RESUMEN
This review summarizes current advances in the role of transcriptional stochasticity in HIV-1 latency, which were possible in a large part due to the development of single-cell approaches. HIV-1 transcription proceeds in bursts of RNA production, which stem from the stochastic switching of the viral promoter between ON and OFF states. This switching is caused by random binding dynamics of transcription factors and nucleosomes to the viral promoter and occurs at several time scales from minutes to hours. Transcriptional bursts are mainly controlled by the core transcription factors TBP, SP1 and NF-κb, the chromatin status of the viral promoter and RNA polymerase II pausing. In particular, spontaneous variability in the promoter chromatin creates heterogeneity in the response to activators such as TNF-α, which is then amplified by the Tat feedback loop to generate high and low viral transcriptional states. This phenomenon is likely at the basis of the partial and stochastic response of latent T cells from HIV-1 patients to latency-reversing agents, which is a barrier for the development of shock-and-kill strategies of viral eradication. A detailed understanding of the transcriptional stochasticity of HIV-1 and the possibility to precisely model this phenomenon will be important assets to develop more effective therapeutic strategies.
Asunto(s)
Seropositividad para VIH , VIH-1 , Humanos , VIH-1/genética , Cromatina , FN-kappa B , NucleosomasRESUMEN
Within the virion, adenovirus DNA associates with the virus-encoded, protamine-like structural protein pVII. Whether this association is organized, and how genome packaging changes during infection and subsequent transcriptional activation is currently unclear. Here, we combined RNA-seq, MNase-seq, ChIP-seq, and single genome imaging during early adenovirus infection to unveil the structure- and time-resolved dynamics of viral chromatin changes as well as their correlation with gene transcription. Our MNase mapping data indicates that the adenoviral genome is arranged in precisely positioned nucleoprotein particles with nucleosome-like characteristics, that we term adenosomes. We identified 238 adenosomes that are positioned by a DNA sequence code and protect about 60-70 bp of DNA. The incoming adenoviral genome is more accessible at early gene loci that undergo additional chromatin de-condensation upon infection. Histone H3.3 containing nucleosomes specifically replaces pVII at distinct genomic sites and at the transcription start sites of early genes. Acetylation of H3.3 is predominant at the transcription start sites and precedes transcriptional activation. Based on our results, we propose a central role for the viral pVII nucleoprotein architecture, which is required for the dynamic structural changes during early infection, including the regulation of nucleosome assembly prior to transcription initiation. Our study thus may aid the rational development of recombinant adenoviral vectors exhibiting sustained expression in gene therapy.
Asunto(s)
Cromatina , Nucleosomas , Nucleosomas/genética , Activación Transcripcional , Cromatina/genética , ADN/metabolismo , Ensamble y Desensamble de Cromatina , Adenoviridae/genéticaRESUMEN
Promoter-proximal pausing of RNA polymerase II is a key process regulating gene expression. In latent HIV-1 cells, it prevents viral transcription and is essential for latency maintenance, while in acutely infected cells the viral factor Tat releases paused polymerase to induce viral expression. Pausing is fundamental for HIV-1, but how it contributes to bursting and stochastic viral reactivation is unclear. Here, we performed single molecule imaging of HIV-1 transcription. We developed a quantitative analysis method that manages multiple time scales from seconds to days and that rapidly fits many models of promoter dynamics. We found that RNA polymerases enter a long-lived pause at latent HIV-1 promoters (>20 minutes), thereby effectively limiting viral transcription. Surprisingly and in contrast to current models, pausing appears stochastic and not obligatory, with only a small fraction of the polymerases undergoing long-lived pausing in absence of Tat. One consequence of stochastic pausing is that HIV-1 transcription occurs in bursts in latent cells, thereby facilitating latency exit and providing a rationale for the stochasticity of viral rebounds.
