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1.  Lentiviral Nef suppresses iron uptake in a strain specific manner through inhibition of Transferrin endocytosis 
Retrovirology  2014;11:1.
Increased cellular iron levels are associated with high mortality in HIV-1 infection. Moreover iron is an important cofactor for viral replication, raising the question whether highly divergent lentiviruses actively modulate iron homeostasis. Here, we evaluated the effect on cellular iron uptake upon expression of the accessory protein Nef from different lentiviral strains.
Surface Transferrin receptor (TfR) levels are unaffected by Nef proteins of HIV-1 and its simian precursors but elevated in cells expressing Nefs from most other primate lentiviruses due to reduced TfR internalization. The SIV Nef-mediated reduction of TfR endocytosis is dependent on an N-terminal AP2 binding motif that is not required for downmodulation of CD4, CD28, CD3 or MHCI. Importantly, SIV Nef-induced inhibition of TfR endocytosis leads to the reduction of Transferrin uptake and intracellular iron concentration and is accompanied by attenuated lentiviral replication in macrophages.
Inhibition of Transferrin and thereby iron uptake by SIV Nef might limit viral replication in myeloid cells. Furthermore, this new SIV Nef function could represent a virus-host adaptation that evolved in natural SIV-infected monkeys.
PMCID: PMC3892060  PMID: 24383984
HIV; SIV; Nef; Iron homeostasis; AIDS; Lentiviral replication; Macrophages
2.  Differentially Expressed Wound Healing-Related microRNAs in the Human Diabetic Cornea 
PLoS ONE  2013;8(12):e84425.
MicroRNAs are powerful gene expression regulators, but their corneal repertoire and potential changes in corneal diseases remain unknown. Our purpose was to identify miRNAs altered in the human diabetic cornea by microarray analysis, and to examine their effects on wound healing in cultured telomerase-immortalized human corneal epithelial cells (HCEC) in vitro. Total RNA was extracted from age-matched human autopsy normal (n=6) and diabetic (n=6) central corneas, Flash Tag end-labeled, and hybridized to Affymetrix® GeneChip® miRNA Arrays. Select miRNAs associated with diabetic cornea were validated by quantitative RT-PCR (Q-PCR) and by in situ hybridization (ISH) in independent samples. HCEC were transfected with human pre-miRTMmiRNA precursors (h-miR) or their inhibitors (antagomirs) using Lipofectamine 2000. Confluent transfected cultures were scratch-wounded with P200 pipette tip. Wound closure was monitored by digital photography. Expression of signaling proteins was detected by immunostaining and Western blot. Using microarrays, 29 miRNAs were identified as differentially expressed in diabetic samples. Two miRNA candidates showing the highest fold increased in expression in the diabetic cornea were confirmed by Q-PCR and further characterized. HCEC transfection with h-miR-146a or h-miR-424 significantly retarded wound closure, but their respective antagomirs significantly enhanced wound healing vs. controls. Cells treated with h-miR-146a or h-miR-424 had decreased p-p38 and p-EGFR staining, but these increased over control levels close to the wound edge upon antagomir treatment. In conclusion, several miRNAs with increased expression in human diabetic central corneas were found. Two such miRNAs inhibited cultured corneal epithelial cell wound healing. Dysregulation of miRNA expression in human diabetic cornea may be an important mediator of abnormal wound healing.
PMCID: PMC3869828  PMID: 24376808
3.  TMPRSS2 Activates the Human Coronavirus 229E for Cathepsin-Independent Host Cell Entry and Is Expressed in Viral Target Cells in the Respiratory Epithelium 
Journal of Virology  2013;87(11):6150-6160.
