21 results on '"Katrien Busschots"'
Search Results
2. Y-box-binding protein 1 supports the early and late steps of HIV replication.
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Caroline Weydert, Bart van Heertum, Lieve Dirix, Stéphanie De Houwer, Flore De Wit, Jan Mast, Steven J Husson, Katrien Busschots, Renate König, Rik Gijsbers, Jan De Rijck, and Zeger Debyser
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Medicine ,Science - Abstract
The human immunodeficiency virus (HIV) depends on cellular proteins, so-called cofactors, to complete its replication cycle. In search for new therapeutic targets we identified the DNA and RNA binding protein Y-box-binding Protein 1 (YB-1) as a cofactor supporting early and late steps of HIV replication. YB-1 depletion resulted in a 10-fold decrease in HIV-1 replication in different cell lines. Dissection of the replication defects revealed that knockdown of YB-1 is associated with a 2- to 5-fold decrease in virion production due to interference with the viral RNA metabolism. Using single-round virus infection experiments we demonstrated that early HIV-1 replication also depends on the cellular YB-1 levels. More precisely, using quantitative PCR and an in vivo nuclear import assay with fluorescently labeled viral particles, we showed that YB-1 knockdown leads to a block between reverse transcription and nuclear import of HIV-1. Interaction studies revealed that YB-1 associates with integrase, although a direct interaction with HIV integrase could not be unambiguously proven. In conclusion, our results indicate that YB-1 affects multiple stages of HIV replication. Future research on the interaction between YB-1 and the virus will reveal whether this protein qualifies as a new antiviral target.
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- 2018
- Full Text
- View/download PDF
3. The BET Family of Proteins Targets Moloney Murine Leukemia Virus Integration near Transcription Start Sites
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Jan De Rijck, Christine de Kogel, Jonas Demeulemeester, Sofie Vets, Sara El Ashkar, Nirav Malani, Frederic D. Bushman, Bart Landuyt, Steven J. Husson, Katrien Busschots, Rik Gijsbers, and Zeger Debyser
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Biology (General) ,QH301-705.5 - Abstract
A hallmark of retroviral replication is integration of the viral genome into host cell DNA. This characteristic makes retrovirus-based vectors attractive delivery vehicles for gene therapy. However, adverse events in gene therapeutic trials, caused by activation of proto-oncogenes due to murine leukemia virus (MLV)-derived vector integration, hamper their application. Here, we show that bromodomain and extraterminal (BET) proteins (BRD2, BRD3, and BRD4) and MLV integrase specifically interact and colocalize within the nucleus of the cell. Inhibition of the BET proteins’ chromatin interaction via specific bromodomain inhibitors blocks MLV virus replication at the integration step. MLV integration site distribution parallels the chromatin binding profile of BET proteins, and expression of an artificial fusion protein of the BET integrase binding domain with the chromatin interaction domain of the lentiviral targeting factor LEDGF/p75 retargets MLV integration away from transcription start sites and into the body of actively transcribed genes, conforming to the HIV integration pattern. Together, these data validate BET proteins as MLV integration targeting factors.
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- 2013
- Full Text
- View/download PDF
4. Bidirectional Allosteric Communication between the ATP-Binding Site and the Regulatory PIF Pocket in PDK1 Protein Kinase
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Ricardo M. Biondi, Sonja Neimanis, Katrien Busschots, Pedro M. Alzari, Jörg O. Schulze, Evelyn Süß, Giorgio Saladino, Dalibor Odadzic, María-Natalia Lisa, Francesco Luigi Gervasio, Stefan Zeuzem, V. Hindie, Amanda K. Herbrand, Universitätsklinikum Frankfurt, University College of London [London] (UCL), Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), German Cancer Research Center - Deutsches Krebsforschungszentrum [Heidelberg] (DKFZ), The work was supported by BMBF GO-Bio (0315102), DFG BI 1044/2-3 and DFG BI 1044/12-1 (to R.M.B.) and in part by the Engineering and Physical Sciences Research Council (grant no. EP/M013898/1)., and Université Paris Diderot - Paris 7 (UPD7)-Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)
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0301 basic medicine ,Clinical Biochemistry ,PIF pocket ,MESH: Allosteric Regulation ,Biochemistry ,MAP2K7 ,Adenosine Triphosphate ,0302 clinical medicine ,Aurora Kinases ,MESH: Adenosine Triphosphate ,Drug Discovery ,MESH: Protein Kinase Inhibitors ,MESH: Allosteric Site ,Aurora ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,protein kinase ,MESH: Pyruvate Dehydrogenase Acetyl-Transferring Kinase ,MESH: Indazoles ,ADENOSINE ,Cell biology ,Molecular Docking Simulation ,ALLOSTERIC REGULATION ,PDK1 ,adenosine ,MESH: HEK293 Cells ,030220 oncology & carcinogenesis ,Molecular Medicine ,PIF POCKET ,CIENCIAS NATURALES Y EXACTAS ,Allosteric Site ,AURORA ,Indazoles ,PROTEIN KINASE ,Otras Ciencias Biológicas ,[PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph] ,Allosteric regulation ,[SDV.BC]Life Sciences [q-bio]/Cellular Biology ,Protein Serine-Threonine Kinases ,Biology ,MESH: Protein-Serine-Threonine Kinases ,Ciencias Biológicas ,MESH: Aurora Kinases ,03 medical and health sciences ,GSK2334470 ,MESH: Molecular Docking Simulation ,[CHIM.CRIS]Chemical Sciences/Cristallography ,Humans ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,ddc:610 ,c-Raf ,Binding site ,Protein kinase A ,Protein Kinase Inhibitors ,Molecular Biology ,Pharmacology ,Binding Sites ,MESH: Humans ,small compounds ,Cyclin-dependent kinase 2 ,Pyruvate Dehydrogenase Acetyl-Transferring Kinase ,AGC kinase ,SMALL COMPOUNDS ,allosteric regulation ,molecular dynamics ,HEK293 Cells ,Pyrimidines ,030104 developmental biology ,MESH: Binding Sites ,Allosteric enzyme ,MESH: Pyrimidines ,biology.protein ,AGC KINASE ,MOLECULAR DYNAMICS ,Cyclin-dependent kinase 9 ,[INFO.INFO-BI]Computer Science [cs]/Bioinformatics [q-bio.QM] - Abstract
Allostery is a phenomenon observed in many proteins where binding of a macromolecular partner or a small-molecule ligand at one location leads to specific perturbations at a site not in direct contact with the region where the binding occurs. The list of proteins under allosteric regulation includes AGC protein kinases. AGC kinases have a conserved allosteric site, the phosphoinositide-dependent protein kinase 1 (PDK1)-interacting fragment (PIF) pocket, which regulates protein ATP-binding, activity, and interaction with substrates. In this study, we identify small molecules that bind to the ATP-binding site and affect the PIF pocket of AGC kinase family members, PDK1 and Aurora kinase. We describe the mechanistic details and show that although PDK1 and Aurora kinase inhibitors bind to the conserved ATP-binding site, they differentially modulate physiological interactions at the PIF-pocket site. Our work outlines a strategy for developing bidirectional small-molecule allosteric modulators of protein kinases and other signaling proteins. Fil: Schulze, Jörg O.. Goethe Universitat Frankfurt; Alemania Fil: Saladino, Giorgio. University College London; Reino Unido Fil: Busschots, Katrien. Goethe Universitat Frankfurt; Alemania Fil: Neimanis, Sonja. Goethe Universitat Frankfurt; Alemania Fil: Süß, Evelyn. Goethe Universitat Frankfurt; Alemania Fil: Odadzic, Dalibor. Goethe Universitat Frankfurt; Alemania Fil: Zeuzem, Stefan. Goethe Universitat Frankfurt; Alemania Fil: Hindie, Valerie. Goethe Universitat Frankfurt; Alemania Fil: Herbrand, Amanda K.. Goethe Universitat Frankfurt; Alemania Fil: Lisa, María Natalia. Instituto Pasteur; Francia. Instituto Pasteur de Montevideo; Uruguay. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Alzari, Pedro M.. Instituto Pasteur; Francia Fil: Gervasio, Francesco L.. University College London; Reino Unido Fil: Biondi, Ricardo Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentina. Goethe Universitat Frankfurt; Alemania. German Cancer Research Center; Alemania
- Published
- 2016
5. Small compounds modulating bi‐directional allostery in protein kinases: a new grip with an old trick
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Francesco Luigi Gervasio, Jörg O. Schulze, Klaus Strebhardt, Monika Raab, Evelyn Süß, Mourad Sanhaji, Ricardo M. Biondi, Larissa Pietsch, Giorgio Saladino, and Katrien Busschots
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Chemistry ,Kinase ,Allosteric regulation ,Genetics ,Molecular Biology ,Biochemistry ,Biotechnology ,Cell biology - Published
- 2018
6. Y-box-binding protein 1 supports the early and late steps of HIV replication
- Author
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Jan De Rijck, Flore De Wit, Renate König, Lieve Dirix, Jan Mast, Caroline Weydert, Rik Gijsbers, Steven J. Husson, Katrien Busschots, Stéphanie De Houwer, Bart van Heertum, and Zeger Debyser
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0301 basic medicine ,RNA viruses ,Small interfering RNA ,Time Factors ,lcsh:Medicine ,RNA-binding protein ,HIV Integrase ,Pathology and Laboratory Medicine ,Virus Replication ,Biochemistry ,chemistry.chemical_compound ,Immunodeficiency Viruses ,STAGE EMBRYONIC-DEVELOPMENT ,Medicine and Health Sciences ,Small interfering RNAs ,lcsh:Science ,IN-VIVO ,GENE-EXPRESSION ,Multidisciplinary ,NUCLEAR EXPRESSION ,VIRAL-RNA ,Cell biology ,Integrase ,Nucleic acids ,Multidisciplinary Sciences ,Medical Microbiology ,Cell Processes ,Viral Pathogens ,Viruses ,HUMAN-IMMUNODEFICIENCY-VIRUS ,293T cells ,Cell lines ,Science & Technology - Other Topics ,RNA, Viral ,Pathogens ,Biological cultures ,MESSENGER-RNA ,Engineering sciences. Technology ,Research Article ,Yellow Fluorescent Protein ,Active Transport, Cell Nucleus ,Biology ,Transfection ,Research and Analysis Methods ,Microbiology ,Virus ,03 medical and health sciences ,Virology ,Retroviruses ,Genetics ,Humans ,Nuclear Import ,Molecular Biology Techniques ,Non-coding RNA ,Microbial Pathogens ,Molecular Biology ,Science & Technology ,Biology and life sciences ,HUMAN-CELLS ,Lentivirus ,lcsh:R ,Organisms ,HIV ,Proteins ,Correction ,Cell Biology ,Reverse Transcription ,Y box binding protein 1 ,Reverse transcriptase ,Viral Replication ,Gene regulation ,Luminescent Proteins ,030104 developmental biology ,Viral replication ,chemistry ,TRANSCRIPTION START SITES ,biology.protein ,HIV-1 ,RNA ,lcsh:Q ,Gene expression ,Y-Box-Binding Protein 1 ,HOST PROTEINS ,DNA ,HeLa Cells - Abstract
