RESUMEN
During the integration step, human immunodeficiency virus type 1 integrase (IN) interacts with viral DNA and the cellular cofactor LEDGF/p75 to effectively integrate the reverse transcript into the host chromatin. Allosteric human immunodeficiency virus type 1 integrase inhibitors (ALLINIs) are a new class of antiviral agents that bind at the dimer interface of the IN catalytic core domain and occupy the binding site of LEDGF/p75. While originally designed to block IN-LEDGF/p75 interactions during viral integration, several of these compounds have been shown to also severely impact viral maturation through an IN multimerization mechanism. In this study, we tested the hypothesis that these dual properties of ALLINIs could be decoupled toward late stage viral replication effects by generating additional contact points between the bound ALLINI and a third subunit of IN. By sequential derivatization at position 7 of a quinoline-based ALLINI scaffold, we show that IN multimerization properties are enhanced by optimizing hydrophobic interactions between the compound and the C-terminal domain of the third IN subunit. These features not only improve the overall antiviral potencies of these compounds but also significantly shift the ALLINIs selectivity toward the viral maturation stage. Thus, we demonstrate that to fully maximize the potency of ALLINIs, the interactions between the inhibitor and all three IN subunits need to be simultaneously optimized.
Asunto(s)
Integrasa de VIH/metabolismo , VIH-1/metabolismo , Quinolinas/farmacología , Regulación Alostérica/efectos de los fármacos , Antivirales/farmacología , Células HEK293 , Integrasa de VIH/fisiología , Inhibidores de Integrasa VIH/metabolismo , Inhibidores de Integrasa VIH/farmacología , VIH-1/efectos de los fármacos , VIH-1/patogenicidad , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Modelos Moleculares , Unión Proteica/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Quinolinas/química , Quinolinas/metabolismo , Integración Viral/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
Currently, used antiretroviral HIV therapy drugs exclusively target critical groups in the enzymes essential for the viral life cycle. Increased mutagenesis of their genes changes these viral enzymes, which once mutated can evade therapeutic targeting, effects which confer drug resistance. To circumvent this, our review addresses a strategy to design and derive HIV-Integrase (HIV-IN) inhibitors which simultaneously target two IN functional domains, rendering it inactive even if the enzyme accumulates many mutations. First we review the enzymatic role of IN to insert the copied viral DNA into a chromosome of the host T lymphocyte, highlighting its main functional and structural features to be subjected to inhibitory action. From a functional and structural perspective we present all classes of HIV-IN inhibitors with their most representative candidates. For each chosen compound we also explain its mechanism of IN inhibition. We use the recently resolved cryo EM IN tetramer intasome DNA complex onto which we dock various reference IN inhibitory chemical scaffolds such as to target adjacent functional IN domains. Pairing compounds with complementary activity, which dock in the vicinity of a IN structural microdomain, we design bifunctional new drugs which may not only be more resilient to IN mutations but also may be more potent inhibitors than their original counterparts. In the end of our review we propose synthesis pathways to link such paired compounds with enhanced synergistic IN inhibitory effects.
Asunto(s)
Inhibidores de Integrasa VIH/farmacología , Integrasa de VIH/química , ADN/metabolismo , Diseño de Fármacos , Integrasa de VIH/metabolismo , Integrasa de VIH/fisiología , Inhibidores de Integrasa VIH/síntesis química , Inhibidores de Integrasa VIH/química , VIH-1/enzimología , Células HeLa , Humanos , Simulación del Acoplamiento Molecular , Unión Proteica/efectos de los fármacos , Dominios ProteicosRESUMEN
Integration is central to HIV-1 replication and helps mold the reservoir of cells that persists in AIDS patients. HIV-1 interacts with specific cellular factors to target integration to interior regions of transcriptionally active genes within gene-dense regions of chromatin. The viral capsid interacts with several proteins that are additionally implicated in virus nuclear import, including cleavage and polyadenylation specificity factor 6, to suppress integration into heterochromatin. The viral integrase protein interacts with transcriptional co-activator lens epithelium-derived growth factor p75 to principally position integration within gene bodies. The integrase additionally senses target DNA distortion and nucleotide sequence to help fine-tune the specific phosphodiester bonds that are cleaved at integration sites. Research into virus-host interactions that underlie HIV-1 integration targeting has aided the development of a novel class of integrase inhibitors and may help to improve the safety of viral-based gene therapy vectors.