Infection with human coronavirus 229E (HCoV-229E) is associated with the common cold and may result in pneumonia in immunocompromised patients. The viral spike (S) protein is incorporated into the viral envelope and mediates infectious entry of HCoV-229E into host cells, a process that depends on the activation of the S-protein by host cell proteases. However, the proteases responsible for HCoV-229E activation are incompletely defined. Here we show that the type II transmembrane serine proteases TMPRSS2 and HAT cleave the HCoV-229E S-protein (229E-S) and augment 229E-S-driven cell-cell fusion, suggesting that TMPRSS2 and HAT can activate 229E-S. Indeed, engineered expression of TMPRSS2 and HAT rendered 229E-S-driven virus-cell fusion insensitive to an inhibitor of cathepsin L, a protease previously shown to facilitate HCoV-229E infection. Inhibition of endogenous cathepsin L or TMPRSS2 demonstrated that both proteases can activate 229E-S for entry into cells that are naturally susceptible to infection. In addition, evidence was obtained that activation by TMPRSS2 rescues 229E-S-dependent cell entry from inhibition by IFITM proteins. Finally, immunohistochemistry revealed that TMPRSS2 is coexpressed with CD13, the HCoV-229E receptor, in human airway epithelial (HAE) cells, and that CD13+ TMPRSS2+ cells are preferentially targeted by HCoV-229E, suggesting that TMPRSS2 can activate HCoV-229E in infected humans. In sum, our results indicate that HCoV-229E can employ redundant proteolytic pathways to ensure its activation in host cells. In addition, our observations and previous work suggest that diverse human respiratory viruses are activated by TMPRSS2, which may constitute a target for antiviral intervention.
PMCID: PMC3648130  PMID: 23536651
4.  A Simple Alkaline Method for Decellularizing Human Amniotic Membrane for Cell Culture 
PLoS ONE  2013;8(11):e79632.
Human amniotic membrane is a standard substratum used to culture limbal epithelial stem cells for transplantation to patients with limbal stem cell deficiency. Various methods were developed to decellularize amniotic membrane, because denuded membrane is poorly immunogenic and better supports repopulation by dissociated limbal epithelial cells. Amniotic membrane denuding usually involves treatment with EDTA and/or proteolytic enzymes; in many cases additional mechanical scraping is required. Although ensuring limbal cell proliferation, these methods are not standardized, require relatively long treatment times and can result in membrane damage. We propose to use 0.5 M NaOH to reliably remove amniotic cells from the membrane. This method was used before to lyse cells for DNA isolation and radioactivity counting. Gently rubbing a cotton swab soaked in NaOH over the epithelial side of amniotic membrane leads to nearly complete and easy removal of adherent cells in less than a minute. The denuded membrane is subsequently washed in a neutral buffer. Cell removal was more thorough and uniform than with EDTA, or EDTA plus mechanical scraping with an electric toothbrush, or n-heptanol plus EDTA treatment. NaOH-denuded amniotic membrane did not show any perforations compared with mechanical or thermolysin denuding, and showed excellent preservation of immunoreactivity for major basement membrane components including laminin α2, γ1-γ3 chains, α1/α2 and α6 type IV collagen chains, fibronectin, nidogen-2, and perlecan. Sodium hydroxide treatment was efficient with fresh or cryopreserved (10% dimethyl sulfoxide or 50% glycerol) amniotic membrane. The latter method is a common way of membrane storage for subsequent grafting in the European Union. NaOH-denuded amniotic membrane supported growth of human limbal epithelial cells, immortalized corneal epithelial cells, and induced pluripotent stem cells. This simple, fast and reliable method can be used to standardize decellularized amniotic membrane preparations for expansion of limbal stem cells in vitro before transplantation to patients.
PMCID: PMC3827346  PMID: 24236148
5.  The Spike Protein of the Emerging Betacoronavirus EMC Uses a Novel Coronavirus Receptor for Entry, Can Be Activated by TMPRSS2, and Is Targeted by Neutralizing Antibodies 
Journal of Virology  2013;87(10):5502-5511.
