmid DNA was investigated. Provision of ATRX inhibited HCMV IE expression, and the presence of the HCMV-specified virion phosphoprotein pp71 overcame the repression. The experiments demonstrate that ATRX can act as a cellular intrinsic antiviral defense in U2OS cells by blocking gene expression from incoming HCMV genomes. In contrast, ATRX did not affect the replication of herpes simplex virus type 1, showing that there are differences in the way U2OS cells respond to the presence of the herpesviral genomes. Copyright © 2011 Elsevier B.V. All rights reserved.

  1. Human Cytomegalovirus-Encoded Human Interleukin-10 (IL-10) Homolog Amplifies Its Immunomodulatory Potential by Upregulating Human IL-10 in Monocytes

    PubMed Central

    Avdic, Selmir; McSharry, Brian P.; Steain, Megan; Poole, Emma; Sinclair, John; Abendroth, Allison

    2016-01-01

    ABSTRACT The human cytomegalovirus (HCMV) gene UL111A encodes cytomegalovirus-encoded human interleukin-10 (cmvIL-10), a homolog of the potent immunomodulatory cytokine human interleukin 10 (hIL-10). This viral homolog exhibits a range of immunomodulatory functions, including suppression of proinflammatory cytokine production and dendritic cell (DC) maturation, as well as inhibition of major histocompatibility complex (MHC) class I and class II. Here, we present data showing that cmvIL-10 upregulates hIL-10, and we identify CD14+ monocytes and monocyte-derived macrophages and DCs as major sources of hIL-10 secretion in response to cmvIL-10. Monocyte activation was not a prerequisite for cmvIL-10-mediated upregulation of hIL-10, which was dose dependent and controlled at the transcriptional level. Furthermore, cmvIL-10 upregulated expression of tumor progression locus 2 (TPL2), which is a regulator of the positive hIL-10 feedback loop, whereas expression of a negative regulator of the hIL-10 feedback loop, dual-specificity phosphatase 1 (DUSP1), remained unchanged. Engagement of the hIL-10 receptor (hIL-10R) by cmvIL-10 led to upregulation of heme oxygenase 1 (HO-1), an enzyme linked with suppression of inflammatory responses, and this upregulation was required for cmvIL-10-mediated upregulation of hIL-10. We also demonstrate an important role for both phosphatidylinositol 3-kinase (PI3K) and STAT3 in the upregulation of HO-1 and hIL-10 by cmvIL-10. In addition to upregulating hIL-10, cmvIL-10 could exert a direct immunomodulatory function, as demonstrated by its capacity to upregulate expression of cell surface CD163 when hIL-10 was neutralized. This study identifies a mechanistic basis for cmvIL-10 function, including the capacity of this viral cytokine to potentially amplify its immunosuppressive impact by upregulating hIL-10 expression. IMPORTANCE Human cytomegalovirus (HCMV) is a large, double-stranded DNA virus that causes significant human disease

  2. Cellular homeoproteins, SATB1 and CDP, bind to the unique region between the human cytomegalovirus UL127 and major immediate-early genes

    SciTech Connect

    Lee Jialing; Klase, Zachary; Gao Xiaoqi; Caldwell, Jeremy S.; Stinski, Mark F.; Kashanchi, Fatah; Chao, S.-H.

    2007-09-15

    An AT-rich 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 (UR). It has been shown that the UR represses activation of transcription from the UL127 promoter and functions as a boundary between the divergent UL127 and MIE genes during human CMV infection [Angulo, A., Kerry, D., Huang, H., Borst, E.M., Razinsky, A., Wu, J., Hobom, U., Messerle, M., Ghazal, P., 2000. Identification of a boundary domain adjacent to the potent human cytomegalovirus enhancer that represses transcription of the divergent UL127 promoter. J. Virol. 74 (6), 2826-2839; Lundquist, C.A., Meier, J.L., Stinski, M.F., 1999. A strong negative transcriptional regulatory region between the human cytomegalovirus UL127 gene and the major immediate-early enhancer. J. Virol. 73 (11), 9039-9052]. A putative forkhead box-like (FOX-like) site, AAATCAATATT, was identified in the UR and found to play a key role in repression of the UL127 promoter in recombinant virus-infected cells [Lashmit, P.E., Lundquist, C.A., Meier, J.L., Stinski, M.F., 2004. Cellular repressor inhibits human cytomegalovirus transcription from the UL127 promoter. J. Virol. 78 (10), 5113-5123]. However, the cellular factors which associate with the UR and FOX-like region remain to be determined. We reported previously that pancreatic-duodenal homeobox factor-1 (PDX1) bound to a 45-bp element located within the UR [Chao, S.H., Harada, J.N., Hyndman, F., Gao, X., Nelson, C.G., Chanda, S.K., Caldwell, J.S., 2004. PDX1, a Cellular Homeoprotein, Binds to and Regulates the Activity of Human Cytomegalovirus Immediate Early Promoter. J. Biol. Chem. 279 (16), 16111-16120]. Here we demonstrate that two additional cellular homeoproteins, special AT-rich sequence binding protein 1 (SATB1) and CCAAT displacement protein (CDP), bind to the human CMV UR in vitro and in vivo. Furthermore, CDP is identified as a FOX-like binding protein

  3. Human Cytomegalovirus IE2 Protein Disturbs Brain Development by the Dysregulation of Neural Stem Cell Maintenance and the Polarization of Migrating Neurons.

    PubMed

    Han, Dasol; Byun, Sung-Hyun; Kim, Juwan; Kwon, Mookwang; Pleasure, Samuel J; Ahn, Jin-Hyun; Yoon, Keejung

    2017-09-01

    Despite the high incidence of severe defects in the central nervous system caused by human cytomegalovirus (HCMV) congenital infection, the mechanism of HCMV neuropathogenesis and the roles of individual viral genes have not yet been fully determined. In this study, we show that the immediate-early 2 (IE2) protein may play a key role in HCMV-caused neurodevelopmental disorders. IE2-transduced neural progenitor cells gave rise to neurospheres with a lower frequency and produced smaller neurospheres than control cells in vitro, indicating reduction of self-renewal and expansion of neural progenitors by IE2. At 2 days after in utero electroporation into the ventricle of the developing brain, a dramatically lower percentage of IE2-expressing cells was detected in the ventricular zone (VZ) and cortical plate (CP) compared to control cells, suggesting that IE2 concurrently dysregulates neural stem cell maintenance in the VZ and neuronal migration to the CP. In addition, most IE2(+) cells in the lower intermediate zone either showed multipolar morphology with short neurites or possessed nonradially oriented processes, whereas control cells had long, radially oriented monopolar or bipolar neurites. IE2(+) callosal axons also failed to cross the midline to form the corpus callosum. Furthermore, we provide molecular evidence that the cell cycle arrest and DNA binding activities of IE2 appear to be responsible for the increased neural stem cell exit from the VZ and cortical migrational defects, respectively. Collectively, our results demonstrate that IE2 disrupts the orderly process of brain development in a stepwise manner to further our understanding of neurodevelopmental HCMV pathogenesis.IMPORTANCE HCMV brain pathogenesis has been studied in limited experimental settings, such as in vitro HCMV infection of neural progenitor cells or in vivo murine CMV infection of the mouse brain. Here, we show that IE2 is a pivotal factor that contributes to HCMV-induced abnormalities in

  4. Immunobiology of congenital cytomegalovirus infection of the central nervous system—the murine cytomegalovirus model.

    PubMed

    Slavuljica, Irena; Kveštak, Daria; Huszthy, Peter Csaba; Kosmac, Kate; Britt, William J; Jonjić, Stipan

    2015-03-01

    Congenital human cytomegalovirus infection is a leading infectious cause of long-term neurodevelopmental sequelae, including mental retardation and hearing defects. Strict species specificity of cytomegaloviruses has restricted the scope of studies of cytomegalovirus infection in animal models. To investigate the pathogenesis of congenital human cytomegalovirus infection, we developed a mouse cytomegalovirus model that recapitulates the major characteristics of central nervous system infection in human infants, including the route of neuroinvasion and neuropathological findings. Following intraperitoneal inoculation of newborn animals with mouse cytomegalovirus, the virus disseminates to the central nervous system during high-level viremia and replicates in the brain parenchyma, resulting in a focal but widespread, non-necrotizing encephalitis. Central nervous system infection is coupled with the recruitment of resident and peripheral immune cells as well as the expression of a large number of pro-inflammatory cytokines. Although infiltration of cellular constituents of the innate immune response characterizes the early immune response in the central nervous system, resolution of productive infection requires virus-specific CD8(+) T cells. Perinatal mouse cytomegalovirus infection results in profoundly altered postnatal development of the mouse central nervous system and long-term motor and sensory disabilities. Based on an enhanced understanding of the pathogenesis of this infection, prospects for novel intervention strategies aimed to improve the outcome of congenital human cytomegalovirus infection are proposed.

  5. Human Cytomegaloviruses Expressing Yellow Fluorescent Fusion Proteins - Characterization and Use in Antiviral Screening

    PubMed Central

    Straschewski, Sarah; Warmer, Martin; Frascaroli, Giada; Hohenberg, Heinrich; Mertens, Thomas; Winkler, Michael

    2010-01-01

    Recombinant viruses labelled with fluorescent proteins are useful tools in molecular virology with multiple applications (e.g., studies on intracellular trafficking, protein localization, or gene activity). We generated by homologous recombination three recombinant cytomegaloviruses carrying the enhanced yellow fluorescent protein (EYFP) fused with the viral proteins IE-2, ppUL32 (pp150), and ppUL83 (pp65). In growth kinetics, the three viruses behaved all like wild type, even at low multiplicity of infection (MOI). The expression of all three fusion proteins was detected, and their respective localizations were the same as for the unmodified proteins in wild-type virus–infected cells. We established the in vivo measurement of fluorescence intensity and used the recombinant viruses to measure inhibition of viral replication by neutralizing antibodies or antiviral substances. The use of these viruses in a pilot screen based on fluorescence intensity and high-content analysis identified cellular kinase inhibitors that block viral replication. In summary, these viruses with individually EYFP-tagged proteins will be useful to study antiviral substances and the dynamics of viral infection in cell culture. PMID:20161802

  6. Detection of human cytomegalovirus in clinical specimens by centrifugation culture with a nonhuman cell line.

    PubMed Central

    Gleaves, C A; Hursh, D A; Meyers, J D

    1992-01-01

    The sensitivities of MRC-5 and mink lung (ML) cells in centrifugation culture were compared simultaneously for the detection of cytomegalovirus (CMV) IE antigen (immediate-early antigen) from clinical specimens. Of 413 samples assayed, 51 (12%) were positive for CMV by both centrifugation and standard cell culture. At 20 h postinoculation (p.i.), 46 of 51 (90.2%) CMV-positive specimens were detected in ML cells. At 40 h p.i., 50 of 51 (98.0%) CMV-positive specimens were detected in ML cells, compared with 48 of 51 (94.0%) in MRC-5 cells. There was no significant difference in the detection of CMV in either cell line by centrifugation culture. However, in 19 of 23 positive samples that had countable foci at 20 h p.i., there was a 25% increase in the number of positive foci observed for ML cells compared with MRC-5 cells. Less toxicity was also noted for ML cells than for MRC cells, particularly in viral blood specimens. These data suggest that ML cells are comparable to MRC-5 cells for the rapid detection of CMV by centrifugation culture. PMID:1315330

  7. Dynamic and Nucleolin-Dependent Localization of Human Cytomegalovirus UL84 to the Periphery of Viral Replication Compartments and Nucleoli

    PubMed Central

    Bender, Brian J.

    2014-01-01

    ABSTRACT Protein-protein and protein-nucleic acid interactions within subcellular compartments are required for viral genome replication. To understand the localization of the human cytomegalovirus viral replication factor UL84 relative to other proteins involved in viral DNA synthesis and to replicating viral DNA in infected cells, we created a recombinant virus expressing a FLAG-tagged version of UL84 (UL84FLAG) and used this virus in immunofluorescence assays. UL84FLAG localization differed at early and late times of infection, transitioning from diffuse distribution throughout the nucleus to exclusion from the interior of replication compartments, with some concentration at the periphery of replication compartments with newly labeled DNA and the viral DNA polymerase subunit UL44. Early in infection, UL84FLAG colocalized with the viral single-stranded DNA binding protein UL57, but colocalization became less prominent as infection progressed. A portion of UL84FLAG also colocalized with the host nucleolar protein nucleolin at the peripheries of both replication compartments and nucleoli. Small interfering RNA (siRNA)-mediated knockdown of nucleolin resulted in a dramatic elimination of UL84FLAG from replication compartments and other parts of the nucleus and its accumulation in the cytoplasm. Reciprocal coimmunoprecipitation of viral proteins from infected cell lysates revealed association of UL84, UL44, and nucleolin. These results indicate that UL84 localization during infection is dynamic, which is likely relevant to its functions, and suggest that its nuclear and subnuclear localization is highly dependent on direct or indirect interactions with nucleolin. IMPORTANCE The protein-protein interactions among viral and cellular proteins required for replication of the human cytomegalovirus (HCMV) DNA genome are poorly understood. We sought to understand how an enigmatic HCMV protein critical for virus replication, UL84, localizes relative to other viral and

  8. Diagnosis of Human Congenital Cytomegalovirus Infection by Amplification of Viral DNA from Dried Blood Spots on Perinatal Cards

    PubMed Central

    Scanga, Lori; Chaing, Shu; Powell, Cynthia; Aylsworth, Arthur S.; Harrell, Lizzie J.; Henshaw, Nancy G.; Civalier, Chris J.; Thorne, Leigh B.; Weck, Karen; Booker, Jessica; Gulley, Margaret L.

    2006-01-01

    Congenital human cytomegalovirus (HCMV) infection affects 1% of children and is the most common infectious cause of sensorineural hearing loss. Due to the difficulty of diagnosing deafness and other neurological disorders in infants, affected individuals may not be recognized until much later when active infection has resolved and culture is no longer informative. To overcome this problem, congenital HCMV infection was diagnosed retrospectively by testing residual blood samples collected from newborns and dried on perinatal cards as part of the North Carolina Newborn Screening Program. We modified the Qiagen method for purifying DNA from dried blood spots to increase the sample size and recovery of the lysate. A multiplex, real-time TaqMan polymerase chain reaction assay on an ABI 7900 instrument measured a highly conserved segment of the HCMV polymerase gene and the APOB human control gene. HCMV DNA was detected in blood dried on perinatal cards from all seven infants with culture-proven congenital infection, and all 24 negative control cases lacked detectable HCMV DNA. Our findings suggest that it is possible to diagnose congenital HCMV infection using dried blood collected up to 20 months earlier. Further studies are warranted on patients with hearing loss or other neurological deficits to determine the percentage that is attributable to congenital HCMV infection. PMID:16645211

  9. Molecular Imprint of Exposure to Naturally Occurring Genetic Variants of Human Cytomegalovirus on the T cell Repertoire

    NASA Astrophysics Data System (ADS)

    Smith, Corey; Gras, Stephanie; Brennan, Rebekah M.; Bird, Nicola L.; Valkenburg, Sophie A.; Twist, Kelly-Anne; Burrows, Jacqueline M.; Miles, John J.; Chambers, Daniel; Bell, Scott; Campbell, Scott; Kedzierska, Katherine; Burrows, Scott R.; Rossjohn, Jamie; Khanna, Rajiv

    2014-02-01

    Exposure to naturally occurring variants of herpesviruses in clinical settings can have a dramatic impact on anti-viral immunity. Here we have evaluated the molecular imprint of variant peptide-MHC complexes on the T-cell repertoire during human cytomegalovirus (CMV) infection and demonstrate that primary co-infection with genetic variants of CMV was coincident with development of strain-specific T-cell immunity followed by emergence of cross-reactive virus-specific T-cells. Cross-reactive CMV-specific T cells exhibited a highly conserved public T cell repertoire, while T cells directed towards specific genetic variants displayed oligoclonal repertoires, unique to each individual. T cell recognition foot-print and pMHC-I structural analyses revealed that the cross-reactive T cells accommodate alterations in the pMHC complex with a broader foot-print focussing on the core of the peptide epitope. These findings provide novel molecular insight into how infection with naturally occurring genetic variants of persistent human herpesviruses imprints on the evolution of the anti-viral T-cell repertoire.

  10. The antiviral restriction factors IFITM1, 2 and 3 do not inhibit infection of human papillomavirus, cytomegalovirus and adenovirus.

    PubMed

    Warren, Cody J; Griffin, Laura M; Little, Alexander S; Huang, I-Chueh; Farzan, Michael; Pyeon, Dohun

    2014-01-01

    Type I interferons (IFN-α and β) induce dynamic host defense mechanisms to inhibit viral infections. It has been recently recognized that the interferon-inducible transmembrane proteins (IFITM) 1, 2 and 3 can block entry of a broad spectrum of RNA viruses. However, no study to date has focused on the role of IFITM proteins in DNA virus restriction. Here, we demonstrate that IFN-α or -β treatment of keratinocytes substantially decreases human papillomavirus 16 (HPV16) infection while robustly inducing IFITM1, 2 and 3 expression. However, IFITM1, 2 and 3 overexpression did not inhibit HPV16 infection; rather, IFITM1 and IFITM3 modestly enhanced HPV16 infection in various cell types including primary keratinocytes. Moreover, IFITM1, 2 and 3 did not inhibit infection by two other DNA viruses, human cytomegalovirus (HCMV) and adenovirus type 5 (Ad5). Taken together, we reveal that the entry of several DNA viruses, including HPV, HCMV, and Ad5 is not affected by IFITM1, 2 and 3 expression. These results imply that HPV, and other DNA viruses, may bypass IFITM restriction during intracellular trafficking.

  11. Novel human cytomegalovirus viral chemokines, vCXCL-1s, display functional selectivity for neutrophil signaling and function

    PubMed Central

    Heo, Jinho; Dogra, Pranay; Masi, Tom J.; Pitt, Elisabeth A.; de Kruijf, Petra; Smit, Martine J.; Sparer, Tim E.

    2015-01-01

    Human cytomegalovirus (HCMV) uses members of the hematopoietic system including neutrophils for dissemination throughout the body. HCMV encodes a viral chemokine, vCXCL-1, that is postulated to attract neutrophils for dissemination within the host. The gene encoding vCXCL-1, UL146, is one of the most variable genes in the HCMV genome. Why HCMV has evolved this hypervariability and how this affects the virus’ dissemination/pathogenesis is unknown. Because the vCXCL-1 hypervariability maps to important binding and activation domains, we hypothesized that vCXCL-1s differentially activate neutrophils, which could contribute to HCMV dissemination and/or pathogenesis. In order to test whether these viral chemokines affect neutrophil function, we generated vCXCL-1 proteins from 11 different clades from clinical isolates from HCMV-congenitally infected infants. All vCXCL-1s were able to induce calcium flux at a concentration of 100 nM and integrin expression on human peripheral blood neutrophils (PBNs) in spite of differences in affinity for the CXCR1 and CXCR2 receptors. In fact their affinity for CXCR1 or CXCR2 did not directly correlate with chemotaxis, G protein-dependent and independent (β-arrestin2) activation, or secondary chemokine (CCL22) expression. Our data suggest that vCXCL-1 polymorphisms impact the binding affinity, receptor usage, and differential PBN activation that could contribute to HCMV dissemination and/or pathogenesis. PMID:25987741

  12. Interaction of the human cytomegalovirus particle with the host cell induces hypoxia-inducible factor 1 alpha

    SciTech Connect

    McFarlane, Steven; Nicholl, Mary Jane; Sutherland, Jane S.; Preston, Chris M.

    2011-05-25

    The cellular protein hypoxia-inducible factor 1 alpha (HIF-1{alpha}) was induced after infection of human fibroblasts with human cytomegalovirus (HCMV). HCMV irradiated with ultraviolet light (uv-HCMV) also elicited the effect, demonstrating that the response was provoked by interaction of the infecting virion with the cell and that viral gene expression was not required. Although induction of HIF-1{alpha} was initiated by an early event, accumulation of the protein was not detected until 9 hours post infection, with levels increasing thereafter. Infection with uv-HCMV resulted in increased abundance of HIF-1{alpha}-specific RNA, indicating stimulation of transcription. In addition, greater phosphorylation of the protein kinase Akt was observed, and the activity of this enzyme was required for induction of HIF-1{alpha} to occur. HIF-1{alpha} controls the expression of many cellular gene products; therefore the findings reveal new ways in which interaction of the HCMV particle with the host cell may cause significant alterations to cellular physiology.

  13. Circulating endothelial giant cells permissive for human cytomegalovirus (HCMV) are detected in disseminated HCMV infections with organ involvement.

    PubMed Central

    Percivalle, E; Revello, M G; Vago, L; Morini, F; Gerna, G

    1993-01-01

    Giant cells fully permissive for human cytomegalovirus (HCMV) were found to circulate, at a variable proportion, in peripheral blood of 21 out of 25 immunocompromised patients with disseminated HCMV infection. Circulating endothelial giant cells (EGC) were identified by a specific monoclonal antibody of endothelial origin and shown to express immediate-early, early, and late viral proteins. Immunostaining patterns of different viral proteins were comparable to those detected in vitro in cultured human umbilical vein endothelial cells. EGC counts > 10 were associated with high levels (> 100) of HCMV viremia and antigenemia, as well as with an overt clinical syndrome in transplanted patients, and to an untreated long lasting organ localization in AIDS patients. On the other hand, EGC counts were < 10 during disseminated HCMV infections of both transplant recipients with no apparent organ syndrome and AIDS patients with recent organ involvement. In tissue sections from AIDS patients, infected endothelial cells were found to progressively enlarge till detaching from the small vessel wall and entering blood stream. HCMV-infected EGC represent a new systemic parameter suitable for the diagnosis of HCMV organ involvement and for the study of the pathogenesis of disseminated infections. Images PMID:8394385

  14. Preparation and identification of HLA-A*1101 tetramer loading with human cytomegalovirus pp65 antigen peptide.

    PubMed

    Li, Fengyao; Xu, Lihui; Zha, Qingbing; Chi, Xiaoyun; Jia, Qiantao; He, Xianhui

    2007-04-01

    MHC/peptide tetramer technology has been widely used to study antigen-specific T cells, especially for identifying virus-specific CD8+ T cells in humans. The tetramer molecule is composed of HLA heavy chain, beta2-microglobulin (beta2m), an antigenic peptide, and fluorescent-labeled streptavidin. To further investigate the HLA-A*1101-restricted CD8+ T cell responses against human cytomegalovirus (HCMV), we established an approach to prepare HLA-A*1101 tetramer complexed with a peptide from HCMV. The cDNA encoding HLA-A*1101 heavy chain was cloned and the prokaryotic expression vector for the ectodomain of HLA-A*1101 fused with a BirA substrate peptide (HLA-A*1101-BSP) at its carboxyl terminus was constructed. The fusion protein was highly expressed as inclusion bodies under optimized conditions in Escherichia coli. Moreover, HLA-A*1101-BSP protein was refolded in the presence of beta2m and an HCMV peptide pp65(16-24) (GPISGHVLK, GPI). Soluble HLA-A*1101-GPI monomer was biotinylated and purified to a purity of 95%, which was subsequently combined with streptavidin to form tetramers at a yield of > 80%. The HLA-A*1101-GPI tetramers could bind to virus-specific CD8+ T cells, suggesting soluble HLA-A*1101-GPI tetramers were biologically functional. This study provides the basis for further evaluation of HLA-A*1101-restricted CD8+ T cell responses against HCMV infection.

  15. A novel flow cytometry-based tool for determining the efficiency of human cytomegalovirus infection in THP-1 derived macrophages.

    PubMed

    Li, Huifen; Mao, Genxiang; Carlson, Joshua; Leng, Sean X

    2015-09-01

    Human cytomegalovirus (hCMV) is a ubiquitous pathogen that causes congenital infection and severe infections in immunocompromised patients. Chronic hCMV infection may also play an important role in immunosenescence and adverse health outcomes in older adults. THP-1, a human monocytic cell line and its derived macrophages serve as a useful cell culture model for mechanistic studies of hCMV infection and its underlying biology. A major methodological challenge is the lack of a quick and reliable tool to accurately determine the efficiency of hCMV infection in THP-1 derived macrophages. In this study, we developed a flow cytometry based method using commercially available monoclonal antibody (MAb) against hCMV immediate early (IE) antigen that can accurately determine infection efficiency. We used 0.5% formaldehyde for fixation, 90% methanol for permeabilization, and incubation with FITC conjugated MAb at 37°C. The method was tested by hCMV infection with laboratory Towne strain in the presence or absence of hydrocortisone. It was also compared with the routine flow cytometry protocol using Cytofix/Cytoperm solution and with immunofluorescence. The results indicate that this new method is reliable and time saving for accurate determination of infection efficiency. It may facilitate further investigations into the underlying biological mechanisms of hCMV infection.

  16. Clinical correlations of human cytomegalovirus strains and viral load in kidney transplant recipients.

    PubMed

    Nogueira, Eliana; Ozaki, Kikumi Suzete; Tomiyama, Helena; Câmara, Niels Olsen Saraiva; Granato, Celso Francisco Hernandes

    2009-01-01

    Little is known about clinical differences associated with cytomegalovirus (CMV) infection by distinct strains in renal transplant patients. Different clinical pictures may be associated with specific viral genotypes, viral load, as well as host factors. The objective of this study was to identify CMV strains to determine viral load (antigenemia), and their correlation with clinical data in renal transplant recipients. Seventy-one patients were enrolled, comprising 91 samples. After selection, polymorphonuclear cells were used to amplify and sequence the gB region of CMV DNA. The sequences were analyzed to ascertain the frequency of different genotypes. Additionally, the results of this study showed that the gB coding gene presents a great variability, revealing a variety of patterns: classical gB1 (1.4%), gB1V (46.4%), classical gB2 (35.2%), gB2V (2.8%), gB3 (1.4%), classical gB4 (4.9%) and gB4V (4.9%). The mean viral load in kidney transplant patient was 75.1 positive cells (1-1000). A higher viral load was observed in patients with genotype 4 infection. Statistically significant differences were detected between gB1 and gB4 (p=0.010), and between gB2 and gB4 (p=0.021). The average numbers of positive cells in relation to clinical presentation were: 34.5 in asymptomatic, 49.5 in CMV associated syndrome and 120.7 in patients with invasive disease (p=0.048). As a group, gB1 was the most frequent strain and revealed a potential risk for developing invasive disease. Viral load also seemed to be important as a marker associated with clinical presentation of the disease.

  17. Comparison of CD8+ T Cell responses to cytomegalovirus between human fetuses and their transmitter mothers.

    PubMed

    Pedron, Beatrice; Guerin, Valerie; Jacquemard, Francois; Munier, Aline; Daffos, Fernand; Thulliez, Philippe; Aujard, Yannick; Luton, Dominique; Sterkers, Ghislaine

    2007-10-01

    The mechanisms responsible for the increased susceptibility of fetuses to cytomegalovirus (CMV) were studied by comparing CD8(+) T cell responses to the virus in susceptible fetuses to those in their comparatively more resistant mothers. Included in the study were 16 transmitter mothers who underwent seroconversion during the first trimester of pregnancy as well as their fetuses, who were positive for CMV in amniotic fluid by polymerase chain reaction at 17-19 weeks of gestation. Fetal and maternal blood samples were collected between the 22nd and 39th week of gestation. Cytotoxic T lymphocytes (CTLs) that had activated (HLA-DR(+)), effector/memory (CD28(-)), and memory (CD18(high)) phenotypes; that stained with the HLA-A2/pp65 or the HLA-B7/pp65 multimer; and that secreted interferon (IFN)- gamma were enumerated by flow cytometry. Viral loads were determined simultaneously. The results showed (1) similar levels of activated, effector/memory, and memory CTLs in fetuses and mothers but a smaller pp65-specific CTL pool in fetuses (median, 0.015% vs. 0.99%; P=.003); (2) similar percentages of CTLs secreting IFN- gamma after stimulation with ionomycin/phorbol myristate acetate in fetuses and mothers but lower percentages of CTLs secreting IFN- gamma after stimulation with a CD3 monoclonal antibody in fetuses (median, 1% vs. 14%; P=.01); and (3) higher viral loads (mean, 17,290 vs. <250 genome equivalents/mL) in fetuses. Impaired viral clearance might be related to a defective expansion of the pp65-specific CTL pool and/or to the immaturity of IFN- gamma -secreting cells in fetuses.