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Regulación Viral de la Expresión Génica , Infecciones por VIH/genética , VIH-1/genética , Regiones Promotoras Genéticas/genética , Latencia del Virus/genética , Algoritmos , ARN Polimerasas Dirigidas por ADN/metabolismo , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , VIH-1/fisiología , Células HeLa , Humanos , Modelos Genéticos , Procesos Estocásticos , Factores de Tiempo , Activación Viral/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Transport of mRNAs is an important step of gene expression, which brings the genetic message from the DNA in the nucleus to a precise cytoplasmic location in a regulated fashion. Perturbation of this process can lead to pathologies such as developmental and neurological disorders. In this review, we discuss recent advances in the field of mRNA transport made using single molecule fluorescent imaging approaches. We present an overview of these approaches in fixed and live cells and their input in understanding the key steps of mRNA journey: transport across the nucleoplasm, export through the nuclear pores and delivery to its final cytoplasmic location. This review puts a particular emphasis on the coupling of mRNA transport with translation, such as localization-dependent translational regulation and translation-dependent mRNA localization. We also highlight the recently discovered translation factories, and how cellular and viral RNAs can hijack membrane transport systems to travel in the cytoplasm.
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Núcleo Celular/metabolismo , Citoplasma/metabolismo , Biosíntesis de Proteínas , Transporte de ARN , ARN Mensajero/metabolismo , Transcripción Genética , Animales , Núcleo Celular/genética , Citoplasma/genética , Humanos , ARN Mensajero/genéticaRESUMEN
In eukaryotes, trimethylation of lysine 9 on histone H3 (H3K9) is associated with transcriptional silencing of transposable elements (TEs). In drosophila ovaries, this heterochromatic repressive mark is thought to be deposited by SetDB1 on TE genomic loci after the initial recognition of nascent transcripts by PIWI-interacting RNAs (piRNAs) loaded on the Piwi protein. Here, we show that the nucleosome remodeler Mi-2, in complex with its partner MEP-1, forms a subunit that is transiently associated, in a MEP-1 C-terminus-dependent manner, with known Piwi interactors, including a recently reported SUMO ligase, Su(var)2-10. Together with the histone deacetylase Rpd3, this module is involved in the piRNA-dependent TE silencing, correlated with H3K9 deacetylation and trimethylation. Therefore, drosophila piRNA-mediated transcriptional silencing involves three epigenetic effectors, a remodeler, Mi-2, an eraser, Rpd3 and a writer, SetDB1, in addition to the Su(var)2-10 SUMO ligase.
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Adenosina Trifosfatasas/metabolismo , Autoantígenos/metabolismo , Proteínas de Drosophila/metabolismo , Heterocromatina/química , Histona Desacetilasa 1/metabolismo , Nucleosomas/metabolismo , ARN Interferente Pequeño/metabolismo , Animales , Proteínas Argonautas/metabolismo , Drosophila melanogaster , Epigénesis Genética , Femenino , Regulación de la Expresión Génica , Silenciador del Gen , Histonas/química , Ovario/metabolismo , Proteínas Inhibidoras de STAT ActivadosRESUMEN
mRNA transport restricts translation to specific subcellular locations, which is the basis for many cellular functions. However, the precise process of mRNA sorting to synapses in neurons remains elusive. Here we use Rgs4 mRNA to investigate 3'-UTR-dependent transport by MS2 live-cell imaging. The majority of observed RNA granules display 3'-UTR independent bidirectional transport in dendrites. Importantly, the Rgs4 3'-UTR causes an anterograde transport bias, which requires the Staufen2 protein. Moreover, the 3'-UTR mediates dynamic, sustained mRNA recruitment to synapses. Visualization at high temporal resolution enables us to show mRNA patrolling dendrites, allowing transient interaction with multiple synapses, in agreement with the sushi-belt model. Modulation of neuronal activity by either chemical silencing or local glutamate uncaging regulates both the 3'-UTR-dependent transport bias and synaptic recruitment. This dynamic and reversible mRNA recruitment to active synapses would allow translation and synaptic remodeling in a spatially and temporally adaptive manner.
