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Thomas Stamminger - One of the best experts on this subject based on the ideXlab platform.

  • the ul69 Transactivator Protein of human cytomegalovirus interacts with dexd h box rna helicase uap56 to promote cytoplasmic accumulation of unspliced rna
    Molecular and Cellular Biology, 2006
    Co-Authors: Peter Lischka, Zsolt Toth, Marco Thomas, Regina Mueller, Thomas Stamminger
    Abstract:

    The UL69 gene product of human cytomegalovirus belongs to a family of regulatory Proteins conserved among all herpesviruses that have in part been characterized as posttranscriptional Transactivators participating in the nuclear export of RNA. Recent experiments suggested that pUL69 also acts as a posttranscriptional activator since it was demonstrated that nucleocytoplasmic shuttling via a CRM1-independent nuclear export signal is a prerequisite for its stimulatory effect on gene expression. Based on these findings we initiated studies to investigate the role of pUL69 in mRNA export and demonstrate that pUL69 efficiently promotes the cytoplasmic accumulation of unspliced RNA. Furthermore, we show that this pUL69 activity is linked to the cellular mRNA export machinery by direct Protein interaction with the highly related DEXD/H-box RNA helicases UAP56 and URH49. Particularly, we identified a 12-amino-acid domain within the N terminus of pUL69 which is required for binding to UAP56 and URH49, and we could demonstrate that UAP56 interaction and nucleocytoplasmic shuttling are both prerequisites for pUL69-mediated mRNA export. Thus, we identified a novel cellular target which provides a herpesviral regulatory Protein with access to a conserved cellular transport system in order to promote nuclear export of unspliced RNA.

  • rna binding of the human cytomegalovirus Transactivator Protein ul69 mediated by arginine rich motifs is not required for nuclear export of unspliced rna
    Nucleic Acids Research, 2006
    Co-Authors: Zsolt Toth, Peter Lischka, Thomas Stamminger
    Abstract:

    The human cytomegalovirus Protein pUL69 belongs to a family of regulatory factors that is conserved within the Herpesviridae and includes the Proteins ICP27 of herpes simplex virus type 1 and EB2 of Epstein–Barr virus. ICP27 and EB2 have been shown to facilitate the nuclear export of viral mRNAs via interacting with the cellular mRNA export factor REF. Furthermore, direct RNA-binding of these Proteins was found to be essential for their stimulating effects on mRNA export. Recently, we demonstrated that pUL69 shares common features with ICP27 and EB2 such as (i) nucleocytoplasmic shuttling and (ii) stimulation of nuclear RNA export via binding to the cellular mRNA export machinery. Here, we demonstrate that pUL69 can also interact with RNA both in vivo and in vitro via a complex N-terminal RNA-binding domain consisting of three arginine-rich motifs. Interestingly, the RNA-binding domain of pUL69 overlaps with both the NLS and the binding site of the cellular mRNA export factors UAP56 and URH49. While the deletion of the UAP56/URH49-binding site abolished pUL69-mediated RNA export, an RNAbinding deficient pUL69 mutant which still interacts with UAP56/URH49 retained its RNA export activity. This surprising finding suggests that, in contrast to its homologues, RNA-binding is not a prerequisite for pUL69-mediated nuclear RNA export.

  • efficient and tightly regulated expression systems for the human cytomegalovirus major Transactivator Protein ie2p86 in permissive cells
    Virus Research, 2002
    Co-Authors: Martina Kronschnabl, Manfred Marschall, Thomas Stamminger
    Abstract:

    The 86-kDa IE2 Protein (IE2p86) of human cytomegalovirus is a pleiotropic regulatory polypeptide that is essential for activation of viral early promoters and thus, for the entire viral replication cycle. Moreover, this Protein modulates cellular gene expression and contributes to the pathogenic features of HCMV. The full spectrum of IE2p86 mediated effects on cellular gene expression has not been defined yet, since efficient expression systems for this Protein in HCMV permissive cells are, so far, limited. Here, we report the establishment of two efficient model systems that allow a tightly regulated expression of IE2p86 in various permissive cell types including primary human fibroblasts, primary endothelial cells and U373MG cells. Firstly, we generated a tetracycline-regulated U373MG cell line, which expresses high levels of IE2p86 upon tetracycline removal from the culture medium. Secondly, a recombinant baculovirus was constructed, which expresses IE2p86 under the control of the HCMV major immediate early enhancer/promoter upon transduction of various cell types. Importantly, IE2p86 was functional in both systems, since strong transactivation of luciferase promoter constructs could be measured. Furthermore, a cell cycle arrest was detectable after infection of primary human fibroblasts with IE2p86-expressing baculoviruses. Both expression systems represent useful tools to fully define the effects of this pleiotropic Transactivator on cellular gene expression and to establish screening systems for novel antiviral drugs targeting this critical immediate early Protein of HCMV.

