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Frederic D Bushman - One of the best experts on this subject based on the ideXlab platform.
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the bet family of proteins targets moloney murine leukemia virus Integration near transcription start sites
Cell Reports, 2013Co-Authors: Jan De Rijck, Christine De Kogel, Jonas Demeulemeester, Sofie Vets, Sara El Ashkar, Nirav Malani, Frederic D Bushman, Bart Landuyt, Steven J HussonAbstract:Summary 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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bromodomain and extra terminal bet proteins target moloney murine leukemia virus Integration to transcription start sites
Retrovirology, 2013Co-Authors: Jan De Riick, Christine De Kogel, Jonas Demeulemeester, Sofie Vets, Nirav Malani, Frederic D Bushman, Katrien Busschots, Steven Husson, Rik Gijsbers, Zeger DebyserAbstract: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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HIV dna Integration
Cold Spring Harbor Perspectives in Medicine, 2012Co-Authors: Robert Craigie, Frederic D BushmanAbstract:Retroviruses are distinguished from other viruses by two characteristic steps in the viral replication cycle. The first is reverse transcription, which results in the production of a double-stranded DNA copy of the viral RNA genome, and the second is Integration, which results in covalent attachment of the DNA copy to host cell DNA. The initial catalytic steps of the Integration reaction are performed by the virus-encoded integrase (IN) protein. The chemistry of the IN-mediated DNA breaking and joining steps is well worked out, and structures of IN-DNA complexes have now clarified how the overall complex assembles. Methods developed during these studies were adapted for identification of IN inhibitors, which received FDA approval for use in patients in 2007. At the chromosomal level, HIV Integration is strongly favored in active transcription units, which may promote efficient viral gene expression after Integration. HIV IN binds to the cellular factor LEDGF/p75, which promotes efficient infection and tethers IN to favored target sites. The HIV Integration machinery must also interact with many additional host factors during infection, including nuclear trafficking and pore proteins during nuclear entry, histones during initial target capture, and DNA repair proteins during completion of the DNA joining steps. Models for some of the molecular mechanisms involved have been proposed, but important details remain to be clarified.
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HIV Integration site distributions in resting and activated cd4 t cells infected in culture
AIDS, 2009Co-Authors: Troy Brady, Nirav Malani, Charles C Berry, Luis M Agosto, Una Odoherty, Frederic D BushmanAbstract:Objective The goal of this study was to investigate whether the location of HIV Integration differs in resting versus activated T cells, a feature that could contribute to the formation of latent viral reservoirs via effects on Integration targeting.
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role of psip1 ledgf p75 in lentiviral infectivity and Integration targeting
PLOS ONE, 2007Co-Authors: Heather Marshall, Keshet Ronen, Manuel Llano, Eric M Poeschla, Charles C Berry, Dyana T Saenz, Heidi G Sutherland, Wendy A Bickmore, Frederic D BushmanAbstract:Background To replicate, lentiviruses such as HIV must integrate DNA copies of their RNA genomes into host cell chromosomes. Lentiviral Integration is favored in active transcription units, which allows efficient viral gene expression after Integration, but the mechanisms directing Integration targeting are incompletely understood. A cellular protein, PSIP1/LEDGF/p75, binds tightly to the lentiviral-encoded integrase protein (IN), and has been reported to be important for HIV infectivity and Integration targeting. Methodology Here we report studies of lentiviral Integration targeting in 1) human cells with intensified RNAi knockdowns of PSIP1/LEDGF/p75, and 2) murine cells with homozygous gene trap mutations in the PSIP1/LEDGF/p75 locus. Infections with vectors derived from equine infections anemia virus (EIAV) and HIV were compared. Integration acceptor sites were analyzed by DNA bar coding and pyrosequencing. Conclusions/Significance In both PSIP1/LEDGF/p75-depleted cell lines, reductions were seen in lentiviral infectivity compared to controls. For the human cells, Integration was reduced in transcription units in the knockdowns, and this reduction was greater than in our previous studies of human cells less completely depleted for PSIP1/LEDGF/p75. For the homozygous mutant mouse cells, similar reductions in Integration in transcription units were seen, paralleling a previous study of a different mutant mouse line. Integration did not become random, however-Integration in transcription units in both cell types was still favored, though to a reduced degree. New trends also appeared, including favored Integration near CpG islands. In addition, we carried out a bioinformatic study of 15 HIV Integration site data sets in different cell types, which showed that the frequency of Integration in transcription units was correlated with the cell-type specific levels of PSIP1/LEDGF/p75 expression.
