scholarly journals Direct tracking of single proviruses reveals HIV-1/LEDGF complexes excluded from virus-induced membraneless organelles

2020 ◽  
Author(s):  
Viviana Scoca ◽  
Marion Louveaux ◽  
Renaud Morin ◽  
Dmitry Ershov ◽  
Jean-Yves Tinevez ◽  
...  
Keyword(s):  
Nuclear Periphery ◽  
Computational Imaging ◽  
Integration Step ◽  
Imaging Analysis ◽  
Rna Fish ◽  
Viral Progeny ◽  
Is Integration ◽  
Direct Tracking ◽  
Hiv 1 ◽  
Host Chromatin

AbstractThe ultimate goal of HIV-1 is integration into the host chromatin to optimize the release of high levels of viral progeny and discretely coexist with the host. To uncover the HIV-1 DNA fate in the nuclear landscape we directly tracked the viral DNA (vDNA) and the viral RNA (vRNA) by coupling HIV-1 ANCHOR technology with RNA FISH or MCP-MS2 RNA-tagging bacterial system. Our computational imaging analysis revealed that proviral forms are early located in proximity of the nuclear periphery of mitotic and non-mitotic cells. We also observed that HIV-1 infection prompts clustering formation of the host factor CPSF6 restructuring membraneless organelles enriched in both viral proteins and speckle factors. Interestingly, we observed that integrase proteins are retained in CPSF6 clusters, while the late retrotranscribed DNA was excluded from HIV-induced membranelless organelles (HIV-1 MLOs), indicating that those structures are not proviral sites, but orchestrate viral events prior to the integration step. HIV-1 MLOs are in the vicinity of pre-existing LEDGF clusters. Importantly, we identified actively transcribing proviruses localize, outside HIV-1 MLOs, in LEDGF-abundant regions, known to be active chromatin sites. This study highlights single functional host-proviral complexes in their nuclear landscape, which is markedly restructured by HIV-1 to favor viral replication.

scholarly journals Critical Contribution of Tyr15 in the HIV-1 Integrase (IN) in Facilitating IN Assembly and Nonenzymatic Function through the IN Precursor Form with Reverse Transcriptase

2016 ◽  
Vol 91 (1) ◽  
Author(s):  
Tatsuro Takahata ◽  
Eri Takeda ◽  
Minoru Tobiume ◽  
Kenzo Tokunaga ◽  
Masaru Yokoyama ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Hydrophobic Interaction ◽  
Critical Role ◽  
Integration Step ◽  
Strand Transfer ◽  
Aromatic Side Chain ◽  
Lethal Mutations ◽  
Subunit Interface ◽  
Severe Impairment ◽  
Hiv 1

ABSTRACT Nonenzymatic roles for HIV-1 integrase (IN) at steps prior to the enzymatic integration step have been reported. To obtain structural and functional insights into the nonenzymatic roles of IN, we performed genetic analyses of HIV-1 IN, focusing on a highly conserved Tyr15 in the N-terminal domain (NTD), which has previously been shown to regulate an equilibrium state between two NTD dimer conformations. Replacement of Tyr15 with alanine, histidine, or tryptophan prevented HIV-1 infection and caused severe impairment of reverse transcription without apparent defects in reverse transcriptase (RT) or in capsid disassembly kinetics after entry into cells. Cross-link analyses of recombinant IN proteins demonstrated that lethal mutations of Tyr15 severely impaired IN structure for assembly. Notably, replacement of Tyr15 with phenylalanine was tolerated for all IN functions, demonstrating that a benzene ring of the aromatic side chain is a key moiety for IN assembly and functions. Additional mutagenic analyses based on previously proposed tetramer models for IN assembly suggested a key role of Tyr15 in facilitating the hydrophobic interaction among IN subunits, together with other proximal residues within the subunit interface. A rescue experiment with a mutated HIV-1 with RT and IN deleted (ΔRT ΔIN) and IN and RT supplied in trans revealed that the nonenzymatic IN function might be exerted through the IN precursor conjugated with RT (RT-IN). Importantly, the lethal mutations of Tyr15 significantly reduced the RT-IN function and assembly. Taken together, Tyr15 seems to play a key role in facilitating the proper assembly of IN and RT on viral RNA through the RT-IN precursor form. IMPORTANCE Inhibitors of the IN enzymatic strand transfer function (INSTI) have been applied in combination antiretroviral therapies to treat HIV-1-infected patients. Recently, allosteric IN inhibitors (ALLINIs) that interact with HIV-1 IN residues, the locations of which are distinct from the catalytic sites targeted by INSTI, have been discovered. Importantly, ALLINIs affect the nonenzymatic role(s) of HIV-1 IN, providing a rationale for the development of next-generation IN inhibitors with a mechanism that is distinct from that of INSTI. Here, we demonstrate that Tyr15 in the HIV-1 IN NTD plays a critical role during IN assembly by facilitating the hydrophobic interaction of the NTD with the other domains of IN. Importantly, we found that the functional assembly of IN through its fusion form with RT is critical for IN to exert its nonenzymatic function. Our results provide a novel mechanistic insight into the nonenzymatic function of HIV-1 IN and its prevention.