The novel human coronavirus EMC (hCoV-EMC), which recently emerged in Saudi Arabia, is highly pathogenic and could pose a significant threat to public health. The elucidation of hCoV-EMC interactions with host cells is critical to our understanding of the pathogenesis of this virus and to the identification of targets for antiviral intervention. Here we investigated the viral and cellular determinants governing hCoV-EMC entry into host cells. We found that the spike protein of hCoV-EMC (EMC-S) is incorporated into lentiviral particles and mediates transduction of human cell lines derived from different organs, including the lungs, kidneys, and colon, as well as primary human macrophages. Expression of the known coronavirus receptors ACE2, CD13, and CEACAM1 did not facilitate EMC-S-driven transduction, suggesting that hCoV-EMC uses a novel receptor for entry. Directed protease expression and inhibition analyses revealed that TMPRSS2 and endosomal cathepsins activate EMC-S for virus-cell fusion and constitute potential targets for antiviral intervention. Finally, EMC-S-driven transduction was abrogated by serum from an hCoV-EMC-infected patient, indicating that EMC-S-specific neutralizing antibodies can be generated in patients. Collectively, our results indicate that hCoV-EMC uses a novel receptor for protease-activated entry into human cells and might be capable of extrapulmonary spread. In addition, they define TMPRSS2 and cathepsins B and L as potential targets for intervention and suggest that neutralizing antibodies contribute to the control of hCoV-EMC infection.
PMCID: PMC3648152  PMID: 23468491
7.  Long-Term Results after Treatment of Very Low-, Low-, and High-Risk Thyroid Cancers in a Combined Setting of Thyroidectomy and Radio Ablation Therapy in Euthyroidism 
Introduction. Differentiated thyroid cancer treatment usually consists of thyroidectomy and radio ablation in hypothyroidism 4-6 weeks after surgery. Replacing hypothyroidism by recombinant human thyroid stimulating hormone can facilitate radio ablation in euthyroidism within one week after surgery. The outcome of this approach was investigated. Methods. This is a prospective randomized trial to compare thyroidectomy and radio ablation within a few days after preconditioning with recombinant human thyroid stimulating hormone versus thyroidectomy and radio ablation separated by four weeks of L-T4 withdrawal. Tumors were graded into very low-, low- , or high-risk tumors. Recurrence-free survival was confirmed at follow-up controls by neck ultrasound and serum thyroglobulin. Suspected tumor recurrence was treated by additional radio ablation or surgery. Quality-of-life questionnaires with additional evaluation of job performance and sick-leave time were used in all patients. Results. Radio ablation in euthyroidism in quick succession after thyroidectomy did not lead to higher tumor recurrence rates of differentiated thyroid cancers in any risk category and was significantly advantageous with respect to quality-of-life (P < 0.001), sick-leave time (P < 0.001), and job performance (P = 0.002). Conclusion. Recombinant human thyroid stimulating hormone can be used safely and with good efficacy to allow radio ablation under sustained euthyroidism within one week after thyroidectomy.
PMCID: PMC3723358  PMID: 23935620
8.  Ligation of Left Renal Vein for Spontaneous Splenorenal Shunt to Prevent Portal Hypoperfusion after Orthotopic Liver Transplantation 
We report a case of recovered portal flow by ligation of the left renal vein on the first postoperative day after orthotopic liver transplantation of a 54-year-old female with alcoholic liver cirrhosis, chronic kidney failure, and spontaneous splenorenal shunt. After reperfusion, Doppler ultrasonography showed almost total diversion of the portal flow into the existing splenorenal shunt, but because of severe coagulopathy and diffuse bleeding, ligation of the shunt was not attempted. A programmed relaparotomy was performed on the first postoperative day, and the left renal vein was ligated just to the left of the inferior vena cava. Portal flows subsequently increased to 37 cm/sec, and the patient presented a good and stable liver function. We conclude that patients with known preoperative splenorenal shunts should be closely monitored, and if the portal flow becomes insufficient, ligation of the left renal vein should be attempted in order to optimize the portal perfusion of the liver.
PMCID: PMC3600271  PMID: 23533923
9.  Functional Cooperation between Human Adenovirus Type 5 Early Region 4, Open Reading Frame 6 Protein, and Cellular Homeobox Protein HoxB7 
Journal of Virology  2012;86(15):8296-8308.