Asunto(s)
Marcación de Gen , Integrasa de VIH/fisiología , VIH-1/fisiología , Integración Viral/fisiología , VIH-1/genética , Interacciones Huésped-Patógeno/genética , Humanos , Internalización del Virus , Latencia del Virus/fisiologíaRESUMEN
The bacterial defense system CRISPR (clustered regularly interspaced short palindromic repeats) has been explored as a powerful tool to edit genomic elements. In this study, we test the potential of CRISPR Csy4 RNA endoribonuclease for targeting HIV-1. We fused human codon-optimized Csy4 endoribonuclease with VPR, a HIV-1 viral preintegration complex protein. An HIV-1 cell model was modified to allow quantitative detection of active virus production. We found that the trans-expressing VPR-Csy4 almost completely blocked viral infection in two target cell lines (SupT1, Ghost). In the MAGI cell assay, where the HIV-1 LTR ß-galactosidase is expressed under the control of the tat gene from an integrated provirus, VPR-Csy4 significantly blocked the activity of the provirus-activated HIV-1 reporter. This proof-of-concept study demonstrates that Csy4 endoribonuclease is a promising tool that could be tailored further to target HIV-1.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Asociadas a CRISPR/genética , Endorribonucleasas/genética , Infecciones por VIH/prevención & control , VIH-1/fisiología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Células HEK293 , Integrasa de VIH/fisiología , Humanos , Ingeniería de Proteínas , Ensamble de Virus , Integración ViralRESUMEN
Multimeric HIV-1 integrase (IN) plays an essential, multifunctional role in virus replication and serves as an important therapeutic target. Structural and biochemical studies have revealed the importance of the ordered interplay between IN molecules for its function. In the presence of viral DNA ends, individual IN subunits assemble into a tetramer and form a stable synaptic complex (SSC), which mediates integration of the reverse transcribed HIV-1 genome into chromatin. Cellular chromatin-associated protein LEDGF/p75 engages the IN tetramer in the SSC and directs HIV-1 integration into active genes. A mechanism to deregulate the productive interplay between IN subunits with small molecule inhibitors has recently received considerable attention. Most notably, allosteric IN inhibitors (ALLINIs) have been shown to bind to the IN dimer interface at the LEDGF/p75 binding pocket, stabilize interacting IN subunits, and promote aberrant, higher order IN multimerization. Consequently, these compounds impair formation of the SSC and associated LEDGF/p75-independent IN catalytic activities as well as inhibit LEDGF/p75 binding to the SSC in vitro. However, in infected cells, ALLINIs more potently impaired correct maturation of virus particles than the integration step. ALLINI treatments induced aberrant, higher order IN multimerization in virions and resulted in eccentric, non-infectious virus particles. These studies have suggested that the correctly ordered IN structure is important for virus particle morphogenesis and highlighted IN multimerization as a plausible therapeutic target for developing new inhibitors to enhance treatment options for HIV-1-infected patients.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Inhibidores de Integrasa VIH/uso terapéutico , Integrasa de VIH/fisiología , Multimerización de Proteína/efectos de los fármacos , Integrasa de VIH/química , Humanos , Subunidades de ProteínaRESUMEN
HIV integrase (IN) catalyzes the insertion into the genome of the infected human cell of viral DNA produced by the retrotranscription process. The discovery of raltegravir validated the existence of the IN, which is a new target in the field of anti-HIV drug research. The mechanism of catalysis of IN is depicted, and the characteristics of the inhibitors of the catalytic site of this viral enzyme are reported. The role played by the resistance is elucidated, as well as the possibility of bypassing this problem. New approaches to block the integration process are depicted as future perspectives, such as development of allosteric IN inhibitors, dual inhibitors targeting both IN and other enzymes, inhibitors of enzymes that activate IN, activators of IN activity, as well as a gene therapy approach.
