RÉSUMÉ
Retroviral integration is mediated by intasome nucleoprotein complexes wherein a pair of viral DNA ends are bridged together by a multimer of integrase (IN). Atomic-resolution structures of HIV-1 intasomes provide detailed insights into the mechanism of integration and inhibition by clinical IN inhibitors. However, previously described HIV-1 intasomes are highly heterogeneous and have the tendency to form stacks, which is a limiting factor in determining high-resolution cryo-EM maps. We have assembled HIV-1 intasomes in the presence of excess IN C-terminal domain protein, which was readily incorporated into the intasomes. The purified intasomes were largely homogeneous and exhibited minimal stacking tendencies. The cryo-EM map resolution was further improved to 2.01 Å, which will greatly facilitate structural studies of IN inhibitor action and drug resistance mechanisms. The C-terminal 18 residues of HIV-1 IN, which are critical for virus replication and integration in vitro, have not been well resolved in previous intasome structures, and its function remains unclear. We show that the C-terminal tail participates in intasome assembly, resides within the intasome core, and forms a small alpha helix (residues 271-276). Mutations that disrupt alpha helix integrity impede IN activity in vitro and disrupt HIV-1 infection at the step of viral DNA integration.
Sujet(s)
Cryomicroscopie électronique , Intégrase du VIH , VIH-1 (Virus de l'Immunodéficience Humaine de type 1) , Intégration virale , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/génétique , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/physiologie , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/enzymologie , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/composition chimique , Intégrase du VIH/métabolisme , Intégrase du VIH/composition chimique , Intégrase du VIH/génétique , Humains , Domaines protéiques , Modèles moléculaires , ADN viral/génétique , ADN viral/métabolismeRÉSUMÉ
Integrase strand transfer inhibitors (INSTIs) are the most prescribed anchor drug in antiretroviral therapy. Today, there is an increasing need for long-acting treatment of HIV-1 infection. Improving drug pharmacokinetics and anti-HIV-1 activity are key to developing more robust inhibitors suitable for long-acting formulations, but 2nd-generation INSTIs have chiral centers, making it difficult to conduct further exploration. In this study, we designed aza-tricyclic and aza-bicyclic carbamoyl pyridone scaffolds which are devoid of the problematic hemiaminal stereocenter present in dolutegravir (DTG). This scaffold hopping made it easy to introduce several substituents, and evolving structure-activity studies using these scaffolds resulted in several leads with promising properties.
Sujet(s)
Conception de médicament , Inhibiteurs de l'intégrase du VIH , Intégrase du VIH , VIH-1 (Virus de l'Immunodéficience Humaine de type 1) , Pyridones , Humains , Composés aza/composition chimique , Composés aza/pharmacologie , Composés aza/synthèse chimique , Relation dose-effet des médicaments , Intégrase du VIH/métabolisme , Inhibiteurs de l'intégrase du VIH/pharmacologie , Inhibiteurs de l'intégrase du VIH/composition chimique , Inhibiteurs de l'intégrase du VIH/synthèse chimique , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/effets des médicaments et des substances chimiques , Structure moléculaire , Pyridones/composition chimique , Pyridones/pharmacologie , Pyridones/synthèse chimique , Relation structure-activité , Integrases/composition chimique , Integrases/métabolisme , Integrases/pharmacocinétiqueRÉSUMÉ
We report herein the design and discovery of novel allosteric HIV-1 integrase inhibitors. Our design concept utilized the spirocyclic moiety to restrain the flexibility of the conformation of the lipophilic part of the inhibitor. Compound 5 showed antiviral activity by binding to the nuclear lens epithelium-derived growth factor (LEDGF/p75) binding site of HIV-1 integrase (IN). The introduction of a lipophilic amide substituent into the central benzene ring resulted in a significant increase in antiviral activity against HIV-1 WT X-ray crystallography of compound 15 in complex with the integrase revealed the presence of a hydrogen bond between the oxygen atom of the amide of compound 15 and the hydroxyl group of the T125 side chain. Chiral compound 17 showed high antiviral activity, good bioavailability, and low clearance in rats.