The human immunodeficiency virus (HIV) depends on cellular proteins, so-called cofactors, to complete its replication cycle. In search for new therapeutic targets we identified the DNA and RNA binding protein Y-box-binding Protein 1 (YB-1) as a cofactor supporting early and late steps of HIV replication. YB-1 depletion resulted in a 10-fold decrease in HIV-1 replication in different cell lines. Dissection of the replication defects revealed that knockdown of YB-1 is associated with a 2- to 5-fold decrease in virion production due to interference with the viral RNA metabolism. Using single-round virus infection experiments we demonstrated that early HIV-1 replication also depends on the cellular YB-1 levels. More precisely, using quantitative PCR and an in vivo nuclear import assay with fluorescently labeled viral particles, we showed that YB-1 knockdown leads to a block between reverse transcription and nuclear import of HIV-1. Interaction studies revealed that YB-1 associates with integrase, although a direct interaction with HIV integrase could not be unambiguously proven. In conclusion, our results indicate that YB-1 affects multiple stages of HIV replication. Future research on the interaction between YB-1 and the virus will reveal whether this protein qualifies as a new antiviral target. ispartof: PLOS ONE vol:13 issue:7 ispartof: location:United States status: published
- Published
- 2017
7. Molecular Mechanism of Regulation of the Atypical Protein Kinase C by N-terminal Domains and an Allosteric Small Compound
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Ricardo M. Biondi, Katrien Busschots, Stefan Zeuzem, Ewgen Proschak, Jörg O. Schulze, Holger Stark, Jose M. Arencibia, Laura A. Lopez-Garcia, Sabine Amon, Hua Zhang, Thomas J. D. Jørgensen, Matthias Engel, Angelika F. Bauer, Sonja Neimanis, and Adriana Stroba
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Models, Molecular ,Clinical Biochemistry ,Allosteric regulation ,Biology ,Inhibitory postsynaptic potential ,Biochemistry ,Structure-Activity Relationship ,Mediator ,Allosteric Regulation ,Drug Discovery ,Humans ,Transferase ,Protein Kinase Inhibitors ,Molecular Biology ,Protein Kinase C ,Pharmacology ,Molecular Structure ,Kinase ,Biphenyl Compounds ,General Medicine ,Protein Structure, Tertiary ,Cell biology ,Drug development ,Allosteric enzyme ,Cinnamates ,Helix ,biology.protein ,Molecular Medicine - Abstract
SummaryProtein kinases play important regulatory roles in cells and organisms. Therefore, they are subject to specific and tight mechanisms of regulation that ultimately converge on the catalytic domain and allow the kinases to be activated or inhibited only upon the appropriate stimuli. AGC protein kinases have a pocket in the catalytic domain, the PDK1-interacting fragment (PIF)-pocket, which is a key mediator of the activation. We show here that helix αC within the PIF-pocket of atypical protein kinase C (aPKC) is the target of the interaction with its inhibitory N-terminal domains. We also provide structural evidence that the small compound PS315 is an allosteric inhibitor that binds to the PIF-pocket of aPKC. PS315 exploits the physiological dynamics of helix αC for its binding and allosteric inhibition. The results will support research on allosteric mechanisms and selective drug development efforts against PKC isoforms.
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- 2014
8. 2-(3-Oxo-1,3-diphenylpropyl)malonic Acids as Potent Allosteric Ligands of the PIF Pocket of Phosphoinositide-Dependent Kinase-1: Development and Prodrug Concept
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Adriana Wilhelm, Jörg O. Schulze, L.A. Lopez-Garcia, Frauke Maurer, Stefan Boettcher, Matthias Engel, Wolfgang Fröhner, Katrien Busschots, Ricardo M. Biondi, and Hua Zhang
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Models, Molecular ,animal structures ,Stereochemistry ,Immunoblotting ,Allosteric regulation ,Plasma protein binding ,Protein Serine-Threonine Kinases ,Structure-Activity Relationship ,Enzyme activator ,Drug Discovery ,medicine ,Humans ,Structure–activity relationship ,Prodrugs ,Binding site ,Protein kinase A ,Protein Kinase Inhibitors ,Muscle Cells ,Binding Sites ,Molecular Structure ,Chemistry ,DNA Helicases ,Pyruvate Dehydrogenase Acetyl-Transferring Kinase ,Prodrug ,Malonates ,Enzyme Activation ,Mechanism of action ,Drug Design ,Molecular Medicine ,medicine.symptom ,Allosteric Site ,Protein Binding - Abstract
The protein kinase C-related kinase 2 (PRK2)-interacting fragment (PIF) pocket of phosphoinositide-dependent kinase-1 (PDK1) was proposed as a novel target site for allosteric modulators. In the present work, we describe the design, synthesis, and structure-activity relationship of a series of 2-(3-oxo-1,3-diphenylpropyl)malonic acids as potent allosteric activators binding to the PIF pocket. Some congeners displayed AC(50) values for PDK1 activation in the submicromolar range. The potency of the best compounds to stabilize PDK1 in a thermal stability shift assay was in the same order of magnitude as that of the PIF pocket binding peptide PIFtide, suggesting comparable binding affinities to the PIF pocket. The crystal structure of PDK1 in complex with compound 4h revealed that additional ionic interactions are mainly responsible for the increased potency compared to the monocarboxylate analogues. Notably, several compounds displayed high selectivity for PDK1. Employing a prodrug strategy, we were able to corroborate the novel mechanism of action in cells.