Asunto(s)
Integrasa de VIH/fisiología , VIH/fisiología , Regulación Alostérica , Animales , Dominio Catalítico , Farmacorresistencia Viral , Activadores de Enzimas/química , Activadores de Enzimas/farmacología , Activadores de Enzimas/uso terapéutico , Terapia Genética , VIH/efectos de los fármacos , Infecciones por VIH/terapia , Infecciones por VIH/virología , Integrasa de VIH/química , Integrasa de VIH/genética , Inhibidores de Integrasa VIH/química , Inhibidores de Integrasa VIH/farmacología , Inhibidores de Integrasa VIH/uso terapéutico , Humanos , Modelos Moleculares , Conformación Proteica , Multimerización de Proteína , Integración 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
Integrating viral vectors are efficient gene transfer tools, but their integration patterns have been associated with genotoxicity and oncogenicity. The recent development of highly specific designer nucleases has enabled target DNA modification and site-specific gene insertion at desired genomic loci. However, a lack of consensus exists regarding a perfect genomic safe harbour (GSH) that would allow transgenes to be stably and reliably expressed without adversely affecting endogenous gene structure and function. Ribosomal DNA (rDNA) has many advantages as a GSH, but efficient means to target integration to this locus are currently lacking. We tested whether lentivirus vector integration can be directed to rDNA by using fusion proteins consisting of the Human Immunodeficiency Virus 1 (HIV-1) integrase (IN) and the homing endonuclease I-PpoI, which has natural cleavage sites in the rDNA. A point mutation (N119A) was introduced into I-PpoI to abolish unwanted DNA cleavage by the endonuclease. The vector-incorporated IN-I-PpoIN119A fusion protein targeted integration into rDNA significantly more than unmodified lentivirus vectors, with an efficiency of 2.7%. Our findings show that IN-fusion proteins can be used to modify the integration pattern of lentivirus vectors, and to package site-specific DNA-recognizing proteins into vectors to obtain safer transgene integration.
Asunto(s)
ADN Ribosómico/genética , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Integrasa de VIH/genética , Mutagénesis Insercional/métodos , Proteínas Recombinantes de Fusión/genética , Clonación Molecular , Roturas del ADN de Doble Cadena , Desoxirribonucleasas de Localización Especificada Tipo II/biosíntesis , Desoxirribonucleasas de Localización Especificada Tipo II/fisiología , Vectores Genéticos , Células HEK293 , Integrasa de VIH/biosíntesis , Integrasa de VIH/fisiología , VIH-1/enzimología , Células HeLa , Humanos , Lentivirus/genética , Physarum polycephalum/enzimología , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/fisiología , Transducción GenéticaRESUMEN
HIV integrase became an important target for drug development more than twenty years ago. However, progress has been hampered by the lack of assays suitable for high throughput screening, a reliable crystal structure or pharmacophore. Thus, a real breakthrough was only observed in 2007 with the introduction of the first integrase inhibitor, raltegravir, into treatment. To date, the armament of integrase inhibitors is broad and covers several drugs from different classes that are under clinical trials. Among them, quinoline-based compounds and analogues occupy an important place. This review is focused on those compounds that have a quinoline scaffold and attempts to answer the question of whether quinoline is privileged for these activities. In fact, quinoline has been claimed as a privileged structure several times for different fields of activities. A closer look at its structural features may reveal the prerequisites responsible for the popularity of quinoline-based inhibitors of HIV integrase.