Zeger Debyser - One of the best experts on this subject based on the ideXlab platform.
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insight in HIV Integration site selection provides a block and lock strategy for a functional cure of HIV infection
Viruses, 2018Co-Authors: Zeger Debyser, Gerlinde Vansant, Anne Bruggemans, Julie Janssens, Frauke ChristAbstract:Despite significant improvements in therapy, the HIV/AIDS pandemic remains an important threat to public health. Current treatments fail to eradicate HIV as proviral DNA persists in long-living cellular reservoirs, leading to viral rebound whenever treatment is discontinued. Hence, a better understanding of viral reservoir establishment and maintenance is required to develop novel strategies to destroy latently infected cells, and/or to durably silence the latent provirus in infected cells. Whereas the mechanism of Integration has been well studied from a catalytic point of view, it remains unknown how Integration site selection and transcription are linked. In recent years, evidence has grown that lens epithelium-derived growth factor p75 (LEDGF/p75) is the main determinant of HIV Integration site selection and that the Integration site affects the transcriptional state of the provirus. LEDGINs have been developed as small molecule inhibitors of the interaction between LEDGF/p75 and integrase. Recently, it was shown that LEDGIN treatment in cell culture shifts the residual integrated provirus towards the inner nuclear compartment and out of transcription units in a dose dependent manner. This LEDGIN-mediated retargeting increased the proportion of provirus with a transcriptionally silent phenotype and the residual reservoir proved refractory to reactivation in vitro. LEDGINs provide us with a research tool to study the link between Integration and transcription, a quintessential question in retrovirology. LEDGIN-mediated retargeting of the residual reservoirs provides a novel potential “block-and-lock” strategy as a functional cure of HIV infection.
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bromodomain and extra terminal bet proteins target moloney murine leukemia virus Integration to transcription start sites
Retrovirology, 2013Co-Authors: Jan De Riick, Christine De Kogel, Jonas Demeulemeester, Sofie Vets, Nirav Malani, Frederic D Bushman, Katrien Busschots, Steven Husson, Rik Gijsbers, Zeger DebyserAbstract: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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phage display directed discovery of ledgf p75 binding cyclic peptide inhibitors of HIV replication
Molecular Therapy, 2012Co-Authors: Belete Ayele Desimmie, Zeger Debyser, Sofie Vets, Rik Gijsbers, Michael Humbert, Eveline Lescrinier, Jelle Hendrix, Ruth M Ruprecht, Ursula Dietrich, Frauke ChristAbstract:The interaction between the human immunodeficiency virus (HIV) integrase (IN) and its cellular cofactor lens epithelium-derived growth factor (LEDGF/p75) is crucial for HIV replication. While recently discovered LEDGINs inhibit HIV-1 replication by occupying the LEDGF/p75 pocket in IN, it remained to be demonstrated whether LEDGF/p75 by itself can be targeted. By phage display we identified cyclic peptides (CPs) as the first LEDGF/p75 ligands that inhibit the LEDGF/p75–IN interaction. The CPs inhibit HIV replication in different cell lines without overt toxicity. In accord with the role of LEDGF/p75 in HIV Integration and its inhibition by LEDGINs, CP64, and CP65 block HIV replication primarily by inhibiting the Integration step. The CPs retained activity against HIV strains resistant to raltegravir or LEDGINs. Saturation transfer difference (STD) NMR showed residues in CP64 that strongly interact with LEDGF/p75 but not with HIV IN. Mutational analysis identified tryptophan as an important residue responsible for the activity of the peptides. Serial passaging of virus in the presence of CPs did not yield resistant strains. Our work provides proof-of-concept for direct targeting of LEDGF/p75 as novel therapeutic strategy and the CPs thereby serve as scaffold for future development of new HIV therapeutics.