scholarly journals Overexpression of the Lens Epithelium-Derived Growth Factor/p75 Integrase Binding Domain Inhibits Human Immunodeficiency Virus Replication

2006 ◽  
Vol 80 (23) ◽  
pp. 11498-11509 ◽  
Author(s):  
Jan De Rijck ◽  
Linos Vandekerckhove ◽  
Rik Gijsbers ◽  
Anneleen Hombrouck ◽  
Jelle Hendrix ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Growth Factor ◽  
Cell Lines ◽  
Fusion Proteins ◽  
Binding Domain ◽  
Lens Epithelium ◽  
Integration Step ◽  
Hiv Replication ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT We initially identified lens epithelium-derived growth factor/p75 (LEDGF/p75) as a binding partner of human immunodeficiency virus type 1 (HIV-1) integrase. To investigate the role of LEDGF/p75 in HIV replication and its potential as a new antiviral target, we stably overexpressed two different fragments containing the integrase binding domain (IBD) of LEDGF/p75 fused to enhanced green fluorescent protein (eGFP). HIV-1 replication was severely inhibited by overexpression of the eGFP-IBD fusion proteins, while no inhibition was observed in cell lines overexpressing the interaction-deficient D366A mutant. Quantitative PCR pinpointed the block to the integration step, whereas nuclear import was not affected. Competition of the IBD fusion proteins with endogenous LEDGF/p75 for binding to integrase led to a potent defect in HIV-1 replication in both HeLaP4- and MT-4-derived cell lines. A previously described diketo acid-resistant HIV-1 strain remained fully susceptible to inhibition, suggesting that this strategy will also work in patients who harbor strains resistant to the current experimental integrase inhibitors. These data support LEDGF/p75 as an important cofactor for HIV replication and provide proof of concept for the LEDGF/p75-integrase interaction as a novel target for treating HIV-1 infection.

Defective HIV-1 Provirus Encoding a Multitarget-Ribozyme Inhibits Accumulation of Spliced and Unspliced HIV-1 mRNAs, Reduces Infectivity of Viral Progeny, and Protects the Cells from Pathogenesis

1997 ◽  
Vol 8 (9) ◽  
pp. 1115-1124 ◽  
Author(s):  
Soon-Young Paik ◽  
Akhil Banerjea ◽  
Chang-Jie Chen ◽  
Zhiping Ye ◽  
George G. Harmison ◽  
...  
Keyword(s):  
Viral Progeny ◽  
Hiv 1

scholarly journals Multispectral immunofluorescence and computational imaging analysis define the prognostic role of T-cell infiltrates in gastric cancer

2019 ◽  
Vol 30 ◽  
pp. xi2
Author(s):  
B.R. Challoner ◽  
K. von Loga ◽  
A. Woolston ◽  
B. Griffiths ◽  
L. Hewitt ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
T Cell ◽  
Computational Imaging ◽  
Imaging Analysis ◽  
Prognostic Role

scholarly journals HIV-1 Pre-Integration Complexes Selectively Target Decondensed Chromatin in the Nuclear Periphery

PLoS ONE ◽  
2008 ◽  
Vol 3 (6) ◽  
pp. e2413 ◽  
Author(s):  
Alberto Albanese ◽  
Daniele Arosio ◽  
Mariaelena Terreni ◽  
Anna Cereseto
Keyword(s):  
Nuclear Periphery ◽  
Decondensed Chromatin ◽  
Hiv 1

scholarly journals CPSF6-Dependent Targeting of Speckle-Associated Domains Distinguishes Primate from Nonprimate Lentiviral Integration

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Wen Li ◽  
Parmit K. Singh ◽  
Gregory A. Sowd ◽  
Gregory J. Bedwell ◽  
Sooin Jang ◽  
...  
Keyword(s):  
Target Genes ◽  
Binding Protein ◽  
Nuclear Periphery ◽  
Dependent Manner ◽  
Nuclear Speckles ◽  
Retroviral Integration ◽  
Integration Sites ◽  
Primate Lentiviruses ◽  
Lentiviral Integration ◽  
Hiv 1