Asunto(s)
Inhibidores de Integrasa VIH/farmacología , Quinolonas/farmacología , Integrasa de VIH/química , Integrasa de VIH/fisiología , Inhibidores de Integrasa VIH/química , Quinolonas/química , Relación Estructura-ActividadRESUMEN
An attenuation of the HIV-1 replication capacity (RC) has been observed for immune-mediated escape mutations in Gag restricted by protective HLA alleles. However, the extent to which escape mutations affect other viral proteins during natural infection is not well understood. We generated recombinant viruses encoding plasma HIV-1 RNA integrase sequences from antiretroviral-naïve individuals with early (n = 88) and chronic (n = 304) infections and measured the in vitro RC of each. In contrast to data from previous studies of Gag, we observed little evidence that host HLA allele expression was associated with integrase RC. A modest negative correlation was observed between the number of HLA-B-associated integrase polymorphisms and RC in chronic infection (R = -0.2; P = 0.003); however, this effect was not driven by mutations restricted by protective HLA alleles. Notably, the integrase variants S119R, G163E, and I220L, which represent uncommon polymorphisms associated with HLA-C*05, -A*33, and -B*52, respectively, correlated with lower RC (all q < 0.2). We identified a novel C*05-restricted epitope (HTDNGSNF(114-121)) that likely contributes to the selection of the S119R variant, the polymorphism most significantly associated with lower RC in patient sequences. An NL4-3 mutant encoding the S119R polymorphism displayed a ~35%-reduced function that was rescued by a single compensatory mutation of A91E. Together, these data indicate that substantial HLA-driven attenuation of integrase is not a general phenomenon during HIV-1 adaptation to host immunity. However, uncommon polymorphisms selected by HLA alleles that are not conventionally regarded to be protective may be associated with impaired protein function. Vulnerable epitopes in integrase might therefore be considered for future vaccine strategies.
Asunto(s)
Infecciones por VIH/inmunología , Integrasa de VIH/fisiología , VIH-1/enzimología , Evasión Inmune , Replicación Viral , Estudios de Cohortes , Infecciones por VIH/genética , Infecciones por VIH/virología , Integrasa de VIH/genética , Integrasa de VIH/inmunología , VIH-1/genética , VIH-1/inmunología , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Datos de Secuencia MolecularRESUMEN
Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections including HIV-1. Despite more than two decades of HIV-1 research, there is still no efficacious HIV-1 vaccine, and the scientific community appears sceptical about the short or long-term feasibility of developing a vaccine that has the ability to induce sterilizing immunity against HIV-1. In this setting, microbicide research has been developed. Among the promising candidate microbicides, the integrase inhibitors are the most recently developed compounds. In fact, since the beginning reverse transcriptase, fusion and entry inhibitors were identified as possible HIV-specific candidate microbicides along with the non-specific topical microbicides. In the case of integrase inhibitors, only a few have demonstrated to block HIV-1 infection in models that mimic sexual transmission of the virus. These compounds have been tested in in vitro and ex vivo assays to determine their efficacy in pre- and/or post-exposure prophylactic settings. In particular, the naphthyridinecarboxyamide L-870,812 has been shown to block viral infection in pre- and post-exposure studies obtaining comparable results to the reverse transcriptase inhibitor PMPA. The purpose of this article is to provide an overview of integrase inhibitors as potential topical microbicides and their comparative evaluation with HIV-specific and non-specific microbicides.
Asunto(s)
Antiinfecciosos Locales/uso terapéutico , Infecciones por VIH/prevención & control , Inhibidores de Integrasa VIH/uso terapéutico , Administración Tópica , Ensayos Clínicos como Asunto , Infecciones por VIH/enzimología , Integrasa de VIH/química , Integrasa de VIH/fisiología , Inhibidores de Integrasa VIH/química , HumanosRESUMEN
Reverse transcription of retroviral RNA into double stranded DNA is a characteristic feature of rertoviruses including human immunodeficiency virus type I (HIV-1). There has been accumulating evidence for the involvement of retroviral integrase (IN) in the reverse transcription of viral RNA. Here, we summarized recent our studies demonstrating direct functional roles of IN and its binding partner of host factor, Gemin2 in the reverse transcription. We established new in vitro cell-free assay to mimic natural reverse transcription and found that HIV-1 IN and host factor, Gemin2 synergistically stimulate reverse transcriptase (RT) activity. Analysis of intracellular stability and multimer formation of IN suggest that that high-ordered structures, especially tetramer formation of IN is critical for the function. In addition, Gemin2 might have a role to keep the higher-order structure of IN. Thus, we provide new aspects of reverse transcription of HIV-1 through IN and host factors in addition to RT.
