scholarly journals Integrase-Specific Enhancement and Suppression of Retroviral DNA Integration by Compacted Chromatin Structure In Vitro

2004 ◽  
Vol 78 (11) ◽  
pp. 5848-5855 ◽  
Author(s):  
Konstantin D. Taganov ◽  
Isabel Cuesta ◽  
René Daniel ◽  
Lisa Ann Cirillo ◽  
Richard A. Katz ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Chromatin Structure ◽  
Site Selection ◽  
Integration Site ◽  
Histone H1 ◽  
Detectable Effect ◽  
Host Chromosome ◽  
Dna Integration ◽  
Hiv 1

ABSTRACT Integration of viral DNA into the host chromosome is an obligatory step in retroviral replication and is dependent on the activity of the viral enzyme integrase. To examine the influence of chromatin structure on retroviral DNA integration in vitro, we used a model target comprising a 13-nucleosome extended array that includes binding sites for specific transcription factors and can be compacted into a higher-ordered structure. We found that the efficiency of in vitro integration catalyzed by human immunodeficiency virus type 1 (HIV-1) integrase was decreased after compaction of this target with histone H1. In contrast, integration by avian sarcoma virus (ASV) integrase was more efficient after compaction by either histone H1 or a high salt concentration, suggesting that the compacted structure enhances this reaction. Furthermore, although site-specific binding of transcription factors HNF3 and GATA4 blocked ASV DNA integration in extended nucleosome arrays, local opening of H1-compacted chromatin by HNF3 had no detectable effect on integration, underscoring the preference of ASV for compacted chromatin. Our results indicate that chromatin structure affects integration site selection of the HIV-1 and ASV integrases in opposite ways. These distinct properties of integrases may also affect target site selection in vivo, resulting in an important bias against or in favor of integration into actively transcribed host DNA.

scholarly journals Genomic Sites of Human Immunodeficiency Virus Type 2 (HIV-2) Integration: Similarities to HIV-1 In Vitro and Possible Differences In Vivo

2006 ◽  
Vol 80 (15) ◽  
pp. 7316-7321 ◽  
Author(s):  
Adam MacNeil ◽  
Jean-Louis Sankalé ◽  
Seema Thakore Meloni ◽  
Abdoulaye Dieng Sarr ◽  
Souleymane Mboup ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Site Selection ◽  
Integration Site ◽  
Proviral Integration ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Integration Site Selection

ABSTRACT Retroviruses have distinct preferences in integration site selection in the host cell genome during in vitro infection, with human immunodeficiency virus type 1 (HIV-1) integration strongly favoring transcriptional units. Additionally, studies with HIV-1 have shown that the genomic site of proviral integration may impact viral replication, with integration in heterochromatin associated with a block in viral transcription. HIV-2 is less pathogenic than HIV-1 and is believed to have a lower replication rate in vivo. Although differences in integration site selection between HIV-2 and HIV-1 could potentially explain the attenuated pathogenicity of HIV-2, no studies have characterized integration site selection by HIV-2. In this study, we mapped 202 HIV-2 integration sites during in vitro infection of peripheral blood mononuclear cells with a primary HIV-2 isolate. In addition, we assayed for in vivo proviral integration within heterochromatin in 21 HIV-1-infected subjects and 23 HIV-2-infected subjects, using an alphoid repeat PCR assay. During in vitro infection, HIV-2 displayed integration site preferences similar to those previously reported for HIV-1. Notably, 82% of HIV-2 integrations mapped to Refseq genes, and integration strongly favored regions of the genome with high gene density and high GC content. Though rare, the proportion of HIV-2 subjects with evidence of proviral integration within heterochromatin in vivo was higher than that of HIV-1-infected subjects. It is therefore possible that integration site selection may play a role in the differences in HIV-1 and HIV-2 in vivo pathogenesis.

scholarly journals Pin1 promotes histone H1 dephosphorylation and stabilizes its binding to chromatin