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Regiones no Traducidas 3'/genética , Dendritas/genética , Hipocampo/metabolismo , Transporte de ARN/fisiología , ARN Mensajero/genética , Sinapsis/metabolismo , Animales , Línea Celular , Células HEK293 , Humanos , Proteínas RGS/genética , Proteínas de Unión al ARN/genética , Ratas , Ratas Sprague-DawleyRESUMEN
RNA polymerase (RNAP) is, in its elongation phase, an emblematic example of a molecular motor whose activity is highly sensitive to DNA supercoiling. After a review of DNA supercoiling basic features, we discuss how supercoiling controls polymerase velocity, while being itself modified by polymerase activity. This coupling is supported by single-molecule measurements. Physical modeling allows us to describe quantitatively how supercoiling and torsional constraints mediate a mechanical coupling between adjacent polymerases. On this basis, we obtain a description that may explain the existence and functioning of RNAP convoys.
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ADN Superhelicoidal/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Fenómenos Biomecánicos , Imagen Individual de Molécula , TorqueRESUMEN
Translation is an essential step in gene expression. In this study, we used an improved SunTag system to label nascent proteins and image translation of single messenger ribonucleoproteins (mRNPs) in human cells. Using a dedicated reporter RNA, we observe that translation of single mRNPs stochastically turns on and off while they diffuse through the cytoplasm. We further measure a ribosome density of 1.3 per kilobase and an elongation rate of 13-18 amino acids per second. Tagging the endogenous POLR2A gene revealed similar elongation rates and ribosomal densities and that nearly all messenger RNAs (mRNAs) are engaged in translation. Remarkably, tagging of the heavy chain of dynein 1 (DYNC1H1) shows this mRNA accumulates in foci containing three to seven RNA molecules. These foci are translation sites and thus represent specialized translation factories. We also observe that DYNC1H1 polysomes are actively transported by motors, which may deliver the mature protein at appropriate cellular locations. The SunTag should be broadly applicable to study translational regulation in live single cells.
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Dineínas Citoplasmáticas/biosíntesis , Microscopía Confocal , Polirribosomas/metabolismo , ARN Polimerasa II/biosíntesis , Ribonucleoproteínas/metabolismo , Citoplasma/metabolismo , Dineínas Citoplasmáticas/genética , Difusión , Recuperación de Fluorescencia tras Fotoblanqueo , Células HeLa , Humanos , Procesamiento de Imagen Asistido por Computador , Extensión de la Cadena Peptídica de Translación , Polirribosomas/genética , Transporte de Proteínas , ARN Polimerasa II/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Ribonucleoproteínas/genética , Factores de Tiempo , TransfecciónRESUMEN
Live-cell imaging has revealed unexpected features of gene expression. Here using improved single-molecule RNA microscopy, we show that synthesis of HIV-1 RNA is achieved by groups of closely spaced polymerases, termed convoys, as opposed to single isolated enzymes. Convoys arise by a Mediator-dependent reinitiation mechanism, which generates a transient but rapid succession of polymerases initiating and escaping the promoter. During elongation, polymerases are spaced by few hundred nucleotides, and physical modelling suggests that DNA torsional stress may maintain polymerase spacing. We additionally observe that the HIV-1 promoter displays stochastic fluctuations on two time scales, which we refer to as multi-scale bursting. Each time scale is regulated independently: Mediator controls minute-scale fluctuation (convoys), while TBP-TATA-box interaction controls sub-hour fluctuations (long permissive/non-permissive periods). A cellular promoter also produces polymerase convoys and displays multi-scale bursting. We propose that slow, TBP-dependent fluctuations are important for phenotypic variability of single cells.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Imagen Individual de Molécula/métodos , Transcripción Genética , Secuencia de Bases , Supervivencia Celular , Productos del Gen tat , VIH-1/genética , Células HeLa , Humanos , Cinética , Modelos Biológicos , Regiones Promotoras Genéticas/genética , ARN/metabolismo , TATA Box/genética , Proteína de Unión a TATA-Box/metabolismoRESUMEN