Asunto(s)
Genoma Viral/genética , Integrasa de VIH/fisiología , Transcriptasa Inversa del VIH/fisiología , VIH-1/enzimología , VIH-1/genética , Transcripción Reversa , Proteínas del Complejo SMN/fisiología , Sistema Libre de Células , ADN , ADN Viral , ARN Viral/genética , Replicación Viral/genéticaRESUMEN
The assembly mechanism for the human immunodeficiency virus type 1 (HIV) synaptic complex (SC) capable of concerted integration is unknown. Molecular and structural studies have established that the HIV SC and prototype foamy virus (PFV) intasome contain a tetramer of integrase (IN) that catalyzes concerted integration. HIV IN purified in the presence of 1 mM EDTA and 10 mM MgSO(4) was predominately a monomer. IN efficiently promoted concerted integration of micromolar concentrations of 3'-OH recessed and blunt-ended U5 long terminal repeat (LTR) oligonucleotide (ODN) substrates (19-42 bp) into circular target DNA. Varying HIV IN to U5 DNA showed that an IN dimer:DNA end molar ratio of 1 was optimal for concerted integration. Integration activities decreased with an increasing length of the ODN, starting from the recessed 18/20 or 19/21 bp set to the 31/33 and 40/42 bp set. Under these conditions, the average fidelity for the HIV 5 bp host site duplication with recessed and blunt-ended substrates was 56%. Modifications of U5 LTR sequences beyond 21 bp from the terminus on longer DNA (1.6 kb) did not alter the ~32 bp DNaseI protective footprint, suggesting viral sequences beyond 21 bp were not essential for IN binding. The results suggest IN binds differentially to an 18/20 bp than to a 40/42 bp ODN substrate for concerted integration. The HIV IN monomer may be a suitable candidate for attempting crystallization of an IN-DNA complex in the absence or presence of strand transfer inhibitors.
Asunto(s)
Integrasa de VIH/química , Integrasa de VIH/fisiología , Duplicado del Terminal Largo de VIH/fisiología , VIH-1/fisiología , Integración Viral/fisiología , Secuencia de Bases , Integrasa de VIH/genética , Duplicado del Terminal Largo de VIH/genética , VIH-1/genética , Humanos , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Integración Viral/genéticaRESUMEN
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
Raltegravir resistance is conferred by mutations at integrase codons 143, 148, and 155 together with associated secondary mutations. The N155H mutants emerge first, and are eventually replaced by Q148H mutants, usually in combination with G140S. These mutations have different effects on susceptibility and replication capacity, but data on the relative fitness of RAL-resistant viruses are limited. To understand the impact of the different RAL resistance pathways on viral fitness, mutations at integrase codons 74, 92, 138, 140, 148, 155, and/or 163 were introduced into HIV-1NL4-3 by site-directed mutagenesis and expressed in recombinant viruses. Relative fitness and drug susceptibility were determined in the absence or presence of RAL. In the absence of drug, RAL-resistant mutants were less fit than wild type, and the Q148H mutant was significantly less fit than the N155H mutant. Fitness was partially restored by the presence of additional RAL resistance mutations at positions G140S and E92Q or E138K, respectively. In the presence of RAL, the N155H mutant remained fitter than the Q148H mutant, but the G140S/Q148H double mutant was fitter than single mutants or the E92Q/N155H double mutant. These findings correspond well with the clinical trials data and help explain the temporal pattern of RAL resistance evolution.