Sujet(s)
Conception de médicament , Inhibiteurs de l'intégrase du VIH , Intégrase du VIH , VIH-1 (Virus de l'Immunodéficience Humaine de type 1) , Spiranes , Inhibiteurs de l'intégrase du VIH/pharmacologie , Inhibiteurs de l'intégrase du VIH/synthèse chimique , Inhibiteurs de l'intégrase du VIH/composition chimique , Intégrase du VIH/métabolisme , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/effets des médicaments et des substances chimiques , Cristallographie aux rayons X , Rats , Relation structure-activité , Spiranes/composition chimique , Spiranes/pharmacologie , Spiranes/synthèse chimique , Animaux , Humains , Régulation allostérique/effets des médicaments et des substances chimiques , Structure moléculaire , Modèles moléculaires , Sites de fixationRÉSUMÉ
Nanotechnology offers promising avenues for enhancing drug delivery systems, particularly in HIV-1 treatment. This study investigates a nanoemulsified formulation combining epigallocatechin gallate (EGCG) with dolutegravir (DTG) for managing HIV-1 infection. The combinatorial interaction between EGCG and DTG was explored through cellular, enzymatic, and molecular studies. In vitro assays demonstrated the potential of a dual drug-loaded nanoemulsion, NE-DTG-EGCG, in inhibiting HIV-1 replication, with EGCG serving as a supplementary treatment containing DTG. In silico molecular interaction studies highlighted EGCG's multifaceted inhibitory potential against HIV-1 integrase and reverse transcriptase enzymes. Further investigations are needed to validate the formulation's efficacy across diverse contexts. Overall, by integrating nanotechnology into drug delivery systems, this study represents a significant advancement in managing HIV-1 infection.
Sujet(s)
Catéchine , VIH-1 (Virus de l'Immunodéficience Humaine de type 1) , Composés hétérocycliques 3 noyaux , Oxazines , Pipérazines , Pyridones , Réplication virale , Catéchine/analogues et dérivés , Catéchine/pharmacologie , Catéchine/composition chimique , Pyridones/pharmacologie , Pyridones/composition chimique , Composés hétérocycliques 3 noyaux/pharmacologie , Composés hétérocycliques 3 noyaux/composition chimique , Pipérazines/pharmacologie , Pipérazines/composition chimique , Oxazines/pharmacologie , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/effets des médicaments et des substances chimiques , Humains , Réplication virale/effets des médicaments et des substances chimiques , Émulsions , Intégrase du VIH/métabolisme , Inhibiteurs de l'intégrase du VIH/pharmacologie , Inhibiteurs de l'intégrase du VIH/composition chimique , Agents antiVIH/pharmacologie , Agents antiVIH/composition chimique , Nanoparticules/composition chimique , Infections à VIH/traitement médicamenteux , Infections à VIH/virologieRÉSUMÉ
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.
Sujet(s)
Intégrase du VIH , VIH-1 (Virus de l'Immunodéficience Humaine de type 1) , Intégration virale , Intégrase du VIH/métabolisme , Intégrase du VIH/composition chimique , Intégrase du VIH/génétique , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/métabolisme , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/physiologie , Humains , ADN viral/métabolisme , ADN viral/génétique , Protéines et peptides de signalisation intercellulaire/métabolisme , Protéines et peptides de signalisation intercellulaire/composition chimiqueRÉSUMÉ
Structural organization of HIV-1 integrase is based on a tetramer formed by two protein dimers. Within this tetramer, the catalytic domain of one subunit of the first dimer interacts with the N-terminal domain of the second dimer subunit. It is the tetrameric structure that allows both ends of the viral DNA to be correctly positioned relative to the cellular DNA and to realize catalytic functions of integrase, namely 3'-processing and strand transfer. However, during the HIV-1 replicative cycle, integrase is responsible not only for the integration stage, it is also involved in reverse transcription and is necessary at the stage of capsid formation of the newly formed virions. It has been suggested that HIV-1 integrase is a structurally dynamic protein and its biological functions depend on its structure. Accordingly, studying interactions between the domains of integrase that provide its tetrameric structure is important for understanding its multiple functions. In this work, we investigated the role of three amino acids of the catalytic domain, I182, R187, and K188, located in the contact region of two integrase dimers in the tetramer structure, in reverse transcription and integration. It has been shown that the R187 residue is extremely important for formation of the correct integrase structure, which is necessary at all stages of its functional activity. The I182 residue is necessary for successful integration and is not important for reverse transcription, while the K188 residue, on the contrary, is involved in formation of the integrase structure, which is important for the effective reverse transcription.