Human adenovirus type 5 (HAdV5) E4orf6 (early region 4 open reading frame 6 protein) is a multifunctional early viral protein promoting efficient replication and progeny production. E4orf6 complexes with E1B-55K to assemble cellular proteins into a functional E3 ubiquitin ligase complex that not only mediates proteasomal degradation of host cell substrates but also facilitates export of viral late mRNA to promote efficient viral protein expression and host cell shutoff. Recent findings defined the role of E4orf6 in RNA splicing independent of E1B-55K binding. To reveal further functions of the early viral protein in infected cells, we used a yeast two-hybrid system and identified the homeobox transcription factor HoxB7 as a novel E4orf6-associated protein. Using a HoxB7 knockdown cell line, we observed a positive role of HoxB7 in adenoviral replication. Our experiments demonstrate that the absence of HoxB7 leads to inefficient viral progeny production, as HAdV5 gene expression is highly regulated by HoxB7-mediated activation of various adenoviral promoters. We have thus identified a novel role of E4orf6 in HAdV5 gene transcription via regulation of homeobox protein-dependent modulation of viral promoter activity.
PMCID: PMC3421667  PMID: 22553335
10.  The Human Cytomegalovirus DNA Polymerase Processivity Factor UL44 Is Modified by SUMO in a DNA-Dependent Manner 
PLoS ONE  2012;7(11):e49630.
During the replication of human cytomegalovirus (HCMV) genome, the viral DNA polymerase subunit UL44 plays a key role, as by binding both DNA and the polymerase catalytic subunit it confers processivity to the holoenzyme. However, several lines of evidence suggest that UL44 might have additional roles during virus life cycle. To shed light on this, we searched for cellular partners of UL44 by yeast two-hybrid screenings. Intriguingly, we discovered the interaction of UL44 with Ubc9, an enzyme involved in the covalent conjugation of SUMO (Small Ubiquitin-related MOdifier) to cellular and viral proteins. We found that UL44 can be extensively sumoylated not only in a cell-free system and in transfected cells, but also in HCMV-infected cells, in which about 50% of the protein resulted to be modified at late times post-infection, when viral genome replication is accomplished. Mass spectrometry studies revealed that UL44 possesses multiple SUMO target sites, located throughout the protein. Remarkably, we observed that binding of UL44 to DNA greatly stimulates its sumoylation both in vitro and in vivo. In addition, we showed that overexpression of SUMO alters the intranuclear distribution of UL44 in HCMV-infected cells, and enhances both virus production and DNA replication, arguing for an important role for sumoylation in HCMV life cycle and UL44 function(s). These data report for the first time the sumoylation of a viral processivity factor and show that there is a functional interplay between the HCMV UL44 protein and the cellular sumoylation system.
PMCID: PMC3499415  PMID: 23166733
11.  Impact of Salinomycin on human cholangiocarcinoma: induction of apoptosis and impairment of tumor cell proliferation in vitro 
BMC Cancer  2012;12:466.
Cholangiocarcinoma (CC) is a primary liver cancer with increasing incidence worldwide. Despite all efforts made in past years, prognosis remains to be poor. At least in part, this might be explained by a pronounced resistance of CC cells to undergo apoptosis. Thus, new therapeutic strategies are imperatively required. In this study we investigated the effect of Salinomycin, a polyether ionophore antibiotic, on CC cells as an appropriate agent to treat CC. Salinomycin was quite recently identified to induce apoptosis in cancer stem cells and to overcome apoptosis-resistance in several leukemia-cells and other cancer cell lines of different origin.
To delineate the effects of Salinomycin on CC, we established an in vitro cell culture model using three different human CC cell lines. After treatment apoptosis as well as migration and proliferation behavior was assessed and additional cell cycle analyses were performed by flowcytometry.
By demonstrating Annexin V and TUNEL positivity of human CC cells, we provide evidence that Salinomycin reveals the capacity to break apoptosis-resistance in CC cells. Furthermore, we are able to demonstrate that the non-apoptotic cell fraction is characterized by sustainable impaired migration and proliferation. Cell cycle analyses revealed G2-phase accumulation of human CC cells after treatment with Salinomycin. Even though apoptosis is induced in two of three cell lines of CC cells, one cell line remained unaffected in regard of apoptosis but revealed as the other CC cells decreased proliferation and migration.