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- 2012
9. In Vitro DNA Tethering of HIV-1 Integrase by the Transcriptional Coactivator LEDGF/p75
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Melanie Gérard, Eline Boons, Melissa McNeely, Katrien Busschots, Jelle Hendrix, Zeger Debyser, Angélique Deleersnijder, and Frauke Christ
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musculoskeletal diseases ,Nuclear Localization Signals ,Mutant ,Oligonucleotides ,Enzyme-Linked Immunosorbent Assay ,HIV Integrase ,Plasma protein binding ,Biology ,Models, Biological ,Structure-Activity Relationship ,03 medical and health sciences ,chemistry.chemical_compound ,Structural Biology ,Humans ,NLS ,skin and connective tissue diseases ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,030302 biochemistry & molecular biology ,DNA ,AT Rich Sequence ,Molecular biology ,biological factors ,Long terminal repeat ,Protein Structure, Tertiary ,3. Good health ,Integrase ,Chromatin ,Kinetics ,Solubility ,nervous system ,chemistry ,HIV-1 ,biology.protein ,Intercellular Signaling Peptides and Proteins ,Biological Assay ,Mutant Proteins ,sense organs ,Nuclear localization sequence ,Protein Binding - Abstract
Although LEDGF/p75 is believed to act as a cellular cofactor of lentiviral integration by tethering integrase (IN) to chromatin, there is no good in vitro model to analyze this functionality. We designed an AlphaScreen assay to study how LEDGF/p75 modulates the interaction of human immunodeficiency virus type 1 IN with DNA. IN bound with similar affinity to DNA mimicking the long terminal repeat or to random DNA. While LEDGF/p75 bound DNA strongly, a mutant of LEDGF/p75 with compromised nuclear localization signal (NLS)/AT hook interacted weakly, and the LEDGF/p75 PWWP domain did not interact, corroborating previous reports on the role of NLS and AT hooks in charge-dependent DNA binding. LEDGF/p75 stimulated IN binding to DNA 10-fold to 30-fold. Stimulation of IN-DNA binding required a direct interaction between IN and the C-terminus of LEDGF/p75. Addition of either the C-terminus of LEDGF/p75 (amino acids 325-530) or LEDGF/p75 mutated in the NLS/AT hooks interfered with IN binding to DNA. Our results are consistent with an in vitro model of LEDGF/p75-mediated tethering of IN to DNA. The inhibition of IN-DNA interaction by the LEDGF/p75 C-terminus may provide a novel strategy for the inhibition of HIV IN activity and may explain the potent inhibition of HIV replication observed after the overexpression of C-terminal fragments in cell culture. ispartof: JOURNAL OF MOLECULAR BIOLOGY vol:410 issue:5 pages:811-830 ispartof: location:Netherlands status: published
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- 2011
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10. LEDGF/p75 and transportin-SR2 are cellular cofactors of HIV integrase and novel targets for antiviral therapy
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Katrien Busschots, Melissa McNeely, Arnout Voet, Wannes Thys, Zeger Debyser, and Frauke Christ
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medicine.medical_treatment ,Dermatology ,Computational biology ,Protein–protein interaction ,03 medical and health sciences ,Acquired immunodeficiency syndrome (AIDS) ,Viral entry ,Virology ,Drug Discovery ,medicine ,Pharmacology (medical) ,030304 developmental biology ,Pharmacology ,0303 health sciences ,biology ,Growth factor ,030302 biochemistry & molecular biology ,medicine.disease ,Reverse transcriptase ,3. Good health ,Integrase ,Infectious Diseases ,Viral replication ,biology.protein ,Nuclear transport - Abstract
The HIV replication cycle is an elaborate interplay between the viral machinery and cellular proteins. In this review we propose that protein-protein interactions between cellular proteins and HIV integrase are new targets for future antiviral therapy. We focus on the early steps of HIV replication, namely viral entry, uncoating, reverse transcription, trafficking, nuclear import and integration, and the host cell proteins involved herein. We then discuss the feasibility of developing small-molecule protein-protein interaction inhibitors as antiviral agents. Next, we review the HIV integrase cofactors described in the literature highlighting two validated cofactors, lens epitheliumderived growth factor/p75 and transportin-SR2, which are discussed in detail. Finally, a speculative viewpoint is given on small-molecule protein-protein interaction inhibitors as future HIV inhibitors. © 2009 Future Medicine Ltd. ispartof: HIV Therapy vol:3 issue:2 pages:171-188 status: published
- Published
- 2009
11. Differential Interaction of HIV-1 Integrase and JPO2 with the C Terminus of LEDGF/p75
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Frauke Christ, Katrien Busschots, Linda Desender, Stéphane Emiliani, Rik Gijsbers, Richard Benarous, Jan De Rijck, Koen Bartholomeeusen, and Zeger Debyser
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musculoskeletal diseases ,medicine.medical_treatment ,Molecular Sequence Data ,Mutant ,Gene Expression ,HIV Integrase ,Biology ,Virus Replication ,Binding, Competitive ,law.invention ,Structural Biology ,Transcription (biology) ,law ,medicine ,Animals ,Humans ,Amino Acid Sequence ,Molecular Biology ,Genetics ,C-terminus ,Growth factor ,Alternative splicing ,Integrase ,Repressor Proteins ,Zinc ,Multiprotein Complexes ,HIV-1 ,biology.protein ,Recombinant DNA ,Hiv 1 integrase ,Intercellular Signaling Peptides and Proteins ,HeLa Cells ,Protein Binding - Abstract
The transcriptional co-activator lens epithelium-derived growth factor (LEDGF) has been shown to protect cells against environmental stress. The protein has been implicated in auto-immunity and cancer, and is present in cells as the p52 or p75 splice variant. Recently, LEDGF/p75, but not p52, was identified as the prominent interaction partner of human immunodeficiency virus type 1 (HIV-1) integrase. This interaction of HIV-1 integrase with the C-terminal integrase-binding domain of LEDGF/p75 is crucial for HIV-1 replication. To gain insight into the cell biology of LEDGF/p75, we were interested in identifying cellular binding partners of its C-terminal domain. By yeast-two-hybrid screening with a CEMC7 cDNA-library, we were able to identify JPO2 as a binding partner of the C-terminal part of LEDGF/p75. The specific interaction between JPO2 and LEDGF/p75 was verified by pull-down, AlphaScreen, and co-immunoprecipitation. Competition assays using recombinant proteins show a mutually exclusive binding of either JPO2 or HIV-1 integrase to LEDGF/p75. However, differing mechanisms of binding were suggested by continuing interaction of JPO2 with some LEDGF/p75 mutants (I365A, D366A, F406A) that are totally defective for interaction with HIV-1 integrase. This finding is of significance for the development of specific inhibitors targeting only the interaction between LEDGF/p75 and HIV-1 integrase, without disturbing interaction with other cellular factors. Over-expression of JPO2 resulted in a modest but reproducible inhibition of HIV-1 replication, consistent with competition between integrase and JPO2 for binding to LEDGF/p75. Furthermore, JPO2 over-expression activated transcription from the HIV-1 LTR.
