RESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections initiate in the bronchi of the upper respiratory tract and are able to disseminate to the lower respiratory tract, where infections can cause an acute respiratory distress syndrome with a high degree of mortality in elderly patients. We used reconstituted primary bronchial epithelia from adult and child donors to follow the SARS-CoV-2 infection dynamics. We show that, in epithelia from adult donors, infections initiate in multiciliated cells and spread within 24 to 48 h throughout the whole epithelia. Syncytia formed of ciliated and basal cells appeared at the apical side of the epithelia within 3 to 4 d and were released into the apical lumen, where they contributed to the transmittable virus dose. A small number of reconstituted epithelia were intrinsically more resistant to virus infection, limiting virus spread to different degrees. This phenotype was more frequent in epithelia derived from children versus adults and correlated with an accelerated release of type III interferon. Treatment of permissive adult epithelia with exogenous type III interferon restricted infection, while type III interferon gene knockout promoted infection. Furthermore, a transcript analysis revealed that the inflammatory response was specifically attenuated in children. Taken together, our findings suggest that apical syncytia formation is an underappreciated source of virus propagation for tissue or environmental dissemination, whereas a robust type III interferon response such as commonly seen in young donors restricted SARS-CoV-2 infection. Thus, the combination of interferon restriction and attenuated inflammatory response in children might explain the epidemiological observation of age-related susceptibility to COVID-19.
Asunto(s)
Bronquios , COVID-19 , Células Gigantes , Interferones , Mucosa Respiratoria , SARS-CoV-2 , Anciano , Bronquios/inmunología , Bronquios/virología , COVID-19/inmunología , COVID-19/virología , Niño , Susceptibilidad a Enfermedades , Células Gigantes/inmunología , Células Gigantes/virología , Humanos , Interferones/inmunología , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/virología , SARS-CoV-2/inmunología , Interferón lambdaRESUMEN
G-quadruplexes (G4s) are four-stranded nucleic acid structures formed by the stacking of G-tetrads. Here we investigated their formation and function during HIV-1 infection. Using bioinformatics and biophysics analyses we first searched for evolutionary conserved G4-forming sequences in HIV-1 genome. We identified 10 G4s with conservation rates higher than those of HIV-1 regulatory sequences such as RRE and TAR. We then used porphyrin-based G4-binders to probe the formation of the G4s during infection of human cells by native HIV-1. The G4-binders efficiently inhibited HIV-1 infectivity, which is attributed to the formation of G4 structures during HIV-1 replication. Using a qRT-PCR approach, we showed that the formation of viral G4s occurs during the first 2 h post-infection and their stabilization by the G4-binders prevents initiation of reverse transcription. We also used a G4-RNA pull-down approach, based on a G4-specific biotinylated probe, to allow the direct detection and identification of viral G4-RNA in infected cells. Most of the detected G4-RNAs contain crucial regulatory elements such as the PPT and cPPT sequences as well as the U3 region. Hence, these G4s would function in the early stages of infection when the viral RNA genome is being processed for the reverse transcription step.
Asunto(s)
G-Cuádruplex , VIH-1 , Humanos , ARN/química , VIH-1/genética , Secuencias Reguladoras de Ácidos Nucleicos , Secuencia ConservadaRESUMEN
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
Currently, an increasing number of drugs are becoming available to clinics for the treatment of 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 concerns. Accordingly, it is important to continue to discover small molecules that have a novel mechanism of inhibition. In this work, HIV integrase inhibitors were selected by high-throughput screening. Chemical structure comparisons enabled the identification of stilbene disulfonic acids as a potential new chemotype. Biochemical characterization of the lead compound stilbenavir (NSC34931) and a few derivatives was performed. Stilbene disulfonic acid derivatives exhibit low to sub-micromolar antiviral activity, and they inhibit integrase through DNA-binding inhibition. They probably bind to the C-terminal domain of integrase, in the cavity normally occupied by the noncleaved strand of the viral DNA substrate. Because of this original mode of action compared to active site strand transfer inhibitors, they do not exhibit cross-resistance to the three main resistance pathways to integrase inhibitors (G140S-Q148H, N155H, and Y143R). Further structure-activity optimization should enable the development of more active and less toxic derivatives with potential clinical relevance.