Sujet(s)
Domaine catalytique , Intégrase du VIH , VIH-1 (Virus de l'Immunodéficience Humaine de type 1) , Transcription inverse , Intégration virale , Intégrase du VIH/métabolisme , Intégrase du VIH/composition chimique , Intégrase du VIH/génétique , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/enzymologie , HumainsRÉSUMÉ
People living with human immunodeficiency virus (HIV) receiving integrase strand transfer inhibitors (INSTIs) have been reported to experience virological failure in the absence of resistance mutations in integrase. To elucidate INSTI resistance mechanisms, we propagated HIV-1 in the presence of escalating concentrations of the INSTI dolutegravir. HIV-1 became resistant to dolutegravir by sequentially acquiring mutations in the envelope glycoprotein (Env) and the nucleocapsid protein. The selected Env mutations enhance the ability of the virus to spread via cell-cell transfer, thereby increasing the multiplicity of infection (MOI). While the selected Env mutations confer broad resistance to multiple classes of antiretrovirals, the fold resistance is ~2 logs higher for INSTIs than for other classes of drugs. We demonstrate that INSTIs are more readily overwhelmed by high MOI than other classes of antiretrovirals. Our findings advance the understanding of how HIV-1 can evolve resistance to antiretrovirals, including the potent INSTIs, in the absence of drug-target gene mutations.
Sujet(s)
Inhibiteurs de l'intégrase du VIH , Intégrase du VIH , VIH-1 (Virus de l'Immunodéficience Humaine de type 1) , Humains , Raltégravir de potassium/pharmacologie , Inhibiteurs de l'intégrase du VIH/pharmacologie , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/génétique , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/métabolisme , Intégrase du VIH/génétique , Intégrase du VIH/métabolisme , MutationRÉSUMÉ
The cellular SFPQ protein is involved in several stages of the HIV-1 life cycle, but the detailed mechanism of its involvement is not yet fully understood. Here, the role of SFPQ in the early stages of HIV-1 replication has been studied. It is found that changes in the intracellular level of SFPQ affect the integration of viral DNA, but not reverse transcription, and SFPQ is a positive factor of integration. A study of the SFPQ interaction with HIV-1 integrase (IN) has revealed two diRGGX1-4 motifs in the N-terminal region of SFPQ, which are involved in IN binding. Substitution of a single amino acid residue in any of these regions led to a decrease in binding efficiency, while mutations in both motifs almost completely disrupted the SFPQ interaction with IN. The effect of the SFPQ mutants with impaired ability to bind IN on viral replication has been analyzed. Unlike the wild-type protein, the SFPQ mutants did not affect viral integration. This confirms that SFPQ influences the integration stage through direct interaction with IN. Our results indicate that the SFPQ/IN complex can be considered as a potential therapeutic target for the development of new inhibitors of HIV replication.
Sujet(s)
Intégrase du VIH , VIH-1 (Virus de l'Immunodéficience Humaine de type 1) , Facteur d'épissage associé à PTB , Intégration virale , Réplication virale , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/métabolisme , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/physiologie , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/génétique , Humains , Intégrase du VIH/métabolisme , Intégrase du VIH/génétique , Facteur d'épissage associé à PTB/métabolisme , Facteur d'épissage associé à PTB/génétique , Liaison aux protéines , Mutation , Cellules HEK293RÉSUMÉ
Allosteric HIV-1 Integrase (IN) Inhibitors or ALLINIs bind at the dimer interface of the IN, away from the enzymatic catalytic site, and disable viral replication by inducing over-multimerization of IN. Interestingly, these inhibitors are capable of impacting both the early and late stages of viral replication. To better understand the important binding features of multi-substituted quinoline-based ALLINIs, we have surveyed published studies on IN multimerization and antiviral properties of various substituted quinolines at the 4, 6, 7, and 8 positions. Here we show how the efficacy of these inhibitors can be modulated by the nature of the substitutions at those positions. These features not only improve the overall antiviral potencies of these compounds but also significantly shift the selectivity toward the viral maturation stage. Thus, to fully maximize the potency of ALLINIs, the interactions between the inhibitor and multiple IN subunits need to be simultaneously optimized.