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- 2007
12. Identification of the LEDGF/p75 Binding Site in HIV-1 Integrase
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Zeger Debyser, Jean-Christophe Rain, Marc De Maeyer, Richard Benarous, Katrien Busschots, Arnout Voet, Linda Desender, Frauke Christ, and Stéphane Emiliani
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Models, Molecular ,Molecular Sequence Data ,Mutant ,HIV Integrase ,Plasma protein binding ,Crystallography, X-Ray ,Virus Replication ,medicine.disease_cause ,Protein Structure, Secondary ,Protein–protein interaction ,Structure-Activity Relationship ,Protein structure ,Structural Biology ,Protein Interaction Mapping ,medicine ,Humans ,Amino Acid Sequence ,Binding site ,Molecular Biology ,Mutation ,Binding Sites ,biology ,Chemistry ,Mutagenesis ,Molecular biology ,Recombinant Proteins ,Protein Structure, Tertiary ,Integrase ,DNA, Viral ,HIV-1 ,biology.protein ,Intercellular Signaling Peptides and Proteins ,Mutant Proteins ,Protein Binding - Abstract
Lens epithelium-derived growth factor (LEDGF)/p75 is an important cellular co-factor for human immunodeficiency virus (HIV) replication. We originally identified LEDGF/p75 as a binding partner of integrase (IN) in human cells. The interaction has been mapped to the integrase-binding domain (IBD) of LEDGF/p75 located in the C-terminal part. We have subsequently shown that IN carrying the Q168A mutation remains enzymatically active but is impaired for interaction with LEDGF/p75. To map the integrase/LEDGF interface in more detail, we have now identified and characterized two regions within the enzyme involved in the interaction with LEDGF/p75. The first region centers around residues W131 and W132 while the second extends from I161 up to E170. For the different IN mutants the interaction with LEDGF/p75 and the enzymatic activities were determined. IN(W131A), IN(I161A), IN(R166A), IN(Q168A) and IN(E170A) are impaired for interaction with LEDGF/p75, but retain 3' processing and strand transfer activities. Due to impaired integration, an HIV-1 strain containing the W131A mutation in IN displays reduced replication capacity, whereas virus carrying IN(Q168A) is replication defective. Comparison of the wild-type IN-LEDGF/p75 co-crystal structure with that of the modelled structure of the IN(Q168A) and IN(W131A) mutant integrases corroborated our experimental data.
- Published
- 2007
13. The Interaction of LEDGF/p75 with Integrase Is Lentivirus-specific and Promotes DNA Binding
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Stéphane Emiliani, Jo Vercammen, Katrien Busschots, Yves Engelborghs, Richard Benarous, Zeger Debyser, and Frauke Christ
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Rous sarcoma virus ,Feline immunodeficiency virus ,Binding Sites ,Integrases ,biology ,Oligonucleotide ,Lentivirus ,Cell Biology ,biology.organism_classification ,Biochemistry ,Molecular biology ,law.invention ,Integrase ,PSIP1 ,law ,DNA, Viral ,Murine leukemia virus ,Recombinant DNA ,biology.protein ,Humans ,Intercellular Signaling Peptides and Proteins ,Protein Isoforms ,Binding site ,Molecular Biology - Abstract
We have previously shown that the p75 isoform of the transcriptional co-activator lens epithelium-derived growth factor (LEDGF) interacts tightly with human immunodeficiency virus (HIV)-1 integrase (IN) and is essential for nuclear targeting of this protein in human cells (Cherepanov, P., Maertens, G., Proost, P., Devreese, B., Van Beeumen, J., Engelborghs, Y., De Clercq, E., and Debyser, Z. (2003) J. Biol. Chem. 278, 372-381; Maertens, G., Cherepanov, P., Pluymers, W., Busschots, K., De Clercq, E., Debyser, Z., and Engelborghs, Y. (2003) J. Biol. Chem. 278, 33528-33539). Here the interaction between recombinant LEDGF/p75 and HIV-1 IN was examined in a pull-down binding test. LEDGF/p75 was shown to increase the solubility of HIV-1 IN. Next, fluorescent correlation spectroscopy was used to measure the interaction of LEDGF/p75 or the complex of HIV-1 IN and LEDGF/p75 with a specific double-stranded DNA oligonucleotide. Whereas LEDGF/p75 displayed only a moderate affinity for DNA, it strongly promoted the binding of HIV-1 IN to DNA. This effect was specific for the p75 isoform of LEDGF and was not seen with p52. In the pull-down assay LEDGF/p75 interacted with HIV-1, HIV-2, and feline immunodeficiency virus IN, but not with the IN of human T-cell lymphotropic virus type 2, Moloney murine leukemia virus, or Rous sarcoma virus. These results strongly suggest that the interaction of LEDGF/p75 with IN is specific to lentiviridae. LEDGF/p75 stimulated the binding of HIV-1 and HIV-2 IN, but not Moloney murine leukemia virus or Rous sarcoma virus IN, to an aspecific DNA. These results provide supporting evidence for our hypothesis that LEDGF/p75 plays a role in the tethering of lentiviral IN to the chromosomal DNA.