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small molecule inhibitors of the ledgf p75 binding site of integrase block HIV replication and modulate integrase multimerization
Antimicrobial Agents and Chemotherapy, 2012Co-Authors: Frauke Christ, Jonas Demeulemeester, Stephen M Shaw, Belete Ayele Desimmie, Arnaud Marchand, Scott L Butler, Wim Smets, Patrick Chaltin, Mike Westby, Zeger DebyserAbstract:Targeting the HIV integrase (HIV IN) is a clinically validated approach for designing novel anti-HIV therapies. We have previously described the discovery of a novel class of Integration inhibitors, 2-(quinolin-3-yl)acetic acid derivatives, blocking HIV replication at a low micromolar concentration through binding in the LEDGF/p75 binding pocket of HIV integrase, hence referred to as LEDGINs. Here we report the detailed characterization of their mode of action. The design of novel and more potent analogues with nanomolar activity enabled full virological evaluation and a profound mechanistic study. As allosteric inhibitors, LEDGINs bind to the LEDGF/p75 binding pocket in integrase, thereby blocking the interaction with LEDGF/p75 and interfering indirectly with the catalytic activity of integrase. Detailed mechanism-of-action studies reveal that the allosteric mode of inhibition is likely caused by an effect on HIV-1 integrase oligomerization. The multimodal inhibition by LEDGINs results in a block in HIV Integration and in a replication deficiency of progeny virus. The allosteric nature of LEDGINs leads to synergy in combination with the clinically approved active site HIV IN strand transfer inhibitor (INSTI) raltegravir, and cross-resistance profiling proves the distinct mode of action of LEDGINs and INSTIs. The allosteric nature of inhibition and compatibility with INSTIs underline an interest in further (clinical) development of LEDGINs.
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pharmacophore based discovery of small molecule inhibitors of protein protein interactions between HIV 1 integrase and cellular cofactor ledgf p75
ChemMedChem, 2009Co-Authors: Laura De Luca, Maria Letizia Barreca, Stefania Ferro, Frauke Christ, Nunzio Iraci, Rosaria Gitto, Anna Maria Monforte, Zeger Debyser, Alba ChimirriAbstract:: The cellular protein lens epithelium-derived growth factor, or transcriptional coactivator p75 (LEDGF/p75), plays a crucial role in HIV Integration. The protein-protein interactions (PPIs) between HIV-1 integrase (IN) and its cellular cofactor LEDGF/p75 may therefore serve as targets for the development of new anti-HIV drugs. In this work, a structure-based pharmacophore model for potential small-molecule inhibitors of HIV-1 IN-LEDGF/p75 interaction was developed using the LigandScout software. The 3D model obtained was used for virtual screening of our in-house chemical database, CHIME, leading to the identification of compound CHIBA-3002 as an interesting hit for further optimization. The rational design, synthesis and biological evaluation of four derivatives were then carried out. Our studies resulted in the discovery of a new and more potent small molecule (7, CHIBA-3003) that is able to interfere with the HIV-1 IN-LEDGF/p75 interaction at micromolar concentration, representing one of the first compounds to show activity against these specific PPIs. Docking simulations were subsequently performed in order to investigate the possible binding mode of our new lead compound to HIV-1 IN. This study is a valid starting point for the identification of anti-HIV agents with a different mechanism of action from currently available antiviral drugs.
Frauke Christ - One of the best experts on this subject based on the ideXlab platform.
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insight in HIV Integration site selection provides a block and lock strategy for a functional cure of HIV infection
Viruses, 2018Co-Authors: Zeger Debyser, Gerlinde Vansant, Anne Bruggemans, Julie Janssens, Frauke ChristAbstract:Despite significant improvements in therapy, the HIV/AIDS pandemic remains an important threat to public health. Current treatments fail to eradicate HIV as proviral DNA persists in long-living cellular reservoirs, leading to viral rebound whenever treatment is discontinued. Hence, a better understanding of viral reservoir establishment and maintenance is required to develop novel strategies to destroy latently infected cells, and/or to durably silence the latent provirus in infected cells. Whereas the mechanism of Integration has been well studied from a catalytic point of view, it remains unknown how Integration site selection and transcription are linked. In recent years, evidence has grown that lens epithelium-derived growth factor p75 (LEDGF/p75) is the main determinant of HIV Integration site selection and that the Integration site affects the transcriptional state of the provirus. LEDGINs have been developed as small molecule inhibitors of the interaction between LEDGF/p75 and integrase. Recently, it was shown that LEDGIN treatment in cell culture shifts the residual integrated provirus towards the inner nuclear compartment and out of transcription units in a dose dependent manner. This LEDGIN-mediated retargeting increased the proportion of provirus with a transcriptionally silent phenotype and the residual reservoir proved refractory to reactivation in vitro. LEDGINs provide us with a research tool to study the link between Integration and transcription, a quintessential question in retrovirology. LEDGIN-mediated retargeting of the residual reservoirs provides a novel potential “block-and-lock” strategy as a functional cure of HIV infection.