  18. Differential decay kinetics of human cytomegalovirus glycoprotein B genotypes following antiviral chemotherapy☆

    PubMed Central

    Emery, Vincent C.; Manuel, Oriol; Asberg, Anders; Pang, Xiaoli; Kumar, Deepali; Hartmann, Anders; Preiksaitis, Jutta K.; Pescovitz, Mark D.; Rollag, Halvor; Jardine, Alan G.; Gahlemann, Christoph G.; Humar, Atul

    2012-01-01

    Background The impact of different cytomegalovirus (HCMV) glycoprotein B (gB) genotypes on pathogenesis remains controversial. Objectives To investigate the effect of gB genotypes either as single infections or as part of multiple infections on the early kinetics of response to ganciclovir therapy. Methods Patients (n = 239) enrolled in a study of intravenous ganciclovir or valganciclovir for the treatment of HCMV disease were analysed by a gB genotype specific PCR to quantify the amount of each gB genotype present at initiation of therapy (baseline, day 0) and at days 3, 7, 14 and 21 post therapy. Results and conclusions In all gB groups (individual gB genotype infections and mixed genotype infections) there was a biphasic decline in viral load after therapy. The first phase half life (days 0–3) was ≤1 day and was followed over the next 18 days by a slower second phase decline with half lives ranging from 3.4 to 4.4 days. The 1st phase rapid decline in viral load was dependent upon gB genotype whereas the ultimate viral load reduction at day 21 was relatively insensitive to gB genotype. A strong correlation between 1st phase decline and extent of viral load reduction at day 21 was observed (r = 0.37; p = 0.002). These data imply that early reductions in HCMV load after therapy may be useful in predicting the duration of drug therapy needed to control HCMV replication. PMID:22410132

  19. Multidrug resistance conferred by novel DNA polymerase mutations in human cytomegalovirus isolates.

    PubMed

    Scott, Gillian M; Weinberg, Adriana; Rawlinson, William D; Chou, Sunwen

    2007-01-01

    The emergence of antiviral-resistant cytomegalovirus (CMV) strains is a continuing clinical problem, with increased numbers of immunocompromised patients given longer-duration antiviral prophylaxis. Two previously unrecognized CMV DNA polymerase mutations (N408K and A834P) identified separately and together in at-risk lung and kidney transplant recipients and a third mutation (L737M) identified in a liver transplant recipient were characterized by marker transfer to antiviral-sensitive laboratory strains AD169 and Towne. Subsequent phenotypic analyses of recombinant strains demonstrated the ability of mutation N408K to confer ganciclovir (GCV) and cidofovir (CDV) resistance and of mutation A834P to confer GCV, foscarnet, and CDV resistance. Mutation L737M did not confer resistance to any of the antiviral agents tested. A recombinant strain containing both N408K and A834P demonstrated increased GCV and CDV resistance compared to the levels of resistance of the virus containing only the A834P mutation. The addition of mutation N408K in combination with A834P also partially reconstituted the replication impairment of recombinant virus containing only A834P. This suggests that perturbation of both DNA polymerization (A834P) and exonuclease (N408K) activities contributes to antiviral resistance and altered replication kinetics in these mutant strains. The identification of these multidrug-resistant CMV strains in at-risk seronegative recipients of organs from seropositive donors suggests that improved prophylactic and treatment strategies are required. The additive effect of multiple mutations on antiviral susceptibility suggests that increasing antiviral-resistant phenotypes can result from different virus-antiviral interactions.

  20. A Myeloid Progenitor Cell Line Capable of Supporting Human Cytomegalovirus Latency and Reactivation, Resulting in Infectious Progeny

    PubMed Central

    O'Connor, Christine M.

    2012-01-01

    Human cytomegalovirus (HCMV) is a herpesvirus that establishes a lifelong, latent infection within a host. At times when the immune system is compromised, the virus undergoes a lytic reactivation producing infectious progeny. The identification and understanding of the biological mechanisms underlying HCMV latency and reactivation are not completely defined. To this end, we have developed a tractable in vitro model system to investigate these phases of viral infection using a clonal population of myeloid progenitor cells (Kasumi-3 cells). Infection of these cells results in maintenance of the viral genome with restricted viral RNA expression that is reversed with the addition of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA, also known as PMA). Additionally, a latent viral transcript (LUNA) is expressed at times where viral lytic transcription is suppressed. Infected Kasumi-3 cells initiate production of infectious virus following TPA treatment, which requires cell-to-cell contact for efficient transfer of virus to other cell types. Importantly, lytically infected fibroblast, endothelial, or epithelial cells can transfer virus to Kasumi-3 cells, which fail to initiate lytic replication until stimulated with TPA. Finally, inflammatory cytokines, in addition to the pharmacological agent TPA, are sufficient for transcription of immediate-early (IE) genes following latent infection. Taken together, our findings argue that the Kasumi-3 cell line is a tractable in vitro model system with which to study HCMV latency and reactivation. PMID:22761372

  1. Correlation between different genotypes of human cytomegalovirus and Epstein-Barr virus and peri-implant tissue status.

    PubMed

    Jankovic, S; Aleksic, Z; Dimitrijevic, B; Lekovic, V; Milinkovic, I; Kenney, B

    2011-12-01

    The purpose of this study was to estimate the prevalence of different genotypes of human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) in peri-implantitis and mucositis sites, and to evaluate the correlation between herpesvirus presence and clinical parameters. A total of 80 dental implants (mean time of loading, 4.16 ± 1.8 years) were evaluated during the course of the study (30 peri-implantitis, 25 mucositis and 25 healthy peri-implant sites). The following clinical parameters were assessed: visible plaque index, bleeding on probing, suppuration and probing depth. A polymerase chain reaction (PCR) assay was used to identify the presence of different HCMV and EBV genotypes in peri-implant tissue plaque samples. HCMV-2 was detected in 53.3% and EBV-1 in 46.6% of the 30 peri-implantitis sites evaluated. By contrast, HCMV-2 was not detected in healthy periodontal sites and EBV-1 was detected in one healthy site. A statistically significant correlation was found between the presence of HCMV-2 and EBV-1 genotypes and clinical parameters of peri-implantitis. The results from the present study confirmed the high prevalence of HCMV-2 and EBV-1 in the peri-implant tissue plaque of peri-implantitis sites and suggests a possible active pathogenic role of the viruses in peri-implantitis. © 2011 Australian Dental Association.

  2. Analysis of the role of autophagy inhibition by two complementary human cytomegalovirus BECN1/Beclin 1-binding proteins

    PubMed Central

    Mouna, Lina; Hernandez, Eva; Bonte, Dorine; Brost, Rebekka; Amazit, Larbi; Delgui, Laura R.; Brune, Wolfram; Geballe, Adam P.; Beau, Isabelle; Esclatine, Audrey

    2016-01-01

    ABSTRACT Autophagy is activated early after human cytomegalovirus (HCMV) infection but, later on, the virus blocks autophagy. Here we characterized 2 HCMV proteins, TRS1 and IRS1, which inhibit autophagy during infection. Expression of either TRS1 or IRS1 was able to block autophagy in different cell lines, independently of the EIF2S1 kinase, EIF2AK2/PKR. Instead, TRS1 and IRS1 interacted with the autophagy protein BECN1/Beclin 1. We mapped the BECN1-binding domain (BBD) of IRS1 and TRS1 and found it to be essential for autophagy inhibition. Mutant viruses that express only IRS1 or TRS1 partially controlled autophagy, whereas a double mutant virus expressing neither protein stimulated autophagy. A mutant virus that did not express IRS1 and expressed a truncated form of TRS1 in which the BBD was deleted, failed to control autophagy. However, this mutant virus had similar replication kinetics as wild-type virus, suggesting that autophagy inhibition is not critical for viral replication. In fact, using pharmacological modulators of autophagy and inhibition of autophagy by shRNA knockdown, we discovered that stimulating autophagy enhanced viral replication. Conversely, inhibiting autophagy decreased HCMV infection. Thus, our results demonstrate a new proviral role of autophagy for a DNA virus. PMID:26654401

  3. Human cytomegalovirus encoded chemokine receptor US28 activates the HIF-1α/PKM2 axis in glioblastoma cells

    PubMed Central

    van Senten, Jeffrey R.; Fraile-Ramos, Alberto; Siderius, Marco; Smit, Martine J.

    2016-01-01

    The human cytomegalovirus (HCMV) encoded chemokine receptor US28 promotes tumorigenesis through activation of various proliferative and angiogenic signaling pathways. Upon infection, US28 displays constitutive activity and signals in a G protein-dependent manner, hijacking the host's cellular machinery. In tumor cells, the hypoxia inducible factor-1α/pyruvate kinase M2 (HIF-1α/PKM2) axis plays an important role by supporting proliferation, angiogenesis and reprogramming of energy metabolism. In this study we show that US28 signaling results in activation of the HIF-1α/PKM2 feedforward loop in fibroblasts and glioblastoma cells. The constitutive activity of US28 increases HIF-1 protein stability through a Gαq-, CaMKII- and Akt/mTOR-dependent mechanism. Furthermore, we found that VEGF and lactate secretion are increased and HIF-1 target genes, glucose transporter type 1 (GLUT1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), involved in glucose metabolism, are upregulated in US28 expressing cells. In addition, PKM2 is phosphorylated and found to be in a tumor-associated dimeric state upon US28 expression. Also in HCMV-infected cells HIF-1 activity is enhanced, which in part is US28-dependent. Finally, increased proliferation of cells expressing US28 is abolished upon inhibition of the HIF-1α/PKM2 cascade. These data highlight the importance of HIF-1α and PKM2 in US28-induced proliferation, angiogenesis and metabolic reprogramming. PMID:27602585

  4. Cloning and sequencing of a highly productive, endotheliotropic virus strain derived from human cytomegalovirus TB40/E.

    PubMed

    Sinzger, Christian; Hahn, Gabriele; Digel, Margarete; Katona, Ruth; Sampaio, Kerstin Laib; Messerle, Martin; Hengel, Hartmut; Koszinowski, Ulrich; Brune, Wolfram; Adler, Barbara

    2008-02-01

    Human cytomegalovirus (HCMV) strain TB40/E, replicates efficiently, exhibits a broad cell tropism and is widely used for infection of endothelial cells and monocyte-derived cells yet has not been available in a phenotypically homogeneous form compatible with genetic analysis. To overcome this problem, we cloned the TB40/E strain into a bacterial artificial chromosome (BAC) vector. Both highly endotheliotropic and poorly endotheliotropic virus clones, representing three distinct restriction fragment patterns, were reconstituted after transfection of BAC clones derived from previously plaque-purified strain TB40/E. For one of the highly endotheliotropic clones, TB40-BAC4, we provide the genome sequence. Two BACs with identical restriction fragment patterns but different cell tropism were further analysed in the UL128-UL131A gene region. Sequence analysis revealed one coding-relevant adenine insertion at position 332 of UL128 in the BAC of the poorly endotheliotropic virus, which caused a frameshift in the C-terminal part of the coding sequence. Removal of this insertion by markerless mutagenesis restored the highly endotheliotropic phenotype, indicating that the loss of endothelial cell tropism was caused by this insertion. In conclusion, HCMV strain TB40/E, which combines the high endothelial cell tropism of a clinical isolate with the high titre growth of a cell culture adapted strain, is now available as a BAC clone suitable for genetic engineering. The results also suggest BAC cloning as a suitable method for selection of genetically defined virus clones.

  5. Characterization of Human Cytomegalovirus Genome Diversity in Immunocompromised Hosts by Whole-Genome Sequencing Directly From Clinical Specimens.

    PubMed

    Hage, Elias; Wilkie, Gavin S; Linnenweber-Held, Silvia; Dhingra, Akshay; Suárez, Nicolás M; Schmidt, Julius J; Kay-Fedorov, Penelope C; Mischak-Weissinger, Eva; Heim, Albert; Schwarz, Anke; Schulz, Thomas F; Davison, Andrew J; Ganzenmueller, Tina

    2017-06-01

    Advances in next-generation sequencing (NGS) technologies allow comprehensive studies of genetic diversity over the entire genome of human cytomegalovirus (HCMV), a significant pathogen for immunocompromised individuals. Next-generation sequencing was performed on target enriched sequence libraries prepared directly from a variety of clinical specimens (blood, urine, breast milk, respiratory samples, biopsies, and vitreous humor) obtained longitudinally or from different anatomical compartments from 20 HCMV-infected patients (renal transplant recipients, stem cell transplant recipients, and congenitally infected children). De novo-assembled HCMV genome sequences were obtained for 57 of 68 sequenced samples. Analysis of longitudinal or compartmental HCMV diversity revealed various patterns: no major differences were detected among longitudinal, intraindividual blood samples from 9 of 15 patients and in most of the patients with compartmental samples, whereas a switch of the major HCMV population was observed in 6 individuals with sequential blood samples and upon compartmental analysis of 1 patient with HCMV retinitis. Variant analysis revealed additional aspects of minor virus population dynamics and antiviral-resistance mutations. In immunosuppressed patients, HCMV can remain relatively stable or undergo drastic genomic changes that are suggestive of the emergence of minor resident strains or de novo infection.

  6. Fast Screening Procedures for Random Transposon Libraries of Cloned Herpesvirus Genomes: Mutational Analysis of Human Cytomegalovirus Envelope Glycoprotein Genes

    PubMed Central

    Hobom, Urs; Brune, Wolfram; Messerle, Martin; Hahn, Gabriele; Koszinowski, Ulrich H.

    2000-01-01

    We have cloned the human cytomegalovirus (HCMV) genome as an infectious bacterial artificial chromosome (BAC) in Escherichia coli. Here, we have subjected the HCMV BAC to random transposon (Tn) mutagenesis using a Tn1721-derived insertion sequence and have provided the conditions for excision of the BAC cassette. We report on a fast and efficient screening procedure for a Tn insertion library. Bacterial clones containing randomly mutated full-length HCMV genomes were transferred into 96-well microtiter plates. A PCR screening method based on two Tn primers and one primer specific for the desired genomic position of the Tn insertion was established. Within three consecutive rounds of PCR a Tn insertion of interest can be assigned to a specific bacterial clone. We applied this method to retrieve mutants of HCMV envelope glycoprotein genes. To determine the infectivities of the mutant HCMV genomes, the DNA of the identified BACs was transfected into permissive fibroblasts. In contrast to BACs with mutations in the genes coding for gB, gH, gL, and gM, which did not yield infectious virus, BACs with disruptions of open reading frame UL4 (gp48) or UL74 (gO) were viable, although gO-deficient viruses showed a severe growth deficit. Thus, gO (UL74), a component of the glycoprotein complex III, is dispensable for viral growth. We conclude that our approach of PCR screening for Tn insertions will greatly facilitate the functional analysis of herpesvirus genomes. PMID:10933677

  7. Activation of Langerhans-Type Dendritic Cells Alters Human Cytomegalovirus Infection and Reactivation in a Stimulus-Dependent Manner

    PubMed Central

    Coronel, Roxanne; Jesus, Desyree M.; Dalle Ore, Lucia; Mymryk, Joe S.; Hertel, Laura

    2016-01-01

    Oral mucosal Langerhans cells (LC) are likely to play important roles in host defense against infection by human cytomegalovirus (CMV). We previously showed that in vitro-differentiated immature LC (iLC) populations contain smaller amounts of infected cells but produce higher yields than mature LC (mLC) cultures, obtained by iLC stimulation with fetal bovine serum (FBS), CD40 ligand (CD40L) and lipopolysaccharide (LPS). Here, we sought to determine if exposure to select stimuli can improve LC permissiveness to infection, if specific components of the mLC cocktail are responsible for lowering viral yields, if this is due to defects in progeny production or release, and if these restrictions are also effective against reactivated virus. None of the stimuli tested extended the proportion of infected cells to 100%, suggesting that the block to infection onset cannot be fully removed. While CD40L and FBS exerted positive effects on viral progeny production per cell, stimulation with LPS alone or in combination with CD40L was detrimental. Reductions in viral titers were not due to defects in progeny release, and the permissive or restrictive intracellular environment established upon exposure to each stimulus appeared to act in a somewhat similar way toward lytic and latent infections. PMID:27683575

  8. Activation of Innate and Adaptive Immunity by a Recombinant Human Cytomegalovirus Strain Expressing an NKG2D Ligand

    PubMed Central

    Tomić, Adriana; Varanasi, Pavankumar R.; Golemac, Mijo; Malić, Suzana; Riese, Peggy; Borst, Eva M.; Guzmán, Carlos A.; Krmpotić, Astrid

    2016-01-01

    Development of an effective vaccine against human cytomegalovirus (HCMV) is a need of utmost medical importance. Generally, it is believed that a live attenuated vaccine would best provide protective immunity against this tenacious pathogen. Here, we propose a strategy for an HCMV vaccine that aims at the simultaneous activation of innate and adaptive immune responses. An HCMV strain expressing the host ligand ULBP2 for the NKG2D receptor was found to be susceptible to control by natural killer (NK) cells, and preserved the ability to stimulate HCMV-specific T cells. Infection with the ULBP2-expressing HCMV strain caused diminished cell surface levels of MHC class I molecules. While expression of the NKG2D ligand increased the cytolytic activity of NK cells, NKG2D engagement in CD8+ T cells provided co-stimulation and compensated for lower MHC class I expression. Altogether, our data indicate that triggering of both arms of the immune system is a promising approach applicable to the generation of a live attenuated HCMV vaccine. PMID:27907183

  9. Control of human cytomegalovirus gene expression by differential histone modifications during lytic and latent infection of a monocytic cell line.

    PubMed

    Ioudinkova, Elena; Arcangeletti, Maria Cristina; Rynditch, Alla; De Conto, Flora; Motta, Federica; Covan, Silvia; Pinardi, Federica; Razin, Sergey V; Chezzi, Carlo

    2006-12-15

    Non-differentiated THP-1 cells can be infected by human cytomegalovirus (HCMV) Towne strain, which persists in these cells in a non-active (latent) form without undergoing a productive cycle. The same cells become permissive for HCMV lytic infection after induction of cell differentiation by treatment with 12-O-tetradecanoylphorbol-13-acetate. We used this cellular model to study the possible role of histone modifications in the control of HCMV latency. Using chromatin immunoprecipitation with antibodies against histone H3 acetylated or dimethylated in position K9, we demonstrated that in lytically infected cells the HCMV enhancer was associated with heavy acetylated but not dimethylated H3. In the case of latent infection, the HCMV enhancer was associated with neither acetylated nor dimethylated H3. HCMV genes encoding DNA polymerase (early), pp65 (early-late) and pp150 (late) proteins were associated preferentially with acetylated H3 in lytically infected cells and with dimethylated H3 in latently infected cells. These data strongly suggest that K9 methylation of H3 is involved in HCMV gene repression, while association of the above genes with acetylated histones is likely to be necessary for active transcription. It can be postulated that the same histone modifications are used to mark active and repressed genes in both cellular and viral chromatin.

  10. Active human cytomegalovirus infection and glycoprotein b genotypes in brazilian pediatric renal or hematopoietic stem cell transplantation patients

    PubMed Central

    de Campos Dieamant, Débora; Bonon, Sandra Helena Alves; Prates, Liliane Cury; Belangelo, Vera Maria Santoro; Pontes, Erika R.; Costa, Sandra Cecília Botelho

    2010-01-01

    A prospective analysis of active Human Cytomegalovirus infection (HCMV) was conducted on 33 pediatric renal or hematopoietic stem cell post-transplant patients. The HCMV-DNA positive samples were evaluated for the prevalence of different gB subtypes and their subsequent correlation with clinical signs. The surveillance of HCMV active infection was based on the monitoring of antigenemia (AGM) and on a nested polymerase chain reaction (N-PCR) for the detection of HCMV in the patients studied. Using restriction analysis of the gB gene sequence by PCR-RFLP (Restriction Fragment Length Polymorphism), different HCMV strains could be detected and classified in at least four HCMV genotypes. Thirty-three pediatric recipients of renal or bone marrow transplantation were monitored. Twenty out of thirty-three (60.6%) patients demonstrated active HCMV infection. gB1 and gB2 genotypes were more frequent in this population. In this study, we observed that gB2 had correlation with reactivation of HCMV infection and that patients with mixture of genotypes did not show any symptoms of HCMV disease. Future studies has been made to confirm this. PMID:24031463

  11. Effect of human cytomegalovirus (HCMV) US27 on CXCR4 receptor internalization measured by fluorogen-activating protein (FAP) biosensors

    PubMed Central

    Boeck, Jordan M.; Spencer, Juliet V.

    2017-01-01

    Human cytomegalovirus (HCMV) is a widespread pathogen and a member of the Herpesviridae family. HCMV has a large genome that encodes many genes that are non-essential for virus replication but instead play roles in manipulation of the host immune environment. One of these is the US27 gene, which encodes a protein with homology to the chemokine receptor family of G protein-coupled receptors (GPCRs). The US27 protein has no known chemokine ligands but can modulate the signaling activity of host receptor CXCR4. We investigated the mechanism for enhanced CXCR4 signaling in the presence of US27 using a novel biosensor system comprised of fluorogen activating proteins (FAPs). FAP-tagged CXCR4 and US27 were used to explore receptor internalization and recovery dynamics, and the results demonstrate that significantly more CXCR4 internalization was observed in the presence of US27 compared to CXCR4 alone upon stimulation with CXCL12. While ligand-induced endocytosis rates were higher, steady state internalization of CXCR4 was not affected by US27. Additionally, US27 underwent rapid endocytosis at a rate that was independent of either CXCR4 expression or CXCL12 stimulation. These results demonstrate that one mechanism by which US27 can enhance CXCR4 signaling is to alter receptor internalization dynamics, which could ultimately have the effect of promoting virus dissemination by increasing trafficking of HCMV-infected cells to tissues where CXCL12 is highly expressed. PMID:28207860

  12. A collaborative study to establish the 1st WHO International Standard for human cytomegalovirus for nucleic acid amplification technology.

    PubMed

    Fryer, Jacqueline F; Heath, Alan B; Minor, Philip D

    2016-07-01

    Variability in the performance of nucleic acid amplification technology (NAT)-based assays presents a significant problem in the diagnosis and management of human cytomegalovirus (HCMV) infections. Here we describe a collaborative study to evaluate the suitability of candidate reference materials to harmonize HCMV viral load measurements in a wide range of NAT assays. Candidate materials comprised lyophilized Merlin virus, liquid Merlin virus, liquid AD169 virus, and purified HCMV Merlin DNA cloned into a bacterial artificial chromosome. Variability in the laboratory mean HCMV concentrations determined for virus samples across the different assays was 2 log10. Variability for the purified DNA sample was higher (>3 log10). The agreement between laboratories was markedly improved when the potencies of the liquid virus samples were expressed relative to the lyophilized virus candidate. In contrast, the agreement between laboratories for the purified DNA sample was not improved. Results indicated the suitability of the lyophilized Merlin virus preparation as the 1st WHO International Standard for HCMV for NAT. It was established in October 2010, with an assigned potency of 5 × 10(6) International Units (IU) (NIBSC code 09/162). It is intended to be used to calibrate secondary references, used in HCMV NAT assays, in IU.

  13. Effect of human cytomegalovirus (HCMV) US27 on CXCR4 receptor internalization measured by fluorogen-activating protein (FAP) biosensors.

    PubMed

    Boeck, Jordan M; Spencer, Juliet V

    2017-01-01

    Human cytomegalovirus (HCMV) is a widespread pathogen and a member of the Herpesviridae family. HCMV has a large genome that encodes many genes that are non-essential for virus replication but instead play roles in manipulation of the host immune environment. One of these is the US27 gene, which encodes a protein with homology to the chemokine receptor family of G protein-coupled receptors (GPCRs). The US27 protein has no known chemokine ligands but can modulate the signaling activity of host receptor CXCR4. We investigated the mechanism for enhanced CXCR4 signaling in the presence of US27 using a novel biosensor system comprised of fluorogen activating proteins (FAPs). FAP-tagged CXCR4 and US27 were used to explore receptor internalization and recovery dynamics, and the results demonstrate that significantly more CXCR4 internalization was observed in the presence of US27 compared to CXCR4 alone upon stimulation with CXCL12. While ligand-induced endocytosis rates were higher, steady state internalization of CXCR4 was not affected by US27. Additionally, US27 underwent rapid endocytosis at a rate that was independent of either CXCR4 expression or CXCL12 stimulation. These results demonstrate that one mechanism by which US27 can enhance CXCR4 signaling is to alter receptor internalization dynamics, which could ultimately have the effect of promoting virus dissemination by increasing trafficking of HCMV-infected cells to tissues where CXCL12 is highly expressed.

  14. Comparative evaluation of the cytomegalovirus DNA load in polymorphonuclear leukocytes and plasma of human immunodeficiency virus-infected subjects.

    PubMed

    Boivin, G; Handfield, J; Toma, E; Murray, G; Lalonde, R; Bergeron, M G

    1998-02-01

    The cytomegalovirus (CMV) DNA load was determined in polymorphonuclear leukocytes (PMNL) and plasma samples from 106 human immunodeficiency virus-infected subjects at risk of developing CMV disease (group 1) and from 27 AIDS patients with documented CMV disease (group 2). For both groups, the number of CMV copies in PMNL was significantly higher than in plasma when results were derived from an equivalent blood volume (P < .001, PMNL vs. plasma). Additionally, group 2 (symptomatic) patients had a greater viral DNA load than group 1 (asymptomatic) subjects (P < .001 for both PMNL and plasma). The sensitivity, specificity, and positive and negative predictive values of qualitative polymerase chain reaction using PMNL (PCR-PMNL) for the presence of CMV disease were 100%, 58%, 38%, and 100%, respectively, compared with 70%, 93%, 74%, and 92% for qualitative PCR-plasma and 93%, 92%, 76%, and 98% for quantitative PCR-PMNL using a cutoff of 16,000 copies/mL. Thus, the best strategy for diagnosing CMV disease in these individuals relies on quantitative assessment of the viral DNA load in PMNL.

  15. The highly conserved human cytomegalovirus UL136 ORF generates multiple Golgi-localizing protein isoforms through differential translation initiation.

    PubMed

    Liao, Huanan; Lee, Jung-Hyun; Kondo, Rikita; Katata, Marei; Imadome, Ken-Ichi; Miyado, Kenji; Inoue, Naoki; Fujiwara, Shigeyoshi; Nakamura, Hiroyuki

    2014-01-22

    The UL133-UL138 locus in the unique long b' (ULb') region of the human cytomegalovirus (HCMV) genome is considered to play certain roles in viral replication, dissemination and latency in a host cell type-dependent manner. Here we characterized the proteins encoded by UL136, one of the open reading frames (ORFs) in the locus. Comparative sequence analysis of UL136 among clinical isolates and laboratory strains indicates that its predicted amino-acid sequence is highly conserved. A polyclonal antibody against UL136 proteins (pUL136s) was raised against its carboxy-terminal region and this antibody specifically recognized at least five UL136-encoded protein isoforms of 29-17 kDa both in HCMV-infected cells and in cells transfected with a construct expressing pUL136. Immunofluorescence analysis with this antibody revealed localization of pUL136 in the Golgi apparatus. Analysis of several pUL136 mutants indicated that the putative transmembrane domain of pUL136 is required for its Golgi localization. Mutational analysis of multiple AUG codons in UL136 demonstrated that translation initiation from these AUG codons contributes in the generation of pUL136 isoforms.