  • a novel transferable nuclear export signal mediates crm1 independent nucleocytoplasmic shuttling of the human cytomegalovirus Transactivator Protein pul69
    The EMBO Journal, 2001
    Co-Authors: Peter Lischka, Olaf Rosorius, Erik Trommer, Thomas Stamminger
    Abstract:

    The best studied nuclear export processes are mediated by classical leucine-rich nuclear export signals that specify recognition by the CRM1 export receptor. However, details concerning alternative nuclear export signals and pathways are beginning to emerge. Within the family of Herpesviridae, a set of homologous regulatory Proteins that are exemplified by the ICP27 of herpes simplex virus were described recently as nucleocytoplasmic shuttling Proteins. Here we report that pUL69 of the β-herpesvirus human cytomegalovirus is a nuclear Protein that is able to shuttle between the nucleus and the cytoplasm independently of virus-encoded cofactors. In contrast to Proteins containing a leucine-rich export signal, the shuttling activity of pUL69 was not affected by leptomycin B, indicating that pUL69 trafficking is not mediated by the export receptor CRM1. Importantly, we identified and characterized a novel type of transferable, leptomycin B-insensitive export signal that is distinct from other export signals described previously and is required for pUL69-mediated activation of gene expression. These data suggest that pUL69 is exported via a novel nuclear export pathway, based on a so far unique nuclear export signal of 28 amino acids.

  • covalent modification of the Transactivator Protein ie2 p86 of human cytomegalovirus by conjugation to the ubiquitin homologous Proteins sumo 1 and hsmt3b
    Journal of Virology, 2000
    Co-Authors: Heike Hofmann, Stefan Floss, Thomas Stamminger
    Abstract:

    The 86-kDa IE2 Protein (IE2-p86) of human cytomegalovirus (HCMV) is a potent Transactivator of viral as well as cellular promoters. Several lines of evidence indicate that this broad transactivation spectrum is mediated by Protein-Protein interactions. To identify novel cellular binding partners, we performed a yeast two-hybrid screen using a N-terminal deletion mutant of IE2-p86 comprising amino acids 135 to 579 as a bait. Here, we report the isolation of two ubiquitin-homologous Proteins, SUMO-1 and hSMT3b, as well as their conjugating activity hUBC9 (human ubiquitin-conjugating enzyme 9) as specific interaction partners of HCMV IE2. The polypeptides SUMO-1 and hSMT3b have previously been shown to be covalently coupled to a subset of nuclear Proteins such as the nuclear domain 10 (ND10) Proteins PML and Sp100 in a manner analogous to ubiquitinylation, which we call SUMOylation. By Western blot analysis, we were able to show that the IE2-p86 Protein can be partially converted to a 105-kDa isoform in a dose-dependent manner after cotransfection of an epitope-tagged SUMO-1. Immunoprecipitation experiments of the conjugated isoforms using denaturing conditions further confirmed the covalent coupling of SUMO-1 or hSMT3b to IE2-p86 both after transient transfection and after lytic infection of human primary fibroblasts. Moreover, we defined two modification sites within IE2, located in an immediate vicinity at amino acid positions 175 and 180, which appear to be used alternatively for coupling. By using a SUMOylation-defective mutant, we showed that the targeting of IE2-p86 to ND10 occurs independent of this modification. However, a strong reduction of IE2-mediated transactivation of two viral early promoters and a heterologous promoter was observed in cotransfection analysis with the SUMOylation-defective mutant. This suggests a functional relevance of covalent modification by ubiquitin-homologous Proteins for IE2-mediated transactivation, possibly by providing an additional interaction motif for cellular cofactors.

Aleem Siddiqui - One of the best experts on this subject based on the ideXlab platform.

