RESUMEN
As ZIKV continues to spread, many "unknowns" remain and research is needed to advance the understanding of this important pathogen. Viral RNA dependent-RNA polymerases (RdRp) are validated targets for inhibitors of the replication of several viruses. Several studies have set up in vitro enzymatic assays of the RdRp of the Zika virus for testing of candidate inhibitors. While most of these studies use short synthetic polymers, we have shown in a previous work that the Zika polymerase domain is capable of a de novo synthesis of the viral genome using the natural viral RNA as template. Here we have studied the role of the sequences at the 3'end of the minus-strand RNA in the initiation of the RNA synthesis by the Zika isolated RdRp. Our results strongly suggest that the region containing the 105 first nucleotides from the 3' end of the minus-strand RNA is important for initiation of the positive RNA synthesis. This indicates that this region displays all the primary and secondary structures to be efficiently recognized by the recombinant RdRp in vitro. Moreover, we show that the 46 nucleotides are sufficient to initiate RNA synthesis. In addition, the ZIKV polymerase domain poorly replicated the RNA of other RNA viruses and appeared highly selective for its own RNA.
Asunto(s)
ARN Polimerasa Dependiente del ARN , Infección por el Virus Zika , Virus Zika , Humanos , ARN Viral/genética , ARN Polimerasa Dependiente del ARN/genética , Replicación Viral , Virus Zika/enzimología , Virus Zika/genética , Virus Zika/fisiologíaRESUMEN
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.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Cromatina/metabolismo , Integrasa de VIH/genética , Histonas/genética , Interacciones Huésped-Patógeno/genética , Factores de Transcripción/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Cromatina/química , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Regulación de la Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Integrasa de VIH/metabolismo , Histonas/metabolismo , Humanos , Células K562 , Cultivo Primario de Células , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Linfocitos T/metabolismo , Linfocitos T/virología , Factores de Transcripción/metabolismoRESUMEN
Multiple viral targets are now available in the clinic to fight HIV infection. Even if this targeted therapy is highly effective at suppressing viral replication, caregivers are facing growing therapeutic failures in patients due to resistance, with or without treatment-adherence glitches. Accordingly, it is important to better understand how HIV and other retroviruses replicate in order to propose alternative antiviral strategies. Recent studies have shown that multiple cellular factors are implicated during the integration step and, more specifically, that integrase can be regulated through post-translational modifications. We have shown that integrase is phosphorylated by GCN2, a cellular protein kinase of the integrated stress response, leading to a restriction of HIV replication. In addition, we found that this mechanism is conserved among other retroviruses. Accordingly, we developed an in vitro interaction assay, based on the AlphaLISA technology, to monitor the integrase-GCN2 interaction. From an initial library of 133 FDA-approved molecules, we identified nine compounds that either inhibited or stimulated the interaction between GCN2 and HIV integrase. In vitro characterization of these nine hits validated this pilot screen and demonstrated that the GCN2-integrase interaction could be a viable solution for targeting integrase out of its active site.
Asunto(s)
Infecciones por VIH/terapia , Integrasa de VIH/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Replicación Viral/efectos de los fármacos , Dominio Catalítico , Evaluación Preclínica de Medicamentos , VIH , Integrasa de VIH/genética , Ensayos Analíticos de Alto Rendimiento , Humanos , Modelos Moleculares , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Retroviridae , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-Actividad , Replicación Viral/genéticaRESUMEN
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.
Asunto(s)
Integrasa de VIH/metabolismo , VIH-1/metabolismo , Histonas/metabolismo , Nucleosomas/metabolismo , Integración Viral , Línea Celular Transformada , Cromatina/virología , ADN Viral/metabolismo , Células HEK293 , VIH-1/genética , Histonas/química , Interacciones Huésped-Parásitos/fisiología , Humanos , Unión ProteicaRESUMEN
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.