  16. Differential kinetics of human cytomegalovirus load and antibody responses in primary infection of the immunocompetent and immunocompromised host.

    PubMed

    Gerna, Giuseppe; Lilleri, Daniele; Fornara, Chiara; Bruno, Francesca; Gabanti, Elisa; Cane, Ilaria; Furione, Milena; Revello, M Grazia

    2015-02-01

    The comparative long-term kinetics of human cytomegalovirus (HCMV) load and HCMV-specific antibody responses in the immunocompetent and immunocompromised solid-organ transplanted host during primary HCMV infection was investigated. In total, 40 immunocompetent subjects and 17 transplanted patients were examined for viral load as well as for IgG antibody responses to HCMV glycoproteins gH/gL/pUL128L, gH/gL and gB, and neutralizing antibodies in ARPE-19 epithelial cells and human fibroblasts. In parallel, the CD4(+) and CD8(+) HCMV-specific T-cell responses were determined by cytokine flow cytometry. Transplanted patients reached significantly higher viral DNA peaks, which persisted longer than in immunocompetent subjects. The ELISA-IgG responses to the pentamer, gH/gL and gB were significantly higher in primary infections of the immunocompetent until six months after onset, with the two antibody levels then overlapping from six to 12 months. Antibody levels neutralizing infection of epithelial cells were significantly higher in transplanted patients after six months, persisting for up to a year after transplantation. This trend was not observed for antibodies neutralizing infection of human fibroblasts, which showed higher titres in the immunocompetent over the entire one-year follow-up. In conclusion, in immunocompromised patients the viral load peak was much higher, while the neutralizing antibody response exceeded that detected in the immunocompetent host starting six months after onset of follow-up, often concomitantly with a lack of specific CD4(+) T cells. In this setting, the elevated antibody response occurred in the presence of differentiated follicular helper T cells in the blood, which decreased in number as did antibody titres upon reappearance of HCMV-specific CD4(+) T cells. © 2015 The Authors.

  17. Characterization of phosphoproteins and protein kinase activity of virions, noninfectious enveloped particles, and dense bodies of human cytomegalovirus.

    PubMed Central

    Roby, C; Gibson, W

    1986-01-01

    Phosphorylation of the proteins of human cytomegalovirus (CMV) virions, noninfectious enveloped particles (NIEPs), and dense bodies was investigated. Analyses of particles phosphorylated in vivo showed the following. Virions contain three predominant phosphoproteins (i.e., basic phosphoprotein and upper and lower matrix proteins) and at least nine minor phosphorylated species. NIEPs contain all of these and one additional major species, the assembly protein. Dense bodies contain only one (i.e., lower matrix) of the predominant and four of the minor virion phosphoproteins. Two-dimensional (charge-size) separations in denaturing polyacrylamide gels showed that the relative net charges of the predominant phosphorylated species ranged from the basic phosphoprotein to the more neutral upper matrix protein. In vitro assays showed that purified virions of human CMV have an associated protein kinase activity. The activity was detected only after disrupting the envelope; it had a pH optimum of approximately 9 to 9.5 and required a divalent cation, preferring magnesium to manganese. In vitro, this activity catalyzed phosphorylation of the virion proteins observed to be phosphorylated in vivo. Peptide comparisons indicated that the sites phosphorylated in vitro are a subset of those phosphorylated in vivo, underscoring the probable biological relevance of the kinase activity. Casein, phosvitin, and to a minor extent lysine-rich histones served as exogenous phosphate acceptors. Arginine-rich and lysine-rich histones and protamine sulfate, as well as the polyamines spermine and spermidine, stimulated incorporation of phosphate into the endogenous viral proteins. Virions of all human and simian CMV strains tested showed this activity. Analyses of other virus particles, including three intracellular capsid forms (i.e., A, B, and C capsids), NIEPs, and dense bodies, indicated that the active enzyme was not present in the capsid. Rate-velocity sedimentation of disrupted virions

  18. Evaluation of the AMPLICOR cytomegalovirus test with specimens from human immunodeficiency virus-infected subjects.

    PubMed

    Boivin, G; Handfield, J; Toma, E; Murray, G; Lalonde, R; Tevere, V J; Sun, R; Bergeron, M G

    1998-09-01

    The AMPLICOR cytomegalovirus (CMV) test, a new qualitative assay for the detection of CMV DNA in plasma, was compared to conventional methods and quantitative PCR (Q-PCR) assays by using leukocytes and plasma from 179 blood samples from subjects with AIDS. For the diagnosis of CMV disease, cell-based assays such as a Q-PCR with polymorphonuclear leukocytes (Q-PCR-PMNL) and a pp65 antigenemia assay had the highest sensitivities but suffered from a lack of specificity. The best agreement between the results of the Q-PCR-PMNL assay and those of the AMPLICOR test was found when a threshold diagnostic value of 690 copies per 10(5) cells was selected for the Q-PCR-PMNL assay. In that context, the AMPLICOR CMV test had a sensitivity of 96.4% and a specificity of 95.3% when results were compared to results of the cell-based PCR assay. This threshold was close to the one described as associated with the best sensitivity and specificity for the diagnosis of CMV disease in a recently published study (4). Blood samples that tested positive by the Q-PCR-PMNL assay but negative by the AMPLICOR CMV test were associated with viral loads (mean, 785 copies, median, 96 copies per 10(5) leukocytes) lower than the viral loads of blood samples that tested positive by both assays (mean, 21,452 copies; median, 9,784 copies per 10(5) leukocytes) (P = 0.003). The AMPLICOR CMV test gave positive results at least 48 days before the development of symptomatic CMV disease in a longitudinal analysis of a limited subset of patients (n = 6) from whom sequential specimens were available for testing. In conclusion, the AMPLICOR CMV test is a very convenient assay combining rapidity, simplicity, and the possibility of batch testing. A positive result by this test seems particularly important since this implies, in most instances, the presence or the imminence of CMV disease, although a negative test result does not rule out disease.

  19. Evaluation of the AMPLICOR Cytomegalovirus Test with Specimens from Human Immunodeficiency Virus-Infected Subjects

    PubMed Central

    Boivin, Guy; Handfield, Julie; Toma, Emil; Murray, Gilles; Lalonde, Richard; Tevere, Vincent J.; Sun, Rita; Bergeron, Michel G.

    1998-01-01

    The AMPLICOR cytomegalovirus (CMV) test, a new qualitative assay for the detection of CMV DNA in plasma, was compared to conventional methods and quantitative PCR (Q-PCR) assays by using leukocytes and plasma from 179 blood samples from subjects with AIDS. For the diagnosis of CMV disease, cell-based assays such as a Q-PCR with polymorphonuclear leukocytes (Q-PCR-PMNL) and a pp65 antigenemia assay had the highest sensitivities but suffered from a lack of specificity. The best agreement between the results of the Q-PCR-PMNL assay and those of the AMPLICOR test was found when a threshold diagnostic value of 690 copies per 105 cells was selected for the Q-PCR-PMNL assay. In that context, the AMPLICOR CMV test had a sensitivity of 96.4% and a specificity of 95.3% when results were compared to results of the cell-based PCR assay. This threshold was close to the one described as associated with the best sensitivity and specificity for the diagnosis of CMV disease in a recently published study (4). Blood samples that tested positive by the Q-PCR-PMNL assay but negative by the AMPLICOR CMV test were associated with viral loads (mean, 785 copies, median, 96 copies per 105 leukocytes) lower than the viral loads of blood samples that tested positive by both assays (mean, 21,452 copies; median, 9,784 copies per 105 leukocytes) (P = 0.003). The AMPLICOR CMV test gave positive results at least 48 days before the development of symptomatic CMV disease in a longitudinal analysis of a limited subset of patients (n = 6) from whom sequential specimens were available for testing. In conclusion, the AMPLICOR CMV test is a very convenient assay combining rapidity, simplicity, and the possibility of batch testing. A positive result by this test seems particularly important since this implies, in most instances, the presence or the imminence of CMV disease, although a negative test result does not rule out disease. PMID:9705384

  20. Vaccine-Derived Neutralizing Antibodies to the Human Cytomegalovirus gH/gL Pentamer Potently Block Primary Cytotrophoblast Infection

    PubMed Central

    Chiuppesi, Flavia; Wussow, Felix; Johnson, Erica; Bian, Chao; Zhuo, Meng; Rajakumar, Augustine; Barry, Peter A.; Britt, William J.; Chakraborty, Rana

    2015-01-01

    ABSTRACT Human cytomegalovirus (HCMV) elicits neutralizing antibodies (NAb) of various potencies and cell type specificities to prevent HCMV entry into fibroblasts (FB) and epithelial/endothelial cells (EpC/EnC). NAb targeting the major essential envelope glycoprotein complexes gB and gH/gL inhibit both FB and EpC/EnC entry. In contrast to FB infection, HCMV entry into EpC/EnC is additionally blocked by extremely potent NAb to conformational epitopes of the gH/gL/UL128/130/131A pentamer complex (PC). We recently developed a vaccine concept based on coexpression of all five PC subunits by a single modified vaccinia virus Ankara (MVA) vector, termed MVA-PC. Vaccination of mice and rhesus macaques with MVA-PC resulted in a high titer and sustained NAb that blocked EpC/EnC infection and lower-titer NAb that inhibited FB entry. However, antibody function responsible for the neutralizing activity induced by the MVA-PC vaccine is uncharacterized. Here, we demonstrate that MVA-PC elicits NAb with cell type-specific neutralization potency and antigen recognition pattern similar to human NAb targeting conformational and linear epitopes of the UL128/130/131A subunits or gH. In addition, we show that the vaccine-derived PC-specific NAb are significantly more potent than the anti-gH NAb to prevent HCMV spread in EpC and infection of human placental cytotrophoblasts, cell types thought to be of critical importance for HCMV transmission to the fetus. These findings further validate MVA-PC as a clinical vaccine candidate to elicit NAb that resembles those induced during HCMV infection and provide valuable insights into the potency of PC-specific NAb to interfere with HCMV cell-associated spread and infection of key placental cells. IMPORTANCE As a consequence of the leading role of human cytomegalovirus (HCMV) in causing permanent birth defects, developing a vaccine against HCMV has been assigned a major public health priority. We have recently introduced a vaccine strategy based

  1. Vaccine-Derived Neutralizing Antibodies to the Human Cytomegalovirus gH/gL Pentamer Potently Block Primary Cytotrophoblast Infection.

    PubMed

    Chiuppesi, Flavia; Wussow, Felix; Johnson, Erica; Bian, Chao; Zhuo, Meng; Rajakumar, Augustine; Barry, Peter A; Britt, William J; Chakraborty, Rana; Diamond, Don J

    2015-12-01

    Human cytomegalovirus (HCMV) elicits neutralizing antibodies (NAb) of various potencies and cell type specificities to prevent HCMV entry into fibroblasts (FB) and epithelial/endothelial cells (EpC/EnC). NAb targeting the major essential envelope glycoprotein complexes gB and gH/gL inhibit both FB and EpC/EnC entry. In contrast to FB infection, HCMV entry into EpC/EnC is additionally blocked by extremely potent NAb to conformational epitopes of the gH/gL/UL128/130/131A pentamer complex (PC). We recently developed a vaccine concept based on coexpression of all five PC subunits by a single modified vaccinia virus Ankara (MVA) vector, termed MVA-PC. Vaccination of mice and rhesus macaques with MVA-PC resulted in a high titer and sustained NAb that blocked EpC/EnC infection and lower-titer NAb that inhibited FB entry. However, antibody function responsible for the neutralizing activity induced by the MVA-PC vaccine is uncharacterized. Here, we demonstrate that MVA-PC elicits NAb with cell type-specific neutralization potency and antigen recognition pattern similar to human NAb targeting conformational and linear epitopes of the UL128/130/131A subunits or gH. In addition, we show that the vaccine-derived PC-specific NAb are significantly more potent than the anti-gH NAb to prevent HCMV spread in EpC and infection of human placental cytotrophoblasts, cell types thought to be of critical importance for HCMV transmission to the fetus. These findings further validate MVA-PC as a clinical vaccine candidate to elicit NAb that resembles those induced during HCMV infection and provide valuable insights into the potency of PC-specific NAb to interfere with HCMV cell-associated spread and infection of key placental cells. As a consequence of the leading role of human cytomegalovirus (HCMV) in causing permanent birth defects, developing a vaccine against HCMV has been assigned a major public health priority. We have recently introduced a vaccine strategy based on a widely used

  2. Baculoviruses deficient in ie1 gene function abrogate viral gene expression in transduced mammalian cells

    SciTech Connect

    Efrose, Rodica; Swevers, Luc; Iatrou, Kostas

    2010-10-25

    One of the newest niches for baculoviruses-based technologies is their use as vectors for mammalian cell transduction and gene therapy applications. However, an outstanding safety issue related to such use is the residual expression of viral genes in infected mammalian cells. Here we show that infectious baculoviruses lacking the major transcriptional regulator, IE1, can be produced in insect host cells stably transformed with IE1 expression constructs lacking targets of homologous recombination that could promote the generation of wt-like revertants. Such ie1-deficient baculoviruses are unable to direct viral gene transcription to any appreciable degree and do not replicate in normal insect host cells. Most importantly, the residual viral gene expression, which occurs in mammalian cells infected with wt baculoviruses is reduced 10 to 100 fold in cells infected with ie1-deficient baculoviruses. Thus, ie1-deficient baculoviruses offer enhanced safety features to baculovirus-based vector systems destined for use in gene therapy applications.

  3. Human cytomegalovirus decreases constitutive transcription of MHC class II genes in mature Langerhans cells by reducing CIITA transcript levels.

    PubMed

    Lee, Andrew W; Wang, Nan; Hornell, Tara M C; Harding, James J; Deshpande, Chetan; Hertel, Laura; Lacaille, Vashti; Pashine, Achal; Macaubas, Claudia; Mocarski, Edward S; Mellins, Elizabeth D

    2011-05-01

    Human cytomegalovirus (HCMV) productively infects CD34(+) progenitor-derived, mature Langerhans-type dendritic cells (matLC) and reduces surface expression of MHC class II complexes (MHC II) by increasing intracellular retention of these molecules. To determine whether HCMV also inhibits MHC II expression by other mechanisms, we assessed mRNA levels of the class II transcriptional regulator, CIITA, and several of its target genes in infected matLC. Levels of CIITA, HLA-DRA (DRA) and DRB transcripts, and new DR protein synthesis were compared in mock-infected and HCMV-infected cells by quantitative PCR and pulse-chase immunoprecipitation analyses, respectively. CIITA mRNA levels were significantly lower in HCMV-infected matLC as compared to mock-infected cells. When assessed in the presence of Actinomycin D, the stability of CIITA transcripts was not diminished by HCMV. Analysis of promoter-specific CIITA isoforms revealed that types I, III and IV all were decreased by HCMV, a result that differs from changes after incubation of these cells with lipopolysaccharide (LPS). Exposure to UV-inactivated virus failed to reduce CIITA mRNA levels, implicating de novo viral gene expression in this effect. HCMV-infected matLC also expressed lower levels of DR transcripts and reduced DR protein synthesis rates compared to mock-infected matLC. In summary, we demonstrate that HCMV infection of a human dendritic cell subset inhibits constitutive CIITA expression, most likely at the transcriptional level, resulting in reduced MHC II biosynthesis. We suggest this represents a new mechanism of modulation of mature LC by HCMV.

  4. Inactivation of retinoblastoma protein does not overcome the requirement for human cytomegalovirus UL97 in lamina disruption and nuclear egress.

    PubMed

    Reim, Natalia I; Kamil, Jeremy P; Wang, Depeng; Lin, Alison; Sharma, Mayuri; Ericsson, Maria; Pesola, Jean M; Golan, David E; Coen, Donald M

    2013-05-01

    Human cytomegalovirus (HCMV) encodes one conventional protein kinase, UL97. During infection, UL97 phosphorylates the retinoblastoma tumor suppressor protein (pRb) on sites ordinarily phosphorylated by cyclin-dependent kinases (CDK), inactivating the ability of pRb to repress host genes required for cell cycle progression to S phase. UL97 is important for viral DNA synthesis in quiescent cells, but this function can be replaced by human papillomavirus type 16 E7, which targets pRb for degradation. However, viruses in which E7 replaces UL97 are still defective for virus production. UL97 is also required for efficient nuclear egress of viral nucleocapsids, which is associated with disruption of the nuclear lamina during infection, and phosphorylation of lamin A/C on serine 22, which antagonizes lamin polymerization. We investigated whether inactivation of pRb might overcome the requirement of UL97 for these roles, as pRb inactivation induces CDK1, and CDK1 phosphorylates lamin A/C on serine 22. We found that lamin A/C serine 22 phosphorylation during HCMV infection correlated with expression of UL97 and was considerably delayed in UL97-null mutants, even when E7 was expressed. E7 failed to restore gaps in the nuclear lamina seen in wild-type but not UL97-null virus infections. In electron microscopy analyses, a UL97-null virus expressing E7 was as impaired as a UL97-null mutant in cytoplasmic accumulation of viral nucleocapsids. Our results demonstrate that pRb inactivation is insufficient to restore efficient viral nuclear egress of HCMV in the absence of UL97 and instead argue further for a direct role of UL97 in this stage of the infectious cycle.

  5. Human cytomegalovirus inhibits apoptosis by proteasome-mediated degradation of Bax at endoplasmic reticulum-mitochondrion contacts.

    PubMed

    Zhang, Aiping; Hildreth, Richard L; Colberg-Poley, Anamaris M

    2013-05-01

    Human cytomegalovirus (HCMV) encodes the UL37 exon 1 protein (pUL37x1), which is the potent viral mitochondrion-localized inhibitor of apoptosis (vMIA), to increase survival of infected cells. HCMV vMIA traffics from the endoplasmic reticulum (ER) to ER subdomains, which are physically linked to mitochondria known as mitochondrion-associated membranes (MAM), and to mitochondria. The antiapoptotic function of vMIA is thought to primarily result from its ability to inhibit Bax-mediated permeabilization of the outer mitochondrial membrane (OMM). Here, we establish that vMIA retargets Bax to the MAM as well as to the OMM from immediate early through late times of infection. However, MAM localization of Bax results in its increased ubiquitination and proteasome-mediated degradation. Surprisingly, HCMV infection does not increase OMM-associated degradation (OMMAD) of Bax, even though the ER and mitochondria are physically connected at the MAM. It was recently found that lipid rafts at the plasma membrane can connect extrinsic and intrinsic apoptotic pathways and can serve as sites of apoptosome assembly. In transfected permissive human fibroblasts, vMIA mediates, through its cholesterol affinity, association of Bax and apoptosome components with MAM lipid rafts. While Bax association with MAM lipid rafts was detected in HCMV-infected cells, association of apoptosome components was not. These results establish that Bax recruitment to the MAM and its MAM-associated degradation (MAMAD) are a newly described antiapoptotic mechanism used by HCMV infection to increase cell survival for its growth.

  6. Human cytomegalovirus TRS1 protein associates with the 7-methylguanosine mRNA cap and facilitates translation.

    PubMed

    Ziehr, Benjamin; Lenarcic, Erik; Vincent, Heather A; Cecil, Chad; Garcia, Benjamin; Shenk, Thomas; Moorman, Nathaniel J

    2015-06-01

    Viruses rely on the host translation machinery for the synthesis of viral proteins. Human cells have evolved sensors that recognize viral RNAs and inhibit mRNA translation in order to limit virus replication. Understanding how viruses manipulate the host translation machinery to gain access to ribosomes and disable the antiviral response is therefore a critical aspect of the host/pathogen interface. In this study, we used a proteomics approach to identify human cytomegalovirus (HCMV) proteins that might contribute to viral mRNA translation. The HCMV TRS1 protein (pTRS1) associated with the 7-methylguanosine mRNA cap, increased the total level of protein synthesis, and colocalized with mRNAs undergoing translation initiation during infection. pTRS1 stimulated translation of a nonviral reporter gene and increased the translation of a reporter containing an HCMV 5' untranslated region (5'UTR) to a greater extent. The preferential effect of pTRS1 on translation of an mRNA containing a viral 5'UTR required the pTRS1 RNA and double-stranded RNA-dependent protein kinase (PKR)-binding domains, and was likely the result of PKR inhibition. However, pTRS1 also stimulated the total level of protein synthesis and translation directed by an HCMV 5'UTR in cells lacking PKR. Thus our results demonstrate that pTRS1 stimulates translation through both PKR-dependent and PKR-independent mechanisms. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Characterization of human cytomegalovirus UL145 and UL136 genes in low-passage clinical isolates from infected Chinese infants

    PubMed Central

    Wang, Bo; Hu, Jing-Jing; Yan, Cui-Fang; Su, Hai-Hao; Ding, Jun-Cai; Guo, Yuan-Yuan; Ye, Ning; Huang, Shui-Qing; Zhang, Xiao-Zhuang; Zhou, Shu-Feng

    2011-01-01

    Summary Background Human cytomegalovirus (HCMV) is a leading cause of morbidity and mortality in immunocompromised individuals. The unique long b’ (ULB’) region of HCMV contains at least 19 open reading frames (ORFs); however, little is known about the function of UL145 and UL136. We characterized UL145 and UL136 in low-passage clinical isolates from Chinese infants. Material/Methods The clinical strains of HCMV were recovered from the urine from HCMV-infected infants. Human embryonic lung fibroblasts (HELFs) were infected with clinical isolates of HCMV, and the viral DNA and mRNA for UL145 and UL136 were analyzed by polymerase chain reaction (PCR) and sequencing techniques. We also predicted the structure and function of UL145 and UL136 proteins. Results Sixty-two Chinese infants infected with HCMV were recruited into this study and the clinical isolates were recovered from the urine. Two strains among the low-passage isolates, D2 and D3, were obtained. The UL145 and UL136 sequences were deposited with GenBank under accession numbers of DQ180367, DQ180381, DQ180377, and DQ180389. The mRNA expression of both UL145 and UL136 was confirmed by reverse transcription (RT-PCR) assays. UL145 was predicted to contain 1 protein kinase C phosphorylation site, 2 casein kinase II phosphorylation sites and a zinc finger structure. UL136 was predicted to contain a protein kinase C phosphorylation site, N-myristoylation site, cAMP- and cGMP-dependent protein kinase phosphorylation site and tyrosine kinase II phosphorylation site. Both UL145 and UL136 are highly conserved. Conclusions UL145 may act as an intranuclear regulating factor by direct binding to DNA, while UL136 may be a membrane receptor involving signal transduction. PMID:21804461

  8. Human cytomegalovirus interleukin-10 polarizes monocytes toward a deactivated M2c phenotype to repress host immune responses.

    PubMed

    Avdic, Selmir; Cao, John Z; McSharry, Brian P; Clancy, Leighton E; Brown, Rebecca; Steain, Megan; Gottlieb, David J; Abendroth, Allison; Slobedman, Barry

    2013-09-01

    Several human cytomegalovirus (HCMV) genes encode products that modulate cellular functions in a manner likely to enhance viral pathogenesis. This includes UL111A, which encodes homologs of human interleukin-10 (hIL-10). Depending upon signals received, monocytes and macrophages become polarized to either classically activated (M1 proinflammatory) or alternatively activated (M2 anti-inflammatory) subsets. Skewing of polarization toward an M2 subset may benefit the virus by limiting the proinflammatory responses to infection, and so we determined whether HCMV-encoded viral IL-10 influenced monocyte polarization. Recombinant viral IL-10 protein polarized CD14(+) monocytes toward an anti-inflammatory M2 subset with an M2c phenotype, as demonstrated by high expression of CD163 and CD14 and suppression of major histocompatibility complex (MHC) class II. Significantly, in the context of productive HCMV infection, viral IL-10 produced by infected cells polarized uninfected monocytes toward an M2c phenotype. We also assessed the impact of viral IL-10 on heme oxygenase 1 (HO-1), which is an enzyme linked with suppression of inflammatory responses. Polarization of monocytes by viral IL-10 resulted in upregulation of HO-1, and inhibition of HO-1 function resulted in a loss of capacity of viral IL-10 to suppress tumor necrosis factor alpha (TNF-α) and IL-1β, implicating HO-1 in viral IL-10-induced suppression of proinflammatory cytokines by M2c monocytes. In addition, a functional consequence of monocytes polarized with viral IL-10 was a decreased capacity to activate CD4(+) T cells. This study identifies a novel role for viral IL-10 in driving M2c polarization, which may limit virus clearance by restricting proinflammatory and CD4(+) T cell responses at sites of infection.

  9. Human cytomegalovirus decreases constitutive transcription of MHC class II genes in mature Langerhans cells by reducing CIITA transcript levels

    PubMed Central

    Lee, Andrew W.; Wang, Nan; Hornell, Tara M.C.; Harding, James J.; Deshpande, Chetan; Hertel, Laura; Lacaille, Vashti; Pashine, Achal; Macaubas, Claudia; Mocarski, Edward S.; Mellins, Elizabeth D.

    2011-01-01

    Human cytomegalovirus (HCMV) productively infects CD34+ progenitor-derived, mature Langerhans-type dendritic cells (matLC) and reduces surface expression of MHC class II complexes (MHC II) by increasing intracellular retention of these molecules. To determine whether HCMV also inhibits MHC II expression by other mechanisms, we assessed mRNA levels of the class II transcriptional regulator, CIITA, and several of its target genes in infected matLC. Levels of CIITA, HLA-DRA (DRA) and DRB transcripts, and new DR protein synthesis were compared in mock-infected and HCMV-infected cells by quantitative PCR and pulse-chase immunoprecipitation analyses, respectively. CIITA mRNA levels were significantly lower in HCMV-infected matLC as compared to mock-infected cells. When assessed in the presence of Actinomycin D, the stability of CIITA transcripts was not diminished by HCMV. Analysis of promoter-specific CIITA isoforms revealed that types I, III and IV all were decreased by HCMV, a result that differs from changes after incubation of these cells with lipopolysaccharide (LPS). Exposure to UV-inactivated virus failed to reduce CIITA mRNA levels, implicating de novo viral gene expression in this effect. HCMV-infected matLC also expressed lower levels of DR transcripts and reduced DR protein synthesis rates compared to mock-infected matLC. In summary, we demonstrate that HCMV infection of a human dendritic cell subset inhibits constitutive CIITA expression, most likely at the transcriptional level, resulting in reduced MHC II biosynthesis. We suggest this represents a new mechanism of modulation of mature LC by HCMV. PMID:21458073

  10. An Endothelial Cell-Specific Requirement for the UL133-UL138 Locus of Human Cytomegalovirus for Efficient Virus Maturation

    PubMed Central

    Bughio, Farah; Elliott, David A.