Asunto(s)
Inhibidores de Integrasa VIH/farmacología , Integrasa de VIH/genética , VIH-1/genética , Pirrolidinonas/farmacología , Línea Celular , Farmacorresistencia Viral/genética , Farmacorresistencia Viral/fisiología , Integrasa de VIH/efectos de los fármacos , Integrasa de VIH/fisiología , VIH-1/crecimiento & desarrollo , VIH-1/fisiología , Humanos , Pruebas de Sensibilidad Microbiana , Mutagénesis Sitio-Dirigida , Mutación/efectos de los fármacos , Mutación/genética , Raltegravir PotásicoRESUMEN
An essential step in the life cycle of human immunodeficiency virus type 1 (HIV-1) is integration of the double-stranded retroviral DNA into the genome of the host cell. HIV-1 integrase, the enzyme that inserts the vital DNA into the host chromosome, is an attractive and rational target for anti-AIDS drug design because it is essential for HIV replication and there are no known counterparts in the host cell. Inhibitors of this enzyme have a great potential to complement the therapeutic use of HIV protease and reverse transcriptase inhibitors. Natural products have provided a source of new drug candidates for anti-AIDS therapy. Dicaffeoylquinic acids, isolated from traditional medicinal plants, are a novel class of integrase inhibitors. These compounds are potent inhibitors of HIV-1 replication in cultured cell lines and catalytic activities of integrase in vitro. They are therefore promising compounds for developing new anti-AIDS drugs. To understand how the inhibitors work and therefore design more potent and specific inhibitors, we have used molecular modeling techniques to investigate the binding modes of 3,4-dicaffeoylquinic acid. Our computational modeling study demonstrated that the inhibitor of this compound on HIV integrase is likely to proceed by two different but equivalent mechanisms with one bound to the active site region of the enzyme and another docked into the binding pocket located on the other side of the catalytic site. Our study will be of help to design new pharmaceuticals for the treatment of AIDS.
Asunto(s)
Integrasa de VIH/fisiología , VIH-1/fisiología , Inhibidores de Integrasa/farmacología , Ácido Quínico/análogos & derivados , Replicación Viral/efectos de los fármacos , Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Dominio Catalítico/efectos de los fármacos , Dominio Catalítico/fisiología , Biología Computacional , Diseño de Fármacos , Unión Proteica , Ácido Quínico/antagonistas & inhibidores , Relación Estructura-Actividad , Integración Viral/efectos de los fármacos , Replicación Viral/fisiologíaRESUMEN
After membrane fusion with a target cell, the core of human immunodeficiency virus type 1 (HIV-1) enters into the cytoplasm, where uncoating occurs. The cone-shaped core is composed of the viral capsid protein (CA), which disassembles during uncoating. The underlying factors and mechanisms governing uncoating are poorly understood. Several CA mutations can cause changes in core stability and a block at reverse transcription, demonstrating the requirement for optimal core stability during viral replication. HIV-1 integrase (IN) catalyzes the insertion of the viral cDNA into the host genome, and certain IN mutations are pleiotropic. Similar to some CA mutants, two IN mutants, one with a complete deletion of IN (NL-DeltaIN) and the other with a Cys-to-Ser substitution (NL-C130S), were noninfectious, with a replication block at reverse transcription. Compared to the wild type (WT), the cytoplasmic CA levels of the IN mutants in infected cells were reduced, suggesting accelerated uncoating. The role of IN during uncoating was examined by isolating and characterizing cores from NL-DeltaIN and NL-C130S. Both IN mutants could form functional cores, but the core yield and stability were decreased. Also, virion incorporation of cyclophilin A (CypA), a cellular peptidyl-prolyl isomerase that binds specifically to CA, was decreased in the IN mutants. Cores isolated from WT virus depleted of CypA had an unstable-core phenotype, confirming a role of CypA in promoting optimal core stability. Taken together, our results indicate that IN is required during uncoating for maintaining CypA-CA interaction, which promotes optimal stability of the viral core.
