Integrase-LEDGF/p75 complex triggers the formation of biomolecular condensates that modulate HIV-1 integration efficiency in vitro.

The pre-integration steps of the HIV-1 viral cycle are some of the most valuable targets of recent therapeutic innovations. HIV-1 integrase (IN) displays multiple functions, thanks to its considerable conformational flexibility. Recently, such flexible proteins have been characterized by their ability to form biomolecular condensates as a result of Liquid-Liquid-Phase-Separation (LLPS), allowing them to evolve in a restricted microenvironment within cells called membrane-less organelles (MLO). The LLPS context constitutes a more physiological approach to study the integration of molecular mechanisms performed by intasomes (complexes containing viral DNA, IN, and its cellular cofactor LEDGF/p75). We investigated here if such complexes can form LLPS in vitro and if IN enzymatic activities were affected by this LLPS environment. We observed that the LLPS formed by IN-LEDGF/p75 functional complexes modulate the in vitro IN activities. While the 3'-processing of viral DNA ends was drastically reduced inside LLPS, viral DNA strand transfer was strongly enhanced. These two catalytic IN activities appear thus tightly regulated by the environment encountered by intasomes.

Mutations in the 3'-PPT Lead to HIV-1 Replication without Integration.

Integration of the reverse-transcribed genome is a critical step of the retroviral life cycle. Strand-transfer inhibitors (INSTIs) used for antiretroviral therapy inhibit integration but can lead to resistance mutations in the integrase gene, the enzyme involved in this reaction. A significant proportion of INSTI treatment failures, particularly those with second-generation INSTIs, show no mutation in the integrase gene. Here, we show that replication of a selected dolutegravir-resistant virus with mutations in the 3'-PPT (polypurine tract) was effective, although no integrated viral DNA was detected, due to the accumulation of unintegrated viral DNA present as 1-LTR circles. Our results show that mutation in the 3'-PPT leads to 1-LTR circles and not linear DNA as classically reported. In conclusion, our data provide a molecular basis to explain a new mechanism of resistance to INSTIs, without mutation of the integrase gene and highlights the importance of unintegrated viral DNA in HIV-1 replication. IMPORTANCE Our work highlights the role of HIV-1 unintegrated viral DNA in viral replication. A virus, resistant to strand-transfer inhibitors, has been selected in vitro. This virus highlights a mutation in the 3'PPT region and not in the integrase gene. This mutation modifies the reverse transcription step leading to the accumulation of 1-LTR circles and not the linear DNA. This accumulation of 1-LTR circles leads to viral replication without integration of the viral genome.

Cassette recruitment in the chromosomal Integron of Vibrio cholerae.

Integrons confer a rapid adaptation capability to bacteria. Integron integrases are able to capture and shuffle novel functions embedded in cassettes. Here, we investigated cassette recruitment in the Vibrio cholerae chromosomal integron during horizontal transfer. We demonstrated that the endogenous integrase expression is sufficiently triggered, after SOS response induction mediated by the entry of cassettes during conjugation and natural transformation, to mediate significant cassette insertions. These insertions preferentially occur at the attIA site, despite the presence of about 180 attC sites in the integron array. Thanks to the presence of a promoter in the attIA site vicinity, all these newly inserted cassettes are expressed and prone to selection. We also showed that the RecA protein is critical for cassette recruitment in the V. cholerae chromosomal integron but not in mobile integrons. Moreover, unlike the mobile integron integrases, that of V. cholerae is not active in other bacteria. Mobile integrons might have evolved from the chromosomal ones by overcoming host factors, explaining their large dissemination in bacteria and their role in antibioresistance expansion.

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.