2013 ◽  
Vol 203 (1) ◽  
pp. 57-71 ◽  
Author(s):  
Nikhil Raghuram ◽  
Hilmar Strickfaden ◽  
Darin McDonald ◽  
Kylie Williams ◽  
He Fang ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Chromatin Structure ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Histone H1 ◽  
Dependent Manner ◽  
Prolyl Isomerase ◽  
Proline Isomerization ◽  
The Stability

Histone H1 plays a crucial role in stabilizing higher order chromatin structure. Transcriptional activation, DNA replication, and chromosome condensation all require changes in chromatin structure and are correlated with the phosphorylation of histone H1. In this study, we describe a novel interaction between Pin1, a phosphorylation-specific prolyl isomerase, and phosphorylated histone H1. A sub-stoichiometric amount of Pin1 stimulated the dephosphorylation of H1 in vitro and modulated the structure of the C-terminal domain of H1 in a phosphorylation-dependent manner. Depletion of Pin1 destabilized H1 binding to chromatin only when Pin1 binding sites on H1 were present. Pin1 recruitment and localized histone H1 phosphorylation were associated with transcriptional activation independent of RNA polymerase II. We thus identify a novel form of histone H1 regulation through phosphorylation-dependent proline isomerization, which has consequences on overall H1 phosphorylation levels and the stability of H1 binding to chromatin.

scholarly journals Insight in HIV Integration Site Selection Provides a Block-and-Lock Strategy for a Functional Cure of HIV Infection

Viruses ◽  
2018 ◽  
Vol 11 (1) ◽  
pp. 12 ◽  
Author(s):  
Zeger Debyser ◽  
Gerlinde Vansant ◽  
Anne Bruggemans ◽  
Julie Janssens ◽  
Frauke Christ
Keyword(s):  
Hiv Infection ◽  
Site Selection ◽  
Integration Site ◽  
Viral Reservoir ◽  
Dependent Manner ◽  
Functional Cure ◽  
Infected Cells ◽  
Hiv Integration ◽  
Integration Site Selection

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.

scholarly journals Functional Interaction between the CoactivatorDrosophila CREB-Binding Protein and ASH1, a Member of the Trithorax Group of Chromatin Modifiers

2000 ◽  
Vol 20 (24) ◽  
pp. 9317-9330 ◽  
Author(s):  
Frédéric Bantignies ◽  
Richard H. Goodman ◽  
Sarah M. Smolik
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Chromatin Structure ◽  
Binding Protein ◽  
Polytene Chromosomes ◽  
Open Chromatin ◽  
Creb Binding Protein ◽  
Trithorax Group ◽  
Chromatin Modifiers

ABSTRACT CREB-binding protein (CBP) is a coactivator for multiple transcription factors that transduce a variety of signaling pathways. Current models propose that CBP enhances gene expression by bridging the signal-responsive transcription factors with components of the basal transcriptional machinery and by augmenting the access of transcription factors to DNA through the acetylation of histones. To define the pathways and proteins that require CBP function in a living organism, we have begun a genetic analysis of CBP in flies. We have overproduced Drosophila melanogaster CBP (dCBP) in a variety of cell types and obtained distinct adult phenotypes. We used an uninflated-wing phenotype, caused by the overexpression of dCBP in specific central nervous system cells, to screen for suppressors of dCBP overactivity. Two genes with mutant versions that act as dominant suppressors of the wing phenotype were identified: thePKA-C1/DCO gene, encoding the catalytic subunit of cyclic AMP protein kinase, and ash1, a member of thetrithorax group (trxG) of chromatin modifiers. Using immunocolocalization, we showed that the ASH1 protein is specifically expressed in the majority of the dCBP-overexpressing cells, suggesting that these proteins have the potential to interact biochemically. This model was confirmed by the findings that the proteins interact strongly in vitro and colocalize at specific sites on polytene chromosomes. The trxG proteins are thought to maintain gene expression during development by creating domains of open chromatin structure. Our results thus implicate a second class of chromatin-associated proteins in mediating dCBP function and imply that dCBP might be involved in the regulation of higher-order chromatin structure.