Asunto(s)
Infecciones por VIH/tratamiento farmacológico , Inhibidores de Integrasa VIH/química , Integrasa de VIH/genética , VIH/efectos de los fármacos , Antivirales/química , Antivirales/farmacología , Dominio Catalítico/efectos de los fármacos , Farmacorresistencia Viral , VIH/enzimología , VIH/patogenicidad , Infecciones por VIH/enzimología , Infecciones por VIH/virología , Inhibidores de Integrasa VIH/farmacología , Ensayos Analíticos de Alto Rendimiento , Humanos , Mutación , Replicación Viral/efectos de los fármacosRESUMEN
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
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
Several RNA interactions are thought to play a role in the regulation of the hepatitis C virus (HCV) life cycle. Most of these interactions involve the 5BSL3.2 domain and therefore occur at the 3' end of the viral genomic RNA. A long-range interaction has also been described between 5BSL3.2 and the 5' untranslated region (UTR). Another interaction involves the SLVI stem loop of the core coding region and the 5'UTR. We aimed to analyse the role of this SLVI domain, which likely interferes with others interactions. By evaluating RNA stability, translation and RNA synthesis, we showed that the SLVI stem loop extensively modulates the effect of the interactions mediated by the 5BSL3.2 domain and strongly affects the IIId/5BSL3.2 interaction. Numerous interactions in HCV genomic RNA have been described in the UTRs and the coding sequence but their roles are poorly understood. We showed that the SLVI domain located in the core coding sequence plays an important role in the translation of the polyprotein, but also in the modulation of long-range RNA interactions centred on the 5BSL3.2 domain. The SLVI domain has been absent from most studies, especially from the extensively used subgenomic replicon; our data highlight the importance of this domain in the studies of these long-range interactions in the HCV life cycle.
Asunto(s)
Regulación Viral de la Expresión Génica , Hepacivirus/genética , Conformación de Ácido Nucleico , ARN Viral/química , ARN Viral/genética , Proteínas del Núcleo Viral/genética , Emparejamiento Base , Biosíntesis de Proteínas , Estabilidad del ARN , ARN Viral/biosíntesis , Transcripción GenéticaRESUMEN
The impact of the amino-acid side-chain length on peptide-RNA binding events has been investigated using HIV-1 Tat derived peptides as ligands and the HIV-1 TAR RNA element as an RNA model. Our studies demonstrate that increasing the length of all peptide side-chains improves unexpectedly the binding affinity (KD) but reduces the degree of compactness of the peptide-RNA complex. Overall, the side-chain length appears to modulate in an unpredictable way the ability of the peptide to compete with the cognate TAR RNA partner. Beyond the establishment of non-intuitive fundamental relationships, our results open up new perspectives in the design of effective RNA ligand competitors, since a large number of them have already been identified but few studies report on the modulation of the biological activity by modifying in the same way the length of all chains connecting RNA recognition motives to the central scaffold of a ligand.
Asunto(s)
VIH-1/genética , Péptidos/metabolismo , ARN Viral/metabolismo , Secuencia de Aminoácidos , Duplicado del Terminal Largo de VIH/genética , Humanos , Simulación de Dinámica Molecular , Péptidos/química , Transición de Fase/efectos de la radiación , Unión Proteica , ARN Viral/química , Espectrofotometría Ultravioleta , Espectroscopía Infrarroja por Transformada de Fourier , Temperatura , Termodinámica , Rayos UltravioletaRESUMEN
G-rich nucleic acids can form non-canonical G-quadruplex structures (G4s) in which four guanines fold in a planar arrangement through Hoogsteen hydrogen bonds. Although many biochemical and structural studies have focused on DNA sequences containing successive, adjacent guanines that spontaneously fold into G4s, evidence for their in vivo relevance has recently begun to accumulate. Complete sequencing of the human genome highlighted the presence of â¼300,000 sequences that can potentially form G4s. Likewise, the presence of putative G4-sequences has been reported in various viruses genomes [e.g., Human immunodeficiency virus (HIV-1), Epstein-Barr virus (EBV), papillomavirus (HPV)]. Many studies have focused on telomeric G4s and how their dynamics are regulated to enable telomere synthesis. Moreover, a role for G4s has been proposed in cellular and viral replication, recombination and gene expression control. In parallel, DNA aptamers that form G4s have been described as inhibitors and diagnostic tools to detect viruses [e.g., hepatitis A virus (HAV), EBV, cauliflower mosaic virus (CaMV), severe acute respiratory syndrome virus (SARS), simian virus 40 (SV40)]. Here, special emphasis will be given to the possible role of these structures in a virus life cycle as well as the use of G4-forming oligonucleotides as potential antiviral agents and innovative tools.
Asunto(s)
Antivirales/química , ADN Viral/química , G-Cuádruplex , ARN Viral/química , Genoma Humano , HumanosRESUMEN
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
Nucleic acid sequences containing guanine tracts are able to adopt noncanonical four-stranded nucleic acid structures called G-quadruplexes (G4s). These structures are based on the stacking of two or more G-tetrads; each tetrad is a planar association of four guanines held together by eight hydrogen bonds. In this study, we analyzed a conserved G-rich region from HIV-1 promoter that is known to regulate the transcription of the HIV-1 provirus. Strikingly, our analysis of an alignment of 1684 HIV-1 sequences from this region showed a high conservation of the ability to form G4 structures despite a lower conservation of the nucleotide primary sequence. Using NMR spectroscopy, we determined the G4 topology adopted by a DNA sequence from this region (HIV-PRO1: 5' TGGCCTGGGCGGGACTGGG 3'). This DNA fragment formed a stable two G-tetrad antiparallel G4 with an additional Watson-Crick CG base pair. This hybrid structure may be critical for HIV-1 gene expression and is potentially a novel target for anti-HIV-1 drug development.