  • hepatitis b virus Transactivator Protein hbx associates with the components of tfiih and stimulates the dna helicase activity of tfiih
    Proceedings of the National Academy of Sciences of the United States of America, 1996
    Co-Authors: Ishtiaq Qadri, J W Conaway, R C Conaway, Jerome Schaack, Aleem Siddiqui
    Abstract:

    Abstract Human hepatitis B virus genome encodes a Protein, termed HBx, that is widely recognized as a transcriptional Transactivator. While HBx does not directly bind cis-acting transcriptional control elements, it has been shown to associate with cellular Proteins that bind DNA. Because HBx transactivated a large number of viral/cellular transcriptional control elements, we looked for its targets within the components of the basal transcriptional machinery. This search led to the identification of its interactions with TFIIH. Here, we show that HBx interacts with yeast and mammalian TFIIH complexes both in vitro and in vivo. These interactions between HBx and the components of TFIIH are supported by several lines of evidence including results from immunoprocedures and direct methods of measuring interactions. We have identified ERCC3 and ERCC2 DNA helicase subunits of holoenzyme TFIIH as targets of HBx interactions. Furthermore, the DNA helicase activity of purified TFIIH from rat liver and, individually, the ERCC2 component of TFIIH is stimulated in the presence of HBx. These observations suggest a role for HBx in transcription and DNA repair.

  • the hepatitis b virus Transactivator Protein hbx interacts with single stranded dna ssdna biochemical characterizations of the hbx ssdna interactions
    Journal of Biological Chemistry, 1996
    Co-Authors: Ishtiaq Qadri, Marilyn E Ferrari, Aleem Siddiqui
    Abstract:

    Abstract Human hepatitis B virus X Protein, HBx, is widely acknowledged as a transcriptional Transactivator. While HBx has been shown to increase gene expression in trans, it is generally believed that it does not bind double-stranded DNA. Using several experimental approaches, we show that HBx interacts with single-stranded DNA in a manner that is not sequence-specific. Various heterologous single-stranded DNA (ssDNA) oligonucleotides were able to compete in HBx-ssDNA interactions in gel shift assays. Escherichia coli non-sequence-specific, single-stranded DNA binding Protein, E. coli SSB, displaced the HBx-ssDNA interactions, confirming the ability of HBx to interact with single-stranded DNA in a non-sequence-specific manner. We have further characterized the HBx-ssDNA interactions under various biochemical conditions. These include the effects of mono- and divalent cations, the effect of cardiolipin and heparin, pH and temperature dependence, and variations in the incubation time. HBx bound more tightly to d(pyrimidines)25 than to d(purines)25, a property that is characteristic of other single-stranded DNA-binding Proteins (SSBs). Collectively the results presented here provide the first evidence of HBx's interaction with ssDNA. The biochemical parameters of these interactions were similar to those of known viral and cellular SSBs.

  • hepatitis b virus Transactivator Protein x interacts with the tata binding Protein
    Proceedings of the National Academy of Sciences of the United States of America, 1995
    Co-Authors: Ishtiaq Qadri, Hugh F Maguire, Aleem Siddiqui
    Abstract:

    Several viral transcriptional activators have been shown to interact with the basal transcription factor TATA-binding Protein (TBP). These associations have been implicated in facilitating the assembly of the transcriptional preinitiation complex. We report here that the hepatitis B virus Protein X (pX) specifically binds to TBP in vitro. While truncations of the highly conserved carboxyl terminus of TBP abolished this binding, amino-terminal deletions had no effect. Deletion analysis suggests that a domain consisting of 71 aa in the highly conserved carboxyl-terminal region of TBP is necessary for its interaction with pX. The minimal region in pX sufficient for its interaction with TBP includes aa 110-143. Furthermore, TBP from phylogenetically distinct species including Arabidopsis thaliana, Saccharomyces cerevisiae, Drosophila melanogaster, and Solanum tuberosum (potato) bound to pX. The pX-TBP interaction was inhibited in the presence of nonhydrolyzable analogs of ATP, suggesting a requirement for ATP. These results provide an explanation for the promiscuous behavior of pX in the transactivation of a large repertoire of cellular promoters. This study further implicates a fundamental role for pX in modulating transcriptional regulatory pathways by interacting with the basal transcription factor TBP.

Rakesh Kumar - One of the best experts on this subject based on the ideXlab platform.