    2013-01-01

    Human cytomegalovirus (HCMV) infects a variety of cell types in humans, resulting in a varied pathogenesis in the immunocompromised host. Endothelial cells (ECs) are considered an important target of HCMV infection that may contribute to viral pathogenesis. Although the viral determinants important for entry into ECs are well defined, the molecular determinants regulating postentry tropism in ECs are not known. We previously identified the UL133-UL138 locus encoded within the clinical strain-specific ULb′ region of the HCMV genome as important for the latent infection in CD34+ hematopoietic progenitor cells (HPCs). Interestingly, this locus, while dispensable for replication in fibroblasts, was required for efficient replication in ECs infected with the TB40E or fusion-inducing factor X (FIX) HCMV strains. ECs infected with a virus lacking the entire locus (UL133-UL138NULL virus) complete the immediate-early and early phases of infection but are defective for infectious progeny virus production. ECs infected with UL133-UL138NULL virus exhibited striking differences in the organization of intracellular membranes and in the assembly of mature virions relative to ECs infected with wild-type (WT) virus. In UL133-UL138NULL virus-infected ECs, Golgi stacks were disrupted, and the viral assembly compartment characteristic of HCMV infection failed to form. Further, progeny virions in UL133-UL138NULL virus-infected ECs inefficiently acquired the virion tegument and secondary envelope. These defects were specific to infection in ECs and not observed in fibroblasts infected with UL133-UL138NULL virus, suggesting an EC-specific requirement for the UL133-UL138 locus for late stages of replication. To our knowledge, the UL133-UL138 locus represents the first cell-type-dependent, postentry tropism determinant required for viral maturation. PMID:23283945

  11. Human cytomegalovirus infant infection adversely affects growth and development in maternally HIV-exposed and unexposed infants in Zambia.

    PubMed

    Gompels, U A; Larke, N; Sanz-Ramos, M; Bates, M; Musonda, K; Manno, D; Siame, J; Monze, M; Filteau, S

    2012-02-01

    Human immunodeficiency virus (HIV) and human cytomegalovirus (HCMV) coinfections have been shown to increase infant morbidity, mortality, and AIDS progression. In HIV-endemic regions, maternal HIV-exposed but HIV-uninfected infants, which is the majority of children affected by HIV, also show poor growth and increased morbidity. Although nutrition has been examined, the effects of HCMV infection have not been evaluated. We studied the effects of HCMV infection on the growth, development, and health of maternally HIV-exposed and unexposed infants in Zambia. Infants were examined in a cohort recruited to a trial of micronutrient-fortified complementary foods. HIV-infected mothers and infants had received perinatal antiretroviral therapy to prevent mother-to-child HIV transmission. Growth, development, and morbidity were analyzed by linear regression analyses in relation to maternal HIV exposure and HCMV infection, as screened by sera DNA for viremia at 6 months of age and by antibody for infection at 18 months. All HCMV-seropositive infants had decreased length-for-age by 18 months compared with seronegative infants (standard deviation [z]-score difference: -0.44 [95% confidence interval {CI}, -.72 to -.17]; P = .002). In HIV-exposed infants, those who were HCMV positive compared with those who were negative, also had reduced head size (mean z-score difference: -0.72 [95% CI, -1.23 to -.22]; P = .01) and lower psychomotor development (Bayley test score difference: -4.1 [95% CI, -7.8 to -.5]; P = .03). HIV-exposed, HCMV-viremic infants were more commonly referred for hospital treatment than HCMV-negative infants. The effects of HCMV were unaffected by micronutrient fortification. HCMV affects child growth, development, and morbidity of African infants, particularly in those maternally exposed to HIV. HCMV is therefore a risk factor for child health in this region.

  12. Nucleosome maps of the human cytomegalovirus genome reveal a temporal switch in chromatin organization linked to a major IE protein.

    PubMed

    Zalckvar, Einat; Paulus, Christina; Tillo, Desiree; Asbach-Nitzsche, Alexandra; Lubling, Yaniv; Winterling, Carla; Strieder, Nicholas; Mücke, Katrin; Goodrum, Felicia; Segal, Eran; Nevels, Michael

    2013-08-06

    Human CMV (hCMV) establishes lifelong infections in most of us, causing developmental defects in human embryos and life-threatening disease in immunocompromised individuals. During productive infection, the viral >230,000-bp dsDNA genome is expressed widely and in a temporal cascade. The hCMV genome does not carry histones when encapsidated but has been proposed to form nucleosomes after release into the host cell nucleus. Here, we present hCMV genome-wide nucleosome occupancy and nascent transcript maps during infection of permissive human primary cells. We show that nucleosomes occupy nuclear viral DNA in a nonrandom and highly predictable fashion. At early times of infection, nucleosomes associate with the hCMV genome largely according to their intrinsic DNA sequence preferences, indicating that initial nucleosome formation is genetically encoded in the virus. However, as infection proceeds to the late phase, nucleosomes redistribute extensively to establish patterns mostly determined by nongenetic factors. We propose that these factors include key regulators of viral gene expression encoded at the hCMV major immediate-early (IE) locus. Indeed, mutant virus genomes deficient for IE1 expression exhibit globally increased nucleosome loads and reduced nucleosome dynamics compared with WT genomes. The temporal nucleosome occupancy differences between IE1-deficient and WT viruses correlate inversely with changes in the pattern of viral nascent and total transcript accumulation. These results provide a framework of spatial and temporal nucleosome organization across the genome of a major human pathogen and suggest that an hCMV major IE protein governs overall viral chromatin structure and function.

  13. Identification and characterization of a putative baculoviral transcriptional factor IE-1 from Choristoneura fumiferana granulovirus.

    PubMed

    Rashidan, Kianoush Khajeh; Nassoury, Nasha; Merzouki, Abderrazzak; Guertin, Claude

    2002-11-30

    A gene that encodes a protein homologue to baculoviral IE-1 was identified and sequenced in the genome of the Choristoneura fumiferana granulovirus (ChfuGV). The gene has an 1278 nucleotide (nt) open-reading frame (ORF) that encodes 426 amino acids with an estimated molecular weight of 50.33 kDa. At the nucleotide level, several cis-acting regulatory elements were detected within the promoter region of the ie-1 gene of ChfuGV along with other studied granuloviruses (GVs). Two putative CCAAT elements were detected within the noncoding leader region of this gene; one was located on the opposite strand at -92 and the other at -420 nt from the putative start triplet. Two baculoviral late promoter motifs (TAAG) were also detected within the promoter region of the ie-1 gene of ChfuGV. A single polyadenylation signal, AATAAA, was located 18nt downstream of the putative translational stop codon of ie-1 from ChfuGV. At the protein level, the amino acid sequence data that was derived from the nucleotide sequence in ChfuGV IE-1 was compared to those of the Cydia pomonella granulovirus (CpGV), Xestia c-nigrum granulovirus (XcGV) and Plutella xylostella granulovirus (PxGV). The C-terminal regions of the granuloviral IE-1 sequences appeared to be more conserved when compared to the N-terminal regions. A domain, similar to the basic helix-loop-helix like (bHLH-like) domain in NPVs, was detected at the C-terminal region of IE-1 from ChfuGV (residues 387 to 414). A phylogenetic tree for baculoviral IE-1 was constructed using a maximum parsimony analysis. A phylogenetic estimation demonstrates that ChfuGV IE-1 is most closely related to that of CpGV.

  14. Human cytomegalovirus glycoprotein complex gH/gL/gO uses PDGFR-α as a key for entry.

    PubMed

    Wu, Yiquan; Prager, Adrian; Boos, Simone; Resch, Moritz; Brizic, Ilija; Mach, Michael; Wildner, Sabrina; Scrivano, Laura; Adler, Barbara

    2017-04-01

    Herpesvirus gH/gL envelope glycoprotein complexes are key players in virus entry as ligands for host cell receptors and by promoting fusion of viral envelopes with cellular membranes. Human cytomegalovirus (HCMV) has two alternative gH/gL complexes, gH/gL/gO and gH/gL/UL128,130,131A which both shape the HCMV tropism. By studying binding of HCMV particles to fibroblasts, we could for the first time show that virion gH/gL/gO binds to platelet-derived growth factor-α (PDGFR-α) on the surface of fibroblasts and that gH/gL/gO either directly or indirectly recruits gB to this complex. PDGFR-α functions as an entry receptor for HCMV expressing gH/gL/gO, but not for HCMV mutants lacking the gH/gL/gO complex. PDGFR-α-dependent entry is not dependent on activation of PDGFR-α. We could also show that the gH/gL/gO-PDGFR-α interaction starts the predominant entry pathway for infection of fibroblasts with free virus. Cell-associated virus spread is either driven by gH/gL/gO interacting with PDGFR-α or by the gH/gL/UL128,130,131A complex. PDGFR-α-positive cells may thus be preferred first target cells for infections with free virus which might have implications for the design of future HCMV vaccines or anti-HCMV drugs.

  15. [Epidemiological evaluations of human immunodeficiency virus, herpes simplex virus type 1 and 2 and cytomegalovirus infections in drug addicts].

    PubMed

    Sagnelli, E; Filippini, P; Guarino, M; Borrelli, G; Aprea, L; Malafronte, G; Felaco, F M; Piccinino, F; Giusti, G

    1989-01-01

    Eighty-eight drug addicts from the "BAN Center" in Torre Annunziata (Naples) and 88 normal subjects pair-matched for age and sex were tested for IgG to human immunodeficiency virus (HIV), herpes simplex virus (HSV) type 1 and 2 and cytomegalovirus (CMV). A high prevalence of subjects with antibodies to HSV-1 and CMV (80.7% and 65.9%) were recorded in the control group testifying to the high level of these infections in Campania. Prevalences were higher in drug addicts, and drug abuse was identified as a risk factor for the acquisition of CMV infection (odds ratio = 2.3). Moreover, drug addiction is also a risk factor for HSV-2 and HIV infection as demonstrated by the observation that drug abusers were anti-HSV-2 (9.1 vs. 1.1%, odds ratio = 6.16) or anti-HIV (11.4 vs. 0%, odds ratio = 23.6) positive more frequently than normal controls. Thus, drug addiction is a risk factor for the acquisition of HIV, HSV-2 and CMV infections. This is probably due to similar habits, frequent among drug addicts from our geographic area and uncommon in the normal population, such as tattooing, needle-sharing needlestick and unsafe sex. Some of these habits, such as unsafe sex and tattooing, seem to be, per se, risk factors for the acquisition of both HIV and CMV infections. The data also suggest that HIV infection was probably introduced in Campania more recently than in northern and central Italy where the prevalence of anti-HIV positive cases among drug addicts is definitely higher.

  16. The ULb′ Region of the Human Cytomegalovirus Genome Confers an Increased Requirement for the Viral Protein Kinase UL97

    PubMed Central

    Wang, Depeng; Li, Gang; Schauflinger, Martin; Nguyen, Christopher C.; Hall, Ellie D.; Yurochko, Andrew D.; von Einem, Jens

    2013-01-01

    We report a requirement for the viral protein kinase UL97 in human cytomegalovirus (HCMV) replication that maps to the ULb′ region of the viral genome. A UL97-null (Δ97) mutant of strain TB40/E, which encodes a full-length ULb′ region, exhibited replication defects, particularly in production of cell-free virus, that were more severe than those seen with a Δ97 mutant of laboratory strain AD169, which harbors extensive deletions in its ULb′ region. These differences were recapitulated with additional HCMV strains by treatment with a UL97 kinase inhibitor, 1-(β-l-ribofuranosyl)-2-isopropylamino-5,6-dichlorobenzimidazole (maribavir). We observed lower levels of viral DNA synthesis and an increased requirement for UL97 in viral late gene expression in strains with full-length ULb′ regions. Analysis of UL97-deficient TB40/E infections by electron microscopy revealed fewer C-capsids in nuclei, unusual viral particles in the cytoplasmic assembly compartment, and defective viral nuclear egress. Partial inhibition of viral DNA synthesis caused defects in production of cell-free virus that were up to ∼100-fold greater than those seen with cell-associated virus in strains TB40/E and TR, suggesting that UL97-dependent defects in cell-free virus production in strains with full-length ULb′ regions were secondary to DNA synthesis defects. Accordingly, a chimeric virus in which the ULb′ region of TB40/E was replaced with that of AD169 showed reduced effects of UL97 inhibition on viral DNA synthesis, late gene expression, and production of cell-free virus compared to parental TB40/E. Together, these results argue that the ULb′ region encodes a factor(s) which invokes an increased requirement for UL97 during viral DNA synthesis. PMID:23536674

  17. Study of human cytomegalovirus replication in body fluids, placental infection, and miscarriage during the first trimester of pregnancy.

    PubMed

    Yan, Xiao Chuan; Wang, Jian Hua; Wang, Bo; Huang, Li Li; Zhou, Li Qin; Zhu, Bo; Liang, Yun

    2015-06-01

    Intrauterine infection caused by human cytomegalovirus (HCMV) can lead to embryo, fetal, and neonatal damage. The prevalence of HCMV replication in body fluids (blood, urine, and cervicovaginal secretion) was investigated, and its effects on HCMV vertical transmission and miscarriages in early pregnant women were evaluated. HCMV DNA in body fluids was detected in 1,064 early pregnant women (624 normal pregnancies and 440 miscarriages). There were 101 cases who were HCMV DNA positive in cervicovaginal secretion and the rates were 10.9% (48/440 cases) and 8.5% (53/624 cases) in miscarriages and normal pregnancies, respectively (P > 0.05). A total of 101 cases (63 and 38 cases with and without HCMV DNA in cervicovaginal secretion, respectively) were given HCMV DNA detection in placental villi/deciduas. There were five cases (7.9%; two normal pregnancies and three miscarriages) with HCMV DNA in placental villi/deciduas among the 63 cases with HCMV DNA in cervicovaginal secretion, whereas none of the other 38 cases were detected HCMV DNA positive in their placental villi/deciduas. The percentage of HCMV DNA in placental villi/deciduas was higher in miscarriage group (9.1% [3/33]) than that in the normal pregnancy group (6.7% [2/30]), but there was no statistical significance (P > 0.05). Two cases with a higher HCMV loads in cervicovaginal secretion and placental villi/deciduas had miscarriages. These findings suggest that HCMV replication in cervicovaginal secretion can involve in placental HCMV infection, and high HCMV DNA loads in cervicovaginal secretion and placental villi/deciduas are associated with miscarriage. © 2015 Wiley Periodicals, Inc.

  18. Baseline antibody level may help predict the risk of active human cytomegalovirus infection in a HCMV seropositive population.

    PubMed

    Li, T-D; Li, J-J; Huang, X; Wang, H; Guo, X-Y; Ge, S-X; Zhang, J

    2017-05-01

    Recurrent human cytomegalovirus (HCMV) infection during pregnancy could lead to congenital HCMV infection and permanent sequelae. However, knowledge regarding the risk factors associated with recurrent HCMV infection is limited. In the present study, 1,659 paired serum samples from the natural population were collected in Guangxi Province, China, from 2003 to 2004 with a 1-year interval. The serum anti-pp150 titre was quantitatively determined using a homemade recombinant pp150-based ELISA, and the IgG titre that increased at least 4-fold was defined as a recurrent infection. The HCMV seroprevalence was above 98.6% (1,636/1,659) in Guangxi in 2003, and the infection rate during the 1-year follow-up was approximately 10% (171/1,659). The seronegative population has the highest infection risk, while the risk of recurrent infection in the seropositive population was negatively correlated with the baseline anti-pp150 titre. With a cutoff of 1:80 (the baseline anti-pp150 IgG titre), the sensitivity and specificity were 73.1% (125/171) and 85.7% (1,275/1,488) respectively, and the relative risk of infection in the high-risk group compared to the low-risk group was 10.6 (95% CI: 7.7-14.6). In conclusion, the baseline anti-pp150 IgG was negatively correlated with the risk of HCMV infection and could be an excellent predictor of HCMV infection in HCMV seropositive populations.

  19. RhoB is a component of the human cytomegalovirus assembly complex and is required for efficient viral production

    PubMed Central

    Goulidaki, Nektaria; Alarifi, Saud; Alkahtani, Saad H; Al-Qahtani, Ahmed; Spandidos, Demetrios A; Stournaras, Christos; Sourvinos, George

    2015-01-01

    Human Cytomegalovirus (HCMV), an ubiquitous β-herpesvirus, is a significant pathogen that causes medically severe diseases in immunocompromised individuals and in congenitally infected neonates. RhoB belongs to the family of Rho GTPases, which regulates diverse cellular processes. Rho proteins are implicated in the entry and egress from the host cell of mainly α- and γ-herpesviruses, whereas β-herpesviruses are the least studied in this regard. Here, we studied the role of RhoB GTPase during HCMV lytic infection. Microscopy analysis, both in fixed and live infected cells showed that RhoB was translocated to the assembly complex/compartment (AC) of HCMV, a cytoplasmic zone in infected cells where many viral structural proteins are known to accumulate and assembly of new virions takes place. Furthermore, RhoB was localized at the AC even when the expression of the late HCMV AC proteins was inhibited. At the very late stages of infection, cellular projections were formed containing RhoB and HCMV virions, potentially contributing to the successful viral spread. Interestingly, the knockdown of RhoB in HCMV-infected cells resulted in a significant reduction of the virus titer and could also affect the accumulation of AC viral proteins at this subcellular compartment. RhoB knockdown also affected actin fibers' structure. Actin reorganization was observed at late stages of infection originating from the viral AC and surrounding the cellular projections, implying a potential interplay between RhoB and actin during HCMV assembly and egress. In conclusion, our results demonstrate for the first time that RhoB is a constituent of the viral AC and is required for HCMV productive infection. PMID:26114383

  20. TLR9 -1486T/C and 2848C/T SNPs Are Associated with Human Cytomegalovirus Infection in Infants

    PubMed Central

    Paradowska, Edyta; Jabłońska, Agnieszka; Studzińska, Mirosława; Skowrońska, Katarzyna; Suski, Patrycja; Wiśniewska-Ligier, Małgorzata; Woźniakowska-Gęsicka, Teresa; Nowakowska, Dorota; Gaj, Zuzanna; Wilczyński, Jan; Leśnikowski, Zbigniew J.

    2016-01-01

    Toll-like receptor 9 (TLR9) recognizes non-methylated viral CpG-containing DNA and serves as a pattern recognition receptor that signals the presence of human cytomegalovirus (HCMV). Here, we present the genotype distribution of single-nucleotide polymorphisms (SNPs) of the TLR9 gene in infants and the relationship between TLR9 polymorphisms and HCMV infection. Four polymorphisms (-1237T/C, rs5743836; -1486T/C, rs187084; 1174G/A, rs352139; and 2848C/T, rs352140) in the TLR9 gene were genotyped in 72 infants with symptomatic HCMV infection and 70 healthy individuals. SNP genotyping was performed by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Digested fragments were separated and identified by capillary electrophoresis. The HCMV DNA copy number was measured by a quantitative real-time PCR assay. We found an increased frequency of heterozygous genotypes TLR9 -1486T/C and 2848C/T in infants with HCMV infection compared with uninfected cases. Heterozygous variants of these two SNPs increased the risk of HCMV disease in children (P = 0.044 and P = 0.029, respectively). In infants with a mutation present in at least one allele of -1486T/C and 2848C/T SNPs, a trend towards increased risk of cytomegaly was confirmed after Bonferroni’s correction for multiple testing (Pc = 0.063). The rs352139 GG genotype showed a significantly reduced relative risk for HCMV infection (Pc = 0.006). In contrast, the -1237T/C SNP was not related to viral infection. We found no evidence for linkage disequilibrium with the four examined TLR9 SNPs. The findings suggest that the TLR9 -1486T/C and 2848C/T polymorphisms could be a genetic risk factor for the development of HCMV disease. PMID:27105145

  1. Human cytomegalovirus UL7, a homologue of the SLAM-family receptor CD229, impairs cytokine production.

    PubMed

    Engel, Pablo; Pérez-Carmona, Natàlia; Albà, M Mar; Robertson, Kevin; Ghazal, Peter; Angulo, Ana

    2011-10-01

    Human cytomegalovirus (HCMV), the β-herpesvirus prototype, has evolved a wide spectrum of mechanisms to counteract host immunity. Among them, HCMV uses cellular captured genes encoding molecules capable of interfering with the original host function or of fulfilling new immunomodulatory tasks. Here, we report on UL7, a novel HCMV heavily glycosylated transmembrane protein, containing an Ig-like domain that exhibits remarkable amino acid similarity to CD229, a cell-surface molecule of the signalling lymphocyte-activation molecule (SLAM) family involved in leukocyte activation. The UL7 Ig-like domain, which is well-preserved in all HCMV strains, structurally resembles the SLAM-family N-terminal Ig-variable domain responsible for the homophilic and heterophilic interactions that trigger signalling. UL7 is transcribed with early-late kinetics during the lytic infectious cycle. Using a mAb generated against the viral protein, we show that it is constitutively shed, through its mucine-like stalk, from the cell-surface. Production of soluble UL7 is enhanced by PMA and reduced by a broad-spectrum metalloproteinase inhibitor. Although UL7 does not hold the ability to interact with CD229 or other SLAM-family members, it shares with them the capacity to mediate adhesion to leukocytes, specifically to monocyte-derived DCs. Furthermore, we demonstrate that UL7 expression attenuates the production of proinflammatory cytokines TNF, IL-8 and IL-6 in DCs and myeloid cell lines. Thus, the ability of UL7 to interfere with cellular proinflammatory responses may contribute to viral persistence. These results enhance our understanding of those HCMV-encoded molecules involved in sustaining the balance between HCMV and the host immune system.

  2. Latent cytomegalovirus infection enhances anti‐tumour cytotoxicity through accumulation of NKG2C+ NK cells in healthy humans

    PubMed Central

    Bigley, A. B.; Rezvani, K.; Shah, N.; Sekine, T.; Balneger, N.; Pistillo, M.; Agha, N.; Kunz, H.; O'Connor, D. P.; Bollard, C. M.

    2016-01-01

    Summary Cytomegalovirus (CMV) infection markedly expands NKG2C+/NKG2A− NK cells, which are potent killers of infected cells expressing human leucocyte antigen (HLA)‐E. As HLA‐E is also over‐expressed in several haematological malignancies and CMV has been linked to a reduced risk of leukaemic relapse, we determined the impact of latent CMV infection on NK cell cytotoxicity against four tumour target cell lines with varying levels of HLA‐E expression. NK cell cytotoxicity against K562 (leukaemia origin) and U266 (multiple myeloma origin) target cells was strikingly greater in healthy CMV‐seropositive donors than seronegative donors and was associated strongly with target cell HLA‐E and NK cell NKG2C expression. NK cell cytotoxicity against HLA‐E transfected lymphoma target cells (221.AEH) was ∼threefold higher with CMV, while NK cell cytotoxicity against non‐transfected 721.221 cells was identical between the CMV groups. NK cell degranulation (CD107a+) and interferon (IFN)‐γ production to 221.AEH cells was localized almost exclusively to the NKG2C subset, and antibody blocking of NKG2C completely eliminated the effect of CMV on NK cell cytotoxicity against 221.AEH cells. Moreover, 221.AEH feeder cells and interleukin (IL)−15 were found to expand NKG2C+/NKG2A– NK cells preferentially from CMV‐seronegative donors and increase NK cell cytotoxicity against HLA‐E+ tumour cell lines. We conclude that latent CMV infection enhances NK cell cytotoxicity through accumulation of NKG2C+ NK cells, which may be beneficial in preventing the initiation and progression of haematological malignancies characterized by high HLA‐E expression. PMID:26940026

  3. Latent cytomegalovirus infection enhances anti-tumour cytotoxicity through accumulation of NKG2C+ NK cells in healthy humans.

    PubMed

    Bigley, A B; Rezvani, K; Shah, N; Sekine, T; Balneger, N; Pistillo, M; Agha, N; Kunz, H; O'Connor, D P; Bollard, C M; Simpson, R J

    2016-08-01

    Cytomegalovirus (CMV) infection markedly expands NKG2C+/NKG2A- NK cells, which are potent killers of infected cells expressing human leucocyte antigen (HLA)-E. As HLA-E is also over-expressed in several haematological malignancies and CMV has been linked to a reduced risk of leukaemic relapse, we determined the impact of latent CMV infection on NK cell cytotoxicity against four tumour target cell lines with varying levels of HLA-E expression. NK cell cytotoxicity against K562 (leukaemia origin) and U266 (multiple myeloma origin) target cells was strikingly greater in healthy CMV-seropositive donors than seronegative donors and was associated strongly with target cell HLA-E and NK cell NKG2C expression. NK cell cytotoxicity against HLA-E transfected lymphoma target cells (221.AEH) was ∼threefold higher with CMV, while NK cell cytotoxicity against non-transfected 721.221 cells was identical between the CMV groups. NK cell degranulation (CD107a(+) ) and interferon (IFN)-γ production to 221.AEH cells was localized almost exclusively to the NKG2C subset, and antibody blocking of NKG2C completely eliminated the effect of CMV on NK cell cytotoxicity against 221.AEH cells. Moreover, 221.AEH feeder cells and interleukin (IL)-15 were found to expand NKG2C(+) /NKG2A(-) NK cells preferentially from CMV-seronegative donors and increase NK cell cytotoxicity against HLA-E(+) tumour cell lines. We conclude that latent CMV infection enhances NK cell cytotoxicity through accumulation of NKG2C(+) NK cells, which may be beneficial in preventing the initiation and progression of haematological malignancies characterized by high HLA-E expression. © 2016 British Society for Immunology.

  4. Roles of polypyrimidine tract binding proteins in major immediate-early gene expression and viral replication of human cytomegalovirus.

    PubMed

    Cosme, Ruth S Cruz; Yamamura, Yasuhiro; Tang, Qiyi

    2009-04-01

    Human cytomegalovirus (HCMV), a member of the beta subgroup of the family Herpesviridae, causes serious health problems worldwide. HCMV gene expression in host cells is a well-defined sequential process: immediate-early (IE) gene expression, early-gene expression, DNA replication, and late-gene expression. The most abundant IE gene, major IE (MIE) gene pre-mRNA, needs to be spliced before being exported to the cytoplasm for translation. In this study, the regulation of MIE gene splicing was investigated; in so doing, we found that polypyrimidine tract binding proteins (PTBs) strongly repressed MIE gene production in cotransfection assays. In addition, we discovered that the repressive effects of PTB could be rescued by splicing factor U2AF. Taken together, the results suggest that PTBs inhibit MIE gene splicing by competing with U2AF65 for binding to the polypyrimidine tract in pre-mRNA. In intron deletion mutation assays and RNA detection experiments (reverse transcription [RT]-PCR and real-time RT-PCR), we further observed that PTBs target all the introns of the MIE gene, especially intron 2, and affect gene splicing, which was reflected in the variation in the ratio of pre-mRNA to mRNA. Using transfection assays, we demonstrated that PTB knockdown cells induce a higher degree of MIE gene splicing/expression. Consistently, HCMV can produce more viral proteins and viral particles in PTB knockdown cells after infection. We conclude that PTB inhibits HCMV replication by interfering with MIE gene splicing through competition with U2AF for binding to the polypyrimidine tract in MIE gene introns.

  5. The Essential Human Cytomegalovirus Gene UL52 Is Required for Cleavage-Packaging of the Viral Genome▿

    PubMed Central

    Borst, Eva Maria; Wagner, Karen; Binz, Anne; Sodeik, Beate; Messerle, Martin

    2008-01-01

    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. PMID:18077717

  6. Latent infection of myeloid progenitors by human cytomegalovirus protects cells from FAS-mediated apoptosis through the cellular IL-10/PEA-15 pathway.

    PubMed

    Poole, Emma; Lau, Jonathan C H; Sinclair, John

    2015-08-01

    Latent infection of primary CD34(+) progenitor cells by human cytomegalovirus (HCMV) results in their increased survival in the face of pro-apoptotic signals. For instance, we have shown previously that primary myeloid cells are refractory to FAS-mediated killing and that cellular IL-10 (cIL-10) is an important survival factor for this effect. However, how cIL-10 mediates this protection is unclear. Here, we have shown that cIL-10 signalling leading to upregulation of the cellular factor PEA-15 mediates latency-associated protection of CD34(+) progenitor cells from the extrinsic death pathway.