Sujet(s)
Inhibiteurs de l'intégrase du VIH , Intégrase du VIH , VIH-1 (Virus de l'Immunodéficience Humaine de type 1) , Quinoléines , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/métabolisme , Régulation allostérique , Inhibiteurs de l'intégrase du VIH/pharmacologie , Inhibiteurs de l'intégrase du VIH/composition chimique , Intégrase du VIH/métabolisme , Quinoléines/pharmacologie , Multimérisation de protéinesSujet(s)
Agents antiVIH , Infections à VIH , Inhibiteurs de l'intégrase du VIH , Intégrase du VIH , VIH-1 (Virus de l'Immunodéficience Humaine de type 1) , Humains , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/génétique , Transcription inverse , Agents antiVIH/pharmacologie , Agents antiVIH/usage thérapeutique , Inhibiteurs de l'intégrase du VIH/pharmacologie , Inhibiteurs de l'intégrase du VIH/usage thérapeutique , Composés hétéromonocycliques , Mutation , Intégrase du VIH/génétique , Intégrase du VIH/métabolisme , Résistance virale aux médicaments/génétique , Infections à VIH/traitement médicamenteuxRÉSUMÉ
As an important antiviral target, HIV-1 integrase plays a key role in the viral life cycle, and five integrase strand transfer inhibitors (INSTIs) have been approved for the treatment of HIV-1 infections so far. However, similar to other clinically used antiviral drugs, resistance-causing mutations have appeared, which have impaired the efficacy of INSTIs. In the current study, to identify novel integrase inhibitors, a set of molecular docking-based virtual screenings were performed, and indole-2-carboxylic acid was developed as a potent INSTI scaffold. Indole-2-carboxylic acid derivative 3 was proved to effectively inhibit the strand transfer of HIV-1 integrase, and binding conformation analysis showed that the indole core and C2 carboxyl group obviously chelated the two Mg2+ ions within the active site of integrase. Further structural optimizations on compound 3 provided the derivative 20a, which markedly increased the integrase inhibitory effect, with an IC50 value of 0.13 µM. Binding mode analysis revealed that the introduction of a long branch on C3 of the indole core improved the interaction with the hydrophobic cavity near the active site of integrase, indicating that indole-2-carboxylic acid is a promising scaffold for the development of integrase inhibitors.
Sujet(s)
Infections à VIH , Inhibiteurs de l'intégrase du VIH , Intégrase du VIH , VIH-1 (Virus de l'Immunodéficience Humaine de type 1) , Humains , Inhibiteurs de l'intégrase du VIH/pharmacologie , Inhibiteurs de l'intégrase du VIH/composition chimique , Simulation de docking moléculaire , Intégrase du VIH/métabolisme , Infections à VIH/traitement médicamenteux , Indoles/pharmacologie , Indoles/usage thérapeutique , Domaine catalytique , Résistance virale aux médicaments , MutationRÉSUMÉ
HIV-1 infection depends on the integration of viral DNA into host chromatin. Integration is mediated by the viral enzyme integrase and is blocked by integrase strand transfer inhibitors (INSTIs), first-line antiretroviral therapeutics widely used in the clinic. Resistance to even the best INSTIs is a problem, and the mechanisms of resistance are poorly understood. Here, we analyze combinations of the mutations E138K, G140A/S, and Q148H/K/R, which confer resistance to INSTIs. The investigational drug 4d more effectively inhibited the mutants compared with the approved drug Dolutegravir (DTG). We present 11 new cryo-EM structures of drug-resistant HIV-1 intasomes bound to DTG or 4d, with better than 3-Å resolution. These structures, complemented with free energy simulations, virology, and enzymology, explain the mechanisms of DTG resistance involving E138K + G140A/S + Q148H/K/R and show why 4d maintains potency better than DTG. These data establish a foundation for further development of INSTIs that potently inhibit resistant forms in integrase.