I. Demirhan - One of the best experts on this subject based on the ideXlab platform.

  • cross talk between human herpesvirus 8 and the Transactivator Protein in the pathogenesis of kaposi s sarcoma in hiv infected patients
    Anticancer Research, 2003
    Co-Authors: Angelika Chandra, I. Demirhan, Charles Massambu, Pawan Pyakurel, Ephata E Kaaya, Malin Enbom, Willy Urassa, Annika Linde, Thomas Heiden, Peter Biberfeld
    Abstract:

    AIDS-associated Kaposis sarcoma (AKS) is particularly aggressive and it is one of the principal neoplasms in regions of Africa affected by both high endemic HHV8 and epidemic HIV infection. In this study serum samples from 18 patients with Kaposis sarcoma from Tanzania mostly males (n = 15 vs 3) were subjected to analysis with respect to HHV8-DNA load and antibody spectrum against the HIV-1 tat Protein. Of the 18 patients 14 were HIV-1-positive. The median HHV8 virus load in the HlV-1-positive group was 2075 DNA copies/ml compared to 450 copies/ml in the HIV-1-negative group. In the HIV-1-positive group the males had a higher HHV8-DNA virus load as compared to females (median: 4600 vs 1400 genome copies per ml). Since tat can promote AKS development (4-6) by intercellular signalling pathways and these signals can be abolished by anti-tat IgG (7-9) we have examined the anti-tat IgG spectrum in this study. It would be expected that the levels of serum HHV8-DNA are higher in KS patients who have low anti-tat IgG titer or who are anti-tat IgG-negative. In the present study seven out of fifteen AKS patients were positive for anti-tat IgG. Although we have not seen a strict quantitative relationship between serum anti-tat IgG and HHV8-DNA levels our data appear to suggest a correlation between the two parameters. In view of these observations and the published data we suggest that cross-signalling pathways between the tat Protein and HHV8-DNA are involved in the complexity of pathogenesis of Kaposis sarcoma. (authors)

  • docking studies reveal a selective binding of d penicillamine to the Transactivator Protein of human immunodeficiency virus type 1
    FEBS Letters, 2002
    Co-Authors: I. Demirhan, Angelika Chandra, Meena Kanyalkar, Hans Wilhelm Doerr, Evans C Coutinho, Johannes Loewer, Anil Saran, Prakash Chandra
    Abstract:

    Abstract DOCK and Affinity studies were carried out to study the binding of D - and L -penicillamine to the Transactivator Protein (tat) of human immunodeficiency virus type 1 (HIV-1). These studies reveal a selective binding of D -penicillamine to the cysteine-rich region covering amino acid residues 20–38 of the tat Protein. A careful analysis of the components of the binding energy of the D - and L -isomers reveals that the D -isomer has a more favorable van der Waals interaction resulting from an optimal placement of the dimethylthiomethyl side chain in the binding site. This observation matches the experimental data that D -penicillamine is a more potent inhibitor of tat-mediated transactivation than the L -isomer. The docking and experimental data offer an interesting approach to design structural molecules with potential application to block signal functions of the tat Protein in HIV-1 pathogenesis.

  • Antibody spectrum against the viral Transactivator Protein in patients with human immunodeficiency virus type 1 infection and Kaposi's sarcoma.
    Journal of Human Virology, 2000
    Co-Authors: I. Demirhan, Chandra A, Mueller F, Mueller H, Peter Biberfeld, Hasselmayer O, Chandra P
    Abstract:

    We analyzed patterns of antibody response to recombinant Transactivator Protein (human Immunodeficiency virus type 1 [HIV-1] tat) in serum samples from HIV-1-negative subjects (n = 60), HIV-1-infected asymptomatic patients (n = 20), HIV-1-infected patients with Kaposi's sarcoma (n = 25), and patients with Kaposi's sarcoma without HIV-1 infection. None of the healthy subjects possessed anti-tat immunoglobulin G (IgG) in their serum. All asymptomatic patients with HIV-1 infection were anti-tat IgG-positive. Epitope mapping revealed that these sera had anti-tat IgG to all the functional domains of tat Protein. Histochemical studies on lymph nodes from five asymptomatic HIV-1-infected patients showed that, in all cases, tat-positive cells were present within the germinal center at the stage of follicular fragmentation containing immunoblasts and small lymphocytes. Of the 25 HIV-1-infected patients with Kaposi's sarcoma, 4 were anti-tat IgG-positive; however, the epitope analysis revealed that IgG to functional domains of tat Protein--in particular to transactivating response element (TAR)-binding site--were absent. All patients with Kaposi's sarcoma without HIV-1 infection were anti-tat IgG-negative. Presence or absence of anti-tat IgG and a prevalence of different antibody profiles in different groups of patients indicated the pathophysiologic role of tat Protein. Thus, a passive immunization with anti-tat IgG could be a useful strategy to influence the pathophysiologic state of the disease.

