Modulation of the intrinsic chromatin binding property of HIV-1 integrase by LEDGF/p75.

The stable insertion of the retroviral genome into the host chromosomes requires the association between integration complexes and cellular chromatin via the interaction between retroviral integrase and the nucleosomal target DNA. This final association may involve the chromatin-binding properties of both the retroviral integrase and its cellular cofactor LEDGF/p75. To investigate this and better understand the LEDGF/p75-mediated chromatin tethering of HIV-1 integrase, we used a combination of biochemical and chromosome-binding assays. Our study revealed that retroviral integrase has an intrinsic ability to bind and recognize specific chromatin regions in metaphase even in the absence of its cofactor. Furthermore, this integrase chromatin-binding property was modulated by the interaction with its cofactor LEDGF/p75, which redirected the enzyme to alternative chromosome regions. We also better determined the chromatin features recognized by each partner alone or within the functional intasome, as well as the chronology of efficient LEDGF/p75-mediated targeting of HIV-1 integrase to chromatin. Our data support a new chromatin-binding function of integrase acting in concert with LEDGF/p75 for the optimal association with the nucleosomal substrate. This work also provides additional information about the behavior of retroviral integration complexes in metaphase chromatin and the mechanism of action of LEDGF/p75 in this specific context.

Structural basis for retroviral integration into nucleosomes.

Retroviral integration is catalysed by a tetramer of integrase (IN) assembled on viral DNA ends in a stable complex, known as the intasome. How the intasome interfaces with chromosomal DNA, which exists in the form of nucleosomal arrays, is currently unknown. Here we show that the prototype foamy virus (PFV) intasome is proficient at stable capture of nucleosomes as targets for integration. Single-particle cryo-electron microscopy reveals a multivalent intasome-nucleosome interface involving both gyres of nucleosomal DNA and one H2A-H2B heterodimer. While the histone octamer remains intact, the DNA is lifted from the surface of the H2A-H2B heterodimer to allow integration at strongly preferred superhelix location ±3.5 positions. Amino acid substitutions disrupting these contacts impinge on the ability of the intasome to engage nucleosomes in vitro and redistribute viral integration sites on the genomic scale. Our findings elucidate the molecular basis for nucleosome capture by the viral DNA recombination machinery and the underlying nucleosome plasticity that allows integration.

Human H4 tail stimulates HIV-1 integration through binding to the carboxy-terminal domain of integrase.

The integration of the retroviral genome into the chromatin of the infected cell is catalysed by the integrase (IN)•viral DNA complex (intasome). This process requires functional association between the integration complex and the nucleosomes. Direct intasome/histone contacts have been reported to modulate the interaction between the integration complex and the target DNA (tDNA). Both prototype foamy virus (PFV) and HIV-1 integrases can directly bind histone amino-terminal tails. We have further investigated this final association by studying the effect of isolated histone tails on HIV-1 integration. We show here that the binding of HIV-1 IN to a peptide derived from the H4 tail strongly stimulates integration catalysis in vitro. This stimulation was not observed with peptide tails from other variants or with alpha-retroviral (RAV) and spuma-retroviral PFV integrases. Biochemical analyses show that the peptide tail induces both an increase in the IN oligomerization state and affinity for the target DNA, which are associated with substantial structural rearrangements in the IN carboxy-terminal domain (CTD) observed by NMR. Our data indicate that the H4 peptide tail promotes the formation of active strand transfer complexes (STCs) and support an activation step of the incoming intasome at the contact of the histone tail.

Two-long terminal repeat (LTR) DNA circles are a substrate for HIV-1 integrase.