  7. Sequence of protein synthesis in cells infected by human cytomegalovirus: early and late virus-induced polypeptides.

    PubMed Central

    Stinski, M F

    1978-01-01

    At least 10 distinct early virus-induced polypeptides were synthesized within 0 to 6 h after infection of permissive cells with cytomegalovirus. These virus-induced polypeptides were synthesized before and independently of viral DNA replication. A majority of these early virus-induced polypeptides were also synthesized in nonpermissive cells, which do not permit viral DNA replication. The virus-induced polypeptides synthesized before viral DNA replication were hypothesized to be nonstructural proteins coded for by the cytomegalovirus genome. Their synthesis was found to be a sequential process, since three proteins preceded the synthesis of the others. Synthesis of all early cytomegalovirus-induced proteins was a transient process; the proteins reached their highest molar ratios before the onset of viral DNA replication. Late viral proteins were synthesized at the time of the onset of viral DNA replication, which was approximately 15 h after infection. Their synthesis was continuous and increased in molar ratios with the accumulation of newly synthesized viral DNA in the cells. The presence of the amino acid analog canavanine or azetadine during the early stage of infection suppressed viral DNA replication. The amount of viral DNA synthesis was directly correlated to the relative amount of late viral protein synthesis. Because synthesis of late viral proteins depended upon viral DNA replication, the proteins were not detected in permissive cells treated with an inhibitor of viral DNA synthesis or in nonpermissive cells that are restrictive for cytomegalovirus DNA replication. Images PMID:209215

  8. 11 CFR 300.60 - Scope (2 U.S.C. 441i(e)(1)).

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 11 Federal Elections 1 2013-01-01 2012-01-01 true Scope (2 U.S.C. 441i(e)(1)). 300.60 Section 300.60 Federal Elections FEDERAL ELECTION COMMISSION BIPARTISAN CAMPAIGN REFORM ACT OF 2002-(BCRA) REGULATIONS NON-FEDERAL FUNDS Federal Candidates and Officeholders § 300.60 Scope (2 U.S.C. 441i(e)(1)). This...

  9. 11 CFR 300.60 - Scope (2 U.S.C. 441i(e)(1)).

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 11 Federal Elections 1 2012-01-01 2012-01-01 false Scope (2 U.S.C. 441i(e)(1)). 300.60 Section 300.60 Federal Elections FEDERAL ELECTION COMMISSION BIPARTISAN CAMPAIGN REFORM ACT OF 2002-(BCRA) REGULATIONS NON-FEDERAL FUNDS Federal Candidates and Officeholders § 300.60 Scope (2 U.S.C. 441i(e)(1)). This...

  10. 11 CFR 300.60 - Scope (2 U.S.C. 441i(e)(1)).

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 11 Federal Elections 1 2014-01-01 2014-01-01 false Scope (2 U.S.C. 441i(e)(1)). 300.60 Section 300.60 Federal Elections FEDERAL ELECTION COMMISSION BIPARTISAN CAMPAIGN REFORM ACT OF 2002-(BCRA) REGULATIONS NON-FEDERAL FUNDS Federal Candidates and Officeholders § 300.60 Scope (2 U.S.C. 441i(e)(1)). This...

  11. Expression of a human cytomegalovirus late gene is posttranscriptionally regulated by a 3'-end-processing event occurring exclusively late after infection.

    PubMed Central

    Goins, W F; Stinski, M F

    1986-01-01

    A phenomenon of posttranscriptional regulation has been previously identified in cytomegalovirus-infected human fibroblast cells (Wathen and Stinski, J. Virol. 41:462, 1982). A region typifying this phenomenon has been located within the large unique component of the viral genome (map units 0.408 to 0.423). Even though this transcriptional unit was highly transcribed at early times after infection, mRNAs from this region were only detectable on the polyribosomes after viral DNA replication. Thus, this region is believed to code for a late gene. Single-strand-specific nuclease mapping experiments of viral transcripts established that the transcriptional initiation sites and the 5' ends of a downstream exon were identical at early and late times. However, the late transcripts differed from the early transcripts by the processing of the 3' end of the viral RNAs. This involved either the removal of a distinct region of the transcript by the selection of an upstream cleavage and polyadenylation site or the differential splicing of the RNA molecule. The upstream cleavage and polyadenylation site was identified by nuclease mapping analyses and DNA sequencing. The 3'-end processing of these transcripts is necessary for the detection of these viral RNAs within the cytoplasm of the infected cell. We propose that human cytomegalovirus either codes for a factor(s) that is involved in the 3'-end-processing event at late times after infection or stimulates the synthesis of a host cell factor(s) involved in this complex regulatory event. This level of regulation may have an influence on the types of cells that permit productive cytomegalovirus replication. Images PMID:3025644

  12. Pretransplant CD8 T-cell response to IE-1 discriminates seropositive kidney recipients at risk of developing CMV infection posttransplant.

    PubMed

    López-Oliva, Maria Ovidia; Martinez, Virginia; Buitrago, Agueda; Jiménez, Carlos; Rivas, Begoña; Escuin, Fernando; Santana, María José; Selgas, Rafael; Bellón, Teresa

    2014-04-27

    Cytomegalovirus (CMV) infection is an ongoing clinical problem in solid-organ transplantation (SOT). Pretransplant CMV serology is currently the only tool for assessing the risk of CMV infection, although cellular immune responses driven by CMV-specific CD4 and CD8 T lymphocytes are important for controlling viral replication. Therefore, the analysis of CMV-specific T cells may be useful for estimating the risk of infection. This is a prospective study of patients with kidney transplants and no prophylactic treatment for CMV replication. CD4 and CD8 T-cell responses to the major CMV pp65 and IE-1 antigens in 15 seropositive patients at intermediate risk of CMV infection were investigated, according to current algorithms. Intracellular flow cytometry was employed to determine IFN-γ production as a functional readout. The response was analyzed in pretransplant samples and prospectively at 1 and 6 months and at 1 year posttransplant. It was observed that the CD8 responses to IE-1 antigen were practically absent pretransplant in patients who developed CMV infection posttransplant. Within the group of patients free of infection, CD8 responses to IE-1 were detected more frequently and were significantly higher (P=0.0083). In a receiver operating characteristics curve analysis (AUC=0.929; P=0.010; 95% CI: 0.078-1.0), low CD8 responses to IE-1 (≤0.05%) pretransplant predicted the development of CMV infection under the immunosuppressive regime after transplant with 100% specificity and 85.7% sensitivity. Assessment of IE-1-specific CD8 T-cell frequencies pretransplant may be a useful tool for identifying seropositive SOT patients at risk of developing CMV infection posttransplant.

  13. Human Cytomegalovirus Infant Infection Adversely Affects Growth and Development in Maternally HIV-Exposed and Unexposed Infants in Zambia

    PubMed Central

    Larke, N.; Sanz-Ramos, M.; Bates, M.; Musonda, K.; Manno, D.; Siame, J.; Monze, M.; Filteau, S.

    2012-01-01

    Background. Human immunodeficiency virus (HIV) and human cytomegalovirus (HCMV) coinfections have been shown to increase infant morbidity, mortality, and AIDS progression. In HIV-endemic regions, maternal HIV-exposed but HIV-uninfected infants, which is the majority of children affected by HIV, also show poor growth and increased morbidity. Although nutrition has been examined, the effects of HCMV infection have not been evaluated. We studied the effects of HCMV infection on the growth, development, and health of maternally HIV-exposed and unexposed infants in Zambia. Methods. Infants were examined in a cohort recruited to a trial of micronutrient-fortified complementary foods. HIV-infected mothers and infants had received perinatal antiretroviral therapy to prevent mother-to-child HIV transmission. Growth, development, and morbidity were analyzed by linear regression analyses in relation to maternal HIV exposure and HCMV infection, as screened by sera DNA for viremia at 6 months of age and by antibody for infection at 18 months. Results. All HCMV-seropositive infants had decreased length-for-age by 18 months compared with seronegative infants (standard deviation [z]-score difference: −0.44 [95% confidence interval {CI}, −.72 to −.17]; P = .002). In HIV-exposed infants, those who were HCMV positive compared with those who were negative, also had reduced head size (mean z-score difference: −0.72 [95% CI, −1.23 to −.22]; P = .01) and lower psychomotor development (Bayley test score difference: −4.1 [95% CI, −7.8 to −.5]; P = .03). HIV-exposed, HCMV-viremic infants were more commonly referred for hospital treatment than HCMV-negative infants. The effects of HCMV were unaffected by micronutrient fortification. Conclusion. HCMV affects child growth, development, and morbidity of African infants, particularly in those maternally exposed to HIV. HCMV is therefore a risk factor for child health in this region. PMID:22247303

  14. Human Cytomegalovirus Promotes Survival of Infected Monocytes via a Distinct Temporal Regulation of Cellular Bcl-2 Family Proteins

    PubMed Central

    Collins-McMillen, Donna; Kim, Jung Heon; Nogalski, Maciej T.; Stevenson, Emily V.; Caskey, Joshua R.; Cieply, Stephen J.

    2015-01-01

    ABSTRACT Monocytes play a key role in the hematogenous dissemination of human cytomegalovirus (HCMV) to target organ systems. To infect monocytes and reprogram them to deliver infectious virus, HCMV must overcome biological obstacles, including the short life span of monocytes and their antiviral proapoptotic response to infection. We have shown that virally induced upregulation of cellular Mcl-1 promotes early survival of HCMV-infected monocytes, allowing cells to overcome an early apoptotic checkpoint at around 48 h postinfection (hpi). Here, we demonstrate an HCMV-dependent shift from Mcl-1 as the primary antiapoptotic player to the related protein, Bcl-2, later during infection. Bcl-2 was upregulated in HCMV-infected monocytes beginning at 48 hpi. Treatment with the Bcl-2 antagonist ABT-199 only reduced the prosurvival effects of HCMV in target monocytes beginning at 48 hpi, suggesting that Mcl-1 controls survival prior to 48 hpi, while Bcl-2 promotes survival after 48 hpi. Although Bcl-2 was upregulated following viral binding/signaling through cellular integrins (compared to Mcl-1, which is upregulated through binding/activation of epidermal growth factor receptor [EGFR]), it functioned similarly to Mcl-1, adopting the early role of Mcl-1 in preventing caspase-3 cleavage/activation. This distinct, HCMV-induced shift from Mcl-1 to Bcl-2 occurs in response to a cellular upregulation of proapoptotic Bax, as small interfering RNA (siRNA)-mediated knockdown of Bax reduced the upregulation of Bcl-2 in infected monocytes and rescued the cells from the apoptotic effects of Bcl-2 inhibition. Our data demonstrate a distinct survival strategy whereby HCMV induces a biphasic regulation of cellular Bcl-2 proteins to promote host cell survival, leading to viral dissemination and the establishment of persistent HCMV infection. IMPORTANCE Hematogenous dissemination of HCMV via infected monocytes is a crucial component of the viral survival strategy and is required for the

  15. Human Cytomegalovirus Infection of Human Embryonic Stem Cell-Derived Primitive Neural Stem Cells Is Restricted at Several Steps but Leads to the Persistence of Viral DNA

    PubMed Central

    Belzile, Jean-Philippe; Stark, Thomas J.; Yeo, Gene W.

    2014-01-01

    ABSTRACT Congenital human cytomegalovirus (HCMV) infection is a major cause of central nervous system structural anomalies and sensory impairments. It is likely that the stage of fetal development, as well as the state of differentiation of susceptible cells at the time of infection, affects the severity of the disease. We used human embryonic stem (ES) cell-derived primitive prerosette neural stem cells (pNSCs) and neural progenitor cells (NPCs) maintained in chemically defined conditions to study HCMV replication in cells at the early stages of neural development. In contrast to what was observed previously using fetus-derived NPCs, infection of ES cell-derived pNSCs with HCMV was nonprogressive. At a low multiplicity of infection, we observed only a small percentage of cells expressing immediate-early genes (IE) and early genes. IE expression was found to be restricted to cells negative for the anterior marker FORSE-1, and treatment of pNSCs with retinoic acid restored IE expression. Differentiation of pNSCs into NPCs restored IE expression but not the transactivation of early genes. Virions produced in NPCs and pNSCs were exclusively cell associated and were mostly non-neural tropic. Finally, we found that viral genomes could persist in pNSC cultures for up to a month after infection despite the absence of detectable IE expression by immunofluorescence, and infectious virus could be produced upon differentiation of pNSCs to neurons. In conclusion, our results highlight the complex array of hurdles that HCMV must overcome in order to infect primitive neural stem cells and suggest that these cells might act as a reservoir for the virus. IMPORTANCE Human cytomegalovirus (HCMV) is a betaherpesvirus that is highly prevalent in the population. HCMV infection is usually asymptomatic but can lead to severe consequences in immunosuppressed individuals. HCMV is also the most important infectious cause of congenital developmental birth defects. Manifestations of fetal

  16. Analysis of the Bombyx mori nucleopolyhedrovirus ie-1 promoter in insect, mammalian, plant, and bacterial cells.

    PubMed

    Fujita, Ryosuke; Ono, Chikako; Ono, Isamu; Asano, Shin-Ichiro; Bando, Hisanori

    2015-09-04

    The Bombyx mori nucleopolyhedrovirus (BmNPV) ie-1 promoter exhibits strong transcriptional activity and is used in transient foreign gene expression systems in insect cells. In a reporter assay experiment using the BmNPV ie-1 promoter, we found that it exhibited activity even in non-host mammalian BHK cells, plant BY-2 cells, and also bacterial Escherichia coli cells. An analysis using a deletion series of the BmNPV ie-1 promoter demonstrated that the core promoter region of this promoter was sufficient to display promoter activity in BHK cells, BY-2 cells, and E. coli cells, whereas upstream elements were required for higher activity in insect cells. Furthermore, we found that the BmNPV ie-1 promoter exhibited sufficient activity for a β-galactosidase assay in E. coli cells. The results obtained here suggest that the BmNPV ie-1 promoter has potential as a universal promoter for transient expression systems in insect, mammalian, plant, and bacterial cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Transmission of murine cytomegalovirus in breast milk: a model of natural infection in neonates.

    PubMed

    Wu, Carol A; Paveglio, Sara A; Lingenheld, Elizabeth G; Zhu, Li; Lefrançois, Leo; Puddington, Lynn

    2011-05-01

    Vertical transmission of viruses in breast milk can expose neonates to infectious pathogens at a time when the capacity of their immune system to control infections is limited. We developed a mouse model to study the outcomes of acquisition of murine cytomegalovirus (MCMV) when neonates are breastfed by mothers with acute or latent infection. Breast milk leukocytes collected from lactating mice were examined for the presence of MCMV IE-1 mRNA by reverse transcription-PCR (RT-PCR) with Southern analysis. As determined by this criterion, breast milk leukocytes from both acute and latent mothers were positive for MCMV. This mimics the outcome seen in humans with latent cytomegalovirus infection, where reactivation of virus occurs specifically in the lactating mammary gland. Interestingly, intraperitoneal injection of breast milk collected from mothers with latent infection was sufficient to transfer MCMV to neonatal mice, demonstrating that breast milk was a source of virus. Furthermore, we found that MCMV was transmitted from infected mothers to breastfed neonates, with MCMV IE-1 mRNA or infectious virus present in multiple organs, including the brain. In fact, 1 day of nursing was sufficient to transmit MCMV from latent mothers to breastfed neonatal mice. Together, these data validate this mouse model of vertical transmission of MCMV from mothers with acute or latent MCMV infection to breastfed neonates. Its relevance to human disease should prove useful in future studies designed to elucidate the immunological and pathological ramifications of neonatal infection acquired via this natural route.

  18. Replacement of the human cytomegalovirus promoter with fish enhancer and core elements to control the expression of the G gene of viral haemorrhagic septicemia virus (VHSV).

    PubMed

    Martinez-Lopez, A; Chinchilla, B; Encinas, P; Gomez-Casado, E; Estepa, A; Coll, J M

    2012-12-15

    This work explores some of the possibilities to replace human cytomegalovirus (CMV) core and/or enhancer promoter control elements to create new expression vectors for use with fish. The work is relevant to fish vaccination, since DNA vaccines use eukaryotic expression plasmids controlled by the human cytomegalovirus (CMV) promoter to be effective against novirhabdoviruses, such as viral haemorrhagic septicemia virus (VHSV), one of the most devastating fish viral European diseases. To reduce possible homologous recombination with fish genome, core and enhancer sequences from fish origin, such as trout interferon-inducible myxovirus protein (Mx), zebrafish retrovirus long terminal repeat (LTR) and carp β-actin (AE6), were combined with those of CMV to design alternative hybrid promoters. The substitution of CMV core and/or enhancer with the corresponding elements of Mx or the LTR core maintained a similar in vitro protein G expression level than that obtained by using the CMV promoter. Vectors using the dsRNA-inducible Mx enhancer followed either by the LTR or the AE6 cores showed the highest in vitro protein G expression levels. Furthermore, synthetic constructs using the Mx enhancer maintained their polyI:C induction capabilities despite the core used. Some of these hybrid promoters might contribute to the development of all-fish-vectors for DNA vaccines while others might be useful for more basic studies.

  19. Human cytomegalovirus contains a tegument protein that enhances transcription from promoters with upstream ATF and AP-1 cis-acting elements.

    PubMed Central

    Liu, B; Stinski, M F

    1992-01-01

    The tegument proteins of human cytomegalovirus are introduced into cells as components of infectious virus. The tegument proteins may affect viral and cellular transcription prior to the synthesis of the immediate-early viral regulatory proteins. The phosphorylated tegument protein of 71 kDa (pp71) is reported to be encoded by the UL82 gene. The UL82 gene products transactivated promoters containing upstream ATF or AP-1 binding sites. In contrast, the phosphorylated tegument protein of 65 kDa (pp65), encoded by the UL83 gene, had no detectable effect on these promoters. Enhancement by UL82 of downstream transcription was directly proportional to the number of upstream ATF sites. Response to UL82 transactivation was abolished by mutation of the ATF site. Mutation in the carboxy-terminal region of UL82 also eliminated transactivation. Even though the major immediate-early promoter of human cytomegalovirus is a strong enhancer-containing promoter, UL82 further enhanced its transcription as much as 20-fold. The mechanism of UL82 enhancement of transcription from viral or cellular promoters is not known, but the enhancement may be mediated by triggering one of the protein kinase signaling pathways, increasing the affinity of ATF or AP-1 for the target sequence, or stabilizing the complex between the eucaryotic transcription factor and the target sequence. Images PMID:1318413

  20. [The validation of kit of reagents for quantitative detection of DNA of human cytomegalovirus in biological material using polymerase chain reaction technique in real time operation mode].

    PubMed

    Sil'veĭstrova, O Iu; Domonova, É A; Shipulina, O Iu

    2014-04-01

    The validation of kit of reagents destined to detection and quantitative evaluation of DNA of human cytomegalovirus in biological material using polymerase chain reaction technique in real time operation mode was implemented. The comparison was made against international WHO standard--The first WHO international standard for human cytomegalovirus to implement measures the kit of reagents "AmpliSens CMV-screen/monitor-FL" and standard sample of enterprise DNA HCMV (The central research institute of epidemiology of Rospotrebnadzor) was applied. The fivefold dilution of international WHO standard and standard sample of enterprise were carried out in concentrations of DNA HCMV from 106 to 102. The arrangement of polymerase chain reaction and analysis of results were implemented using programed amplifier with system of detection of fluorescent signal in real-time mode "Rotor-Gene Q" ("Qiagen", Germany). In the total of three series of experiments, all stages of polymerase chain reaction study included, the coefficient of translation of quantitative evaluation of DNA HCMV from copy/ml to ME/ml equal to 0.6 was introduced for this kit of reagents.

  1. Cytomegalovirus (For Parents)

    MedlinePlus

    ... 3 Years) Feeding Your 1- to 3-Month-Old Feeding Your 4- to 7-Month-Old Feeding Your 8- to 12-Month-Old Feeding Your 1- to 2-Year-Old Cytomegalovirus (CMV) KidsHealth > For Parents > Cytomegalovirus (CMV) Print ...

  2. Detection of Human Cytomegalovirus pp67 Late Gene Transcripts in Cerebrospinal Fluid of Human Immunodeficiency Virus Type 1-Infected Patients by Nucleic Acid Sequence-Based Amplification

    PubMed Central

    Zhang, Fan; Tetali, Surya; Wang, Xue Ping; Kaplan, Mark H.; Cromme, Frans V.; Ginocchio, Christine C.

    2000-01-01

    This study examined the clinical correlation between the presence of human cytomegalovirus (HCMV) pp67 mRNA in cerebrospinal fluid (CSF) and active HCMV central nervous system (CNS) disease in patients with human immunodeficiency virus type 1 (HIV-1). In total, 76 CSF specimens collected from 65 HIV-1-positive patients diagnosed with HCMV CNS disease, other non-HCMV-related CNS diseases, or no CNS disease were tested for the presence of HCMV pp67 mRNA using the NucliSens cytomegalovirus (CMV) pp67 assay (Organon Teknika, Durham, N.C.). The results were compared to those of a nested PCR for the detection of HCMV glycoprotein B DNA and to those obtained by viral culture (54 samples). CSF specimens collected from patients without HCMV CNS disease yielded the following results: pp67 assay negative, 62 of 62 specimens; culture negative, 41 of 41 specimens; and PCR negative, 56 of 62 specimens (6 specimens were positive). CSF specimens collected from patients with HCMV CNS disease yielded the following results: pp67 assay positive, 9 of 13 specimens; PCR positive, 13 of 13 specimens; and culture positive, 2 of 13 specimens. After resolution of the discordant results, the following positive and negative predictive values (PPV and NPV, respectively) for the diagnosis of HCMV CNS disease were determined. The PPV for PCR, pp67 assay, and culture were 68.4, 100, and 100%, respectively, and the NPV for PCR, pp67 assay, and culture were 100, 97.0, and 82.7%, respectively. The sensitivities for DNA PCR, pp67 assay, and culture for the detection of HCMV were 100, 84.6, and 18%, respectively, and the clinical specificities were 90.5, 100, and 100%, respectively. This study indicates that the detection of HCMV pp67 mRNA in CSF has good correlation with active HCMV CNS disease, whereas CSF culture is insensitive and qualitative DNA PCR may detect latent nonreplicating virus in CSF from patients without HCMV CNS disease. PMID:10790122

  3. Detection of human cytomegalovirus pp67 late gene transcripts in cerebrospinal fluid of human immunodeficiency virus type 1-infected patients by nucleic acid sequence-based amplification.

    PubMed

    Zhang, F; Tetali, S; Wang, X P; Kaplan, M H; Cromme, F V; Ginocchio, C C

    2000-05-01

    This study examined the clinical correlation between the presence of human cytomegalovirus (HCMV) pp67 mRNA in cerebrospinal fluid (CSF) and active HCMV central nervous system (CNS) disease in patients with human immunodeficiency virus type 1 (HIV-1). In total, 76 CSF specimens collected from 65 HIV-1-positive patients diagnosed with HCMV CNS disease, other non-HCMV-related CNS diseases, or no CNS disease were tested for the presence of HCMV pp67 mRNA using the NucliSens cytomegalovirus (CMV) pp67 assay (Organon Teknika, Durham, N.C.). The results were compared to those of a nested PCR for the detection of HCMV glycoprotein B DNA and to those obtained by viral culture (54 samples). CSF specimens collected from patients without HCMV CNS disease yielded the following results: pp67 assay negative, 62 of 62 specimens; culture negative, 41 of 41 specimens; and PCR negative, 56 of 62 specimens (6 specimens were positive). CSF specimens collected from patients with HCMV CNS disease yielded the following results: pp67 assay positive, 9 of 13 specimens; PCR positive, 13 of 13 specimens; and culture positive, 2 of 13 specimens. After resolution of the discordant results, the following positive and negative predictive values (PPV and NPV, respectively) for the diagnosis of HCMV CNS disease were determined. The PPV for PCR, pp67 assay, and culture were 68.4, 100, and 100%, respectively, and the NPV for PCR, pp67 assay, and culture were 100, 97.0, and 82. 7%, respectively. The sensitivities for DNA PCR, pp67 assay, and culture for the detection of HCMV were 100, 84.6, and 18%, respectively, and the clinical specificities were 90.5, 100, and 100%, respectively. This study indicates that the detection of HCMV pp67 mRNA in CSF has good correlation with active HCMV CNS disease, whereas CSF culture is insensitive and qualitative DNA PCR may detect latent nonreplicating virus in CSF from patients without HCMV CNS disease.

  4. Human cytomegalovirus and Epstein-Barr virus infection in inflammatory bowel disease: Need for mucosal viral load measurement

    PubMed Central

    Ciccocioppo, Rachele; Racca, Francesca; Paolucci, Stefania; Campanini, Giulia; Pozzi, Lodovica; Betti, Elena; Riboni, Roberta; Vanoli, Alessandro; Baldanti, Fausto; Corazza, Gino Roberto

    2015-01-01

    AIM: To evaluate the best diagnostic technique and risk factors of the human Cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) infection in inflammatory bowel disease (IBD). METHODS: A cohort of 40 IBD patients (17 refractory) and 40 controls underwent peripheral blood and endoscopic colonic mucosal sample harvest. Viral infection was assessed by quantitative real-time polymerase chain reaction and immunohistochemistry, and correlations with clinical and endoscopic indexes of activity, and risk factors were investigated. RESULTS: All refractory patients carried detectable levels of HCMV and/or EBV mucosal load as compared to 13/23 (56.5%) non-refractory and 13/40 (32.5%) controls. The median DNA value was significantly higher in refractory (HCMV 286 and EBV 5.440 copies/105 cells) than in non-refractory (HCMV 0 and EBV 6 copies/105 cells; P < 0.05 and < 0.001) IBD patients and controls (HCMV and EBV 0 copies/105 cells; P < 0.001 for both). Refractory patients showed DNA peak values ≥ 103 copies/105 cells in diseased mucosa in comparison to non-diseased mucosa (P < 0.0121 for HCMV and < 0.0004 for EBV), while non-refractory patients and controls invariably displayed levels below this threshold, thus allowing us to differentiate viral colitis from mucosal infection. Moreover, the mucosal load positively correlated with the values found in the peripheral blood, whilst no correlation with the number of positive cells at immunohistochemistry was found. Steroid use was identified as a significant risk factor for both HCMV (P = 0.018) and EBV (P = 0.002) colitis. Finally, a course of specific antiviral therapy with ganciclovir was successful in all refractory patients with HCMV colitis, whilst refractory patients with EBV colitis did not show any improvement despite steroid tapering and discontinuation of the other medications. CONCLUSION: Viral colitis appeared to contribute to mucosal lesions in refractory IBD, and its correct diagnosis and management require

  5. Human cytomegalovirus and Epstein-Barr virus infection in inflammatory bowel disease: need for mucosal viral load measurement.