Sujet(s)
Inhibiteurs de l'intégrase du VIH , Intégrase du VIH , Inhibiteurs de l'intégrase du VIH/pharmacologie , Inhibiteurs de l'intégrase du VIH/composition chimique , Oxazines/pharmacologie , Mutation , Intégrase du VIH/génétique , Intégrase du VIH/composition chimique , Intégrase du VIH/métabolismeRÉSUMÉ
HIV-1 integrase-LEDGF allosteric inhibitors (INLAIs) share the binding site on the viral protein with the host factor LEDGF/p75. These small molecules act as molecular glues promoting hyper-multimerization of HIV-1 IN protein to severely perturb maturation of viral particles. Herein, we describe a new series of INLAIs based on a benzene scaffold that display antiviral activity in the single digit nanomolar range. Akin to other compounds of this class, the INLAIs predominantly inhibit the late stages of HIV-1 replication. A series of high-resolution crystal structures revealed how these small molecules engage the catalytic core and the C-terminal domains of HIV-1 IN. No antagonism was observed between our lead INLAI compound BDM-2 and a panel of 16 clinical antiretrovirals. Moreover, we show that compounds retained high antiviral activity against HIV-1 variants resistant to IN strand transfer inhibitors and other classes of antiretroviral drugs. The virologic profile of BDM-2 and the recently completed single ascending dose phase I trial (ClinicalTrials.gov identifier: NCT03634085) warrant further clinical investigation for use in combination with other antiretroviral drugs. Moreover, our results suggest routes for further improvement of this emerging drug class.
Sujet(s)
Infections à VIH , Inhibiteurs de l'intégrase du VIH , Intégrase du VIH , Humains , Réplication virale , Inhibiteurs de l'intégrase du VIH/pharmacologie , Inhibiteurs de l'intégrase du VIH/usage thérapeutique , Antiviraux/pharmacologie , Intégrase du VIH/métabolisme , Infections à VIH/traitement médicamenteux , Régulation allostériqueRÉSUMÉ
Lens epithelial-derived growth factor (LEDGF) increases the efficiency of proviral DNA integration into the host genome by interacting with HIV integrase (IN) and directing it to a chromatin environment that favors viral transcription. Allosteric integrase inhibitors (ALLINIs), such as known 2-(tert-butoxy)acetic acid (1), bind to the LEDGF pocket on the catalytic core domain (CCD) of IN, but exert more potent antiviral activities by inhibition of late-stage HIV-1 replication events than through disruption of proviral integration at an earlier phase. A high-throughput screen (HTS) for compounds that disrupt IN-LEDGF interaction led to the identification of a novel arylsulfonamide series, as exemplified by 2, possessing ALLINI-like properties. Further SAR studies led to more potent compound 21 and provided key chemical biology probes revealing that arylsulfonamides are a novel class of ALLINIs with a distinct binding mode than that of 2-(tert-butoxy)acetic acids.
Sujet(s)
Agents antiVIH , Inhibiteurs de l'intégrase du VIH , Intégrase du VIH , Inhibiteurs de l'intégrase du VIH/pharmacologie , Inhibiteurs de l'intégrase du VIH/composition chimique , Régulation allostérique , Domaine catalytique , Intégrase du VIH/métabolismeRÉSUMÉ
Human immunodeficiency virus (HIV) treatment with antiretroviral regimens containing integrase strand transfer inhibitors such as dolutegravir (DTG) and bictegravir (BIC) offers high levels of protection against the development of drug resistance mutations. Despite this, resistance to DTG and BIC can occur through the development of the R263K integrase substitution. Failure with DTG has also been associated with the emergence of the G118R substitution. G118R and R263K are usually found separately but have been reported together in highly treatment-experienced persons who experienced treatment failure with DTG. We used cell-free strand transfer and DNA binding assays and cell-based infectivity, replicative capacity, and resistance assays to characterize the G118R plus R263K combination of integrase mutations. R263K reduced DTG and BIC susceptibility ~2-fold, in agreement with our previous work. Single-cycle infectivity assays showed that G118R and G118R plus R263K conferred ~10-fold resistance to DTG. G118R alone conferred low levels of resistance to BIC (3.9-fold). However, the G118R plus R263K combination conferred high levels of resistance to BIC (33.7-fold), likely precluding the use of BIC after DTG failure with the G118R plus R263K combination. DNA binding, viral infectivity, and replicative capacity of the double mutant were further impaired, compared to single mutants. We propose that impaired fitness helps to explain the scarcity of the G118R plus R263K combination of integrase substitutions in clinical settings and that immunodeficiency likely contributes to its development.