Integration of the HIV-1 DNA into the host genome is essential for viral replication and is catalyzed by the retroviral integrase. To date, the only substrate described to be involved in this critical reaction is the linear viral DNA produced in reverse transcription. However, during HIV-1 infection, two-long terminal repeat DNA circles (2-LTRcs) are also generated through the ligation of the viral DNA ends by the host cell's nonhomologous DNA end-joining pathway. These DNAs contain all the genetic information required for viral replication, but their role in HIV-1's life cycle remains unknown. We previously showed that both linear and circular DNA fragments containing the 2-LTR palindrome junction can be efficiently cleaved in vitro by recombinant integrases, leading to the formation of linear 3'-processed-like DNA. In this report, using in vitro experiments with purified proteins and DNAs along with DNA endonuclease and in vivo integration assays, we show that this circularized genome can also be efficiently used as a substrate in HIV-1 integrase-mediated integration both in vitro and in eukaryotic cells. Notably, we demonstrate that the palindrome cleavage occurs via a two-step mechanism leading to a blunt-ended DNA product, followed by a classical 3'-processing reaction; this cleavage leads to integrase-dependent integration, highlighted by a 5-bp duplication of the host genome. Our results suggest that 2-LTRc may constitute a reserve supply of HIV-1 genomes for proviral integration.

Structural basis for spumavirus GAG tethering to chromatin.

The interactions between a retrovirus and host cell chromatin that underlie integration and provirus expression are poorly understood. The prototype foamy virus (PFV) structural protein GAG associates with chromosomes via a chromatin-binding sequence (CBS) located within its C-terminal region. Here, we show that the PFV CBS is essential and sufficient for a direct interaction with nucleosomes and present a crystal structure of the CBS bound to a mononucleosome. The CBS interacts with the histone octamer, engaging the H2A-H2B acidic patch in a manner similar to other acidic patch-binding proteins such as herpesvirus latency-associated nuclear antigen (LANA). Substitutions of the invariant arginine anchor residue in GAG result in global redistribution of PFV and macaque simian foamy virus (SFVmac) integration sites toward centromeres, dampening the resulting proviral expression without affecting the overall efficiency of integration. Our findings underscore the importance of retroviral structural proteins for integration site selection and the avoidance of genomic junkyards.

Interactions of Prototype Foamy Virus Capsids with Host Cell Polo-Like Kinases Are Important for Efficient Viral DNA Integration.

Unlike for other retroviruses, only a few host cell factors that aid the replication of foamy viruses (FVs) via interaction with viral structural components are known. Using a yeast-two-hybrid (Y2H) screen with prototype FV (PFV) Gag protein as bait we identified human polo-like kinase 2 (hPLK2), a member of cell cycle regulatory kinases, as a new interactor of PFV capsids. Further Y2H studies confirmed interaction of PFV Gag with several PLKs of both human and rat origin. A consensus Ser-Thr/Ser-Pro (S-T/S-P) motif in Gag, which is conserved among primate FVs and phosphorylated in PFV virions, was essential for recognition by PLKs. In the case of rat PLK2, functional kinase and polo-box domains were required for interaction with PFV Gag. Fluorescently-tagged PFV Gag, through its chromatin tethering function, selectively relocalized ectopically expressed eGFP-tagged PLK proteins to mitotic chromosomes in a Gag STP motif-dependent manner, confirming a specific and dominant nature of the Gag-PLK interaction in mammalian cells. The functional relevance of the Gag-PLK interaction was examined in the context of replication-competent FVs and single-round PFV vectors. Although STP motif mutated viruses displayed wild type (wt) particle release, RNA packaging and intra-particle reverse transcription, their replication capacity was decreased 3-fold in single-cycle infections, and up to 20-fold in spreading infections over an extended time period. Strikingly similar defects were observed when cells infected with single-round wt Gag PFV vectors were treated with a pan PLK inhibitor. Analysis of entry kinetics of the mutant viruses indicated a post-fusion defect resulting in delayed and reduced integration, which was accompanied with an enhanced preference to integrate into heterochromatin. We conclude that interaction between PFV Gag and cellular PLK proteins is important for early replication steps of PFV within host cells.