Asunto(s)
Cromatina/metabolismo , Integrasa de VIH/metabolismo , VIH-1/fisiología , Chaperonas de Histonas/metabolismo , Interacciones Huésped-Patógeno , Integración Viral/fisiología , Células Cultivadas , Ensamble y Desensamble de Cromatina/fisiología , Infecciones por VIH/genética , Infecciones por VIH/virología , VIH-1/genética , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Nucleosomas/metabolismo , Unión ProteicaRESUMEN
Mosquito- and tick-borne pathogens including Chikungunya, Dengue, Japanese encephalitis, West Nile, Yellow fever and Zika virus, represent a new economic and public health challenge. In the absence of effective vaccines and specific therapies, only supportive regimens are administrated for most of these infections. Thus, the development of a targeted therapy is mandatory to stop the rapid progression of these pathogens and preoccupant associated burdens such as Guillain-Barre syndrome, microcephaly. For this, it is essential to develop biochemical tools to help study and target key viral enzymes involved in replication such as helicase complexes, methyl-transferases and RNA-dependent RNA polymerases. Here, we show that a highly purified ZIKV polymerase domain is active in vitro. Importantly, we show that this isolated domain is capable of de novo synthesis of the viral genome and efficient elongation without terminal nucleotide transferase activity. Altogether, this isolated polymerase domain will be a precious tool to screen and optimize specific nucleoside and non-nucleoside inhibitors to fight against Zika infections.
Asunto(s)
ARN Viral/genética , ARN Polimerasa Dependiente del ARN/metabolismo , Transcripción Genética , Infección por el Virus Zika/virología , Virus Zika/fisiología , Catálisis , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Dominios Proteicos , Dominios y Motivos de Interacción de Proteínas , ARN Viral/metabolismo , ARN Polimerasa Dependiente del ARN/química , Replicación ViralRESUMEN
The cellular DNA repair hRAD51 protein has been shown to restrict HIV-1 integration both in vitro and in vivo. To investigate its regulatory functions, we performed a pharmacological analysis of the retroviral integration modulation by hRAD51. We found that, in vitro, chemical activation of hRAD51 stimulates its integration inhibitory properties, whereas inhibition of hRAD51 decreases the integration restriction, indicating that the modulation of HIV-1 integration depends on the hRAD51 recombinase activity. Cellular analyses demonstrated that cells exhibiting high hRAD51 levels prior to de novo infection are more resistant to integration. On the other hand, when hRAD51 was activated during integration, cells were more permissive. Altogether, these data establish the functional link between hRAD51 activity and HIV-1 integration. Our results highlight the multiple and opposite effects of the recombinase during integration and provide new insights into the cellular regulation of HIV-1 replication.
Asunto(s)
VIH-1/fisiología , Recombinasa Rad51/metabolismo , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/química , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/metabolismo , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/metabolismo , Benzamidas/química , Benzamidas/metabolismo , Benzamidas/farmacología , ADN/química , ADN/metabolismo , Reparación del ADN , Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Morfolinas/química , Morfolinas/metabolismo , Morfolinas/farmacología , Unión Proteica , Pirroles/química , Pirroles/metabolismo , Pirroles/farmacología , Recombinasa Rad51/química , Recombinasa Rad51/genética , Estilbenos/química , Estilbenos/metabolismo , Estilbenos/farmacología , Sulfonamidas/química , Sulfonamidas/metabolismo , Sulfonamidas/farmacología , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
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.