- Published
- 2005
14. The BET family of proteins targets moloney murine leukemia virus integration near transcription start sites
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Christine de Kogel, Sara El Ashkar, Frederic D. Bushman, Jan De Rijck, Rik Gijsbers, Bart Landuyt, Steven J. Husson, Nirav Malani, Zeger Debyser, Katrien Busschots, Jonas Demeulemeester, and Sofie Vets
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BRD4 ,Chromosomal Proteins, Non-Histone ,Virus Integration ,viruses ,Molecular Sequence Data ,HIV integration ,Protein Serine-Threonine Kinases ,Virus Replication ,Article ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,Mice ,Retrovirus ,Animals ,Amino Acid Sequence ,lcsh:QH301-705.5 ,Biology ,biology ,Chromatin binding ,Nuclear Proteins ,hemic and immune systems ,3T3 Cells ,Azepines ,Triazoles ,biology.organism_classification ,Virology ,Protein Structure, Tertiary ,Bromodomain ,Integrase ,Chromatin ,Leukemia Virus, Murine ,lcsh:Biology (General) ,DNA, Viral ,biology.protein ,Intercellular Signaling Peptides and Proteins ,Human medicine ,Transcription Initiation Site ,Transcription Factors - Abstract
A hallmark of retroviral replication is integration of the viral genome into host cell DNA. This characteristic makes retrovirus-based vectors attractive delivery vehicles for gene therapy. However, adverse events in gene therapeutic trials, caused by activation of proto-oncogenes due to murine leukemia virus (MLV)-derived vector integration, hamper their application. Here, we show that bromodomain and extraterminal (BET) proteins (BRD2, BRD3, and BRD4) and MLV integrase specifically interact and colocalize within the nucleus of the cell. Inhibition of the BET proteins' chromatin interaction via specific bromodomain inhibitors blocks MLV virus replication at the integration step. MLV integration site distribution parallels the chromatin binding profile of BET proteins, and expression of an artificial fusion protein of the BET integrase binding domain with the chromatin interaction domain of the lentiviral targeting factor LEDGF/p75 retargets MLV integration away from transcription start sites and into the body of actively transcribed genes, conforming to the HIV integration pattern. Together, these data validate BET proteins as MLV integration targeting factors. publisher: Elsevier articletitle: The BET Family of Proteins Targets Moloney Murine Leukemia Virus Integration near Transcription Start Sites journaltitle: Cell Reports articlelink: http://dx.doi.org/10.1016/j.celrep.2013.09.040 content_type: article copyright: Copyright © 2013 The Authors. Published by Elsevier Inc. ispartof: CELL REPORTS vol:5 issue:4 pages:886-894 ispartof: location:United States status: published
- Published
- 2013
15. Substrate-Selective Inhibition of Protein Kinase PDK1 by Small Compounds that Bind to the PIF-Pocket Allosteric Docking Site
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Laura A. Lopez-Garcia, Pedro M. Alzari, Sonja Neimanis, Carmen Lammi, Jose M. Arencibia, Jörg O. Schulze, Katrien Busschots, Stefan Zeuzem, Adriana Stroba, Albrecht Piiper, Ricardo M. Biondi, Matthias Engel, Universitätsklinikum Frankfurt, Universität des Saarlandes [Saarbrücken], Biochimie Structurale, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), We acknowledge support from the Deutsche Krebshilfe (106388), DFG (BI 1044/2-3) and BMBF Go-Bio (0315102) to R.M.B., and Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris]
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Models, Molecular ,Clinical Biochemistry ,01 natural sciences ,Biochemistry ,Substrate Specificity ,Mice ,Chalcones ,MESH: Structure-Activity Relationship ,Drug Discovery ,MESH: Protein Kinase Inhibitors ,Dicarboxylic Acids ,Prodrugs ,MESH: Animals ,MESH: Allosteric Site ,0303 health sciences ,Molecular Structure ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,Kinase ,MESH: Molecular Weight ,General Medicine ,MESH: Pyruvate Dehydrogenase Acetyl-Transferring Kinase ,3. Good health ,MESH: HEK293 Cells ,Molecular Medicine ,Allosteric Site ,MESH: Models, Molecular ,animal structures ,[PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph] ,Allosteric regulation ,MESH: Molecular Structure ,P70-S6 Kinase 1 ,[SDV.BC]Life Sciences [q-bio]/Cellular Biology ,Protein Serine-Threonine Kinases ,Biology ,Models, Biological ,MESH: Protein-Serine-Threonine Kinases ,Cell Line ,Structure-Activity Relationship ,03 medical and health sciences ,MESH: Dicarboxylic Acids ,[CHIM.CRIS]Chemical Sciences/Cristallography ,Animals ,Humans ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Protein kinase A ,Protein Kinase Inhibitors ,Molecular Biology ,Protein kinase B ,MESH: Chalcones ,MESH: Mice ,030304 developmental biology ,Pharmacology ,MESH: Humans ,010405 organic chemistry ,Activator (genetics) ,MESH: Models, Biological ,Pyruvate Dehydrogenase Acetyl-Transferring Kinase ,0104 chemical sciences ,MESH: Cell Line ,Molecular Weight ,HEK293 Cells ,Protein kinase domain ,Docking (molecular) ,Biophysics ,MESH: Substrate Specificity ,MESH: Prodrugs ,[INFO.INFO-BI]Computer Science [cs]/Bioinformatics [q-bio.QM] - Abstract
International audience; The PIF-pocket of AGC protein kinases participates in the physiologic mechanism of regulation by acting as a docking site for substrates and as a switch for the transduction of the conformational changes needed for activation or inhibition. We describe the effects of compounds that bind to the PIF-pocket of PDK1. In vitro, PS210 is a potent activator of PDK1, and the crystal structure of the PDK1-ATP-PS210 complex shows that PS210 stimulates the closure of the kinase domain. However, in cells, the prodrug of PS210 (PS423) acts as a substrate-selective inhibitor of PDK1, inhibiting the phosphorylation and activation of S6K, which requires docking to the PIF-pocket, but not affecting PKB/Akt. This work describes a tool to study the dynamics of PDK1 activity and a potential approach for drug discovery.