Correction: Interactions of Prototype Foamy Virus Capsids with Host Cell Polo-Like Kinases Are Important for Efficient Viral DNA Integration.

[This corrects the article DOI: 10.1371/journal.ppat.1005860.].

Retroviral DNA Integration.

The integration of a DNA copy of the viral RNA genome into host chromatin is the defining step of retroviral replication. This enzymatic process is catalyzed by the virus-encoded integrase protein, which is conserved among retroviruses and LTR-retrotransposons. Retroviral integration proceeds via two integrase activities: 3'-processing of the viral DNA ends, followed by the strand transfer of the processed ends into host cell chromosomal DNA. Herein we review the molecular mechanism of retroviral DNA integration, with an emphasis on reaction chemistries and architectures of the nucleoprotein complexes involved. We additionally discuss the latest advances on anti-integrase drug development for the treatment of AIDS and the utility of integrating retroviral vectors in gene therapy applications.

Modulation of chromatin structure by the FACT histone chaperone complex regulates HIV-1 integration.

BACKGROUND: Insertion of retroviral genome DNA occurs in the chromatin of the host cell. This step is modulated by chromatin structure as nucleosomes compaction was shown to prevent HIV-1 integration and chromatin remodeling has been reported to affect integration efficiency. LEDGF/p75-mediated targeting of the integration complex toward RNA polymerase II (polII) transcribed regions ensures optimal access to dynamic regions that are suitable for integration. Consequently, we have investigated the involvement of polII-associated factors in the regulation of HIV-1 integration. RESULTS: Using a pull down approach coupled with mass spectrometry, we have selected the FACT (FAcilitates Chromatin Transcription) complex as a new potential cofactor of HIV-1 integration. FACT is a histone chaperone complex associated with the polII transcription machinery and recently shown to bind LEDGF/p75. We report here that a tripartite complex can be formed between HIV-1 integrase, LEDGF/p75 and FACT in vitro and in cells. Biochemical analyzes show that FACT-dependent nucleosome disassembly promotes HIV-1 integration into chromatinized templates, and generates highly favored nucleosomal structures in vitro. This effect was found to be amplified by LEDGF/p75. Promotion of this FACT-mediated chromatin remodeling in cells both increases chromatin accessibility and stimulates HIV-1 infectivity and integration. CONCLUSIONS: Altogether, our data indicate that FACT regulates HIV-1 integration by inducing local nucleosomes dissociation that modulates the functional association between the incoming intasome and the targeted nucleosome.

Modulation of the functional association between the HIV-1 intasome and the nucleosome by histone amino-terminal tails.

BACKGROUND: Stable insertion of the retroviral DNA genome into host chromatin requires the functional association between the intasome (integrase·viral DNA complex) and the nucleosome. The data from the literature suggest that direct protein-protein contacts between integrase and histones may be involved in anchoring the intasome to the nucleosome. Since histone tails are candidates for interactions with the incoming intasomes we have investigated whether they could participate in modulating the nucleosomal integration process. RESULTS: We show here that histone tails are required for an optimal association between HIV-1 integrase (IN) and the nucleosome for efficient integration. We also demonstrate direct interactions between IN and the amino-terminal tail of human histone H4 in vitro. Structure/function studies enabled us to identify amino acids in the carboxy-terminal domain of IN that are important for this interaction. Analysis of the nucleosome-binding properties of catalytically active mutated INs confirmed that their ability to engage the nucleosome for integration in vitro was affected. Pseudovirus particles bearing mutations that affect the IN/H4 association also showed impaired replication capacity due to altered integration and re-targeting of their insertion sites toward dynamic regions of the chromatin with lower nucleosome occupancy. CONCLUSIONS: Collectively, our data support a functional association between HIV-1 IN and histone tails that promotes anchoring of the intasome to nucleosomes and optimal integration into chromatin.