ABSTRACT Lentiviral DNA integration favors transcriptionally active chromatin. We previously showed that the interaction of human immunodeficiency virus type 1 (HIV-1) capsid with cleavage and polyadenylation specificity factor 6 (CPSF6) localizes viral preintegration complexes (PICs) to nuclear speckles for integration into transcriptionally active speckle-associated domains (SPADs). In the absence of the capsid-CPSF6 interaction, PICs uncharacteristically accumulate at the nuclear periphery and target heterochromatic lamina-associated domains (LADs) for integration. The integrase-binding protein lens epithelium-derived growth factor (LEDGF)/p75 in contrast to CPSF6 predominantly functions to direct HIV-1 integration to interior regions of transcription units. Though CPSF6 and LEDGF/p75 can reportedly interact with the capsid and integrase proteins of both primate and nonprimate lentiviruses, the extents to which these different viruses target SPADs versus LADs, as well as their dependencies on CPSF6 and LEDGF/p75 for integration targeting, are largely unknown. Here, we mapped 5,489,157 primate and nonprimate lentiviral integration sites in HEK293T and Jurkat T cells as well as derivative cells that were knocked out or knocked down for host factor expression. Despite marked preferences of all lentiviruses to target genes for integration, nonprimate lentiviruses only marginally favored SPADs, with corresponding upticks in LAD-proximal integration. While LEDGF/p75 knockout disrupted the intragenic integration profiles of all lentiviruses similarly, CPSF6 depletion specifically counteracted SPAD integration targeting by primate lentiviruses. CPSF6 correspondingly failed to appreciably interact with nonprimate lentiviral capsids. We conclude that primate lentiviral capsid proteins evolved to interact with CPSF6 to optimize PIC localization for integration into transcriptionally active SPADs. IMPORTANCE Integration is the defining step of the retroviral life cycle and underlies the inability to cure HIV/AIDS through the use of intensified antiviral therapy. The reservoir of latent, replication-competent proviruses that forms early during HIV infection reseeds viremia when patients discontinue medication. HIV cure research is accordingly focused on the factors that guide provirus formation and associated chromatin environments that regulate transcriptional reactivation, and studies of orthologous infectious agents such as nonprimate lentiviruses can inform basic principles of HIV biology. HIV-1 utilizes the integrase-binding protein LEDGF/p75 and the capsid interactor CPSF6 to target speckle-associated domains (SPADs) for integration. However, the extent to which these two host proteins regulate integration of other lentiviruses is largely unknown. Here, we mapped millions of retroviral integration sites in cell lines that were depleted for LEDGF/p75 and/or CPSF6. Our results reveal that primate lentiviruses uniquely target SPADs for integration in a CPSF6-dependent manner.

Computational imaging analysis of glycated fibrin gels reveals aggregated and anisotropic structures

2017 ◽  
Vol 105 (8) ◽  
pp. 2191-2198 ◽  
Author(s):  
David G. Norton ◽  
Natalie K. Fan ◽  
Marcus J. Goudie ◽  
Hitesh Handa ◽  
Manu O. Platt ◽  
...  
Keyword(s):  
Computational Imaging ◽  
Imaging Analysis ◽  
Fibrin Gels ◽  
Anisotropic Structures

scholarly journals Transient and Stable Knockdown of the Integrase Cofactor LEDGF/p75 Reveals Its Role in the Replication Cycle of Human Immunodeficiency Virus

2006 ◽  
Vol 80 (4) ◽  
pp. 1886-1896 ◽  
Author(s):  
Linos Vandekerckhove ◽  
Frauke Christ ◽  
Bénédicte Van Maele ◽  
Jan De Rijck ◽  
Rik Gijsbers ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Cell Lines ◽  
Lentiviral Vector ◽  
Antiviral Drug ◽  
Integration Step ◽  
Hiv Replication ◽  
Immunodeficiency Virus ◽  
Cellular Cofactor ◽  
Interfering Rna ◽  
Hiv 1

ABSTRACT After identifying the interaction between the transcriptional coactivator lens epithelium-derived growth factor (LEDGF/p75) and the human immunodeficiency virus type 1 (HIV-1) integrase (IN), we have now investigated the role of LEDGF/p75 during HIV replication. Transient small interfering RNA-mediated knockdown of LEDGF/p75 in HeLaP4 cells resulted in a three- to fivefold inhibition of HIV-1 (strain NL4.3) replication. Quantitative PCR was used to pinpoint the replication block to the integration step. Next, polyclonal and monoclonal HeLaP4-derived cell lines were selected with a stable knockdown of LEDGF/p75 mediated by a lentiviral vector (lentivector) encoding a short hairpin RNA (shRNA) targeting this protein. Cell lines stably transduced with a lentivector encoding an unrelated hairpin or a double-mismatch hairpin served as controls. Again, a two- to fourfold reduction of HIV-1 replication was observed. The extent of LEDGF/p75 knockdown closely correlated with the reduction of HIV-1 replication. After the back-complementation of LEDGF/p75 in the poly- and monoclonal knockdown cell lines using an shRNA-resistant expression plasmid, viral replication was restored to nearly wild-type levels. The Q168A mutation in integrase has been shown to interfere with the interaction with LEDGF/p75 without reducing the enzymatic activity. Transduction by HIV-1-derived lentivectors carrying the Q168A IN mutant was severely hampered, pointing again to a requirement for LEDGF/p75. Altogether, our data validate LEDGF/p75 as an important cellular cofactor for HIV integration and as a potential target for antiviral drug development.

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