scholarly journals Towards a Functional Cure of HIV-1: Insight Into the Chromatin Landscape of the Provirus

2021 ◽  
Vol 12 ◽  
Author(s):  
Julie Janssens ◽  
Anne Bruggemans ◽  
Frauke Christ ◽  
Zeger Debyser
Keyword(s):  
Site Selection ◽  
Integration Site ◽  
Great Success ◽  
Elite Controllers ◽  
Functional Cure ◽  
Binding Partner ◽  
The Impact ◽  
Transcriptional State ◽  
Hiv 1 ◽  
Integration Site Selection

Despite potent combination antiretroviral therapy, HIV-1 infection persists due to irreversible integration of the virus in long-living cells of the immune system. The main focus of HIV-1 cure strategies has been on HIV-1 eradication, yet without great success so far. Therefore, HIV-1 remission or a functional cure, whereby the virus is silenced rather than eradicated, is considered as an alternative strategy. Elite controllers, individuals who spontaneously control HIV-1, may point us the way toward a functional HIV-1 cure. In order to achieve such a cure, a profound understanding of the mechanisms controlling HIV-1 expression and silencing is needed. In recent years, evidence has grown that the site of integration as well as the chromatin landscape surrounding the integration site affects the transcriptional state of the provirus. Still, at present, the impact of integration site selection on the establishment and maintenance of the HIV-1 reservoirs remains poorly understood. The discovery of LEDGF/p75 as a binding partner of HIV-1 integrase has led to a better understanding of integration site selection. LEDGF/p75 is one of the important determinants of integration site selection and targets integration toward active genes. In this review, we will provide an overview of the most important determinants of integration site selection. Secondly, we will discuss the chromatin landscape at the integration site and its implications on HIV-1 gene expression and silencing. Finally, we will discuss how interventions that affect integration site selection or modifications of the chromatin could yield a functional cure of HIV-1 infection.

scholarly journals DNA Integration by Ty Integrase in yku70Mutant Saccharomyces cerevisiae Cells

2000 ◽  
Vol 20 (23) ◽  
pp. 8836-8844 ◽  
Author(s):  
Markus Kiechle ◽  
Anna A. Friedl ◽  
Palaniyandi Manivasakam ◽  
Friederike Eckardt-Schupp ◽  
Robert H. Schiestl
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasmid Dna ◽  
Integration Site ◽  
Cellular Function ◽  
Target Site ◽  
Target Site Duplication ◽  
Wild Type ◽  
Dna Integration ◽  
Site Preferences

ABSTRACT In the present work we examined nonhomologous integration of plasmid DNA in a yku70 mutant. Ten of 14 plasmids integrated as composite elements, including Ty sequences probably originating from erroneous strand-switching and/or priming events. Three additional plasmids integrated via Ty integrase without cointegrating Ty sequences, as inferred from 5-bp target site duplication and integration site preferences. Ty integrase-mediated integration of non-Ty DNA has never been observed in wild-type cells, although purified integrase is capable of using non-Ty DNA as a substrate in vitro. Hence our data implicate yKu70 as the cellular function preventing integrase from accepting non-Ty DNA as a substrate.

scholarly journals HRP-2 determines HIV-1 integration site selection in LEDGF/p75 depleted cells

Retrovirology ◽  
2012 ◽  
Vol 9 (1) ◽  
pp. 84 ◽  
Author(s):  
Rik Schrijvers ◽  
Sofie Vets ◽  
Jan De Rijck ◽  
Nirav Malani ◽  
Frederic D Bushman ◽  
...  
Keyword(s):  
Site Selection ◽  
Integration Site ◽  
Hiv 1 ◽  
Integration Site Selection

scholarly journals LEDGF/p75 Determines Cellular Trafficking of Diverse Lentiviral but Not Murine Oncoretroviral Integrase Proteins and Is a Component of Functional Lentiviral Preintegration Complexes