Integrase inhibitor reversal dynamics indicate unintegrated HIV-1 dna initiate de novo integration.

BACKGROUND: Genomic integration, an obligate step in the HIV-1 replication cycle, is blocked by the integrase inhibitor raltegravir. A consequence is an excess of unintegrated viral DNA genomes, which undergo intramolecular ligation and accumulate as 2-LTR circles. These circularized genomes are also reliably observed in vivo in the absence of antiviral therapy and they persist in non-dividing cells. However, they have long been considered as dead-end products that are not precursors to integration and further viral propagation. RESULTS: Here, we show that raltegravir action is reversible and that unintegrated viral DNA is integrated in the host cell genome after raltegravir removal leading to HIV-1 replication. Using quantitative PCR approach, we analyzed the consequences of reversing prolonged raltegravir-induced integration blocks. We observed, after RAL removal, a decrease of 2-LTR circles and a transient increase of linear DNA that is subsequently integrated in the host cell genome and fuel new cycles of viral replication. CONCLUSIONS: Our data highly suggest that 2-LTR circles can be used as a reserve supply of genomes for proviral integration highlighting their potential role in the overall HIV-1 replication cycle.

Intasome architecture and chromatin density modulate retroviral integration into nucleosome.

BACKGROUND: Retroviral integration depends on the interaction between intasomes, host chromatin and cellular targeting cofactors as LEDGF/p75 or BET proteins. Previous studies indicated that the retroviral integrase, by itself, may play a role in the local integration site selection within nucleosomal target DNA. We focused our study on this local association by analyzing the intrinsic properties of various retroviral intasomes to functionally accommodate different chromatin structures in the lack of other cofactors. RESULTS: Using in vitro conditions allowing the efficient catalysis of full site integration without these cofactors, we show that distinct retroviral integrases are not equally affected by chromatin compactness. Indeed, while PFV and MLV integration reactions are favored into dense and stable nucleosomes, HIV-1 and ASV concerted integration reactions are preferred into poorly dense chromatin regions of our nucleosomal acceptor templates. Predicted nucleosome occupancy around integration sites identified in infected cells suggests the presence of a nucleosome at the MLV and HIV-1 integration sites surrounded by differently dense chromatin. Further analyses of the relationships between the in vitro integration site selectivity and the structure of the inserted DNA indicate that structural constraints within intasomes could account for their ability to accommodate nucleosomal DNA and could dictate their capability to bind nucleosomes functionally in these specific chromatin contexts. CONCLUSIONS: Thus, both intasome architecture and compactness of the chromatin surrounding the targeted nucleosome appear important determinants of the retroviral integration site selectivity. This supports a mechanism involving a global targeting of the intasomes toward suitable chromatin regions followed by a local integration site selection modulated by the intrinsic structural constraints of the intasomes governing the target DNA bending and dictating their sensitivity toward suitable specific nucleosomal structures and density.

Functional coupling between HIV-1 integrase and the SWI/SNF chromatin remodeling complex for efficient in vitro integration into stable nucleosomes.

Establishment of stable HIV-1 infection requires the efficient integration of the retroviral genome into the host DNA. The molecular mechanism underlying the control of this process by the chromatin structure has not yet been elucidated. We show here that stably associated nucleosomes strongly inhibit in vitro two viral-end integration by decreasing the accessibility of DNA to integrase. Remodeling of the chromatinized template by the SWI/SNF complex, whose INI1 major component interacts with IN, restores and redirects the full-site integration into the stable nucleosome region. These effects are not observed after remodeling by other human remodeling factors such as SNF2H or BRG1 lacking the integrase binding protein INI1. This suggests that the restoration process depends on the direct interaction between IN and the whole SWI/SNF complex, supporting a functional coupling between the remodeling and integration complexes. Furthermore, in silico comparison between more than 40,000 non-redundant cellular integration sites selected from literature and nucleosome occupancy predictions also supports that HIV-1 integration is promoted in the genomic region of weaker intrinsic nucleosome density in the infected cell. Our data indicate that some chromatin structures can be refractory for integration and that coupling between nucleosome remodeling and HIV-1 integration is required to overcome this natural barrier.