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- 2012
16. Assays for Evaluation of HIV-1 Integrase Enzymatic Activity, DNA Binding, and Cofactor Interaction
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Frauke Christ, Katrien Busschots, Yves Engelborghs, Melissa McNeely, Jelle Hendrix, and Zeger Debyser
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chemistry.chemical_classification ,chemistry.chemical_compound ,Enzyme ,Biochemistry ,chemistry ,biology ,Hiv 1 integrase ,biology.protein ,Molecular biology ,Cofactor ,DNA ,Integrase - Published
- 2011
17. In search of small molecules blocking interactions between HIV proteins and intracellular cofactors
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Jan De Rijck, Frauke Christ, Zeger Debyser, and Katrien Busschots
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0303 health sciences ,biology ,HIV Proteins ,Anti-HIV Agents ,Human Immunodeficiency Virus Proteins ,Nanotechnology ,HIV Infections ,Computational biology ,Small molecule ,Cofactor ,3. Good health ,03 medical and health sciences ,0302 clinical medicine ,030220 oncology & carcinogenesis ,biology.protein ,Animals ,Humans ,Small molecule binding ,Site-directed mutagenesis ,Molecular Biology ,Intracellular ,030304 developmental biology ,Biotechnology ,Protein Binding - Abstract
One of the major obstacles to pursue the discovery of small molecule inhibitors targeting protein-protein interactions is the flat nature of their interface. X-Ray structures have indeed shown that a large part of the interaction area is buried with atoms closely packed together, implying a lack of available cavities for small molecule binding. Yet, it has become clear that some protein-protein interfaces have a well-defined compact area, commonly referred to as a hot spot, that plays a major role in the affinity of the interaction. These hot spots define potential targets for the development of small molecule protein-protein interaction inhibitors (SMPPIIs). In this review we discuss the interactions between viral and host proteins that have the potential for the future development of SMPPIIs. In light of the current anti-HIV therapy a short overview of protein-protein interactions that may serve as targets for novel drugs is provided. Our hypothesis will exemplify and discuss the interaction between HIV-1 integrase and its cellular cofactor LEDGF/p75, which, as evidenced by crystallography and site directed mutagenesis, displays favourable properties needed for the development of interaction inhibitors.
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- 2008
18. Cellular co-factors of HIV-1 integration
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Bénédicte Van Maele, Zeger Debyser, Katrien Busschots, Linos Vandekerckhove, and Frauke Christ
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Chromosomal Proteins, Non-Histone ,Virus Integration ,HIV Integrase ,Biochemistry ,DNA-binding protein ,Animals ,Humans ,DNA Integration ,HMGA1a Protein ,Nuclear protein ,Molecular Biology ,Transcription factor ,Genetics ,biology ,Membrane Proteins ,Nuclear Proteins ,SMARCB1 Protein ,Long terminal repeat ,Integrase ,DNA-Binding Proteins ,High-mobility group ,DNA, Viral ,biology.protein ,HIV-1 ,Intercellular Signaling Peptides and Proteins ,Transcription Factors - Abstract
To achieve productive infection, the reverse transcribed cDNA of human immunodeficiency virus type 1 (HIV-1) is inserted in the host cell genome. The main protein responsible for this reaction is the viral integrase. However, studies indicate that the virus is assisted by cellular proteins, or co-factors, to achieve integration into the infected cell. The barrier-to-autointegration factor (BAF) might prevent autointegration. Its ability to bridge DNA and the finding that the nuclear lamina-associated polypeptide-2alpha interacts with BAF suggest a role in nuclear structure organization. Integrase interactor 1 was found to directly interact with HIV-1 integrase and to activate its DNA-joining activity, and the high mobility group chromosomal protein A1 might approximate both long terminal repeat (LTR) ends and facilitate integrase binding by unwinding the LTR termini. Furthermore, the lens-epithelium-derived growth factor (LEDGF; also known as p75) seems to tether HIV-1 integrase to the chromosomes. Although a direct role in integration has only been demonstrated for LEDGF/p75, to date, each validated cellular co-factor for HIV-1 integration could constitute a promising new target for antiviral therapy.
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- 2005
19. Integrase Mutants Defective for Interaction with LEDGF/p75 Are Impairedin Chromosome Tethering and HIV-1Replication
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Myriam Witvrouw, Denis Tempé, Jean-Christophe Rain, Aurelie Mousnier, Lilia Ben-Slama, Katrien Busschots, Zeger Debyser, Catherine Dargemont, Fanny Moisant, Richard Benarous, Frauke Christ, Marlène Maroun, Bénédicte Van Maele, Linos Vandekerckhove, Stéphane Emiliani, Institut de Génétique Moléculaire de Montpellier (IGMM), and Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)
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Glutamine ,Virus Integration ,[SDV]Life Sciences [q-bio] ,Mutant ,HIV integration ,HIV Infections ,HIV Integrase ,Virus Replication ,Biochemistry ,03 medical and health sciences ,Chromosomes, Human ,Humans ,DNA Integration ,Molecular Biology ,ComputingMilieux_MISCELLANEOUS ,030304 developmental biology ,Genetics ,0303 health sciences ,biology ,030302 biochemistry & molecular biology ,virus diseases ,RNA ,Cell Biology ,3. Good health ,Integrase ,Directed mutagenesis ,Viral replication ,biology.protein ,HIV-1 ,Intercellular Signaling Peptides and Proteins ,HeLa Cells - Abstract
The insertion of a DNA copy of its RNA genome into a chromosome of the host cell is mediated by the viral integrase with the help of mostly uncharacterized cellular cofactors. We have recently described that the transcriptional co-activator LEDGF/p75 strongly interacts with HIV-1 integrase. Here we show that interaction of HIV-1 integrase with LEDGF/p75 is important for viral replication. Using multiple approaches including two-hybrid interaction studies, random and directed mutagenesis, we could demonstrate that HIV-1 virus harboring a single mutation that disrupts integrase-LEDGF/p75 interaction, resulted in defective HIV-1 replication. Furthermore, we found that LEDGF/p75 tethers HIV-1 integrase to chromosomes and that this interaction may be important for the integration process and the replication of HIV-1.