Selection of Bis-Indolyl Pyridines and Triphenylamines as New Inhibitors of SARS-CoV-2 Cellular Entry by Modulating the Spike Protein/ACE2 Interfaces.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the infectious agent that has caused the current coronavirus disease (COVID) pandemic. Viral infection relies on the viral S (spike) protein/cellular receptor ACE2 interaction. Disrupting this interaction would lead to early blockage of viral replication. To identify chemical tools to further study these functional interfaces, 139,146 compounds from different chemical libraries were screened through an S/ACE2 in silico virtual molecular model. The best compounds were selected for further characterization using both cellular and biochemical approaches, reiterating SARS-CoV-2 entry and the S/ACE2 interaction. We report here two selected hits, bis-indolyl pyridine AB-00011778 and triphenylamine AB-00047476. Both of these compounds can block the infectivity of lentiviral vectors pseudotyped with the SARS-CoV-2 S protein as well as wild-type and circulating variant SARS-CoV-2 strains in various human cell lines, including pulmonary cells naturally susceptible to infection. AlphaLISA and biolayer interferometry confirmed a direct inhibitory effect of these drugs on the S/ACE2 association. A specific study of the AB-00011778 inhibitory properties showed that this drug inhibits viral replication with a 50% effective concentration (EC50) between 0.1 and 0.5 µM depending on the cell lines. Molecular docking calculations of the interaction parameters of the molecules within the S/ACE2 complex from both wild-type and circulating variants of the virus showed that the molecules may target multiple sites within the S/ACE2 interface. Our work indicates that AB-00011778 constitutes a good tool for modulating this interface and a strong lead compound for further therapeutic purposes.

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.

Carboxylate-functionalized foldamer inhibitors of HIV-1 integrase and Topoisomerase 1: artificial analogues of DNA mimic proteins.

Inspired by DNA mimic proteins, we have introduced aromatic foldamers bearing phosphonate groups as synthetic mimics of the charge surface of B-DNA and competitive inhibitors of some therapeutically relevant DNA-binding enzymes: the human DNA Topoisomerase 1 (Top1) and the human HIV-1 integrase (HIV-1 IN). We now report on variants of these anionic foldamers bearing carboxylates instead of phosphonates. Several new monomers have been synthesized with protecting groups suitable for solid phase synthesis (SPS). Six hexadecaamides have been prepared using SPS. Proof of their resemblance to B-DNA was brought by the first crystal structure of one of these DNA-mimic foldamers in its polyanionic form. While some of the foldamers were found to be as active as, or even more active than, the original phosphonate oligomers, others had no activity at all or could even stimulate enzyme activity in vitro. Some foldamers were found to have differential inhibitory effects on the two enzymes. These results demonstrate a strong dependence of inhibitory activity on foldamer structure and charge distribution. They open broad avenues for the development of new classes of derivatives that could inhibit the interaction of specific proteins with their DNA target thereby influencing the cellular pathways in which they are involved.

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.

DNA minicircles clarify the specific role of DNA structure on retroviral integration.

Chromatin regulates the selectivity of retroviral integration into the genome of infected cells. At the nucleosome level, both histones and DNA structure are involved in this regulation. We propose a strategy that allows to specifically study a single factor: the DNA distortion induced by the nucleosome. This strategy relies on mimicking this distortion using DNA minicircles (MCs) having a fixed rotational orientation of DNA curvature, coupled with atomic-resolution modeling. Contrasting MCs with linear DNA fragments having identical sequences enabled us to analyze the impact of DNA distortion on the efficiency and selectivity of integration. We observed a global enhancement of HIV-1 integration in MCs and an enrichment of integration sites in the outward-facing DNA major grooves. Both of these changes are favored by LEDGF/p75, revealing a new, histone-independent role of this integration cofactor. PFV integration is also enhanced in MCs, but is not associated with a periodic redistribution of integration sites, thus highlighting its distinct catalytic properties. MCs help to separate the roles of target DNA structure, histone modifications and integrase (IN) cofactors during retroviral integration and to reveal IN-specific regulation mechanisms.

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.

The integron integrase efficiently prevents the melting effect of Escherichia coli single-stranded DNA-binding protein on folded attC sites.

Integrons play a major role in the dissemination of antibiotic resistance genes among bacteria. Rearrangement of gene cassettes occurs by recombination between attI and attC sites, catalyzed by the integron integrase. Integron recombination uses an unconventional mechanism involving a folded single-stranded attC site. This site could be a target for several host factors and more precisely for proteins able to bind single-stranded DNA. One of these, Escherichia coli single-stranded DNA-binding protein (SSB), regulates many DNA processes. We studied the influence of this protein on integron recombination. Our results show the ability of SSB to strongly bind folded attC sites and to destabilize them. This effect was observed only in the absence of the integrase. Indeed, we provided evidence that the integrase is able to counterbalance the observed effect of SSB on attC site folding. We showed that IntI1 possesses an intrinsic property to capture attC sites at the moment of their extrusion, stabilizing them and recombining them efficiently. The stability of DNA secondary structures in the chromosome must be restrained to avoid genetic instability (mutations or deletions) and/or toxicity (replication arrest). SSB, which hampers attC site folding in the absence of the integrase, likely plays an important role in maintaining the integrity and thus the recombinogenic functionality of the integron attC sites. We also tested the RecA host factor and excluded any role of this protein in integron recombination.