Asunto(s)
Cromatina/virología , Interacciones Huésped-Patógeno , Nucleosomas/virología , Retroviridae/fisiología , Integración Viral , Cromatina/metabolismo , ADN/metabolismo , Humanos , Integrasas/metabolismo , Nucleosomas/metabolismoRESUMEN
The antiviral efficacy of raltegravir (RAL) has been proven against human immunodeficiency virus type 1 (HIV-1) subtypes B and C but remained to be determined against other subtypes. Therefore, the enzymatic activities as well as RAL resistance of HIV-1 subtype A and CRF01_AE integrases (INs) were investigated. Previously published subtype A and CRF01_AE IN sequences from RAL-naïve patients were aligned to generate consensus sequences for both IN subtypes. Subtype A and CRF01_AE INs encoded by these consensus sequences as well as the corresponding enzymes harbouring the N155H resistance mutation were expressed and purified. Enzymatic activities of subtype A and CRF01_AE INs were analysed with regard to typical 3'-end processing (3'-P) and strand transfer (ST) activities both in the presence and absence of RAL and were compared with subtype B IN as well as with the corresponding INs harbouring the N155H resistance mutation. Subtypes B, A and CRF01_AE INs showed similar 3'-P and ST activities. In the presence of RAL, the three wild-type INs exhibited ST activity IC50 values (50% inhibitory concentrations) of 86.3 ± 32.5, 158.3 ± 99.0 and 100.0 ± 65.7 nM, respectively. Analysis of 3'-P activity in the presence of RAL revealed IC(50) > 10 µM for all three enzymes. The three INs harbouring the N155H mutation presented in vitro low but similar resistance levels to RAL. In conclusion, INs from HIV-1 subtypes B, A and CRF01_AE showed similar responses to RAL in vitro, suggesting the potency of this antiretroviral drug to treat HIV-1 subtype A- and CRF01_AE-infected patients.
Asunto(s)
Fármacos Anti-VIH/farmacología , Integrasa de VIH/metabolismo , VIH-1/efectos de los fármacos , Pirrolidinonas/farmacología , Clonación Molecular , Expresión Génica , Genotipo , Integrasa de VIH/genética , VIH-1/clasificación , VIH-1/genética , VIH-1/aislamiento & purificación , Humanos , Concentración 50 Inhibidora , Pruebas de Sensibilidad Microbiana , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Raltegravir PotásicoRESUMEN
We report herein further insight into the biological activities displayed by a series of 2-hydroxyisoquinoline-1,3(2H,4H)-diones (HIDs). Substitution of the N-hydroxyimide two-metal binding pharmacophore at position 4 by carboxamido side chains was previously shown by us to be fruitful for this scaffold, since strong human immunodeficiency virus type 1 integrase (HIV-1 IN) inhibitors in the low nanomolar range associated with low micromolar anti-HIV activities were obtained. We investigated the influence of substitution at position 7 on biological activity. Introduction of electron-withdrawing functional groups such as the nitro moiety at position 7 led to a noticeable improvement of antiviral activity, down to low nanomolar anti-HIV potencies, with advantageous therapeutic indexes going close to those of the clinically used raltegravir and retained potencies against a panel of IN mutants.
Asunto(s)
Inhibidores de Integrasa VIH/síntesis química , VIH-1/efectos de los fármacos , Isoquinolinas/síntesis química , Hidrocarburo de Aril Hidroxilasas/antagonistas & inhibidores , Línea Celular Tumoral , Citocromo P-450 CYP2C9 , Citocromo P-450 CYP3A , Inhibidores del Citocromo P-450 CYP3A , Farmacorresistencia Viral , Inhibidores de Integrasa VIH/química , Inhibidores de Integrasa VIH/farmacología , VIH-1/genética , Humanos , Isoquinolinas/química , Isoquinolinas/farmacología , Simulación del Acoplamiento Molecular , Mutación , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Productos Biológicos/farmacología , Proteínas de Unión al ADN/antagonistas & inhibidores , Inhibidores de Integrasa VIH/farmacología , Integrasa de VIH/metabolismo , Estilbenos/farmacología , Transposasas/antagonistas & inhibidores , Vitis/química , Productos Biológicos/aislamiento & purificación , Evaluación Preclínica de Medicamentos , Eucariontes/enzimología , Células HEK293 , Inhibidores de Integrasa VIH/aislamiento & purificación , Humanos , Lentivirus/efectos de los fármacos , Lentivirus/fisiología , Estilbenos/aislamiento & purificación , Replicación Viral/efectos de los fármacosRESUMEN
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.