    PubMed

    Ciccocioppo, Rachele; Racca, Francesca; Paolucci, Stefania; Campanini, Giulia; Pozzi, Lodovica; Betti, Elena; Riboni, Roberta; Vanoli, Alessandro; Baldanti, Fausto; Corazza, Gino Roberto

    2015-02-14

    To evaluate the best diagnostic technique and risk factors of the human Cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) infection in inflammatory bowel disease (IBD). A cohort of 40 IBD patients (17 refractory) and 40 controls underwent peripheral blood and endoscopic colonic mucosal sample harvest. Viral infection was assessed by quantitative real-time polymerase chain reaction and immunohistochemistry, and correlations with clinical and endoscopic indexes of activity, and risk factors were investigated. All refractory patients carried detectable levels of HCMV and/or EBV mucosal load as compared to 13/23 (56.5%) non-refractory and 13/40 (32.5%) controls. The median DNA value was significantly higher in refractory (HCMV 286 and EBV 5.440 copies/10(5) cells) than in non-refractory (HCMV 0 and EBV 6 copies/10(5) cells; P < 0.05 and < 0.001) IBD patients and controls (HCMV and EBV 0 copies/10(5) cells; P < 0.001 for both). Refractory patients showed DNA peak values ≥ 10(3) copies/10(5) cells in diseased mucosa in comparison to non-diseased mucosa (P < 0.0121 for HCMV and < 0.0004 for EBV), while non-refractory patients and controls invariably displayed levels below this threshold, thus allowing us to differentiate viral colitis from mucosal infection. Moreover, the mucosal load positively correlated with the values found in the peripheral blood, whilst no correlation with the number of positive cells at immunohistochemistry was found. Steroid use was identified as a significant risk factor for both HCMV (P = 0.018) and EBV (P = 0.002) colitis. Finally, a course of specific antiviral therapy with ganciclovir was successful in all refractory patients with HCMV colitis, whilst refractory patients with EBV colitis did not show any improvement despite steroid tapering and discontinuation of the other medications. Viral colitis appeared to contribute to mucosal lesions in refractory IBD, and its correct diagnosis and management require quantitative real

  6. Structural Basis for Clonal Diversity of the Public T Cell Response to a Dominant Human Cytomegalovirus Epitope*

    PubMed Central

    Yang, Xinbo; Gao, Mingming; Chen, Guobing; Pierce, Brian G.; Lu, Jinghua; Weng, Nan-ping; Mariuzza, Roy A.

    2015-01-01

    Cytomegalovirus (CMV) is a ubiquitous and persistent human pathogen that is kept in check by CD8+ cytotoxic T lymphocytes. Individuals expressing the major histocompatibility complex (MHC) class I molecule HLA-A2 produce cytotoxic T lymphocytes bearing T cell receptors (TCRs) that recognize the immunodominant CMV epitope NLVPMVATV (NLV). The NLV-specific T cell repertoire is characterized by a high prevalence of TCRs that are frequently observed in multiple unrelated individuals. These public TCRs feature identical, or nearly identical, complementarity-determining region 3α (CDR3α) and/or CDR3β sequences. The TCRs may express public CDR3α motifs alone, public CDR3β motifs alone, or dual public CDR3αβ motifs. In addition, the same public CDR3α motif may pair with different CDR3β motifs (and the reverse), giving rise to highly diverse NLV-specific TCR repertoires. To investigate the structural underpinnings of this clonal diversity, we determined crystal structures of two public TCRs (C7 and C25) in complex with NLV·HLA-A2. These TCRs utilize completely different CDR3α and CDR3β motifs that, in addition, can associate with multiple variable α and variable β regions in NLV-specific T cell repertoires. The C7·NLV·HLA-A2 and C25·NLV·HLA-A2 complexes exhibit divergent TCR footprints on peptide-MHC such that C25 is more focused on the central portion of the NLV peptide than is C7. These structures combined with molecular modeling show how the public CDR3α motif of C25 may associate with different variable α regions and how the public CDR3α motif of C7 may pair with different CDR3β motifs. This interchangeability of TCR V regions and CDR3 motifs permits multiple structural solutions to binding an identical peptide-MHC ligand and thereby the generation of a clonally diverse public T cell response to CMV. PMID:26429912

  7. Human Cytomegalovirus gH/gL Forms a Stable Complex with the Fusion Protein gB in Virions.

    PubMed

    Vanarsdall, Adam L; Howard, Paul W; Wisner, Todd W; Johnson, David C

    2016-04-01

    Human cytomegalovirus (HCMV) is a ubiquitous virus that is a major pathogen in newborns and immunocompromised or immunosuppressed patients. HCMV infects a wide variety of cell types using distinct entry pathways that involve different forms of the gH/gL glycoprotein: gH/gL/gO and gH/gL/UL128-131 as well as the viral fusion glycoprotein, gB. However, the minimal or core fusion machinery (sufficient for cell-cell fusion) is just gH/gL and gB. Here, we demonstrate that HCMV gB and gH/gL form a stable complex early after their synthesis and in the absence of other viral proteins. gH/gL can interact with gB mutants that are unable to mediate cell-cell fusion. gB-gH/gL complexes included as much as 16-50% of the total gH/gL in HCMV virus particles. In contrast, only small amounts of gH/gL/gO and gH/gL/UL128-131 complexes were found associated with gB. All herpesviruses express gB and gH/gL molecules and most models describing herpesvirus entry suggest that gH/gL interacts with gB to mediate membrane fusion, although there is no direct evidence for this. For herpes simplex virus (HSV-1) it has been suggested that after receptor binding gH/gL binds to gB either just before, or coincident with membrane fusion. Therefore, our results have major implications for these models, demonstrating that HCMV gB and gH/gL forms stable gB-gH/gL complexes that are incorporated virions without receptor binding or membrane fusion. Moreover, our data is the best support to date for the proposal that gH/gL interacts with gB.

  8. Human cytomegalovirus pUL79 is an elongation factor of RNA polymerase II for viral gene transcription.

    PubMed

    Perng, Yi-Chieh; Campbell, Jessica A; Lenschow, Deborah J; Yu, Dong

    2014-08-01

    In this study, we have identified a unique mechanism in which human cytomegalovirus (HCMV) protein pUL79 acts as an elongation factor to direct cellular RNA polymerase II for viral transcription during late times of infection. We and others previously reported that pUL79 and its homologues are required for viral transcript accumulation after viral DNA synthesis. We hypothesized that pUL79 represented a unique mechanism to regulate viral transcription at late times during HCMV infection. To test this hypothesis, we analyzed the proteome associated with pUL79 during virus infection by mass spectrometry. We identified both cellular transcriptional factors, including multiple RNA polymerase II (RNAP II) subunits, and novel viral transactivators, including pUL87 and pUL95, as protein binding partners of pUL79. Co-immunoprecipitation (co-IP) followed by immunoblot analysis confirmed the pUL79-RNAP II interaction, and this interaction was independent of any other viral proteins. Using a recombinant HCMV virus where pUL79 protein is conditionally regulated by a protein destabilization domain ddFKBP, we showed that this interaction did not alter the total levels of RNAP II or its recruitment to viral late promoters. Furthermore, pUL79 did not alter the phosphorylation profiles of the RNAP II C-terminal domain, which was critical for transcriptional regulation. Rather, a nuclear run-on assay indicated that, in the absence of pUL79, RNAP II failed to elongate and stalled on the viral DNA. pUL79-dependent RNAP II elongation was required for transcription from all three kinetic classes of viral genes (i.e. immediate-early, early, and late) at late times during virus infection. In contrast, host gene transcription during HCMV infection was independent of pUL79. In summary, we have identified a novel viral mechanism by which pUL79, and potentially other viral factors, regulates the rate of RNAP II transcription machinery on viral transcription during late stages of HCMV infection.

  9. Regulation and Gene Expression Profiling of NKG2D Positive Human Cytomegalovirus-Primed CD4+ T-Cells

    PubMed Central

    Jensen, Helle; Folkersen, Lasse; Skov, Søren

    2012-01-01

    NKG2D is a stimulatory receptor expressed by natural killer (NK) cells, CD8+ T-cells, and γδ T-cells. NKG2D expression is normally absent from CD4+ T-cells, however recently a subset of NKG2D+ CD4+ T-cells has been found, which is specific for human cytomegalovirus (HCMV). This particular subset of HCMV-specific NKG2D+ CD4+ T-cells possesses effector-like functions, thus resembling the subsets of NKG2D+ CD4+ T-cells found in other chronic inflammations. However, the precise mechanism leading to NKG2D expression on HCMV-specific CD4+ T-cells is currently not known. In this study we used genome-wide analysis of individual genes and gene set enrichment analysis (GSEA) to investigate the gene expression profile of NKG2D+ CD4+ T-cells, generated from HCMV-primed CD4+ T-cells. We show that the HCMV-primed NKG2D+ CD4+ T-cells possess a higher differentiated phenotype than the NKG2D– CD4+ T-cells, both at the gene expression profile and cytokine profile. The ability to express NKG2D at the cell surface was primarily determined by the activation or differentiation status of the CD4+ T-cells and not by the antigen presenting cells. We observed a correlation between CD94 and NKG2D expression in the CD4+ T-cells following HCMV stimulation. However, knock-down of CD94 did not affect NKG2D cell surface expression or signaling. In addition, we show that NKG2D is recycled at the cell surface of activated CD4+ T-cells, whereas it is produced de novo in resting CD4+ T-cells. These findings provide novel information about the gene expression profile of HCMV-primed NKG2D+ CD4+ T-cells, as well as the mechanisms regulating NKG2D cell surface expression. PMID:22870231

  10. Low levels of Human Cytomegalovirus Infection in Glioblastoma multiforme associates with patient survival; -a case-control study

    PubMed Central

    2012-01-01

    Background Glioblastoma multiforme (GBM) represent the most aggressive brain tumor with a median overall survival of about 12-15 months. Over 90% of GBM tumors have recently been shown to be infected with human cytomegalovirus (HCMV). In this case-control study, we evaluated whether there was an association between the grade of HCMV infection and long-term survival (> 18 months) in GBM patients. Material and methods Brain tumor tissue sections from consecutive GBMs patients who survived more than 18 months (n = 40), and an equal number of GBM patients, matched to date of diagnosis and surgery, operated at Karolinska University Hospital in 2000-2005 were selected. HCMV infection grade was determined by estimation of the number of HCMV positive cells (scored negative or grade 1-4) in tumor tissue specimens. Using Chi-Square test and logistic regression analysis, we analyzed whether there was an association between long-term survival and HCMV low-grade infection or other clinical parameters known to be associated with prolonged survival of GBM patients; age under 50 years, radical surgery or low recursive partition analysis (RPA) subclass. Results HCMV infection was detected in tumor samples from 79 of 80 patients (99%). Among patients surviving > 18 months, HCMV infection grade 1 in the GBM tumor was predominant. A low grade HCMV infection was found in 19 patients, of these 16 survived > 18 months. Thus, 16 of 40 (40%) GBM patients who lived > 18 months had low-grade HCMV infection while only 3 of 40 (8%) GBM patients who lived < 18 months did (p .0006, Chi-Square test). Multiple logistic regression analyses yielded an odds ratio estimate of 6.604 with 95% confidence interval (1.36-32.1) (p .019) for low grade HCMV after adjustment for RPA class III and IV, radical surgery, age and gamma knife treatment. Conclusion In conclusion, we found that low-grade HCMV infection was strongly associated with long-term survival in GBM patients. PMID:22424569

  11. Site-specific glycosylation of the human cytomegalovirus tegument basic phosphoprotein (UL32) at serine 921 and serine 952.

    PubMed Central

    Greis, K D; Gibson, W; Hart, G W

    1994-01-01

    The virion basic phosphoprotein (BPP), UL32, of the human cytomegalovirus (HCMV) is a 149-kDa tegument protein that represents about 15% of the virion protein mass and is modified by O-linked N-acetylglucosamine (O-GlcNAc). O-GlcNAc has been postulated to mediate subunit-subunit interaction in many different types of intracellular protein complexes, while BPP may play a role in viral assembly and/or envelopment. This report describes the identification of the major O-GlcNAc attachment sites on the HCMV (AD169) BPP. Because the amount of BPP isolated from infectious virions was insufficient to determine the site(s) of glycosylation, the full-length protein has been characterized following overexpression in recombinant baculovirus-infected insect cells. The recombinant protein (rBPP) was electrophoretically (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and immunologically (by Western immunoassaying) indistinguishable from the BPP isolated from HCMV virions. In addition, the rBPP was modified by O-GlcNAc, and a comparison of the tryptic glycopeptides from the rBPP and native virion BPP indicated that their O-GlcNAc sites are the same. Furthermore, the major sites of O-GlcNAc attachment to the rBPP were mapped on high-performance liquid chromatography-purified glycopeptides by gas phase microsequencing, manual Edman degradation, and electrospray-mass spectrometry. The results demonstrate that the major sites of O-GlcNAc attachment to the BPP are Ser-921 and Ser-952. Identification of these sites will now enable mutagenesis studies to determine the influence of O-GlcNAc on the intracellular location, protein-protein interaction, and biological function of BPP. Finally, the fidelity of the addition of O-GlcNAc to rBPP in insect cells compared with native virion BPP is documented to demonstrate the possible general applicability of the baculovirus expression system to study O-GlcNAc on other low-abundance proteins. Images PMID:7966627

  12. Human cytomegalovirus resistance to deoxyribosylindole nucleosides maps to a transversion mutation in the terminase subunit-encoding gene UL89.

    PubMed

    Gentry, Brian G; Phan, Quang; Hall, Ellie D; Breitenbach, Julie M; Borysko, Katherine Z; Kamil, Jeremy P; Townsend, Leroy B; Drach, John C

    2015-01-01

    Human cytomegalovirus (HCMV) infection can cause severe illnesses, including encephalopathy and mental retardation, in immunocompromised and immunologically immature patients. Current pharmacotherapies for treating systemic HCMV infections include ganciclovir, cidofovir, and foscarnet. However, long-term administration of these agents can result in serious adverse effects (myelosuppression and/or nephrotoxicity) and the development of viral strains with reduced susceptibility to drugs. The deoxyribosylindole (indole) nucleosides demonstrate a 20-fold greater activity in vitro (the drug concentration at which 50% of the number of plaques was reduced with the presence of drug compared to the number in the absence of drug [EC50] = 0.34 μM) than ganciclovir (EC50 = 7.4 μM) without any observed increase in cytotoxicity. Based on structural similarity to the benzimidazole nucleosides, we hypothesize that the indole nucleosides target the HCMV terminase, an enzyme responsible for packaging viral DNA into capsids and cleaving the DNA into genome-length units. To test this hypothesis, an indole nucleoside-resistant HCMV strain was isolated, the open reading frames of the genes that encode the viral terminase were sequenced, and a G766C mutation in exon 1 of UL89 was identified; this mutation resulted in an E256Q change in the amino acid sequence of the corresponding protein. An HCMV wild-type strain, engineered with this mutation to confirm resistance, demonstrated an 18-fold decrease in susceptibility to the indole nucleosides (EC50 = 3.1 ± 0.7 μM) compared to that of wild-type virus (EC50 = 0.17 ± 0.04 μM). Interestingly, this mutation did not confer resistance to the benzimidazole nucleosides (EC50 for wild-type HCMV = 0.25 ± 0.04 μM, EC50 for HCMV pUL89 E256Q = 0.23 ± 0.04 μM). We conclude, therefore, that the G766C mutation that results in the E256Q substitution is unique for indole nucleoside resistance and distinct from previously discovered substitutions

  13. Single Chain Antibodies Against gp55 of Human Cytomegalovirus (HCMV) for Prophylaxis and Treatment of HCMV Infections

    PubMed Central

    Moazen, Bahareh; Ebrahimi, Elahe; Nejatollahi, Foroogh

    2016-01-01

    Background: Immunotherapy is a promising prospective new treatment for cytomegalovirus (CMV) infections. Neutralizing effects have been reported using monoclonal antibodies. Recombinant single chain antibodies (scFvs) due to their advantages over monoclonal antibodies are potential alternatives and provide valuable clinical agents. Objectives: The aim of this study was to select specific single chain antibodies against gp55 of CMV and to evaluate their neutralizing effects. In the present study, we selected specific single chain antibodies against glycoprotein 55 (gp55) of CMV for their use in treatment and diagnosis. Materials and Methods: Single chain antibodies specific against an epitope located in the C-terminal part of gp55 were selected from a phage antibody display library. After four rounds of panning, twenty clones were amplified by the polymerase chain reaction (PCR) and fingerprinted by MvaI restriction enzyme. The reactivities of the specific clones were tested by the enzyme-linked immunosorbent assay (ELISA) and the neutralizing effects were evaluated by the plaque reduction assay. Results: Fingerprinting of selected clones revealed three specific single chain antibodies (scFv1, scFv2 and scFv3) with frequencies 25%, 20 and 20%. The clones produced positive ELISA with the corresponding peptide. The percentages of plaque reduction for scFv1, scFv2 and scFv3 were 23.7, 68.8 and 11.6, respectively. Conclusions: Gp55 of human CMV is considered as an important candidate for immunotherapy. In this study, we selected three specific clones against gp55. The scFvs reacted only with the corresponding peptide in a positive ELISA. The scFv2 with 68.8% neutralizing effect showed the potential to be considered for prophylaxis and treatment of CMV infections, especially in solid organ transplant recipients, for whom treatment of CMV is urgently needed. The scFv2 with neutralizing effect of 68.8%, has the potential to be considered for treatment of these patients

  14. A “Coiled-Coil” Motif Is Important for Oligomerization and DNA Binding Properties of Human Cytomegalovirus Protein UL77

    PubMed Central

    Dittmer, Alexandra; Lapp, Sara; Bogner, Elke

    2011-01-01

    Human cytomegalovirus (HCMV) UL77 gene encodes the essential protein UL77, its function is characterized in the present study. Immunoprecipitation identified monomeric and oligomeric pUL77 in HCMV infected cells. Immunostaining of purified virions and subviral fractions showed that pUL77 is a structural protein associated with capsids. In silico analysis revealed the presence of a coiled-coil motif (CCM) at the N-terminus of pUL77. Chemical cross-linking of either wild-type pUL77 or CCM deletion mutant (pUL77ΔCCM) implicated that CCM is critical for oligomerization of pUL77. Furthermore, co-immunoprecipitations of infected and transfected cells demonstrated that pUL77 interacts with the capsid-associated DNA packaging motor components, pUL56 and pUL104, as well as the major capsid protein. The ability of pUL77 to bind dsDNA was shown by an in vitro assay. Binding to certain DNA was further confirmed by an assay using biotinylated 36-, 250-, 500-, 1000-meric dsDNA and 966-meric HCMV-specific dsDNA designed for this study. The binding efficiency (BE) was determined by image processing program defining values above 1.0 as positive. While the BE of the pUL56 binding to the 36-mer bio-pac1 containing a packaging signal was 10.0±0.63, the one for pUL77 was only 0.2±0.03. In contrast to this observation the BE of pUL77 binding to bio-500 bp or bio-1000 bp was 2.2±0.41 and 4.9±0.71, respectively. By using pUL77ΔCCM it was demonstrated that this protein could not bind to dsDNA. These data indicated that pUL77 (i) could form homodimers, (ii) CCM of pUL77 is crucial for oligomerization and (iii) could bind to dsDNA in a sequence independent manner. PMID:21998635

  15. Novel Method Based on Real-Time Cell Analysis for Drug Susceptibility Testing of Herpes Simplex Virus and Human Cytomegalovirus

    PubMed Central

    Piret, Jocelyne; Goyette, Nathalie

    2016-01-01

    The plaque reduction assay (PRA) is the gold standard phenotypic method to determine herpes simplex virus (HSV) and human cytomegalovirus (HCMV) susceptibilities to antiviral drugs. However, this assay is subjective and labor intensive. Here, we describe a novel antiviral phenotypic method based on real-time cell analysis (RTCA) that measures electronic impedance over time. The effective drug concentrations that reduced by 50% (EC50s) the cytopathic effects induced by HSV-1 and HCMV were evaluated by both methods. The EC50s of acyclovir and foscarnet against a reference wild-type (WT) HSV-1 strain in Vero cells were, respectively, 0.5 μM and 32.6 μM by PRA and 0.8 μM and 93.6 μM by RTCA. The EC50 ratios for acyclovir against several HSV-1 thymidine kinase (TK) mutants were 101.8×, 73.4×, 28.8×, and 35.4× (PRA) and 18.0×, 52.0×, 5.5×, and 87.8× (RTCA) compared to those for the WT. The EC50 ratios for acyclovir and foscarnet against the HSV-1 TK/DNA polymerase mutant were 182.8× and 9.7× (PRA) and >125.0× and 10.8× (RTCA) compared to the WT. The EC50s of ganciclovir and foscarnet against WT HCMV strain AD169 in fibroblasts were, respectively, 1.6 μM and 27.8 μM by PRA and 5.0 μM and 111.4 μM by RTCA. The EC50 ratios of ganciclovir against the HCMV UL97 mutant were 3.8× (PRA) and 8.2× (RTCA) compared to those for the WT. The EC50 ratios of ganciclovir and foscarnet against the HCMV UL97/DNA polymerase mutant were 17.1× and 12.1× (PRA) and 14.7× and 4.6× (RTCA) compared to those for the WT. RTCA allows objective drug susceptibility testing of HSV and HCMV and could permit high-throughput screening of new antivirals. PMID:27252463

  16. Diagnostic significance and clinical impact of quantitative assays for diagnosis of human cytomegalovirus infection/disease in immunocompromised patients.

    PubMed

    Gerna, G; Percivalle, E; Baldanti, F; Sarasini, A; Zavattoni, M; Furione, M; Torsellini, M; Revello, M G

    1998-07-01

    In recent years several assays have been developed for quantitation of human cytomegalovirus (HCMV) in blood of immunocompromised (transplanted and AIDS) patients. It is currently agreed that the only reliable indication of the degree of dissemination of HCMV infection/disease is the measurement of HCMV in blood. Diagnosis of HCMV end-organ disease (organ localizations) often does not benefit from quantitation of virus in blood, but requires detection and quantification of virus in samples taken locally. The most important and clinically useful diagnostic assays for HCMV quantitation in blood are: i) viremia, quantifying infectious HCMV carried by peripheral blood leukocytes (PBL); ii) pp65-antigenemia, quantifying the number of PBL positive for HCMV pp65 in the nucleus; iii) circulating cytomegalic endothelial cell (CEC) viremia (CEC-viremia) measuring the number of circulating CEC carrying infectious HCMV (during the antigenemia assay); iv) leuko- and plasma-DNAemia, quantifying the number of HCMV genome equivalents present in PBL or plasma, respectively, by quantitative polymerase chain reaction (Q-PCR). Other less widely used assays are: i) determination of immediate early and late gene transcripts (mRNA) to detect active viral infection; ii) in situ hybridization to detect viral nucleic acid (DNA or RNA) in tissue sections or cell smears; iii) in situ PCR to detect a low DNA copy number in single cells. Monitoring of HCMV infection/disease in transplant recipients and AIDS patients has established threshold values for different assays above which HCMV-related clinical symptoms are likely to appear. These values are approximately 10 for viremia, 100 for antigenemia and 1,000 GE for leukoDNAemia, and are valid for both solid organ and bone marrow transplant recipients as well as AIDS patients, whereas presence of even a single circulating CEC is sufficient to suggest the presence of a disseminated HCMV infection with potential organ involvement. Monitoring of

  17. Human Cytomegalovirus gH/gL Forms a Stable Complex with the Fusion Protein gB in Virions

    PubMed Central

    Vanarsdall, Adam L.; Howard, Paul W.; Wisner, Todd W.; Johnson, David C.

    2016-01-01

    Human cytomegalovirus (HCMV) is a ubiquitous virus that is a major pathogen in newborns and immunocompromised or immunosuppressed patients. HCMV infects a wide variety of cell types using distinct entry pathways that involve different forms of the gH/gL glycoprotein: gH/gL/gO and gH/gL/UL128-131 as well as the viral fusion glycoprotein, gB. However, the minimal or core fusion machinery (sufficient for cell-cell fusion) is just gH/gL and gB. Here, we demonstrate that HCMV gB and gH/gL form a stable complex early after their synthesis and in the absence of other viral proteins. gH/gL can interact with gB mutants that are unable to mediate cell-cell fusion. gB-gH/gL complexes included as much as 16–50% of the total gH/gL in HCMV virus particles. In contrast, only small amounts of gH/gL/gO and gH/gL/UL128-131 complexes were found associated with gB. All herpesviruses express gB and gH/gL molecules and most models describing herpesvirus entry suggest that gH/gL interacts with gB to mediate membrane fusion, although there is no direct evidence for this. For herpes simplex virus (HSV-1) it has been suggested that after receptor binding gH/gL binds to gB either just before, or coincident with membrane fusion. Therefore, our results have major implications for these models, demonstrating that HCMV gB and gH/gL forms stable gB-gH/gL complexes that are incorporated virions without receptor binding or membrane fusion. Moreover, our data is the best support to date for the proposal that gH/gL interacts with gB. PMID:27082872

  18. Cytomegalovirus immediate early proteins promote stemness properties in glioblastoma

    PubMed Central

    Soroceanu, Liliana; Matlaf, Lisa; Khan, Sabeena; Akhavan, Armin; Singer, Eric; Bezrookove, Vladimir; Decker, Stacy; Ghanny, Saleena; Hadaczek, Piotr; Bengtsson, Henrik; Ohlfest, John; Luciani-Torres, Maria-Gloria; Harkins, Lualhati; Perry, Arie; Guo, Hong; Soteropoulos, Patricia; Cobbs, Charles S

    2015-01-01

    Glioblastoma (GBM) is the most common and aggressive human brain tumor. Human cytomegalovirus (HCMV) immediate early (IE) proteins that are endogenously expressed in GBM cells are strong viral transactivators with onconcogenic properties. Here, we show how HCMV IE are preferentially expressed in glioma stem-like cells (GSC), where they co-localize with the other GBM stemness markers, CD133, Nestin, and Sox2. In patient-derived GSC that are endogenously infected with HCMV, attenuating IE expression by an RNA-i-based strategy, was sufficient to inhibit tumorsphere formation, Sox2 expression, cell cycle progression, and cell survival. Conversely, HCMV infection of HMCV-negative GSC elicited robust self-renewal and proliferation of cells that could be partially reversed by IE attenuation. In HCMV-positive GSC, IE attenuation induced a molecular program characterized by enhanced expression of mesenchymal markers and pro-inflammatory cytokines, resembling the therapeutically-resistant GBM phenotype. Mechanistically, HCMV/IE regulation of Sox2 occurred via inhibition of miRNA-145, a negative regulator of Sox2 protein expression. In a spontaneous mouse model of glioma, ectopic expression of the IE1 gene (UL123) specifically increased Sox2 and Nestin levels in the IE1-positive tumors, upregulating stemness and proliferation markers in vivo. Similarly, human GSC infected with the HCMV strain Towne but not the IE1-deficient strain CR208 showed enhanced growth as tumorspheres and intracranial tumor xenografts, compared to mock-infected human GSC. Overall, our findings offer new mechanistic insights into how HCMV/IE control stemness properties in glioblastoma cells. PMID:26239477

  19. Cytomegalovirus immune globulin intravenous (human) administration modulates immune response to alloantigens in sensitized renal transplant candidates

    PubMed Central

    Sivasai, K S R; Mohanakumar, T; Phelan, D; Martin, S; Anstey, M E; Brennan, D C

    2000-01-01

    One of the important parameters for prolonged waiting time for potential renal transplant recipients is the presence of preformed antibodies to human leucocyte antigen (HLA) antigens, which is often caused by previous transplants, pregnancy or transfusions. In vivo administration of specific and unselected polyclonal intravenous immunoglobulin (IVIGs) preparations have been shown to inhibit anti-HLA alloantibodies in highly sensitized patients. We sought to determine whether Cytogam (Medimmune Inc., Gaithersburg, MD, USA), a hyperimmune anticytomegalovirus immunoglobulin would (1) effect either in vitro or in vivo alloreactivity, and (2) whether Cytogam therapy could reduce the titre of preformed anti-HLA antibodies in highly sensitized patients. Alloreactivity was assessed by mixed lymphocyte reaction (MLR) and cytotoxic T lymphocyte (CTL) assay. A complement dependent microlymphocytotoxicity assay was done to assess for panel reactive antibody (PRA) status and the presence of anti-idiotypic antibodies in the Cytogam preparation. The MLR was inhibited by Cytogam in vitro in a dose-dependent fashion ranging from 31–92%. Significant inhibition of the MLR responses was not observed in recipients who received Cytogam in vivo (50 mg/kg). This could be a result of adminstration of a low dose of IVIG. However, CTL activity against the alloantigens in all individuals assessed was significantly inhibited after in vivo administration of Cytogam. Three of five individuals experienced a decrease of 5–32% in the PRA status at 4 weeks post administration of Cytogam. Cytogam also blocked the anti-HLA antibody titres in a microlymphocytotoxicity assay, suggesting the presence of anti-idiotypic antibodies. Our study was based on a single prophylactic dose of Cytogam (50 mg/kg), however, higher dose administration could be feasible by removing more fluid at dialysis, but should be given intradialytically to avoid volume overload. Overall, our results suggest that Cytogam can

  20. Human cytomegalovirus alters localization of MHC class II and dendrite morphology in mature Langerhans cells.

    PubMed

    Lee, Andrew W; Hertel, Laura; Louie, Ryan K; Burster, Timo; Lacaille, Vashti; Pashine, Achal; Abate, Davide A; Mocarski, Edward S; Mellins, Elizabeth D

    2006-09-15

    Hemopoietic stem cell-derived mature Langerhans-type dendritic cells (LC) are susceptible to productive infection by human CMV (HCMV). To investigate the impact of infection on this cell type, we examined HLA-DR biosynthesis and trafficking in mature LC cultures exposed to HCMV. We found decreased surface HLA-DR levels in viral Ag-positive as well as in Ag-negative mature LC. Inhibition of HLA-DR was independent of expression of unique short US2-US11 region gene products by HCMV. Indeed, exposure to UV-inactivated virus, but not to conditioned medium from infected cells, was sufficient to reduce HLA-DR on mature LC, implicating particle binding/penetration in this effect. Reduced surface levels reflected an altered distribution of HLA-DR because total cellular HLA-DR was not diminished. Accumulation of HLA-DR was not explained by altered cathepsin S activity. Mature, peptide-loaded HLA-DR molecules were retained within cells, as assessed by the proportion of SDS-stable HLA-DR dimers. A block in egress was implicated, as endocytosis of surface HLA-DR was not increased. Immunofluorescence microscopy corroborated the intracellular retention of HLA-DR and revealed markedly fewer HLA-DR-positive dendritic projections in infected mature LC. Unexpectedly, light microscopic analyses showed a dramatic loss of the dendrites themselves and immunofluorescence revealed that cytoskeletal elements crucial for the formation and maintenance of dendrites are disrupted in viral Ag-positive cells. Consistent with these dendrite effects, HCMV-infected mature LC exhibit markedly reduced chemotaxis in response to lymphoid chemokines. Thus, HCMV impedes MHC class II molecule trafficking, dendritic projections, and migration of mature LC. These changes likely contribute to the reduced activation of CD4+ T cells by HCMV-infected mature LC.