Asunto(s)
Acridinas/farmacología , Fármacos Anti-VIH/farmacología , ADN Viral/efectos de los fármacos , G-Cuádruplex/efectos de los fármacos , VIH-1/efectos de los fármacos , VIH-1/genética , Regiones Promotoras Genéticas/efectos de los fármacos , Acridinas/síntesis química , Acridinas/química , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/química , ADN Viral/química , ADN Viral/genética , VIH-1/química , Ligandos , Regiones Promotoras Genéticas/genética , Relación Estructura-ActividadRESUMEN
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
Nucleic acid sequences containing guanine tracts are able to form non-canonical DNA or RNA structures known as G-quadruplexes (or G4s). These structures, based on the stacking of G-tetrads, are involved in various biological processes such as gene expression regulation. Here, we investigated a G4 forming sequence, HIVpro2, derived from the HIV-1 promoter. This motif is located 60 nucleotides upstream of the proviral Transcription Starting Site (TSS) and overlaps with two SP1 transcription factor binding sites. Using NMR spectroscopy, we determined that HIVpro2 forms a hybrid type G4 structure with a core that is interrupted by a single nucleotide bulge. An additional reverse-Hoogsteen AT base pair is stacked on top of the tetrad. SP1 transcription factor is known to regulate transcription activity of many genes through the recognition of Guanine-rich duplex motifs. Here, the formation of HIVpro2 G4 may modulate SP1 binding sites architecture by competing with the formation of the canonical duplex structure. Such DNA structural switch potentially participates to the regulation of viral transcription and may also interfere with HIV-1 reactivation or viral latency.
Asunto(s)
G-Cuádruplex , VIH-1 , Factor de Transcripción Sp1 , Sitios de Unión , ADN/química , Guanina/química , VIH-1/genética , VIH-1/metabolismo , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp1/metabolismo , Humanos , Regulación Viral de la Expresión GénicaRESUMEN
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
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
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
Despite being simple eukaryotic organisms, the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe have been widely used as a model to study human pathologies and the replication of human, animal, and plant viruses, as well as the function of individual viral proteins. The complete genome of S. cerevisiae was the first of eukaryotic origin to be sequenced and contains about 6,000 genes. More than 75% of the genes have an assigned function, while more than 40% share conserved sequences with known or predicted human genes. This strong homology has allowed the function of human orthologs to be unveiled starting from the data obtained in yeast. RNA plant viruses were the first to be studied in yeast. In this paper, we focus on the use of the yeast model to study the function of the proteins of human immunodeficiency virus type 1 (HIV-1) and the search for its cellular partners. This human retrovirus is the cause of AIDS. The WHO estimates that there are 33.4 million people worldwide living with HIV/AIDS, with 2.7 million new HIV infections per year and 2.0 million annual deaths due to AIDS. Current therapy is able to control the disease but there is no permanent cure or a vaccine. By using yeast, it is possible to dissect the function of some HIV-1 proteins and discover new cellular factors common to this simple cell and humans that may become potential therapeutic targets, leading to a long-lasting treatment for AIDS.
Asunto(s)
VIH-1/fisiología , Levaduras/fisiología , Animales , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiología , VIH-1/genética , Humanos , Proteínas Virales/genética , Proteínas Virales/fisiología , Levaduras/genéticaRESUMEN
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
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
IgG abzymes (Abzs) with different catalytic activities are a distinctive feature of various autoimmune (AI) diseases. At the same time, data concerning IgMs with catalytic activities are very limited. Electrophoretically and immunologically homogeneous IgMs were isolated from the sera of acquired immunodeficiency syndrome (AIDS) patients by chromatography on several affinity sorbents. Several rigid criteria have been applied to show that the integrase (IN)-hydrolyzing activity is an intrinsic property of IgMs from HIV-infected patients but not from healthy donors. We present evidence showing that 22 of 24 (91.7%) IgMs purified from the sera of HIV-infected patients specifically hydrolyze only HIV IN but not many other tested proteins. Usually, proteolytic antibodies of AI patients are serine protease-like or metal dependent. Only 30% of IN-hydrolyzing IgMs were inhibited by specific inhibitors of serine proteases and 60% by inhibitors of metal-dependent proteases. Unusually, a significant reduction of the activity by specific inhibitors of acidic (in 20% of IgM preparations) and thiol proteases (in 100% of IgM preparations) was observed. Although HIV infection leads to formation of antibodies to many viral and human antigens, possible biological roles for most of them are unknown. Since anti-IN IgG can efficiently hydrolyze IN, a positive role of Abzs in counteracting the infection cannot be excluded. In addition, detection of IN-hydrolyzing activity can be useful for diagnostic purposes and for assessment of the immune status in AIDS patients.