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phage display directed discovery of ledgf p75 binding cyclic peptide inhibitors of HIV replication
Molecular Therapy, 2012Co-Authors: Belete Ayele Desimmie, Zeger Debyser, Sofie Vets, Rik Gijsbers, Michael Humbert, Eveline Lescrinier, Jelle Hendrix, Ruth M Ruprecht, Ursula Dietrich, Frauke ChristAbstract:The interaction between the human immunodeficiency virus (HIV) integrase (IN) and its cellular cofactor lens epithelium-derived growth factor (LEDGF/p75) is crucial for HIV replication. While recently discovered LEDGINs inhibit HIV-1 replication by occupying the LEDGF/p75 pocket in IN, it remained to be demonstrated whether LEDGF/p75 by itself can be targeted. By phage display we identified cyclic peptides (CPs) as the first LEDGF/p75 ligands that inhibit the LEDGF/p75–IN interaction. The CPs inhibit HIV replication in different cell lines without overt toxicity. In accord with the role of LEDGF/p75 in HIV Integration and its inhibition by LEDGINs, CP64, and CP65 block HIV replication primarily by inhibiting the Integration step. The CPs retained activity against HIV strains resistant to raltegravir or LEDGINs. Saturation transfer difference (STD) NMR showed residues in CP64 that strongly interact with LEDGF/p75 but not with HIV IN. Mutational analysis identified tryptophan as an important residue responsible for the activity of the peptides. Serial passaging of virus in the presence of CPs did not yield resistant strains. Our work provides proof-of-concept for direct targeting of LEDGF/p75 as novel therapeutic strategy and the CPs thereby serve as scaffold for future development of new HIV therapeutics.
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small molecule inhibitors of the ledgf p75 binding site of integrase block HIV replication and modulate integrase multimerization
Antimicrobial Agents and Chemotherapy, 2012Co-Authors: Frauke Christ, Jonas Demeulemeester, Stephen M Shaw, Belete Ayele Desimmie, Arnaud Marchand, Scott L Butler, Wim Smets, Patrick Chaltin, Mike Westby, Zeger DebyserAbstract:Targeting the HIV integrase (HIV IN) is a clinically validated approach for designing novel anti-HIV therapies. We have previously described the discovery of a novel class of Integration inhibitors, 2-(quinolin-3-yl)acetic acid derivatives, blocking HIV replication at a low micromolar concentration through binding in the LEDGF/p75 binding pocket of HIV integrase, hence referred to as LEDGINs. Here we report the detailed characterization of their mode of action. The design of novel and more potent analogues with nanomolar activity enabled full virological evaluation and a profound mechanistic study. As allosteric inhibitors, LEDGINs bind to the LEDGF/p75 binding pocket in integrase, thereby blocking the interaction with LEDGF/p75 and interfering indirectly with the catalytic activity of integrase. Detailed mechanism-of-action studies reveal that the allosteric mode of inhibition is likely caused by an effect on HIV-1 integrase oligomerization. The multimodal inhibition by LEDGINs results in a block in HIV Integration and in a replication deficiency of progeny virus. The allosteric nature of LEDGINs leads to synergy in combination with the clinically approved active site HIV IN strand transfer inhibitor (INSTI) raltegravir, and cross-resistance profiling proves the distinct mode of action of LEDGINs and INSTIs. The allosteric nature of inhibition and compatibility with INSTIs underline an interest in further (clinical) development of LEDGINs.