In this study, we are able to demonstrate that Salinomycin is an effective agent against previously resistant CC cells and might be a potential candidate for the treatment of CC in the future.
PMCID: PMC3487825  PMID: 23057720
Salinomycin; Cholangiocarcinoma; Apoptosis; Tumor cell migration; Cell cycle
12.  Influenza A Virus Does Not Encode a Tetherin Antagonist with Vpu-Like Activity and Induces IFN-Dependent Tetherin Expression in Infected Cells 
PLoS ONE  2012;7(8):e43337.
The interferon-induced host cell factor tetherin inhibits release of human immunodeficiency virus (HIV) from the plasma membrane of infected cells and is counteracted by the HIV-1 protein Vpu. Influenza A virus (FLUAV) also buds from the plasma membrane and is not inhibited by tetherin. Here, we investigated if FLUAV encodes a functional equivalent of Vpu for tetherin antagonism. We found that expression of the FLUAV protein NS1, which antagonizes the interferon (IFN) response, did not block the tetherin-mediated restriction of HIV release, which was rescued by Vpu. Similarly, tetherin-mediated inhibition of HIV release was not rescued by FLUAV infection. In contrast, FLUAV infection induced tetherin expression on target cells in an IFN-dependent manner. These results suggest that FLUAV escapes the antiviral effects of tetherin without encoding a tetherin antagonist with Vpu-like activity.
PMCID: PMC3428345  PMID: 22952667
13.  A Quantitative Assay for Insulin-expressing Colony-forming Progenitors 
The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 μL of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin βB, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.
PMCID: PMC3308582  PMID: 22143165
14.  Boerhaave syndrome as a complication of colonoscopy preparation: a case report 
Colonoscopy is one of the most frequently performed elective and invasive diagnostic interventions. For every colonoscopy, complete colon preparation is mandatory to provide the best possible endoluminal visibility; for example, the patient has to drink a great volume of a non-resorbable solution to flush out all feces. Despite the known possible nauseating side effects of colonoscopy preparation and despite the knowledge that excessive vomiting can cause rupture of the distal esophagus (Boerhaave syndrome), which is a rare but severe complication with high morbidity and mortality, it is not yet a standard procedure to provide a patient with an anti-emetic medication during a colon preparation process. This is the first report of Boerhaave syndrome induced by colonoscopy preparation, and this case strongly suggests that the prospect of being at risk of a severe complication connected with an elective colonoscopy justifies a non-invasive, inexpensive yet effective precaution such as an anti-emetic co-medication during the colonoscopy preparation process.
Case presentation
A 73-year-old Caucasian woman was scheduled to undergo elective colonoscopy. For the colonoscopy preparation at home she received commercially available bags containing soluble polyethylene glycol powder. No anti-emetic medication was prescribed. After drinking the prepared solution she had to vomit excessively and experienced a sudden and intense pain in her back. An immediate computed tomography (CT) scan revealed a rupture of the distal esophagus (Boerhaave syndrome). After initial conservative treatment by endoluminal sponge vacuum therapy, she was taken to the operating theatre and the longitudinal esophageal rupture was closed by direct suture and gastric fundoplication (Nissen procedure). She recovered completely and was discharged three weeks after the initial event.
To the best of our knowledge, this is the first report of a case of Boerhaave syndrome as a complication of excessive vomiting caused by colonoscopy preparation. The case suggests that patients who are prepared for a colonoscopy by drinking large volumes of fluid should routinely receive an anti-emetic medication during the preparation process, especially when they have a tendency to nausea and vomiting.
PMCID: PMC3220652  PMID: 22054124
15.  Interaction of the Papillomavirus E8∧E2C Protein with the Cellular CHD6 Protein Contributes to Transcriptional Repression▿ †  
Journal of Virology  2010;84(18):9505-9515.