Targeting the Nucleosome Acidic Patch by Viral Proteins: Two Birds with One Stone?

The past decade illuminated the H2A-H2B acidic patch as a cornerstone for both nucleosome recognition and chromatin structure regulation. Higher-order folding of chromatin arrays is mediated by interactions of histone H4 tail with an adjacent nucleosome acidic patch. Dynamic chromatin folding ensures a proper regulation of nuclear functions fundamental to cellular homeostasis. Many cellular factors have been shown to act on chromatin by tethering nucleosomes via an arginine anchor binding to the acidic patch. This tethering mechanism has also been described for several viral proteins. In this minireview, we will discuss the structural basis for acidic patch engagement by viral proteins and the implications during respective viral infections. We will also discuss a model in which acidic patch occupancy by these non-self viral proteins alters the local chromatin state by preventing H4 tail-mediated higher-order chromatin folding.

In Silico, In Vitro and In Cellulo Models for Monitoring SARS-CoV-2 Spike/Human ACE2 Complex, Viral Entry and Cell Fusion.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent responsible for the recent coronavirus disease 2019 (COVID-19) pandemic. Productive SARS-CoV-2 infection relies on viral entry into cells expressing angiotensin-converting enzyme 2 (ACE2). Indeed, viral entry into cells is mostly mediated by the early interaction between the viral spike protein S and its ACE2 receptor. The S/ACE2 complex is, thus, the first contact point between the incoming virus and its cellular target; consequently, it has been considered an attractive therapeutic target. To further characterize this interaction and the cellular processes engaged in the entry step of the virus, we set up various in silico, in vitro and in cellulo approaches that allowed us to specifically monitor the S/ACE2 association. We report here a computational model of the SARS-CoV-2 S/ACE2 complex, as well as its biochemical and biophysical monitoring using pulldown, AlphaLISA and biolayer interferometry (BLI) binding assays. This led us to determine the kinetic parameters of the S/ACE2 association and dissociation steps. In parallel to these in vitro approaches, we developed in cellulo transduction assays using SARS-CoV-2 pseudotyped lentiviral vectors and HEK293T-ACE2 cell lines generated in-house. This allowed us to recapitulate the early replication stage of the infection mediated by the S/ACE2 interaction and to detect cell fusion induced by the interaction. Finally, a cell imaging system was set up to directly monitor the S/ACE2 interaction in a cellular context and a flow cytometry assay was developed to quantify this association at the cell surface. Together, these different approaches are available for both basic and clinical research, aiming to characterize the entry step of the original SARS-CoV-2 strain and its variants as well as to investigate the possible chemical modulation of this interaction. All these models will help in identifying new antiviral agents and new chemical tools for dissecting the virus entry step.

Structural Insights on Retroviral DNA Integration: Learning from Foamy Viruses.

Foamy viruses (FV) are retroviruses belonging to the Spumaretrovirinae subfamily. They are non-pathogenic viruses endemic in several mammalian hosts like non-human primates, felines, bovines, and equines. Retroviral DNA integration is a mandatory step and constitutes a prime target for antiretroviral therapy. This activity, conserved among retroviruses and long terminal repeat (LTR) retrotransposons, involves a viral nucleoprotein complex called intasome. In the last decade, a plethora of structural insights on retroviral DNA integration arose from the study of FV. Here, we review the biochemistry and the structural features of the FV integration apparatus and will also discuss the mechanism of action of strand transfer inhibitors.

Retroviral integration site selection: a running Gag?