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- 2005
20. LEDGF/p75 is essential for nuclear and chromosomal targeting of HIV-1 integrase in human cells
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Katrien Busschots, Yves Engelborghs, Peter Cherepanov, W Pluymers, Erik De Clercq, Zeger Debyser, and Goedele N. Maertens
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Cytoplasm ,Recombinant Fusion Proteins ,HIV integration ,Blotting, Western ,Molecular Sequence Data ,Active Transport, Cell Nucleus ,Mitosis ,HIV Integrase ,Biology ,Transfection ,Biochemistry ,PSIP1 ,Transcription (biology) ,medicine ,Humans ,RNA, Small Interfering ,Fluorescent Antibody Technique, Indirect ,Molecular Biology ,Cells, Cultured ,Cell Nucleus ,Microscopy, Confocal ,Base Sequence ,Models, Genetic ,Cell Biology ,Molecular biology ,Integrase ,Protein Structure, Tertiary ,Cell nucleus ,Alternative Splicing ,Zinc ,medicine.anatomical_structure ,Microscopy, Fluorescence ,Mutation ,biology.protein ,Intercellular Signaling Peptides and Proteins ,Nuclear transport ,Nuclear localization sequence ,HeLa Cells ,Plasmids ,Protein Binding - Abstract
We have reported that human immunodeficiency virus type 1 (HIV-1) integrase (IN) forms a specific nuclear complex with human lens epithelium-derived growth factor/transcription co-activator p75 (LEDGF/p75) protein. We now studied the IN-LEDGF/p75 interaction and nuclear import of IN in living cells using fusions of IN and LEDGF/p75 with enhanced green fluorescent protein and far-red fluorescent protein HcRed1. We show that both the N-terminal zinc binding domain and the central core domains of IN are involved in the interaction with LEDGF/p75. Both domains are essential for nuclear localization of IN as well as for the association of IN with condensed chromosomes during mitosis. However, upon overexpression of LEDGF/p75, the core domain fragment of IN was recruited to the nuclei and mitotic chromosomes with a distribution pattern characteristic of the full-length protein, indicating that it harbors the main determinant for interaction with LEDGF/p75. Although the C-terminal domain of IN was dispensable for nuclear/chromosomal localization, a fusion of the C-terminal IN fragment with enhanced green fluorescent protein was found exclusively in the nucleus, with a diffuse nuclear/nucleolar distribution, suggesting that the C-terminal domain may also play a role in the nuclear import of IN. In contrast to LEDGF/p75, its alternative splice variant, p52, did not interact with HIV-1 IN in vitro and in living cells. Finally, RNA interference-mediated knock-down of endogenous LEDGF/p75 expression abolished nuclear/chromosomal localization of IN. We conclude, therefore, that the interaction with LEDGF/p75 accounts for the karyophilic properties and chromosomal targeting of HIV-1 IN.
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- 2003
21. Bromodomain and extra-terminal (BET) proteins target Moloney murine leukemia virus integration to transcription start sites
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Rik Gijsbers, Jan De Riick, Katrien Busschots, Frederic D. Bushman, Zeger Debyser, Jonas Demeulemeester, Sofie Vets, Nirav Malani, Steven J. Husson, and Christine de Kogel
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BRD4 ,biology ,viruses ,Chromatin binding ,biology.organism_classification ,Virology ,Bromodomain ,Integrase ,Chromatin ,Transduction (genetics) ,Infectious Diseases ,Viral replication ,Murine leukemia virus ,biology.protein ,Oral Presentation - Abstract
A hallmark of retroviral replication is stable integration of the viral genome in the host cell DNA. This characteristic makes retroviral-derived vector particles attractive vehicles for gene therapy. However, retroviral integration is not a random process. Lentiviruses preferentially integrate in the body of active transcription units, while gammaretroviruses, including Moloney Murine Leukemia Virus (MLV), favour transcription start sites and CpG islands. In clinical trials using gammaretroviral vectors for gene therapy, leukemogenesis has been associated with integration of vectors near oncogene transcription start sites. We found that the bromodomain and extra-terminal (BET) proteins (BRD2, BRD3 and BRD4) interact with MLV integrase and direct integration towards transcription start regions. BET proteins specifically bind and co-localize with the gammaretrovirus integrase protein in the nucleus of the cell. The interaction is gammaretroviral-specific and mediated by the integrase C-terminal domain and the BET extraterminal (ET) domain as determined by co-immunoprecipitation assays and in an Alphascreen assay using recombinant proteins. Interfering with chromatin interaction of BET proteins via specific bromodomain inhibitors JQ1 and l-BET decreases MLV virus replication and MLV vector transduction 5-to 10-fold, while HIV vector transduction is not affected. Analysis of viral DNA intermediates by quantitative PCR revealed a block at the integration step. In addition, bromodomain inhibitors do not have an effect on the late steps of viral replication. MLV integration site distribution analysis revealed a strong correlation with the BET protein chromatin binding profile. Finally, expression of an artificial fusion protein that merges the BET integrase binding domain with the chromatin interaction domain of the lentiviral targeting factor LEDGF/p75, retargets MLV integration into the body of actively transcribed genes, paralleling the Human Immunodeficiency Virus (HIV) integration pattern. Our results explain the molecular mechanism behind gammaretroviral integration site targeting and suggest methods for engineering gammaretroviral vectors with a safer integration site profile.
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