Expression of the E6 and E7 oncogenes of high-risk human papillomaviruses (HPV) is controlled by cellular transcription factors and by viral E2 and E8∧E2C proteins, which are both derived from the HPV E2 gene. Both proteins bind to and repress the HPV E6/E7 promoter. Promoter inhibition has been suggested to be due to binding site competition with cellular transcription factors and to interactions of different cellular transcription modulators with the different amino termini of E2 and E8∧E2C. We have now identified the cellular chromodomain helicase DNA binding domain 6 protein (CHD6) as a novel interactor with HPV31 E8∧E2C by using yeast two-hybrid screening. Pull-down and coimmunoprecipitation assays indicate that CHD6 interacts with the HPV31 E8∧E2C protein via the E2C domain. This interaction is conserved, as it occurs also with the E8∧E2C proteins expressed by HPV16 and -18 and with the HPV31 E2 protein. Both RNA knockdown experiments and mutational analyses of the E2C domain suggest that binding of CHD6 to E8∧E2C contributes to the transcriptional repression of the HPV E6/E7 oncogene promoter. We provide evidence that CHD6 is also involved in transcriptional repression but not activation by E2. Taken together our results indicate that the E2C domain not only mediates specific DNA binding but also has an additional role in transcriptional repression by recruitment of the CHD6 protein. This suggests that repression of the E6/E7 promoter by E2 and E8∧E2C involves multiple interactions with host cell proteins through different protein domains.
PMCID: PMC2937640  PMID: 20631145
16.  Human Cytomegaloviruses Expressing Yellow Fluorescent Fusion Proteins - Characterization and Use in Antiviral Screening 
PLoS ONE  2010;5(2):e9174.
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.
PMCID: PMC2820100  PMID: 20161802
17.  RBP-Jκ/SHARP Recruits CtIP/CtBP Corepressors To Silence Notch Target Genes 
Molecular and Cellular Biology  2005;25(23):10379-10390.
Notch is a transmembrane receptor that determines cell fates and pattern formation in all animal species. After ligand binding, proteolytic cleavage steps occur and the intracellular part of Notch translocates to the nucleus, where it targets the DNA-binding protein RBP-Jκ/CBF1. In the absence of Notch, RBP-Jκ represses Notch target genes through the recruitment of a corepressor complex. We and others have identified SHARP as a component of this complex. Here, we functionally demonstrate that the SHARP repression domain is necessary and sufficient to repress transcription and that the absence of this domain causes a dominant negative Notch-like phenotype. We identify the CtIP and CtBP corepressors as novel components of the human RBP-Jκ/SHARP-corepressor complex and show that CtIP binds directly to the SHARP repression domain. Functionally, CtIP and CtBP augment SHARP-mediated repression. Transcriptional repression of the Notch target gene Hey1 is abolished in CtBP-deficient cells or after the functional knockout of CtBP. Furthermore, the endogenous Hey1 promoter is derepressed in CtBP-deficient cells. We propose that a corepressor complex containing CtIP/CtBP facilitates RBP-Jκ/SHARP-mediated repression of Notch target genes.
PMCID: PMC1291242  PMID: 16287852
18.  Human Cytomegalovirus Tegument Protein ppUL35 Is Important for Viral Replication and Particle Formation 
Journal of Virology  2005;79(5):3084-3096.
The tegument proteins ppUL35 and ppUL82 (pp71) of human cytomegalovirus (HCMV) physically interact and cooperatively activate the major immediate-early transcription. While an HCMV mutant lacking UL82 displayed a multiplicity of infection (MOI)-dependent growth, the biological significance of ppUL35 has not been addressed so far. We generated a mutant virus with a deletion of the UL35 gene. Using an MOI of 0.1, the progeny virus yield of this mutant was reduced by a factor of 1,000; however, when infected at a low MOI (0.01), the gene was essential. Characterization of the replication cycle showed that the mutant virus had two defects: when virus inoculum was standardized by the amount of viral DNA, a reduced immediate-early gene expression was observed, leading to a strongly delayed expression of lytic genes. A second defect was apparent in the virus assembly, as fewer enveloped particles and no dense bodies were present in cells infected with the mutant virus. However, the particles produced by wild-type and mutant viruses did not show significant ultrastructural differences. These results suggest an important role for ppUL35 in immediate-early gene expression and virus assembly.
PMCID: PMC548451  PMID: 15709028
19.  Human Cytomegalovirus Tegument Proteins ppUL82 (pp71) and ppUL35 Interact and Cooperatively Activate the Major Immediate-Early Enhancer 
Journal of Virology  2004;78(17):9512-9523.