Asunto(s)
Ciclofilina A/metabolismo , Proteína p24 del Núcleo del VIH/metabolismo , Integrasa de VIH/fisiología , VIH-1/fisiología , Internalización del Virus , Replicación Viral , Sustitución de Aminoácidos/genética , Eliminación de Gen , Integrasa de VIH/genética , Humanos , Unión ProteicaRESUMEN
Long-standing evidence indicates that quiescent human peripheral blood T lymphocytes (PBLs) do not support efficient HIV infection. In resting PBLs, reverse transcription of viral RNA takes longer than in activated cells, partially because formation of the late products of reverse transcription is decreased by RNA binding by apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G). In a subsequent step, integration of the viral complementary DNA that is eventually formed is markedly impaired. Here we show that cellular c-Jun N-terminal kinase (JNK), an enzyme that is not expressed in resting CD4+ T cells, regulates permissiveness to HIV-1 infection, and we unravel a new, sequential post-translational pathway of protein modification that regulates viral DNA integration. We found that, in activated T lymphocytes, viral integrase, which mediates HIV-1 cDNA integration into the host cell genome, is phosphorylated by JNK on a highly conserved serine residue in its core domain. Phosphorylated integrase, in turn, becomes a substrate for the cellular peptidyl prolyl-isomerase enzyme Pin1, which catalyzes a conformational modification of integrase. These concerted activities increase integrase stability and are required for efficient HIV-1 integration and infection. Lack of these modifications restricts viral infection in nonactivated, primary CD4+ T lymphocytes.
Asunto(s)
Linfocitos T CD4-Positivos/virología , VIH-1/fisiología , Activación de Linfocitos/fisiología , MAP Quinasa Quinasa 4/fisiología , Isomerasa de Peptidilprolil/fisiología , Integración Viral/fisiología , Western Blotting , Células Cultivadas , ADN Viral/metabolismo , Infecciones por VIH/inmunología , Infecciones por VIH/virología , Integrasa de VIH/fisiología , Humanos , Peptidilprolil Isomerasa de Interacción con NIMA , Fosforilación , Reacción en Cadena de la PolimerasaRESUMEN
HIV-1 integrase (IN) is an essential enzyme for retroviral replication. There is no analogue for this enzyme in human cells so that inhibition of IN will not bring strong effect on human body. Thus, HIV-1 IN has become a rational target for therapy of AIDS. This review provides a comprehensive report of alpha, gamma-diketo IN inhibitors discovered in recent years. Compilation of such data will prove to be beneficial in developing QSAR, pharmacophore hypothesis generation and validation, virtual screening and synthesis of compounds with higher activity.
Asunto(s)
Fármacos Anti-VIH , Inhibidores de Integrasa VIH , VIH-1/efectos de los fármacos , Cetoácidos , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Integrasa de VIH/química , Integrasa de VIH/fisiología , Inhibidores de Integrasa VIH/síntesis química , Inhibidores de Integrasa VIH/química , Inhibidores de Integrasa VIH/farmacología , Humanos , Cetoácidos/síntesis química , Cetoácidos/química , Cetoácidos/farmacología , Estructura Molecular , Relación Estructura-Actividad CuantitativaRESUMEN
Significant advances have transpired in the human immunodeficiency virus type 1 (HIV-1) integration field in recent years. Considering its essential nature, integrase has long been a target of interest for antiviral drug development. The most significant advance was the approval of the Merck compound raltegravir, the first licensed integrase inhibitor, in October 2007. Another milestone was the identification and characterization of specific nucleoprotein complexes that mediate integrase 3' processing and DNA strand transfer activities in vitro. Genome-wide distribution analyses have furthermore revealed that different retroviruses differentially target distinctive regions of chromatin during integration. For examples, lentiviruses favor actively transcribed genes whereas gammaretroviruses such as Moloney murine leukemia virus prefer transcriptional start sites. Though the underlying mechanisms are unknown for most retroviruses, the lentiviral preference is in large part guided through the interaction with the integrase binding protein lens epithelium-derived growth factor (LEDGF)/p75. Experimental methods that formed the foundations for each of these advances, as well as other techniques topical to the study of HIV-1 integration, are described in this issue of Methods.