Activation of GCN2 upon HIV-1 infection and inhibition of translation.

Higher eukaryotic organisms have a variety of specific and nonspecific defense mechanisms against viral invaders. In animal cells, viral replication may be limited through the decrease in translation. Some viruses, however, have evolved mechanisms that counteract the response of the host. We report that infection by HIV-1 triggers acute decrease in translation. The human protein kinase GCN2 (eIF2AK4) is activated by phosphorylation upon HIV-1 infection in the hours following infection. Thus, infection by HIV-1 constitutes a stress that leads to the activation of GCN2 with a resulting decrease in protein synthesis. We have shown that GCN2 interacts with HIV-1 integrase (IN). Transfection of IN in amino acid-starved cells, where GCN2 is activated, increases the protein synthesis level. These results point to an as yet unknown role of GCN2 as an early mediator in the cellular response to HIV-1 infection, and suggest that the virus is able to overcome the involvement of GCN2 in the cellular response by eliciting methods to maintain protein synthesis.

Integron cassettes integrate into bacterial genomes via widespread non-classical attG sites.

Integrons are genetic elements involved in bacterial adaptation which capture, shuffle and express genes encoding adaptive functions embedded in cassettes. These events are governed by the integron integrase through site-specific recombination between attC and attI integron sites. Using computational and molecular genetic approaches, here we demonstrate that the integrase also catalyses cassette integration into bacterial genomes outside of its known att sites. Once integrated, these cassettes can be expressed if located near bacterial promoters and can be excised at the integration point or outside, inducing chromosomal modifications in the latter case. Analysis of more than 5 × 105 independent integration events revealed a very large genomic integration landscape. We identified consensus recombination sequences, named attG sites, which differ greatly in sequence and structure from classical att sites. These results unveil an alternative route for dissemination of adaptive functions in bacteria and expand the role of integrons in bacterial evolution.

Cassette recombination dynamics within chromosomal integrons are regulated by toxin-antitoxin systems.

Integrons are adaptive bacterial devices that rearrange promoter-less gene cassettes into variable ordered arrays under stress conditions, thereby sampling combinatorial phenotypic diversity. Chromosomal integrons often carry hundreds of silent gene cassettes, with integrase-mediated recombination leading to rampant DNA excision and integration, posing a potential threat to genome integrity. How this activity is regulated and controlled, particularly through selective pressures, to maintain such large cassette arrays is unknown. Here, we show a key role of promoter-containing toxin-antitoxin (TA) cassettes as systems that kill the cell when the overall cassette excision rate is too high. These results highlight the importance of TA cassettes regulating the cassette recombination dynamics and provide insight into the evolution and success of integrons in bacterial genomes.

Proteomic analysis of SARS-CoV-2 particles unveils a key role of G3BP proteins in viral assembly.

Considerable progress has been made in understanding the molecular host-virus battlefield during SARS-CoV-2 infection. Nevertheless, the assembly and egress of newly formed virions are less understood. To identify host proteins involved in viral morphogenesis, we characterize the proteome of SARS-CoV-2 virions produced from A549-ACE2 and Calu-3 cells, isolated via ultracentrifugation on sucrose cushion or by ACE-2 affinity capture. Bioinformatic analysis unveils 92 SARS-CoV-2 virion-associated host factors, providing a valuable resource to better understand the molecular environment of virion production. We reveal that G3BP1 and G3BP2 (G3BP1/2), two major stress granule nucleators, are embedded within virions and unexpectedly favor virion production. Furthermore, we show that G3BP1/2 participate in the formation of cytoplasmic membrane vesicles, that are likely virion assembly sites, consistent with a proviral role of G3BP1/2 in SARS-CoV-2 dissemination. Altogether, these findings provide new insights into host factors required for SARS-CoV-2 assembly with potential implications for future therapeutic targeting.

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.