The tegument protein ppUL82 (pp71) of human cytomegalovirus (HCMV) has previously been shown to activate the immediate-early transcription of HCMV and to enhance the infectivity of viral DNA. This is concordant with its localization adjacent to promyelocytic leukemia oncogenic domains (PODs) immediately after infection. In a yeast two-hybrid screen, we identified the tegument protein ppUL35 as an interacting partner of ppUL82. The interaction could be confirmed in transfected and infected cells. The domain responsible for interaction was narrowed down to amino acids 447 to 516 within ppUL35, thus allowing both forms of ppUL35 to interact with ppUL82. Immunofluorescence experiments showed a relocalization of ppUL35 from a diffuse nuclear pattern when expressed alone to PODs when expressed together with ppUL82. In accordance with this observation and the role of ppUL82 as a transactivator, we observed a cooperative activation of the HCMV major immediate-early enhancer but not of heterologous viral enhancer elements. These results suggest an important role for this interaction in the stimulation of viral immediate-early gene expression and viral infection.
PMCID: PMC506970  PMID: 15308743
20.  A Nonconventional Nuclear Localization Signal within the UL84 Protein of Human Cytomegalovirus Mediates Nuclear Import via the Importin α/β Pathway 
Journal of Virology  2003;77(6):3734-3748.
The open reading frame UL84 of human cytomegalovirus encodes a multifunctional regulatory protein which is required for viral DNA replication and binds with high affinity to the immediate-early transactivator IE2-p86. Although the exact role of pUL84 in DNA replication is unknown, the nuclear localization of this protein is a prerequisite for this function. To investigate whether the activities of pUL84 are modulated by cellular proteins we used the Saccharomyces cerevisiae two-hybrid system to screen a cDNA-library for interacting proteins. Strong interactions were found between pUL84 and four members of the importin α protein family. These interactions could be confirmed in vitro by pull down experiments and in vivo by coimmunoprecipitation analysis from transfected cells. Using in vitro transport assays we showed that the pUL84 nuclear import required importin α, importin β, and Ran, thus following the classical importin-mediated import pathway. Deletion mutagenesis of pUL84 revealed a domain of 282 amino acids which is required for binding to the importin α proteins. Its function as a nuclear localization signal (NLS) was confirmed by fusion to heterologous proteins. Although containing a cluster of basic amino acids similar to classical NLSs, this cluster did not contain the NLS activity. Thus, a complex structure appears to be essential for importin α binding and import activity.
PMCID: PMC149505  PMID: 12610148
21.  Open Reading Frame UL26 of Human Cytomegalovirus Encodes a Novel Tegument Protein That Contains a Strong Transcriptional Activation Domain 
Journal of Virology  2002;76(10):4836-4847.
A selection strategy, the activator trap, was used in order to identify genes of human cytomegalovirus (HCMV) that encode strong transcriptional activation domains in mammalian cells. This approach is based on the isolation of activation domains from a GAL4 fusion library by means of selective plasmid replication, which is mediated in transfected cells by a GAL4-inducible T antigen gene. With this screening strategy, we were able to isolate two types of plasmids encoding transactivating fusion proteins from a library of random HCMV DNA inserts. One plasmid contained the exon 3 of the HCMV IE-1/2 gene region, which has previously been identified as a strong transcriptional activation domain. In the second type of plasmid, the open reading frame (ORF) UL26 of HCMV was fused to the GAL4 DNA-binding domain. By quantitative RNA mapping using S1 nuclease analysis, we were able to classify UL26 as a strong enhancer-type activation domain with no apparent homology to characterized transcriptional activators. Western blot analysis with a specific polyclonal antibody raised against a prokaryotic UL26 fusion protein revealed that two protein isoforms of 21 and 27 kDa are derived from the UL26 ORF in both infected and transfected cells. Both protein isoforms, which arise via alternative usage of two in-frame translational start codons, showed a nuclear localization and could be detected as early as 6 h after infection of primary human fibroblasts. By performing Western blot analysis with purified virions combined with fractionation experiments, we provide evidence that pUL26 is a novel tegument protein of HCMV that is imported during viral infection. Furthermore, we observed transactivation of the HCMV major immediate-early enhancer-promoter by pUL26, whereas several early and late promoters were not affected. Our data suggest that pUL26 is a novel tegument protein of HCMV with a strong transcriptional activation domain that could play an important role during initiation of the viral replicative cycle.