Asunto(s)
VIH-1/patogenicidad , Biosíntesis de Proteínas , Proteínas Serina-Treonina Quinasas/fisiología , Silenciador del Gen , Infecciones por VIH/inmunología , Infecciones por VIH/patología , Infecciones por VIH/virología , Integrasa de VIH/metabolismo , Integrasa de VIH/fisiología , VIH-1/fisiología , Células HeLa , Humanos , Fosforilación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Estrés Fisiológico , Replicación ViralRESUMEN
A series of 2-hydroxy-1,3-dioxoisoquinoline-4-carboxamides featuring an N-hydroxyimide chelating functionality was evaluated for their inhibitory properties against human immunodeficiency virus type 1 integrase (HIV-1 IN). Several derivatives displayed low nanomolar IC50 values comparable to that of the clinically used raltegravir. A marked effect of one compound on both primary IN-catalyzed reactions, strand transfer (ST), and 3' processing (3'-P), emphasizes a novel IN inhibition mechanism establishing it as a potential new generation IN inhibitor. Substitution of the 2-hydroxyisoquinoline-1,3-dione scaffold at position 4 by carboxamido chains was beneficial for antiviral activity since reproducible low micromolar anti-HIV activities were obtained for the first time within this scaffold.
RESUMEN
In contrast to canonical proteases, total immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies (Abs) from HIV-infected patients hydrolyze effectively only HIV integrase (IN), reverse transcriptase (RT), human casein, and serum albumin. Anti-IN IgG and IgM isolated by chromatography on IN-Sepharose hydrolyze specifically only IN but not many other tested proteins. Total Abs from HIV-infected patients hydrolyze not only globular proteins but also different specific and nonspecific tri-, tetra-, and 20- to 25-mer oligopeptides (OPs) with a higher rate than anti-IN Abs isolated using IN-Sepharose. A similar situation was observed for IgG from patients with multiple sclerosis and HIV-infected patients, which after purification on myelin basic protein (MBP)-Sepharose and RT-Sepharose specifically hydrolyze only MBP and RT, respectively. The active sites of all anti-protein abzymes are localized on their light chains, whereas the heavy chain is responsible for the affinity of protein substrates. Interactions of intact globular proteins with both light and heavy chains of abzymes provide the specificity of protein hydrolysis. The affinity of anti-IN and anti-MBP abzymes for intact IN and MBP is approximately 10(2)- to 10(5)-fold higher than for short and long specific and nonspecific OPs. The data suggest that all OPs interact mainly with the light chain of different Abs, which possesses a lower affinity for substrates, and therefore, depending on the OP sequences, their hydrolysis may be less specific or completely nonspecific. The data indicate that the relative activity of Abs not fractionated on specific protein sorbents in the hydrolysis of specific and nonspecific OPs can correspond to an average proteolytic activity of light chains of polyclonal Abs directed against many different proteins.
Asunto(s)
Anticuerpos Catalíticos/sangre , Anticuerpos Antivirales/sangre , Infecciones por VIH/sangre , Integrasa de VIH/inmunología , VIH-1/enzimología , Oligopéptidos/química , Adolescente , Adulto , Secuencia de Aminoácidos , Anticuerpos Catalíticos/aislamiento & purificación , Anticuerpos Antivirales/aislamiento & purificación , Cromatografía de Afinidad , Femenino , Infecciones por VIH/inmunología , Integrasa de VIH/química , Transcriptasa Inversa del VIH/química , Transcriptasa Inversa del VIH/inmunología , Humanos , Hidrólisis , Masculino , Datos de Secuencia Molecular , Péptidos/química , Proteolisis , Homología de Secuencia de Aminoácido , Adulto JovenRESUMEN
Human immunodeficiency virus type 1 integrase (IN) catalyzes integration of a DNA copy of the viral genome into the host genome. It was shown previously that IN preincubation with various oligodeoxynucleotides (ODNs) induces formation of dimers and oligomers of different gyration radii; only specific ODNs stimulate the formation of catalytically active dimers. Here we have shown that preincubation of IN with specific and nonspecific ODNs leads to a significant and comparable decrease in its hydrolysis by chymotrypsin, while nonspecific ODNs protect the enzyme from the hydrolysis by trypsin worse than specific ODNs; all ODNs had little effect on the IN hydrolysis by proteinase K. In contrast to canonical proteweases, IgGs from HIV-infected patients specifically hydrolyze only IN. While d(pT)(n) markedly decreased the IgG-dependent hydrolysis of IN, d(pA)(n) and d(pA)(n) â¢d(pT)(n) demonstrated no detectable protective effect. The best protection from the hydrolysis by IgGs was observed for specific single- and especially double-stranded ODNs. Although IN was considerably protected by specific ODNs, proteolytic IgGs and IgMs significantly suppressed both 3'-processing and integration reaction catalyzed by IN. Since anti-IN IgGs and IgMs can efficiently hydrolyze IN, a positive role of abzymes in counteracting the infection cannot be excluded.