  1. Analysis of memory-like natural killer cells in human cytomegalovirus-infected children undergoing αβ+T and B cell-depleted hematopoietic stem cell transplantation for hematological malignancies.

    PubMed

    Muccio, Letizia; Bertaina, Alice; Falco, Michela; Pende, Daniela; Meazza, Raffaella; Lopez-Botet, Miguel; Moretta, Lorenzo; Locatelli, Franco; Moretta, Alessandro; Della Chiesa, Mariella

    2016-03-01

    We analyzed the impact of human cytomegalovirus infection on the development of natural killer cells in 27 pediatric patients affected by hematological malignancies, who had received a HLA-haploidentical hematopoietic stem cell transplantation, depleted of both α/β+ T cells and B cells. In line with previous studies in adult recipients of umbilical cord blood transplantation, we found that human cytomegalovirus reactivation accelerated the emergence of mature natural killer cells. Thus, most children displayed a progressive expansion of a memory-like natural killer cell subset expressing NKG2C, a putative receptor for human cytomegalovirus, and CD57, a marker of terminal natural killer cell differentiation. NKG2C(+)CD57(+) natural killer cells were detectable by month 3 following hematopoietic stem cell transplantation and expanded until at least month 12. These cells were characterized by high killer Ig-like receptors (KIRs) and leukocyte inhibitory receptor 1 (LIR-1) and low Siglec-7, NKG2A and Interleukin-18Rα expression, killed tumor targets and responded to cells expressing HLA-E (a NKG2C ligand). In addition, they were poor Interferon-γ producers in response to Interleukin-12 and Interleukin-18. The impaired response to these cytokines, together with their highly differentiated profile, may reflect their skewing toward an adaptive condition specialized in controlling human cytomegalovirus. In conclusion, in pediatric patients receiving a type of allograft different from umbilical cord blood transplantation, human cytomegalovirus also induced memory-like natural killer cells, possibly contributing to controlling infections and reinforcing anti-leukemia effects.

  2. Human cytomegalovirus and human umbilical vein endothelial cells: restriction of primary isolation to blood samples and susceptibilities of clinical isolates from other sources to adaptation.

    PubMed

    Gerna, Giuseppe; Percivalle, Elena; Sarasini, Antonella; Revello, M Grazia

    2002-01-01

    In immunocompromised patients with disseminated infection, human cytomegalovirus (HCMV) is widespread in the microvascular endothelium of multiple organs. Human umbilical vein endothelial cells (HUVEC) were used in parallel to human embryonic lung fibroblasts (HELF) to recover HCMV from blood samples of immunocompromised patients. Using the shell vial technique, comparable median numbers of p72-positive HUVEC and HELF cells were found with the 26 HCMV-positive buffy coat samples out of 150 examined. Analysis of other clinical samples inoculated as controls revealed, in the presence of highly infected HELF monolayers, either the presence of very few infected HUVEC with urine specimens (n = 10 samples) or the lack of infected HUVEC with throat washes (n = 3) or amniotic fluid samples (n = 2). Thus, peripheral blood leukocytes (PBL) appear essential for primary isolation of HCMV in HUVEC. In this respect, HCMV strains, recovered from clinical samples other than buffy coats in HELF only, could be readily adapted to growth in HUVEC by coculturing PBL from healthy blood donors with infected HELF and then inoculating infected PBL onto HUVEC. Recently elucidated mechanisms of interaction of leukocytes and HUVEC with bidirectional transfer of virus seem to provide the basis for the restriction of HCMV primary isolation in HUVEC to blood samples. However, virus strains recovered from only HELF could be adapted to growth in HUVEC when inoculated with HELF-derived (either cell-associated or cell-free) HCMV strains upon primary isolation. In conclusion, due to the in vitro selection of virus variants provided with both PBL tropism and HUVEC tropism, HCMV recovery in HUVEC is PBL mediated and substantially restricted to blood samples. Lack of HCMV recovery in HUVEC from clinical samples other than blood leads to the assumption that epithelial cells, such as urinary, amniotic, or pharyngeal cells, do not possess adequate adhesion molecules to establish close contacts with HUVEC.

  3. MORC3, a Component of PML Nuclear Bodies, Has a Role in Restricting Herpes Simplex Virus 1 and Human Cytomegalovirus

    PubMed Central

    Orr, Anne; Everett, Roger D.

    2016-01-01

    ABSTRACT We previously reported that MORC3, a protein associated with promyelocytic leukemia nuclear bodies (PML NBs), is a target of herpes simplex virus 1 (HSV-1) ICP0-mediated degradation (E. Sloan, et al., PLoS Pathog 11:e1005059, 2015, http://dx.doi.org/10.1371/journal.ppat.1005059). Since it is well known that certain other components of the PML NB complex play an important role during an intrinsic immune response to HSV-1 and are also degraded or inactivated by ICP0, here we further investigate the role of MORC3 during HSV-1 infection. We demonstrate that MORC3 has antiviral activity during HSV-1 infection and that this antiviral role is counteracted by ICP0. In addition, MORC3's antiviral role extends to wild-type (wt) human cytomegalovirus (HCMV) infection, as its plaque-forming efficiency increased in MORC3-depleted cells. We found that MORC3 is recruited to sites associated with HSV-1 genomes after their entry into the nucleus of an infected cell, and in wt infections this is followed by its association with ICP0 foci prior to its degradation. The RING finger domain of ICP0 was required for degradation of MORC3, and we confirmed that no other HSV-1 protein is required for the loss of MORC3. We also found that MORC3 is required for fully efficient recruitment of PML, Sp100, hDaxx, and γH2AX to sites associated with HSV-1 genomes entering the host cell nucleus. This study further unravels the intricate ways in which HSV-1 has evolved to counteract the host immune response and reveals a novel function for MORC3 during the host intrinsic immune response. IMPORTANCE Herpesviruses have devised ways to manipulate the host intrinsic immune response to promote their own survival and persistence within the human population. One way in which this is achieved is through degradation or functional inactivation of PML NB proteins, which are recruited to viral genomes in order to repress viral transcription. Because MORC3 associates with PML NBs in uninfected cells and

  4. Cytomegalovirus (CMV) and Pregnancy

    MedlinePlus

    ... Close contact includes activities like changing diapers and kissing. What is congenital cytomegalovirus (congenital CMV)? Pregnant women ... or saliva. Try to avoid mouth-to-mouth kissing with children in day-care. Do not share ...

  5. Glucocorticoids facilitate the transcription from the human cytomegalovirus major immediate early promoter in glucocorticoid receptor- and nuclear factor-I-like protein-dependent manner.

    PubMed

    Inoue-Toyoda, Maki; Kato, Kohsuke; Nagata, Kyosuke; Yoshikawa, Hiroyuki

    2015-02-27

    Human cytomegalovirus (HCMV) is a common and usually asymptomatic virus agent in healthy individuals. Initiation of HCMV productive infection depends on expression of the major immediate early (MIE) genes. The transcription of HCMV MIE genes is regulated by a diverse set of transcription factors. It was previously reported that productive HCMV infection is triggered probably by elevation of the plasma hydroxycorticoid level. However, it is poorly understood whether the transcription of MIE genes is directly regulated by glucocorticoid. Here, we found that the dexamethasone (DEX), a synthetic glucocorticoid, facilitates the transcription of HCMV MIE genes through the MIE promoter and enhancer in a glucocorticoid receptor (GR)-dependent manner. By competitive EMSA and reporter assays, we revealed that an NF-I like protein is involved in DEX-mediated transcriptional activation of the MIE promoter. Thus, this study supports a notion that the increased level of hydroxycorticoid in the third trimester of pregnancy reactivates HCMV virus production from the latent state.

  6. Use of 5-ethynyl-2'-deoxyuridine labelling and flow cytometry to study cell cycle-dependent regulation of human cytomegalovirus gene expression.

    PubMed

    Wiebusch, Lüder; Hagemeier, Christian

    2014-01-01

    The cell cycle position at the time of infection has a profound influence on human cytomegalovirus (HCMV) gene expression and therefore needs consideration in the design and control of HCMV experiments. While G0/G1 cells support the immediate onset of viral transcription, cells progressing through the S and G2 cell cycle phases prevent HCMV from entering the lytic replication cycle. Here, we provide two fast and reliable protocols that allow one to determine the cell cycle distribution of the designated host cells and monitor viral protein expression as a function of the cell cycle state. Both protocols make use of the thymidine analogue 5-ethynyl-2'-deoxyuridine and "click" chemistry to label HCMV-non-permissive S phase cells in a gentle and sensitive way.

  7. The Smallest Capsid Protein Mediates Binding of the Essential Tegument Protein pp150 to Stabilize DNA-Containing Capsids in Human Cytomegalovirus

    PubMed Central

    Dai, Xinghong; Yu, Xuekui; Gong, Hao; Jiang, Xiaohong; Abenes, Gerrado; Liu, Hongrong; Shivakoti, Sakar; Britt, William J.; Zhu, Hua; Liu, Fenyong; Zhou, Z. Hong

    2013-01-01

    Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that causes birth defects in newborns and life-threatening complications in immunocompromised individuals. Among all human herpesviruses, HCMV contains a much larger dsDNA genome within a similarly-sized capsid compared to the others, and it was proposed to require pp150, a tegument protein only found in cytomegaloviruses, to stabilize its genome-containing capsid. However, little is known about how pp150 interacts with the underlying capsid. Moreover, the smallest capsid protein (SCP), while dispensable in herpes simplex virus type 1, was shown to play essential, yet undefined, role in HCMV infection. Here, by cryo electron microscopy (cryoEM), we determine three-dimensional structures of HCMV capsid (no pp150) and virion (with pp150) at sub-nanometer resolution. Comparison of these two structures reveals that each pp150 tegument density is composed of two helix bundles connected by a long central helix. Correlation between the resolved helices and sequence-based secondary structure prediction maps the tegument density to the N-terminal half of pp150. The structures also show that SCP mediates interactions between the capsid and pp150 at the upper helix bundle of pp150. Consistent with this structural observation, ribozyme inhibition of SCP expression in HCMV-infected cells impairs the formation of DNA-containing viral particles and reduces viral yield by 10,000 fold. By cryoEM reconstruction of the resulting “SCP-deficient” viral particles, we further demonstrate that SCP is required for pp150 functionally binding to the capsid. Together, our structural and biochemical results point to a mechanism whereby SCP recruits pp150 to stabilize genome-containing capsid for the production of infectious HCMV virion. PMID:23966856

  8. Detection of human cytomegalovirus and Epstein-Barr Virus in symptomatic and asymptomatic apical periodontitis lesions by real-time PCR

    PubMed Central

    Ozbek, Selcuk M.; Yavuz, Muhammed S.

    2013-01-01

    Objectives: Recent studies have investigated the occurrence of human cytomegalovirus and Epstein-Barr Virus in samples from apical periodontitis lesions and a role in the pathogenesis of this disease has been suggested. Because genotype distribution and seroprevalence of EBV and HCMV differ among populations, it is important to determine the presence of these viruses in endodontic periapical lesions of different populations. The aims of this study were to determine the presence of HCMV and EBV DNAs in samples from Turkish patients with symptomatic and asymptomatic apical periodontitis lesions using real-time polymerase chain reaction method and to evaluate their presence in both symptomatic and asymptomatic apical periodontitis lesions. Study Design: Periapical samples were collected from 12 asymptomatic and 16 symptomatic periapical lesions in conjunction with apicectomy. HCMV and EBV DNAs were identified in the samples by real-time PCR. The chi-squared test with Yates’s correction or the Fisher’s exact test was used to analyse the significance of differences. Results: HCMV DNA was detected in 10 of the 16 (62.5%) symptomatic and in five of the 12 (41.7 %) asymptomatic periapical study lesions. The EBV DNA was identified in seven of the 16 (43.7 %) symptomatic and three of the 12 (25 %) asymptomatic periapical lesions. The difference in occurrence of HCMV and EBV DNA between symptomatic and asymptomatic periapical lesions was not statistically significant. (All comparisons have p > 0.05). Conclusions: Our findings suggest that HCMV and EBV is a frequent inhabitant of both symptomatic and asymptomatic apical periodontitis lesions of endodontic origin in Turkish population. Key words:Human cytomegalovirus, Epstein-Barr Virus, apical periodontitis, Polymerase chain reaction method. PMID:23722135

  9. Specific Residues of a Conserved Domain in the N Terminus of the Human Cytomegalovirus pUL50 Protein Determine Its Intranuclear Interaction with pUL53*

    PubMed Central

    Milbradt, Jens; Auerochs, Sabrina; Sevvana, Madhumati; Muller, Yves A.; Sticht, Heinrich; Marschall, Manfred

    2012-01-01

    Herpesviral capsids are assembled in the host cell nucleus and are subsequently translocated to the cytoplasm. During this process it has been demonstrated that the human cytomegalovirus proteins pUL50 and pUL53 interact and form, together with other viral and cellular proteins, the nuclear egress complex at the nuclear envelope. In this study we provide evidence that specific residues of a conserved N-terminal region of pUL50 determine its intranuclear interaction with pUL53. In silico evaluation and biophysical analyses suggested that the conserved region forms a regular secondary structure adopting a globular fold. Importantly, site-directed replacement of individual amino acids by alanine indicated a strong functional influence of specific residues inside this globular domain. In particular, mutation of the widely conserved residues Glu-56 or Tyr-57 led to a loss of interaction with pUL53. Consistent with the loss of binding properties, mutants E56A and Y57A showed a defective function in the recruitment of pUL53 to the nuclear envelope in expression plasmid-transfected and human cytomegalovirus-infected cells. In addition, in silico analysis suggested that residues 3–20 form an amphipathic α-helix that appears to be conserved among Herpesviridae. Point mutants revealed a structural role of this N-terminal α-helix for pUL50 stability rather than a direct role in the binding of pUL53. In contrast, the central part of the globular domain including Glu-56 and Tyr-57 is directly responsible for the functional interaction with pUL53 and thus determines formation of the basic nuclear egress complex. PMID:22589554

  10. Refining human T-cell immunotherapy of cytomegalovirus disease: a mouse model with 'humanized' antigen presentation as a new preclinical study tool.

    PubMed

    Lemmermann, Niels A W; Reddehase, Matthias J

    2016-12-01

    With the cover headline 'T cells on the attack,' the journal Science celebrated individualized cancer immunotherapy by adoptive transfer of T cells as the 'Breakthrough of the Year' 2013 (J. Couzin-Frankel in Science 342:1432-1433, 2013). It is less well recognized and appreciated that individualized T cell immunotherapy of cytomegalovirus (CMV) infection is approaching clinical application for preventing CMV organ manifestations, interstitial CMV pneumonia in particular. This coincident medical development is particularly interesting as reactivated CMV infection is a major viral complication in the state of transient immunodeficiency after the therapy of hematopoietic malignancies by hematopoietic cell transplantation (HCT). It may thus be attractive to combine T cell immunotherapy of 'minimal residual disease/leukemia (MRD)' and CMV-specific T cell immunotherapy to combat both risks in HCT recipients simultaneously, and ideally with T cells derived from the respective HLA-matched HCT donor. Although clinical trials of human CMV-specific T cell immunotherapy were promising in that the incidence of virus reactivation and disease was found to be reduced with statistical significance, animal models are still instrumental for providing 'proof of concept' by directly documenting the prevention of viral multiple-organ histopathology and organ failure under controlled conditions of the absence versus presence of the therapy, which obviously is not feasible in an individual human patient. Further, animal models can make predictions regarding parameters that determine the efficacy of T cell immunotherapy for improved study design in clinical investigations, and they allow for manipulating host and virus genetics. The latter is of particular value as it opens the possibility for epitope specificity controls that are inherently missing in clinical trials. Here, we review a recently developed new mouse model that is more approximated to human CMV-specific T cell immunotherapy

  11. Prevalence and association of human papillomavirus 16, Epstein-Barr virus, herpes simplex virus-1 and cytomegalovirus infection with human esophageal carcinoma: a case-control study.

    PubMed

    Zhang, Dong-Hong; Zhang, Qing-Ying; Hong, Chao-Qun; Chen, Jiong-Yu; Shen, Zhong-Ying; Zhu, Yi

    2011-06-01

    Recent research shows esophageal carcinoma (EC) as the ninth most common malignancy in the world. The association of viral infection and EC has been reported in the last 30 years. However, geographic variation in infection rates and the key mechanisms of the viral action have yet to be resolved. This study aimed to determine the prevalence and association of human papillomavirus 16 (HPV-16), herpes simplex virus 1 (HSV-1), Epstein-Barr virus (EBV) and cytomegalovirus (CMV) infection in the etiology of EC in the area of Shantou, Guangdong, China. Nested PCR was used to detect viral DNA in the mucosa of 70 cases of EC and in paracancerous tissues, as well as 100 cases of normal esophagus mucosa. Data were analyzed by χ2 test, Fisher's exact test and bivariate correlation analysis. The infection rates of HPV-16, HSV-1 and EBV were 40.0, 30.0 and 30.0%, respectively, in EC mucosa, and were significantly higher than those in normal mucosa. However, no CMV DNA was detected in either EC or normal mucosa. HPV-16 or EBV infection was mainly detected in EC patients 48-58 years old, and the infection rate was positively associated with pathological grade of EC (P<0.05). Tobacco smoking and alcohol consuption were high risk factors for HPV-16 infection for male patients [odds ratio (OR), 5.9; 95% confidence interval (CI), 1.4-24.6; OR = 3.8; 95% CI, 1.1-13.8]. Rates of infection with a mixture of these 3 viruses were all more than 10.0% in cancerous mucosa and closely related to the pathological grade of EC (P = 0.001). Infection with HPV-16, HSV-1 or EBV may be an important etiological factor in EC.

  12. Cytomegalovirus Colitis in Immunocompetent Patients

    PubMed Central

    Hussain, Qulsoom; Shafique, Khurram; Tasleem, Syed H; Hurairah, Abu

    2016-01-01

    Cytomegalovirus colitis is common in immunocompromised patients, but rare in immunocompetent patients. The present study not only represents the colonoscopy and pathological findings, but also applies the method of diagnosing and treating cytomegalovirus colitis in immunocompetent patients. PMID:27980888

  13. Recombinant human monoclonal antibodies to human cytomegalovirus glycoprotein B neutralize virus in a complement-dependent manner.

    PubMed

    Ohta, Akane; Fujita, Ayano; Murayama, Tsugiya; Iba, Yoshitaka; Kurosawa, Yoshikazu; Yoshikawa, Tetsushi; Asano, Yoshizo

    2009-11-01

    Human antibodies specific for HCMV are currently considered as potential anti-HCMV therapeutic agents. In this study, we used a combinatorial human antibody library to isolate and characterize complete human monoclonal antibodies that effectively neutralize HCMV in a complement-dependent manner. One hundred and six clones were isolated in two independent screens using HCMV virions and recombinant glycoprotein B, gB654, as antigens. All of the clones recognized the same molecule gB and were classified into 14 groups based on the amino acid sequence of the V(H) region. Seven representative clones from these 14 groups had a strong gB654 binding affinity by surface plasmon resonance (SPR). A pairwise binding competition analysis suggested that there were three groups based on differences in the gB recognition sites. Although Fab fragments of the seven groups showed strong affinity for gB, none of the Fab fragments neutralized HCMV infectivity in vitro. In contrast, complete human IgG(1) antibodies of at least three groups neutralized HCMV in a complement-dependent manner. These data suggest that potent therapeutic antibodies can be obtained from a human antibody library, including most of the functional antibodies that mediate humoral immunity to the selected pathogen.

  14. High levels of CMV-IE-1-specific memory T cells are associated with less alloimmunity and improved renal allograft function.

    PubMed

    Nickel, Peter; Bold, Gantuja; Presber, Franziska; Biti, Didier; Babel, Nina; Kreutzer, Stephanie; Pratschke, Johann; Schönemann, Constanze; Kern, Florian; Volk, Hans-Dieter; Reinke, Petra

    2009-03-01

    Cytomegalovirus (CMV) infection has been associated with allograft rejection in solid organ transplantation. However, the immunologic mechanisms behind this observation have not been elucidated. One proposed mechanism is direct cross-reactivity of antiviral T-cells with allogeneic MHC/peptide complexes, a process termed heterologous immunity. Another model favours indirect stimulation of alloimmunity by CMV-induced proinflammatory cytokines and upregulation of MHC class II and adhesion molecules. Recently, we found that protection from CMV disease was correlated with high levels of CMV-immediate early-1 (IE-1) specific IFN-gamma-producing T-cell responses in heart and lung transplant recipients. The aim of this study was to define the relation of CMV-specific T-cell responses to acute rejection, donor-reactive memory T cells, and allograft function after kidney transplantation. To address this issue, IFN-gamma-producing T-cell responses following ex-vivo stimulation with pools of overlapping peptides representing the CMV pp65 and IE-1 proteins, as well as donor-reactive IFN-gamma-producing T-cells were determined at multiple time points before (pre-Tx) and during the first 6 months posttransplant (post-Tx) in 36 kidney transplant recipients using an enzyme linked immunoabsorbent spot assay (ELISPOT). CMV-specific T cells were not exclusively detectable in CMV seropositive patients, as 3/12 seronegative patients had significant pre- and post-Tx pp65/IE-1-specific T-cell responses. In patients with detectable anti-CMV antibody or T-cell responses, no difference in CMV-specific T-cell frequencies was found between patients with versus without acute rejection. However, early (week 1, r=0.457, p=0.037) and average IE-1-specific T-cell responses (r=-0.415, p=0.032) during 6 months post-Tx showed a significant inverse correlation with average post-Tx donor-reactive T-cell responses. Furthermore, average post-Tx IE-1-specific T-cell responses correlated significantly with

  15. Diagnosis of Cytomegalovirus Infections

    PubMed Central

    Ross, S.A.; Novak, Z.; Pati, S.; Boppana, S.B.

    2013-01-01

    Cytomegalovirus (CMV) is recognized as the most common congenital viral infection in humans and an important cause of morbidity and mortality in immunocompromised hosts. This recognition of the clinical importance of invasive CMV disease in the setting of immunodeficiency and in children with congenital CMV infection has led to the development of new diagnostic procedures for the rapid identification of immunocompromised individuals with CMV disease, as well as fetuses and infants with congenital infection. Diagnosis of acute maternal CMV infection by the presence of IgM and low IgG avidity requires confirmation of fetal infection which is typically performed by CMV PCR of the amniotic fluid. Viral culture of the urine and saliva obtained within the first two weeks of life continue to be the gold standard for diagnosis of congenitally infected infants. PCR assays of dried blood spots from infants have not been shown to have sufficient sensitivity for the identification of most infants with congenital CMV infection. However, saliva PCR assays are currently being assessed as a useful screening method for congenital CMV infection. In the immunocompromised host, newer rapid diagnostic assays such as pp65 antigenemia and real-time CMV PCR of blood or plasma have allowed for preemptive treatment reducing morbidity and mortality. However, lack of standardized real-time PCR protocols hinders the comparison of the data across different centers and the development of uniform guidelines for the management of invasive CMV infections in immunocompromised individuals. PMID:21827433

  16. Evaluation of T Cell Immunity against Human Cytomegalovirus: Impact on Patient Management and Risk Assessment of Vertical Transmission

    PubMed Central

    Quaranta, Paola; Pistello, Mauro

    2016-01-01

    Cytomegalovirus (CMV) is one of the most common infectious agents, infecting the general population at an early age without causing morbidity most of the time. However, on particular occasions, it may represent a serious risk, as active infection is associated with rejection and disease after solid organ transplantation or fetal transmission during pregnancy. Several methods for CMV diagnosis are available on the market, but because infection is so common, careful selection is needed to discriminate primary infection from reactivation. This review focuses on methods based on CMV-specific T cell reactivity to help monitor the consequences of CMV infection/reactivation in specific categories of patients. This review makes an attempt at discussing the pros and cons of the methods available. PMID:28044143

  17. Development and preclinical evaluation of an alphavirus replicon particle vaccine for cytomegalovirus.

    PubMed

    Reap, Elizabeth A; Morris, John; Dryga, Sergey A; Maughan, Maureen; Talarico, Todd; Esch, Robert E; Negri, Sarah; Burnett, Bruce; Graham, Andrew; Olmsted, Robert A; Chulay, Jeffrey D

    2007-10-16

    We used a replication-incompetent, single-cycle, alphavirus replicon vector system to produce virus-like replicon particles (VRP) expressing the extracellular domain of human cytomegalovirus (CMV) glycoprotein B or a pp65/IE1 fusion protein. Efficient production methods were scaled to produce pilot lots and clinical lots of each alphavirus replicon vaccine component. The vaccine induced high-titered antibody responses in mice and rabbits, as measured by ELISA and CMV neutralization assays, and robust T-cell responses in mice, as measured by IFN-gamma ELISPOT assay. A toxicity study in rabbits showed no adverse effects in any toxicology parameter. These studies support clinical testing of this novel CMV alphavirus replicon vaccine in humans.