Sujet(s)
Infections à VIH , Inhibiteurs de l'intégrase du VIH , Intégrase du VIH , VIH-1 (Virus de l'Immunodéficience Humaine de type 1) , Humains , Inhibiteurs de l'intégrase du VIH/pharmacologie , Inhibiteurs de l'intégrase du VIH/usage thérapeutique , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/génétique , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/métabolisme , Substitution d'acide aminé , Intégrase du VIH/génétique , Intégrase du VIH/métabolisme , Mutation , Composés hétérocycliques 3 noyaux/pharmacologie , Composés hétérocycliques 3 noyaux/usage thérapeutique , Pyridones/pharmacologie , ADN/pharmacologie , ADN/usage thérapeutique , Résistance virale aux médicaments/génétique , Infections à VIH/traitement médicamenteuxRÉSUMÉ
Integrase Strand Transfer Inhibitors (INSTIs) are currently used as the most effective therapy in the treatment of human immunodeficiency virus (HIV) infections. Raltegravir (RAL) and Elvitegravir (EVG), the first generation of INSTIs used successfully in clinical treatment, are susceptible to the emergence of viral resistance and have a high rate of cross-resistance. To counteract these resistant mutants, second-generation INSTI drugs have been developed: Dolutegravir (DTG), Cabotegravir (CAB), and Bictegravir (BIC). However, HIV is also able to develop resistance mechanisms against the second-generation of INSTIs. This review describes the mode of action of INSTIs and then summarizes and evaluates some typical resistance mutations, such as substitution and insertion mutations. The role of unintegrated viral DNA is also discussed as a new pathway involved in conferring resistance to INSTIs. This allows us to have a more detailed understanding of HIV resistance to these inhibitors, which may contribute to the development of new INSTIs in the future.
Sujet(s)
Infections à VIH , Inhibiteurs de l'intégrase du VIH , Intégrase du VIH , Humains , Inhibiteurs de l'intégrase du VIH/pharmacologie , Inhibiteurs de l'intégrase du VIH/usage thérapeutique , Raltégravir de potassium/pharmacologie , Infections à VIH/traitement médicamenteux , Composés hétérocycliques 3 noyaux/pharmacologie , Composés hétérocycliques 3 noyaux/usage thérapeutique , Mutation , Integrases/génétique , Intégrase du VIH/génétique , Intégrase du VIH/métabolisme , Résistance virale aux médicaments/génétiqueRÉSUMÉ
RNA viruses, in pursuit of genome miniaturization, tend to employ cellular proteins to facilitate their replication. HIV-1, one of the most well-studied retroviruses, is not an exception. There is numerous evidence that the exploitation of cellular machinery relies on nucleic acid-protein and protein-protein interactions. Apart from Vpr, Vif, and Nef proteins that are known to regulate cellular functioning via interaction with cell components, another viral protein, integrase, appears to be crucial for proper virus-cell dialog at different stages of the viral life cycle. The goal of this review is to summarize and systematize existing data on known cellular partners of HIV-1 integrase and their role in the HIV-1 life cycle.
Sujet(s)
Intégrase du VIH , VIH-1 (Virus de l'Immunodéficience Humaine de type 1) , Acides nucléiques , Réplication virale , Intégrase du VIH/métabolisme , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/physiologie , Protéines viralesRÉSUMÉ
HIV-1 DNA is preferentially integrated into chromosomal hot spots by the preintegration complex (PIC). To understand the mechanism, we measured the DNA integration activity of PICs-extracted from infected cells-and intasomes, biochemically assembled PIC substructures using a number of relevant target substrates. We observed that PIC-mediated integration into human chromatin is preferred compared to genomic DNA. Surprisingly, nucleosomes lacking histone modifications were not preferred integration compared to the analogous naked DNA. Nucleosomes containing the trimethylated histone 3 lysine 36 (H3K36me3), an epigenetic mark linked to active transcription, significantly stimulated integration, but the levels remained lower than the naked DNA. Notably, H3K36me3-modified nucleosomes with linker DNA optimally supported integration mediated by the PIC but not by the intasome. Interestingly, optimal intasome-mediated integration required the cellular cofactor LEDGF. Unexpectedly, LEDGF minimally affected PIC-mediated integration into naked DNA but blocked integration into nucleosomes. The block for the PIC-mediated integration was significantly relieved by H3K36me3 modification. Mapping the integration sites in the preferred substrates revealed that specific features of the nucleosome-bound DNA are preferred for integration, whereas integration into naked DNA was random. Finally, biochemical and genetic studies demonstrate that DNA condensation by the H1 protein dramatically reduces integration, providing further evidence that features inherent to the open chromatin are preferred for HIV-1 integration. Collectively, these results identify the optimal target substrate for HIV-1 integration, report a mechanistic link between H3K36me3 and integration preference, and importantly, reveal distinct mechanisms utilized by the PIC for integration compared to the intasomes. IMPORTANCE HIV-1 infection is dependent on integration of the viral DNA into the host chromosomes. The preintegration complex (PIC) containing the viral DNA, the virally encoded integrase (IN) enzyme, and other viral/host factors carries out HIV-1 integration. HIV-1 integration is not dependent on the target DNA sequence, and yet the viral DNA is selectively inserted into specific "hot spots" of human chromosomes. A growing body of literature indicates that structural features of the human chromatin are important for integration targeting. However, the mechanisms that guide the PIC and enable insertion of the PIC-associated viral DNA into specific hot spots of the human chromosomes are not fully understood. In this study, we describe a biochemical mechanism for the preference of the HIV-1 DNA integration into open chromatin. Furthermore, our study defines a direct role for the histone epigenetic mark H3K36me3 in HIV-1 integration preference and identify an optimal substrate for HIV-1 PIC-mediated viral DNA integration.