The ability of retroviruses to integrate their genomes into host chromatin is a key step for the completion of their replication cycle. Selection of a suitable chromosomal integration site has been described as a hierarchical mechanism involving both cellular and viral proteins but the exact molecular determinants are still unclear. We recently showed that the spumaretrovirus prototype foamy virus (PFV) Gag protein is acting as a chromatin tether by interacting with the nucleosome acidic patch (Lesbats et al. PNAS 114(21)). Disruption of the nucleosome binding leads to a dramatic delocalization of both the viral particles and the integration sites accompanied with a reduction of integrated genes expression. These data show for the first time a direct interaction between retroviral structural proteins with the host chromosomes, and highlight their importance in the integration sites selection.

Natural stilbenoids isolated from grapevine exhibiting inhibitory effects against HIV-1 integrase and eukaryote MOS1 transposase in vitro activities.

Polynucleotidyl transferases are enzymes involved in several DNA mobility mechanisms in prokaryotes and eukaryotes. Some of them such as retroviral integrases are crucial for pathogenous processes and are therefore good candidates for therapeutic approaches. To identify new therapeutic compounds and new tools for investigating the common functional features of these proteins, we addressed the inhibition properties of natural stilbenoids deriving from resveratrol on two models: the HIV-1 integrase and the eukaryote MOS-1 transposase. Two resveratrol dimers, leachianol F and G, were isolated for the first time in Vitis along with fourteen known stilbenoids: E-resveratrol, E-piceid, E-pterostilbene, E-piceatannol, (+)-E-ε-viniferin, E-ε-viniferinglucoside, E-scirpusin A, quadragularin A, ampelopsin A, pallidol, E-miyabenol C, E-vitisin B, hopeaphenol, and isohopeaphenol and were purified from stalks of Vitis vinifera (Vitaceae), and moracin M from stem bark of Milliciaexelsa (Moraceae). These compounds were tested in in vitro and in vivo assays reproducing the activity of both enzymes. Several molecules presented significant inhibition on both systems. Some of the molecules were found to be active against both proteins while others were specific for one of the two models. Comparison of the differential effects of the molecules suggested that the compounds could target specific intermediate nucleocomplexes of the reactions. Additionally E-pterostilbene was found active on the early lentiviral replication steps in lentiviruses transduced cells. Consequently, in addition to representing new original lead compounds for further modelling of new active agents against HIV-1 integrase, these molecules could be good tools for identifying such reaction intermediates in DNA mobility processes.

Structural and functional role of INI1 and LEDGF in the HIV-1 preintegration complex.

Integration of the HIV-1 cDNA into the human genome is catalyzed by the viral integrase (IN) protein. Several studies have shown the importance of cellular cofactors that interact with integrase and affect viral integration and infectivity. In this study, we produced a stable complex between HIV-1 integrase, viral U5 DNA, the cellular cofactor LEDGF/p75 and the integrase binding domain of INI1 (INI1-IBD), a subunit of the SWI/SNF chromatin remodeling factor. The stoichiometry of the IN/LEDGF/INI1-IBD/DNA complex components was found to be 4/2/2/2 by mass spectrometry and Fluorescence Correlation Spectroscopy. Functional assays showed that INI1-IBD inhibits the 3' processing reaction but does not interfere with specific viral DNA binding. Integration assays demonstrate that INI1-IBD decreases the amount of integration events but inhibits by-product formation such as donor/donor or linear full site integration molecules. Cryo-electron microscopy locates INI1-IBD within the cellular DNA binding site of the IN/LEDGF complex, constraining the highly flexible integrase in a stable conformation. Taken together, our results suggest that INI1 could stabilize the PIC in the host cell, by maintaining integrase in a stable constrained conformation which prevents non-specific interactions and auto integration on the route to its integration site within nucleosomes, while LEDGF organizes and stabilizes an active integrase tetramer suitable for specific vDNA integration. Moreover, our results provide the basis for a novel type of integrase inhibitor (conformational inhibitor) representing a potential new strategy for use in human therapy.