  18. Preliminary exploration of HLA-A 1101-restricted human cytomegalovirus glycoprotein B-specific CD8⁺ T cells in allogeneic stem-cell transplant recipients.

    PubMed

    Liu, Anbing; Hu, Jianhua; Wu, Wei; Huang, Yaping; Liang, Hanying; Wang, Huiqi; Yang, Rong; Fan, Jun

    2014-08-08

    T-cell responses directed against human cytomegalovirus (HCMV) glycoprotein B (gB) contribute to protective immunity against HCMV infection in both animal models and humans. However, the gB-specific human CD8(+) T cell responses remain poorly understood. gB antigen-specific CD8(+) T cells were stained with seven major histocompatibility complex (MHC)-peptide pentamers in 16 human leukocyte antigen (HLA)-A 1101-positive, HCMV-seropositive patients following hematopoietic stem cell transplantation (HSCT). Of these seven pentamers, the most frequent CD8(+) T-cell responses were directed against the gB332-340 peptide. These gB332-340-specific CD8(+) T cells were strongly associated with the presence of plasma HCMV immunoglobulin M in all HSCT recipients and exhibited a probable causal relationship with the level of pp65 antigenemia. Together, these data suggest a role for gB332-340-specific CD8(+) T cells in HCMV reactivation after HSCT. Furthermore, the pentamer assay may be valuable in detecting antigen-specific CD8(+) T cells.

  19. Failure to detect human herpes simplex virus, cytomegalovirus, and Epstein-Barr virus viral genomes in giant cell arteritis biopsy specimens by real-time quantitative polymerase chain reaction.

    PubMed

    Cankovic, Milena; Zarbo, Richard J

    2006-01-01

    A study provided evidence of human herpes simplex virus (HSV) DNA in giant cell arteritis (GCA) biopsy specimens. This prompted us to study our own GCA biopsy specimens using real-time quantitative polymerase chain reaction for the detection of HSV1, cytomegalovirus, and Epstein-Barr virus DNAs. Our study failed to confirm an association between HSV1 and GCA, revealing no viral genome in 35 biopsy specimens of histologically positive temporal arteries.

  20. Natural Killer Cells Can Inhibit the Transmission of Human Cytomegalovirus in Cell Culture by Using Mechanisms from Innate and Adaptive Immune Responses

    PubMed Central

    Wu, Zeguang; Sinzger, Christian; Reichel, Johanna Julia; Just, Marlies

    2014-01-01

    ABSTRACT Human cytomegalovirus (HCMV) transmission within the host is important for the pathogenesis of HCMV diseases. Natural killer (NK) cells are well known to provide a first line of host defense against virus infections. However, the role of NK cells in the control of HCMV transmission is still unknown. Here, we provide the first experimental evidence that NK cells can efficiently control HCMV transmission in different cell types. NK cells engage different mechanisms to control the HCMV transmission both via soluble factors and by cell contact. NK cell-produced interferon gamma (IFN-γ) suppresses HCMV production and induces resistance of bystander cells to HCMV infection. The UL16 viral gene contributes to an immune evasion from the NK cell-mediated control of HCMV transmission. Furthermore, the efficacy of the antibody-dependent NK cell-mediated control of HCMV transmission is dependent on a CD16-158V/F polymorphism. Our findings indicate that NK cells may have a clinical relevance in HCMV infection and highlight the need to consider potential therapeutic strategies based on the manipulation of NK cells. IMPORTANCE Human cytomegalovirus (HCMV) infects 40% to 100% of the human population worldwide. After primary infection, mainly in childhood, the virus establishes a lifelong persistence with possible reactivations. Most infections remain asymptomatic; however, HCMV represents a major health problem since it is the most frequent cause of infection-induced birth defects and is responsible for high morbidity and mortality in immunocompromised patients. The immune system normally controls the infection by antibodies and immune effector cells. One type of effector cells are the natural killer (NK) cells, which provide a rapid response to virus-infected cells. NK cells participate in viral clearance by inducing the death of infected cells. NK cells also secrete antiviral cytokines as a consequence of the interaction with an infected cell. In this study, we

  1. Human Cytomegalovirus Stimulates the Synthesis of Select Akt-Dependent Antiapoptotic Proteins during Viral Entry To Promote Survival of Infected Monocytes

    PubMed Central

    Peppenelli, Megan A.; Arend, Kyle C.; Cojohari, Olesea; Moorman, Nathaniel J.

    2016-01-01

    ABSTRACT Primary peripheral blood monocytes are responsible for the hematogenous dissemination of human cytomegalovirus (HCMV) following a primary infection. To facilitate viral spread, we have previously shown HCMV to extend the short 48-h life span of monocytes. Mechanistically, HCMV upregulated two specific cellular antiapoptotic proteins, myeloid leukemia sequence 1 (Mcl-1) and heat shock protein 27 (HSP27), to block the two proteolytic cleavages necessary for the formation of fully active caspase 3 and the subsequent initiation of apoptosis. We now show that HCMV more robustly upregulated Mcl-1 than normal myeloid growth factors and that Mcl-1 was the only myeloid survival factor to rapidly induce HSP27 prior to the 48-h cell fate checkpoint. We determined that HCMV glycoproteins gB and gH signal through the cellular epidermal growth factor receptor (EGFR) and αvβ3 integrin, respectively, during viral entry in order to drive the increase of Mcl-1 and HSP27 in an Akt-dependent manner. Although Akt is known to regulate protein stability and transcription, we found that gB- and gH-initiated signaling preferentially and cooperatively stimulated the synthesis of Mcl-1 and HSP27 through mTOR-mediated translation. Overall, these data suggest that the unique signaling network generated during the viral entry process stimulates the upregulation of select antiapoptotic proteins allowing for the differentiation of short-lived monocytes into long-lived macrophages, a key step in the viral dissemination strategy. IMPORTANCE Human cytomegalovirus (HCMV) infection is endemic within the human population. Although primary infection is generally asymptomatic in immunocompetent individuals, HCMV is a significant cause of morbidity and mortality in the immunocompromised. The multiorgan inflammatory diseases associated with symptomatic HCMV infection are a direct consequence of the monocyte-mediated systemic spread of the virus. In order for peripheral blood monocytes to

  2. Neutralization of Diverse Human Cytomegalovirus Strains Conferred by Antibodies Targeting Viral gH/gL/pUL128-131 Pentameric Complex

    PubMed Central

    Ha, Sha; Li, Fengsheng; Troutman, Matthew C.; Freed, Daniel C.; Tang, Aimin; Loughney, John W.; Wang, I-Ming; Vlasak, Josef; Nickle, David C.; Rustandi, Richard R.; Hamm, Melissa; DePhillips, Pete A.; Zhang, Ningyan; McLellan, Jason S.; Zhu, Hua; Adler, Stuart P.; McVoy, Michael A.; An, Zhiqiang

    2017-01-01

    ABSTRACT Human cytomegalovirus (HCMV) is the leading cause of congenital viral infection, and developing a prophylactic vaccine is of high priority to public health. We recently reported a replication-defective human cytomegalovirus with restored pentameric complex glycoprotein H (gH)/gL/pUL128-131 for prevention of congenital HCMV infection. While the quantity of vaccine-induced antibody responses can be measured in a viral neutralization assay, assessing the quality of such responses, including the ability of vaccine-induced antibodies to cross-neutralize the field strains of HCMV, remains a challenge. In this study, with a panel of neutralizing antibodies from three healthy human donors with natural HCMV infection or a vaccinated animal, we mapped eight sites on the dominant virus-neutralizing antigen—the pentameric complex of glycoprotein H (gH), gL, and pUL128, pUL130, and pUL131. By evaluating the site-specific antibodies in vaccine immune sera, we demonstrated that vaccination elicited functional antiviral antibodies to multiple neutralizing sites in rhesus macaques, with quality attributes comparable to those of CMV hyperimmune globulin. Furthermore, these immune sera showed antiviral activities against a panel of genetically distinct HCMV clinical isolates. These results highlighted the importance of understanding the quality of vaccine-induced antibody responses, which includes not only the neutralizing potency in key cell types but also the ability to protect against the genetically diverse field strains. IMPORTANCE HCMV is the leading cause of congenital viral infection, and development of a preventive vaccine is a high public health priority. To understand the strain coverage of vaccine-induced immune responses in comparison with natural immunity, we used a panel of broadly neutralizing antibodies to identify the immunogenic sites of a dominant viral antigen—the pentameric complex. We further demonstrated that following vaccination of a replication

  3. High-Throughput Small Interfering RNA Screening Identifies Phosphatidylinositol 3-Kinase Class II Alpha as Important for Production of Human Cytomegalovirus Virions

    PubMed Central

    Polachek, William S.; Moshrif, Hanan F.; Franti, Michael; Coen, Donald M.; Sreenu, Vattipally B.

    2016-01-01

    ABSTRACT High-throughput small interfering RNA (siRNA) screening is a useful methodology to identify cellular factors required for virus replication. Here we utilized a high-throughput siRNA screen based on detection of a viral antigen by microscopy to interrogate cellular protein kinases and phosphatases for their importance during human cytomegalovirus (HCMV) replication and identified the class II phosphatidylinositol 3-kinase class II alpha (PI3K-C2A) as being involved in HCMV replication. Confirming this observation, infected cells treated with either pooled or individual siRNAs targeting PI3K-C2A mRNA produced approximately 10-fold less infectious virus than the controls. Western blotting and quantitative PCR analysis of infected cells treated with siRNAs indicated that depletion of PI3K-C2A slightly reduced the accumulation of late but not immediate early or early viral antigens and had no appreciable effect on viral DNA synthesis. Analysis of siRNA-treated cells by electron microscopy and Western blotting indicated that PI3K-C2A was not required for the production of viral capsids but did lead to increased numbers of enveloped capsids in the cytoplasm that had undergone secondary envelopment and a reduction in the amount of viral particles exiting the cell. Therefore, PI3K-C2A is a factor important for HCMV replication and has a role in the production of HCMV virions. IMPORTANCE There is limited information about the cellular factors required for human cytomegalovirus (HCMV) replication. Therefore, to identify proteins involved in HCMV replication, we developed a methodology to conduct a high-throughput siRNA screen of HCMV-infected cells. From our screening data, we focused our studies on the top hit from our screen, the lipid kinase phosphatidylinositol 3-kinase class II alpha (PI3K-C2A), as its role in HCMV replication was unknown. Interestingly, we found that PI3K-C2A is important for the production of HCMV virions and is involved in virion production

  4. Development and optimization of a sensitive TaqMan® real-time PCR with synthetic homologous extrinsic control for quantitation of Human cytomegalovirus viral load.

    PubMed

    Slavov, Svetoslav Nanev; Otaguiri, Katia Kaori; de Figueiredo, Glauciane Garcia; Yamamoto, Aparecida Yulie; Mussi-Pinhata, Marisa Marcia; Kashima, Simone; Covas, Dimas Tadeu

    2016-09-01

    Human cytomegalovirus (Human herpesvirus 5, HCMV) causes frequent asymptomatic infections in the general population. However, in immunosuppressed patients or congenitally infected infants, HCMV is related to high morbidity and mortality. In such cases, a rapid viral detection is crucial for monitoring the clinical outcome and the antiviral treatment. In this study, we optimized a sensitive biplex TaqMan® real-time PCR for the simultaneous detection and differentiation of a partial HCMV UL97 sequence and homologous extrinsic control (HEC) in the same tube. HEC was represented by a plasmid containing a modified HCMV sequence retaining the original primer binding sites, while the probe sequence was substituted by a phylogenetically divergent one (chloroplast CF0 subunit plant gene). It was estimated that the optimal HEC concentration, which did not influence the HCMV amplification is 1,000 copies/reaction. The optimized TaqMan® PCR demonstrated high analytical sensitivity (6.97 copies/reaction, CI = 95%) and specificity (100%). Moreover, the reaction showed adequate precision (repeatability, CV = 0.03; reproducibility, CV = 0.0027) and robustness (no carry-over or cross-contamination). The diagnostic sensitivity (100%) and specificity (97.8%) were adequate for the clinical application of the molecular platform. The optimized TaqMan® real-time PCR is suitable for HCMV detection and quantitation in predisposed patients and monitoring of the applied antiviral therapy. J. Med. Virol. 88:1604-1612, 2016. © 2016 Wiley Periodicals, Inc.

  5. Human cytomegalovirus specifically controls the levels of the endoplasmic reticulum chaperone BiP/GRP78, which is required for virion assembly.

    PubMed

    Buchkovich, Nicholas J; Maguire, Tobi G; Yu, Yongjun; Paton, Adrienne W; Paton, James C; Alwine, James C

    2008-01-01

    The endoplasmic reticulum (ER) chaperone BiP/GRP78 regulates ER function and the unfolded protein response (UPR). Human cytomegalovirus infection of human fibroblasts induces the UPR but modifies it to benefit viral replication. BiP/GRP78 protein levels are tightly regulated during infection, rising after 36 h postinfection (hpi), peaking at 60 hpi, and decreasing thereafter. To determine the effects of this regulation on viral replication, BiP/GRP78 was depleted using the SubAB subtilase cytotoxin, which rapidly and specifically cleaves BiP/GRP78. Toxin treatment of infected cells for 12-h periods beginning at 36, 48, 60, and 84 hpi caused complete loss of BiP but had little effect on viral protein synthesis. However, progeny virion formation was significantly inhibited, suggesting that BiP/GRP78 is important for virion formation. Electron microscopic analysis showed that infected cells were resistant to the toxin and showed none of the cytotoxic effects seen in uninfected cells. However, all viral activity in the cytoplasm ceased, with nucleocapsids remaining in the nucleus or concentrated in the cytoplasmic space just outside of the outer nuclear membrane. These data suggest that one effect of the controlled expression of BiP/GRP78 in infected cells is to aid in cytoplasmic virion assembly and egress.

  6. Human cytomegalovirus hyperimmune globulin not only neutralizes HCMV infectivity, but also inhibits HCMV-induced intracellular NF-kappaB, Sp1, and PI3-K signaling pathways.

    PubMed

    Andreoni, Kenneth A; Wang, Xin; Huang, Shu-Mei; Huang, Eng-Shang

    2002-05-01

    Inhibition of virus-induced intracellular signaling pathways and viral infectivity are our ultimate goals in the development of effective antiviral agents to control human cytomegalovirus (HCMV) infections. The HCMV hyperimmune globulin may meet such criteria. In a human embryonic lung (HEL) fibroblast culture model, pretreatment of Towne strain HCMV with HCMV hyperimmune globulin was shown to inhibit viral infectivity successfully, as measured by a standard plaque assay. The extracellular viral titers and extracellular viral DNA, as measured by plaque assay and PCR, respectively, were also decreased. In addition, the HCMV hyperimmune globulin prevented HCMV from inducing the intracellular activation of NF-kappaB, Sp-1, and PI3-K signaling pathways. The PI3-K pathway was examined by following phosphorylation (activation) of two of its downstream kinases, Akt and p70S6K. HCMV hyperimmune globulin also prevented the production of immediate early, early, and late viral proteins. These studies show that HCMV hyperimmune globulin neutralization of HCMV prevents the earliest known events observed after viral envelope glycoproteins bind their cell membrane receptors, i.e., NF-kappaB, Sp-1 and PI3-K activation. This suggests that HCMV hyperimmune globulin not only can inhibit viral infectivity, but can also prevent the abnormal cellular signaling that may induce unwanted cellular proliferation or cytokine synthesis. Copyright 2002 Wiley-Liss, Inc.

  7. Importance of Highly Conserved Peptide Sites of Human Cytomegalovirus gO for Formation of the gH/gL/gO Complex

    PubMed Central

    Stegmann, Cora; Abdellatif, Mohamed E. A.; Laib Sampaio, Kerstin; Walther, Paul

    2016-01-01

    ABSTRACT The glycoprotein O (gO) is betaherpesvirus specific. Together with the viral glycoproteins H and L, gO forms a covalent trimeric complex that is part of the viral envelope. This trimer is crucial for cell-free infectivity of human cytomegalovirus (HCMV) but dispensable for cell-associated spread. We hypothesized that the amino acids that are conserved among gOs of different cytomegaloviruses are important for the formation of the trimeric complex and hence for efficient virus spread. In a mutational approach, nine peptide sites, containing all 13 highly conserved amino acids, were analyzed in the context of HCMV strain TB40-BAC4 with regard to infection efficiency and formation of the gH/gL/gO complex. Mutation of amino acids (aa) 181 to 186 or aa 193 to 198 resulted in the loss of the trimer and a complete small-plaque phenotype, whereas mutation of aa 108 or aa 249 to 254 caused an intermediate phenotype. While individual mutations of the five conserved cysteines had little impact, their relevance was revealed in a combined mutation, which abrogated both complex formation and cell-free infectivity. C343 was unique, as it was sufficient and necessary for covalent binding of gO to gH/gL. Remarkably, however, C218 together with C167 rescued infectivity in the absence of detectable covalent complex formation. We conclude that all highly conserved amino acids contribute to the function of gO to some extent but that aa 181 to 198 and cysteines 343, 218, and 167 are particularly relevant. Surprisingly, covalent binding of gO to gH/gL is required neither for its incorporation into virions nor for proper function in cell-free infection. IMPORTANCE Like all herpesviruses, the widespread human pathogen HCMV depends on glycoproteins gB, gH, and gL for entry into target cells. Additionally, gH and gL have to bind gO in a trimeric complex for efficient cell-free infection. Homologs of gO are shared by all cytomegaloviruses, with 13 amino acids being highly conserved

  8. Importance of Highly Conserved Peptide Sites of Human Cytomegalovirus gO for Formation of the gH/gL/gO Complex.

    PubMed

    Stegmann, Cora; Abdellatif, Mohamed E A; Laib Sampaio, Kerstin; Walther, Paul; Sinzger, Christian

    2017-01-01

    The glycoprotein O (gO) is betaherpesvirus specific. Together with the viral glycoproteins H and L, gO forms a covalent trimeric complex that is part of the viral envelope. This trimer is crucial for cell-free infectivity of human cytomegalovirus (HCMV) but dispensable for cell-associated spread. We hypothesized that the amino acids that are conserved among gOs of different cytomegaloviruses are important for the formation of the trimeric complex and hence for efficient virus spread. In a mutational approach, nine peptide sites, containing all 13 highly conserved amino acids, were analyzed in the context of HCMV strain TB40-BAC4 with regard to infection efficiency and formation of the gH/gL/gO complex. Mutation of amino acids (aa) 181 to 186 or aa 193 to 198 resulted in the loss of the trimer and a complete small-plaque phenotype, whereas mutation of aa 108 or aa 249 to 254 caused an intermediate phenotype. While individual mutations of the five conserved cysteines had little impact, their relevance was revealed in a combined mutation, which abrogated both complex formation and cell-free infectivity. C343 was unique, as it was sufficient and necessary for covalent binding of gO to gH/gL. Remarkably, however, C218 together with C167 rescued infectivity in the absence of detectable covalent complex formation. We conclude that all highly conserved amino acids contribute to the function of gO to some extent but that aa 181 to 198 and cysteines 343, 218, and 167 are particularly relevant. Surprisingly, covalent binding of gO to gH/gL is required neither for its incorporation into virions nor for proper function in cell-free infection. Like all herpesviruses, the widespread human pathogen HCMV depends on glycoproteins gB, gH, and gL for entry into target cells. Additionally, gH and gL have to bind gO in a trimeric complex for efficient cell-free infection. Homologs of gO are shared by all cytomegaloviruses, with 13 amino acids being highly conserved. In a mutational

  9. Hearing Loss and Cytomegalovirus.

    ERIC Educational Resources Information Center

    Strauss, Melvin

    1997-01-01

    Cytomegalovirus is the most common cause of congenital virally induced hearing loss. Maternal infection is most often asymptomatic as is the infection in the newborn. Hearing loss occurs in both clinically apparent infection and in the asymptomatic infection. Current methods of detection, treatment, and prevention and research efforts are…

  10. Hearing Loss and Cytomegalovirus.

    ERIC Educational Resources Information Center

    Strauss, Melvin

    1997-01-01

    Cytomegalovirus is the most common cause of congenital virally induced hearing loss. Maternal infection is most often asymptomatic as is the infection in the newborn. Hearing loss occurs in both clinically apparent infection and in the asymptomatic infection. Current methods of detection, treatment, and prevention and research efforts are…

  11. Association of host genetic risk factors with the course of cytomegalovirus retinitis in patients infected with human immunodeficiency virus.

    PubMed

    Sezgin, Efe; van Natta, Mark L; Ahuja, Alka; Lyon, Alice; Srivastava, Sunil; Troyer, Jennifer L; O'Brien, Stephen J; Jabs, Douglas A

    2011-06-01

    To evaluate the effects of previously reported host genetics factors that influence cytomegalovirus (CMV) retinitis incidence, progression to acquired immune deficiency syndrome (AIDS), and efficacy of highly active antiretroviral therapy (HAART) for mortality, retinitis progression, and retinal detachment in patients with CMV retinitis and AIDS in the era of HAART. Prospective, multicenter, observational study. Cox proportional hazards model based genetic association tests examined the influence of IL-10R1_S420L, CCR5-Δ32, CCR2-V64I, CCR5 promoter, and SDF-3'A polymorphisms among patients with mortality, retinitis progression, and retinal detachment. Participants were 203 European-American and 117 African-American patients with AIDS and CMV retinitis. European-American patients with the CCR5 +.P1.+ promoter haplotype showed increased risk for mortality (hazard ratio [HR] = 1.83; 95% confidence interval [CI]: 1.00-3.40; P = .05). Although the same haplotype also trended for increased risk for mortality in African-American patients, the result was not significant (HR = 2.28; 95% CI: 0.93-5.60; P = .07). However, this haplotype was associated with faster retinitis progression in African Americans (HR = 5.22; 95% CI: 1.54-17.71; P = .007). Increased risk of retinitis progression was also evident for African-American patients with the SDF1-3'A variant (HR = 3.89; 95% CI: 1.42-10.60; P = .008). In addition, the SDF1-3'A variant increased the retinal detachment risk in this patient group (HR = 3.05; 95% CI: 1.01-9.16; P = .05). Besides overall immune health, host genetic factors influence mortality, retinitis progression, and retinal detachment in patients with AIDS and CMV retinitis that are receiving HAART. Published by Elsevier Inc.

  12. Epigenetically repressing human cytomegalovirus lytic infection and reactivation from latency in THP-1 model by targeting H3K9 and H3K27 histone demethylases

    PubMed Central

    Yu, Yanyan; Yi, Wei; Zhu, Shanshan; Li, En

    2017-01-01

    Human Cytomegalovirus (hCMV) infects a broad range of the population and establishes life-long latency in the infected individuals. Periodically the latently infected virus can reactivate and becomes a significant cause of morbidity and mortality in immunocompromised individuals. In latent infection, the viral genome is suppressed in a heterochromatic state and viral gene transcription is silenced. Upon reactivation, the repressive chromatin is remodeled to an active form, allowing viral lytic gene transcription, initiated by the expression of viral Immediate Early (IE) genes. During this process, a number of histone modification enzymes, including histone demethylases (HDMs), play important roles in driving IE expression, but the mechanisms involved are not fully understood. To get a better understanding of these mechanisms, we focused on two HDMs, KDM4 and KDM6, which reverse the repressive histone H3-lysine 9 and lysine 27 methylation, respectively. Our studies show that in lytic infection, both demethylases are important in the activation of viral IE gene expression. Simultaneous disruption of both via genetic or chemical methods leads to severely impaired viral IE gene expression and viral replication. Additionally, in an experimental latency-reactivation model in THP-1 cells, the KDM6 family member JMJD3 is induced upon viral reactivation and its knockdown resulted in reduced IE gene transcription. These findings suggest pharmacological inhibition of these HDMs may potentially block hCMV lytic infection and reactivation, and control the viral infection associated diseases, which are of significant unmet medical needs. PMID:28407004

  13. Distinct functional domains within the acidic cluster of tegument protein pp28 required for trafficking and cytoplasmic envelopment of human cytomegalovirus.

    PubMed

    Seo, Jun-Young; Jeon, Hyejin; Hong, Sookyung; Britt, William J

    2016-10-01

    Human cytomegalovirus UL99-encoded tegument protein pp28 contains a 16 aa acidic cluster that is required for pp28 trafficking to the assembly compartment (AC) and the virus assembly. However, functional signals within the acidic cluster of pp28 remain undefined. Here, we demonstrated that an acidic cluster rather than specific sorting signals was required for trafficking to the AC. Recombinant viruses with chimeric pp28 proteins expressing non-native acidic clusters exhibited delayed viral growth kinetics and decreased production of infectious virus, indicating that the native acidic cluster of pp28 was essential for wild-type virus assembly. These results suggested that the acidic cluster of pp28 has distinct functional domains required for trafficking and for efficient virus assembly. The first half (aa 44-50) of the acidic cluster was sufficient for pp28 trafficking, whereas the native acidic cluster consisting of aa 51-59 was required for the assembly of wild-type levels of infectious virus.

  14. Human cytomegalovirus miR-UL36-5p inhibits apoptosis via downregulation of adenine nucleotide translocator 3 in cultured cells.

    PubMed

    Guo, Xin; Huang, Yujing; Qi, Ying; Liu, Zhongyang; Ma, Yanping; Shao, Yaozhong; Jiang, Shujuan; Sun, Zhengrong; Ruan, Qiang

    2015-10-01

    Human cytomegalovirus (HCMV) encodes at least 26 microRNAs (miRNA). These miRNAs are utilized by HCMV to regulate its own genes as well as the genes of the host cell during infection. It has been reported that a cellular gene, solute carrier family 25, member 6 (SLC25A6), which is also designated adenine nucleotide translocator 3 (ANT3), was identified as a candidate target of hcmv-miR-UL36-5p by hybrid PCR. In this study, ANT3 was further demonstrated to be a direct target of hcmv-miR-UL36-5p by luciferase reporter assays. The expression level of ANT3 protein was confirmed, by western blotting, to be directly downregulated by overexpression of hcmv-miR-UL36-5p in HEK293 cells, U373 cells and HELF cells. Moreover, HCMV-infected cells showed a decrease in the ANT3 protein level. Using ANT3-specific small interfering RNA (siRNA) and an inhibitor for hcmv-miR-UL36-5p, it was shown that inhibition of apoptosis by hcmv-miR-UL36-5p in these cells specifically occurred via inhibition of ANT3 expression. These results imply that hcmv-miR-UL36-5 may play the same role during actual HCMV infection in order to establish a balance between the host cell and the virus.

  15. Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane and Detergent Resistant Membrane Fractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts

    PubMed Central

    Williamson, Chad D.; Wong, Daniel S.; Bozidis, Petros; Zhang, Aiping; Colberg-Poley, Anamaris M.

    2015-01-01

    Increasingly mechanistic virology studies require dependable and sensitive methods for isolating purified organelles containing functional cellular sub-domains. The mitochondrial network is, in part, closely apposed to the endoplasmic reticulum (ER). The mitochondria-associated membrane (MAM) fraction provides direct physical contact between the ER and mitochondria. Characterization of the dual localization and trafficking of human