Asunto(s)
Anticuerpos Catalíticos/química , Anticuerpos Anti-VIH/química , Infecciones por VIH/inmunología , Integrasa de VIH/química , VIH-1/inmunología , Inmunoglobulina G/química , Adolescente , Adulto , Catálisis , Quimotripsina/química , Dimerización , Endopeptidasa K/química , Femenino , Integrasa de VIH/metabolismo , VIH-1/enzimología , Humanos , Hidrólisis , Masculino , Oligodesoxirribonucleótidos/químicaRESUMEN
HIV-1 integrase (IN) catalyzes integration of a DNA copy of the viral genome into the host genome. In contrast to canonical proteases (trypsin, chymotrypsin and proteinase K), IgGs and IgMs isolated from HIV-infected patients by affinity chromatography on immobilized IN specifically hydrolyzed only IN but not many other tested intact globular proteins. The sites of IN cleavage determined by MALDI mass spectrometry were localized mainly within seven known immunodominant regions of IN. Thin layer chromatography analysis has shown that the abzymes (Abzs) could also cleave 17 to 22-mer oligopeptides (OPs) corresponding to the immunodominant regions of IN sequence with a much higher rate than non-specific long peptides or three- and tetrapeptides of various sequence. Therefore, a prolonged incubation of IN with AIDS IgGs and IgMs having high catalytic activity usually produces many OPs of different length. Since anti-IN IgGs and IgMs can efficiently hydrolyze IN, a positive role of the Abzs in counteracting the infection is possible.
Asunto(s)
Biocatálisis , Anticuerpos Anti-VIH/inmunología , Antígenos VIH/metabolismo , Integrasa de VIH/inmunología , Integrasa de VIH/metabolismo , Adolescente , Adulto , Femenino , Anticuerpos Anti-VIH/sangre , Antígenos VIH/inmunología , Infecciones por VIH/sangre , Infecciones por VIH/inmunología , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inmunoglobulina M/sangre , Inmunoglobulina M/inmunología , Masculino , Especificidad por Sustrato , Adulto JovenRESUMEN
2-Hydroxyisoquinoline-1,3(2H,4H)-dione was recently discovered as a scaffold for the inhibition of HIV-1 integrase and the ribonuclease H function of HIV-1 reverse transcriptase. First, we investigate its interaction with Mg(2+) and Mn(2+) using different spectroscopic techniques and report that 2-hydroxyisoquinoline-1,3(2H,4H)-dione forms a 1:1 complex with Mg(2+) but a 1:2 complex with Mn(2+). The complex formation requires enolization of the ligand. ESR spectroscopy shows a redox reaction between the ligand and Mn(2+) producing superoxide anions. Second, 2-hydroxyisoquinoline-1,3(2H,4H)-dione, its magnesium complex, and its 4-methyl and 2-hydroxy-4-methoxycarbonylisoquinoline-1,3(2H,4H)-diones were tested as inhibitors of HIV-1 integrase, reverse transcriptase ribonuclease H, and DNA polymerase functions. Their antiviral activities were evaluated and 2-hydroxy-4-methoxycarbonyl-isoquinoline-1,3(2H,4H)-dione was found to inhibit the viral replication of HIV-1 in MT-4 cells. Cross-resistance was measured for this compound on three different viral strains. Experimental data suggest that the antiviral activity of 2-hydroxy-4-methoxycarbonylisoquinoline-1,3(2H,4H)-dione is probably due to the RNase H inhibition.