  • Intercellular traffic of human immunodeficiency virus type 1 Transactivator Protein defined by monoclonal antibodies
    FEBS Letters, 1999
    Co-Authors: I. Demirhan, Angelika Chandra, Oliver Hasselmayer, Prakash Chandra
    Abstract:

    Monoclonal antibodies (mAbs) directed against the amino-terminal region (N-terminal sequence 2–19) of Transactivator Protein (tat) of HIV-1 have been shown to inhibit intercellular transactivation mediated by the extracellular tat Protein. The intracellular transactivation was not significantly affected by anti-tat mAbs. The specificity of anti-tat mAbs in abolishing the transactivating potential of extracellular tat is documented by studies with mAbs to HIV-1 reverse transcriptase, or to a human mammary cancer Protein. None of these antibodies showed any inhibitory effect on intercellular transactivation. Specific interaction of anti-tat IgG with tat Protein expressed in Jurkat cells is further supported by experiments on immunoblotting. Extracellular tat is responsible for signals which induce a variety of biological responses in HIV-infected cells, as well as in uninfected cells. The fact that anti-tat mAbs can abolish the intercellular traffic of tat Protein offers a unique strategy in the development of vaccines against AIDS.

Tri M Buinguyen - One of the best experts on this subject based on the ideXlab platform.

  • stimulation of inducible nitric oxide by hepatitis b virus Transactivator Protein hbx requires mta1 coregulator
    Journal of Biological Chemistry, 2010
    Co-Authors: Tri M Buinguyen, Suresh B Pakala, Divijendranatha Reddy Sirigiri, Emil Martin, Ferid Murad, Rakesh K Kumar
    Abstract:

    Nitric oxide has been implicated in the pathogenesis of inflammatory disorders, including hepatitis B virus-associated hepatocellular carcinoma. Transactivator Protein HBx, a major regulator of cellular responses of hepatitis B virus, is known to induce the expression of MTA1 (metastasis-associated Protein 1) coregulator via NF-κB signaling in hepatic cells. However, the underlying mechanism of HBx regulation of the inducible nitric-oxide synthase (iNOS) pathway remains unknown. Here we provide evidence that MTA1 is a positive regulator of iNOS transcription and plays a mechanistic role in HBx stimulation of iNOS expression and activity. We found that the HBx-MTA1 complex is recruited onto the human iNOS promoter in an NF-κB-dependent manner. Pharmacological inhibition of the NF-κB signaling prevented the ability of HBx to stimulate the transcription, the expression, and the activity of iNOS; nevertheless, these effects could be substantially rescued by MTA1 dysregulation. We further discovered that HBx-mediated stimulation of MTA1 is paralleled by the suppression of miR-661, a member of the small noncoding RNAs, recently shown to target MTA1. We observed that miR-661 controls of MTA1 expression contributed to the expression and activity of iNOS in HBx-expressing HepG2 cells. Accordingly, depletion of MTA1 by either miR-661 or siRNA in HBx-expressing cells severely impaired the ability of HBx to modulate the endogenous levels of iNOS and nitrite production. Together, these findings reveal an inherent role of MTA1 in HBx regulation of iNOS expression and consequently its function in the liver cancer cells.

  • nf κb signaling mediates the induction of mta1 by hepatitis b virus Transactivator Protein hbx
    Oncogene, 2010
    Co-Authors: Tri M Buinguyen, Suresh B Pakala, Reddy Divijendranatha Sirigiri, Mien Chie Hung, Shiv Kumar Sarin, Vijay Kumar, Betty L Slagle, Rakesh Kumar
    Abstract:

    NF-κB signaling mediates the induction of MTA1 by hepatitis B virus Transactivator Protein HBx