Sujet(s)
Chromosomes humains , VIH-1 (Virus de l'Immunodéficience Humaine de type 1) , Code histone , Histone , Nucléosomes , Intégration virale , Chromatine/métabolisme , Chromosomes humains/virologie , ADN viral/génétique , ADN viral/métabolisme , Infections à VIH/virologie , Intégrase du VIH/génétique , Intégrase du VIH/métabolisme , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/génétique , Histone/composition chimique , Histone/métabolisme , Humains , Lysine/génétique , Méthylation , Nucléosomes/génétique , Nucléosomes/métabolisme , Nucléosomes/virologie , Intégration virale/génétiqueRÉSUMÉ
Allosteric integrase (IN) inhibitors (ALLINIs), which are promising preclinical compounds that engage the lens epithelium-derived growth factor (LEDGF)/p75 binding site on IN, can inhibit different aspects of human immunodeficiency virus 1 (HIV-1) replication. During the late phase of replication, ALLINIs induce aberrant IN hyper-multimerization, the consequences of which disrupt IN binding to genomic RNA and virus particle morphogenesis. During the early phase of infection, ALLINIs can suppress HIV-1 integration into host genes, which is also observed in LEDGF/p75-depelted cells. Despite this similarity, the roles of LEDGF/p75 and its paralog hepatoma-derived growth factor like 2 (HDGFL2) in ALLINI-mediated integration retargeting are untested. Herein, we mapped integration sites in cells knocked out for LEDGF/p75, HDGFL2, or both factors, which revealed that these two proteins in large part account for ALLINI-mediated integration retargeting during the early phase of infection. We also determined that ALLINI-treated viruses are defective during the subsequent round of infection for integration into genes associated with speckle-associated domains, which are naturally highly targeted for HIV-1 integration. Class II IN mutant viruses with alterations distal from the LEDGF/p75 binding site moreover shared this integration retargeting phenotype. Altogether, our findings help to inform the molecular bases and consequences of ALLINI action.
Sujet(s)
Agents antiVIH , Inhibiteurs de l'intégrase du VIH , Intégrase du VIH , VIH-1 (Virus de l'Immunodéficience Humaine de type 1) , Agents antiVIH/pharmacologie , Intégrase du VIH/métabolisme , Inhibiteurs de l'intégrase du VIH/composition chimique , Inhibiteurs de l'intégrase du VIH/pharmacologie , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/génétique , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/métabolisme , Humains , Protéines et peptides de signalisation intercellulaire , ARN , Intégration virale , Réplication viraleRÉSUMÉ
Retroviral integrase is a multimeric enzyme that catalyzes the integration of reverse-transcribed viral DNA into the cellular genome. Beyond integration, the Human immunodeficiency virus type 1 (HIV-1) integrase is also involved in many other steps of the viral life cycle, such as reverse transcription, nuclear import, virion morphogenesis and proviral transcription. All these additional functions seem to depend on the action of the integrase C-terminal domain (CTD) that works as a molecular hub, interacting with many different viral and cellular partners. In this review, we discuss structural issues concerning the CTD, with particular attention paid to its interaction with nucleic acids. We also provide a detailed map of post-translational modifications and interaction with molecular partners.