2004 ◽  
Vol 78 (17) ◽  
pp. 9524-9537 ◽  
Author(s):  
Manuel Llano ◽  
Maria Vanegas ◽  
Oliver Fregoso ◽  
Dyana Saenz ◽  
Susan Chung ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Intracellular Trafficking ◽  
Integration Site ◽  
Close Association ◽  
Cellular Trafficking ◽  
Cell Nuclei ◽  
Nuclear Localization Signals ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1), feline immunodeficiency virus (FIV), and Moloney murine leukemia virus (MoMLV) integrases were stably expressed to determine their intracellular trafficking. Each lentiviral integrase localized to cell nuclei in close association with chromatin while the murine oncoretroviral integrase was cytoplasmic. Fusions of pyruvate kinase to the lentiviral integrases did not reveal transferable nuclear localization signals. The intracellular trafficking of each was determined instead by the transcriptional coactivator LEDGF/p75, which was required for nuclear localization. Stable small interfering RNA expression eliminated detectable LEDGF/p75 expression and caused dramatic, stable redistribution of each lentiviral integrase from nucleus to cytoplasm while the distribution of MoMLV integrase was unaffected. In addition, endogenous LEDGF/p75 coimmunoprecipitated specifically with each lentiviral integrase. In vitro integration assays with preintegration complexes (PICs) showed that endogenous LEDGF/p75 is a component of functional HIV-1 and FIV PICs. However, HIV-1 and FIV infection and replication in LEDGF/p75-deficient cells was equivalent to that in control cells, whether cells were dividing or growth arrested. Two-long terminal repeat circle accumulation in nondividing cell nuclei was also equivalent to that of LEDGF/p75 wild-type cells. Virions produced in LEDGF/p75-deficient cells had normal infectivity. We conclude that LEDGF/p75 fully accounts for cellular trafficking of diverse lentiviral, but not oncoretroviral, integrases and is the main lentiviral integrase-to-chromatin tethering factor. While lentiviral PIC nuclear import is unaffected by LEDGF/p75 knockdown, this protein is a component of functional lentiviral PICs. A role in HIV-1 integration site distribution merits investigation.

scholarly journals Parathymosin Affects the Binding of Linker Histone H1 to Nucleosomes and Remodels Chromatin Structure

2005 ◽  
Vol 280 (16) ◽  
pp. 16143-16150 ◽  
Author(s):  
Goran Martic ◽  
Zoe Karetsou ◽  
Katerina Kefala ◽  
Anastasia S. Politou ◽  
Cedric R. Clapier ◽  
...  
Keyword(s):  
Chromatin Structure ◽  
Histone H1 ◽  
Higher Order ◽  
Linker Histone ◽  
Laser Scanning Microscopy ◽  
Micrococcal Nuclease ◽  
Confocal Laser ◽  
Dependent Manner ◽  
Linker Histone H1

Linker histone H1 is the major factor that stabilizes higher order chromatin structure and modulates the action of chromatin-remodeling enzymes. We have previously shown that parathymosin, an acidic, nuclear protein binds to histone H1in vitroandin vivo. Confocal laser scanning microscopy reveals a nuclear punctuate staining of the endogenous protein in interphase cells, which is excluded from dense heterochromatic regions. Using anin vitrochromatin reconstitution system under physiological conditions, we show here that parathymosin (ParaT) inhibits the binding of H1 to chromatin in a dose-dependent manner. Consistent with these findings, H1-containing chromatin assembled in the presence of ParaT has reduced nucleosome spacing. These observations suggest that interaction of the two proteins might result in a conformational change of H1. Fluorescence spectroscopy and circular dichroism-based measurements on mixtures of H1 and ParaT confirm this hypothesis. Human sperm nuclei challenged with ParaT become highly decondensed, whereas overexpression of green fluorescent protein- or FLAG-tagged protein in HeLa cells induces global chromatin decondensation and increases the accessibility of chromatin to micrococcal nuclease digestion. Our data suggest a role of parathymosin in the remodeling of higher order chromatin structure through modulation of H1 interaction with nucleosomes and point to its involvement in chromatin-dependent functions.

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