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pharmacophore based discovery of small molecule inhibitors of protein protein interactions between HIV 1 integrase and cellular cofactor ledgf p75
ChemMedChem, 2009Co-Authors: Laura De Luca, Maria Letizia Barreca, Stefania Ferro, Frauke Christ, Nunzio Iraci, Rosaria Gitto, Anna Maria Monforte, Zeger Debyser, Alba ChimirriAbstract:: The cellular protein lens epithelium-derived growth factor, or transcriptional coactivator p75 (LEDGF/p75), plays a crucial role in HIV Integration. The protein-protein interactions (PPIs) between HIV-1 integrase (IN) and its cellular cofactor LEDGF/p75 may therefore serve as targets for the development of new anti-HIV drugs. In this work, a structure-based pharmacophore model for potential small-molecule inhibitors of HIV-1 IN-LEDGF/p75 interaction was developed using the LigandScout software. The 3D model obtained was used for virtual screening of our in-house chemical database, CHIME, leading to the identification of compound CHIBA-3002 as an interesting hit for further optimization. The rational design, synthesis and biological evaluation of four derivatives were then carried out. Our studies resulted in the discovery of a new and more potent small molecule (7, CHIBA-3003) that is able to interfere with the HIV-1 IN-LEDGF/p75 interaction at micromolar concentration, representing one of the first compounds to show activity against these specific PPIs. Docking simulations were subsequently performed in order to investigate the possible binding mode of our new lead compound to HIV-1 IN. This study is a valid starting point for the identification of anti-HIV agents with a different mechanism of action from currently available antiviral drugs.
Steven J Husson - One of the best experts on this subject based on the ideXlab platform.
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the bet family of proteins targets moloney murine leukemia virus Integration near transcription start sites
Cell Reports, 2013Co-Authors: Jan De Rijck, Christine De Kogel, Jonas Demeulemeester, Sofie Vets, Sara El Ashkar, Nirav Malani, Frederic D Bushman, Bart Landuyt, Steven J HussonAbstract:Summary 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.
Jonas Demeulemeester - One of the best experts on this subject based on the ideXlab platform.
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the bet family of proteins targets moloney murine leukemia virus Integration near transcription start sites
Cell Reports, 2013Co-Authors: Jan De Rijck, Christine De Kogel, Jonas Demeulemeester, Sofie Vets, Sara El Ashkar, Nirav Malani, Frederic D Bushman, Bart Landuyt, Steven J HussonAbstract:Summary 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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bromodomain and extra terminal bet proteins target moloney murine leukemia virus Integration to transcription start sites
Retrovirology, 2013Co-Authors: Jan De Riick, Christine De Kogel, Jonas Demeulemeester, Sofie Vets, Nirav Malani, Frederic D Bushman, Katrien Busschots, Steven Husson, Rik Gijsbers, Zeger DebyserAbstract: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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small molecule inhibitors of the ledgf p75 binding site of integrase block HIV replication and modulate integrase multimerization
Antimicrobial Agents and Chemotherapy, 2012Co-Authors: Frauke Christ, Jonas Demeulemeester, Stephen M Shaw, Belete Ayele Desimmie, Arnaud Marchand, Scott L Butler, Wim Smets, Patrick Chaltin, Mike Westby, Zeger DebyserAbstract:Targeting the HIV integrase (HIV IN) is a clinically validated approach for designing novel anti-HIV therapies. We have previously described the discovery of a novel class of Integration inhibitors, 2-(quinolin-3-yl)acetic acid derivatives, blocking HIV replication at a low micromolar concentration through binding in the LEDGF/p75 binding pocket of HIV integrase, hence referred to as LEDGINs. Here we report the detailed characterization of their mode of action. The design of novel and more potent analogues with nanomolar activity enabled full virological evaluation and a profound mechanistic study. As allosteric inhibitors, LEDGINs bind to the LEDGF/p75 binding pocket in integrase, thereby blocking the interaction with LEDGF/p75 and interfering indirectly with the catalytic activity of integrase. Detailed mechanism-of-action studies reveal that the allosteric mode of inhibition is likely caused by an effect on HIV-1 integrase oligomerization. The multimodal inhibition by LEDGINs results in a block in HIV Integration and in a replication deficiency of progeny virus. The allosteric nature of LEDGINs leads to synergy in combination with the clinically approved active site HIV IN strand transfer inhibitor (INSTI) raltegravir, and cross-resistance profiling proves the distinct mode of action of LEDGINs and INSTIs. The allosteric nature of inhibition and compatibility with INSTIs underline an interest in further (clinical) development of LEDGINs.