PMCID: PMC136153  PMID: 11967300
22.  Matrix Attachment Region-Dependent Function of the Immunoglobulin μ Enhancer Involves Histone Acetylation at a Distance without Changes in Enhancer Occupancy 
Molecular and Cellular Biology  2001;21(1):196-208.
Nuclear matrix attachment regions (MARs), which flank the immunoglobulin μ heavy-chain enhancer on either side, are required for the activation of the distal variable-region (VH) promoter in transgenic mice. Previously, we have shown that the MARs extend a local domain of chromatin accessibility at the μ enhancer to more distal sites. In this report, we examine the influence of MARs on the formation of a nucleoprotein complex at the enhancer and on the acetylation of histones, which have both been implicated in contributing to chromatin accessibility. By in vivo footprint analysis of transgenic μ gene constructs, we show that the occupancy of factor-binding sites at the μ enhancer is similar in transcriptionally active wild-type and transcriptionally inactive ΔMAR genes. Chromatin immunoprecipitation experiments indicate, however, that the acetylation of histones at enhancer-distal nucleosomes is enhanced 10-fold in the presence of MARs, whereas the levels of histone acetylation at enhancer-proximal nucleosomes are similar for wild-type and ΔMAR genes. Taken together, these data indicate that the function of MARs in mediating long-range chromatin accessibility and transcriptional activation of the VH promoter involves the generation of an extended domain of histone acetylation, independent of changes in the occupancy of the μ enhancer.
PMCID: PMC88794  PMID: 11113195
23.  Functional Interaction between Pleiotropic Transactivator pUL69 of Human Cytomegalovirus and the Human Homolog of Yeast Chromatin Regulatory Protein SPT6 
Journal of Virology  2000;74(17):8053-8064.
The phosphoprotein pUL69 of human cytomegalovirus (HCMV), which is a herpesvirus of considerable medical importance in immunosuppressed patients and newborns, has previously been identified as an early-late viral protein that can stimulate several viral and cellular promoters and thus exerts a rather broad activation pattern. To gain insight into the mechanism of this transactivation process, we looked for cellular factors interacting with pUL69 in a yeast two-hybrid screen. Using a B-lymphocyte cDNA library fused to the GAL4 activation domain, we identified 34 clones, 11 of which comprised one distinct gene. Interaction with this gene turned out to be very strong, producing β-galactosidase levels 100-fold greater than the background as measured in an ONPG (o-nitrophenyl-β-d-galactopyranoside) assay. Sequencing identified this gene as the human homolog of the yeast factor SPT6, which is thought to be involved in the regulation of chromatin structure. A direct interaction of pUL69 and the carboxy terminus of hSPT6 could be demonstrated using in vitro pull-down experiments. After having generated a specific antiserum that is able to detect the endogenous hSPT6 protein, we were able to observe an in vivo interaction of both proteins by coimmunoprecipitation analysis. The interaction domain within pUL69 was mapped to a central domain of this viral protein that is conserved within the homologous proteins of other herpesviruses such as the ICP27 protein of herpes simplex virus. Internal deletions within this central domain, as well as a single amino acid exchange at position C495, resulted in a loss of interaction. This correlated with a loss of the transactivation potential of the respective mutants, suggesting that the hSPT6 interaction of pUL69 is essential for stimulating gene expression. Furthermore, we demonstrate that the carboxy terminus of hSPT6 also binds to histon H3 and that this interaction can be antagonized by pUL69. This allows the deduction of a model by which pUL69 acts as an antirepressor by competing for binding of histones to hSPT6, thereby antagonizing the chromatin remodeling function of this cellular protein.
PMCID: PMC112338  PMID: 10933715

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