Asunto(s)
Quelantes/síntesis química , Complejos de Coordinación/síntesis química , Inhibidores de Integrasa VIH/síntesis química , VIH-1/efectos de los fármacos , Isoquinolinas/síntesis química , Magnesio , Ribonucleasa H del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Dominio Catalítico , Línea Celular , Quelantes/química , Quelantes/farmacología , Complejos de Coordinación/química , Complejos de Coordinación/farmacología , Farmacorresistencia Viral , Espectroscopía de Resonancia por Spin del Electrón , Inhibidores de Integrasa VIH/química , Inhibidores de Integrasa VIH/farmacología , VIH-1/enzimología , Humanos , Isomerismo , Isoquinolinas/química , Isoquinolinas/farmacología , Espectroscopía de Resonancia Magnética , Ribonucleasa H del Virus de la Inmunodeficiencia Humana/metabolismo , Espectrofotometría Infrarroja , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacosRESUMEN
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.
Asunto(s)
Proteínas Cromosómicas no Histona/fisiología , Integrasa de VIH/fisiología , Nucleosomas/metabolismo , Nucleosomas/virología , Factores de Transcripción/fisiología , Integración Viral/fisiología , Animales , Transformación Celular Viral/genética , Células Cultivadas , Ensamble y Desensamble de Cromatina/fisiología , Proteínas Cromosómicas no Histona/metabolismo , Eficiencia , Regulación Viral de la Expresión Génica , Infecciones por VIH/genética , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , Integrasa de VIH/metabolismo , Células HeLa , Humanos , Modelos Biológicos , Estabilidad Proteica , Spodoptera , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
We report herein the synthesis of a series of fifteen 2-hydroxyisoquinoline-1,3(2H,4H)-dione derivatives. Alkyl and arylalkyl groups were introduced on position 4 of the basis scaffold. All the compounds presented poor inhibitory properties against HIV-1 reverse transcriptase ribonuclease H (RNase H). Four compounds inhibited HIV-1 integrase at a low micromolar level. A docking study using the later crystallographic data available for PFV integrase allowed us to explain the slightly improved integrase inhibitory activities of 4-pentyl and 4-(3-phenylpropyl)-2-hydroxyisoquinoline-1,3(2H,4H)-diones, when compared to the basis scaffold. Physicochemical studies were consistent with 1:1 and 1:2 (metal/ligand) stoichiometries of the magnesium complexes in solution. Unfortunately all tested compounds exhibited high cellular cytotoxicity in cell culture which limited their applications as antiviral agents. However these identified integrase inhibitors provide a very good basis for the development of new hits.
Asunto(s)
Inhibidores de Integrasa VIH/síntesis química , Inhibidores de Integrasa VIH/farmacología , Integrasa de VIH/metabolismo , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Isoquinolinas/síntesis química , Isoquinolinas/farmacología , Alquilación , Cristalografía por Rayos X , Inhibidores de Integrasa VIH/química , Isoquinolinas/química , Modelos Moleculares , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Resistance to HIV-1 integrase (IN) inhibitor raltegravir (RAL), is encoded by mutations in the IN region of the pol gene. The emergence of the N155H mutation was replaced by a pattern including the Y143R/C/H mutations in three patients with anti-HIV treatment failure. Cloning analysis of the IN gene showed an independent selection of the mutations at loci 155 and 143. Characterization of the phenotypic evolution showed that the switch from N155H to Y143C/R was linked to an increase in resistance to RAL. Wild-type (WT) IN and IN with mutations Y143C or Y143R were assayed in vitro in 3'end-processing, strand transfer and concerted integration assays. Activities of mutants were moderately impaired for 3'end-processing and severely affected for strand transfer. Concerted integration assay demonstrated a decrease in mutant activities using an uncleaved substrate. With 3'end-processing assay, IC(50) were 0.4 microM, 0.9 microM (FC = 2.25) and 1.2 microM (FC = 3) for WT, IN Y143C and IN Y143R, respectively. An FC of 2 was observed only for IN Y143R in the strand transfer assay. In concerted integration, integrases were less sensitive to RAL than in ST or 3'P but mutants were more resistant to RAL than WT.