Dimerization is a unique and vital characteristic of retroviral genomes. It is commonly accepted that genomic RNA (gRNA) must be dimeric at the plasma membrane of the infected cells to be packaged during virus assembly. However, where, when and how HIV-1 gRNA find each other and dimerize in the cell are long-standing questions that cannot be answered using conventional approaches. Here, we combine two state-of-the-art, multicolor RNA labeling strategies with two single-molecule microscopy technologies to address these questions. We used 3D-super-resolution structured illumination microscopy to analyze and quantify the spatial gRNA association throughout the cell and monitored the dynamics of RNA-RNA complexes in living-cells by cross-correlation fluctuation analysis. These sensitive and complementary approaches, combined with trans-complementation experiments, reveal for the first time the presence of interacting gRNA in the cytosol, a challenging observation due to the low frequency of these events and their dilution among the bulk of other RNAs, and allow the determination of the subcellular orchestration of the HIV-1 dimerization process.
Asunto(s)
Dimerización , VIH-1/genética , Imagenología Tridimensional , Microscopía/métodos , Conformación de Ácido Nucleico , ARN Viral/química , Membrana Celular/metabolismo , Supervivencia Celular , Color , Citosol/metabolismo , Genoma Viral , Células HeLa , Humanos , Hibridación Fluorescente in Situ , ARN Viral/genética , Coloración y Etiquetado , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
The human LINE-1 retrotransposon has the ability to mobilize into a new genomic location through an intracellular replication cycle. Immunofluorescence and in situ hybridization experiments have been developed to detect subcellular localization of retrotransposition intermediates (i.e., ORF1p, ORF2p, and L1 mRNA). Currently, these protocols are also used to validate the interaction between retrotransposition complex components and potential cellular partners involved in L1 replication. Here, we describe in details methods for the identification of LINE-1 proteins and/or RNA in cells transfected with vectors expressing engineered human LINE-1 elements.
Asunto(s)
Elementos de Nucleótido Esparcido Largo , Sistemas de Lectura Abierta , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcripción Genética , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Ingeniería Genética , Humanos , Hibridación Fluorescente in Situ , Espacio Intracelular/metabolismo , Transporte de ProteínasRESUMEN
BACKGROUND: Current therapies have succeeded in controlling AIDS pandemic. However, there is a continuing need for new drugs, in particular those acting through new and as yet unexplored mechanisms of action to achieve HIV infection cure. We took advantage of the unique feature of proviral genome to require both activation and inhibition of splicing of viral transcripts to develop molecules capable of achieving long lasting effect on viral replication in humanized mouse models through inhibition of Rev-mediated viral RNA biogenesis. RESULTS: Current HIV therapies reduce viral load during treatment but titers rebound after treatment is discontinued. We devised a new drug that has a long lasting effect after viral load reduction. We demonstrate here that ABX464 compromises HIV replication of clinical isolates of different subtypes without selecting for drug resistance in PBMCs or macrophages. ABX464 alone, also efficiently compromised viral proliferation in two humanized mouse models infected with HIV that require a combination of 3TC, Raltegravir and Tenofovir (HAART) to achieve viral inhibition in current protocols. Crucially, while viral load increased dramatically just one week after stopping HAART treatment, only slight rebound was observed following treatment cessation with ABX464 and the magnitude of the rebound was maintained below to that of HAART for two months after stopping the treatment. Using a system to visualize single HIV RNA molecules in living cells, we show that ABX464 inhibits viral replication by preventing Rev-mediated export of unspliced HIV-1 transcripts to the cytoplasm and by interacting with the Cap Binding Complex (CBC). Deep sequencing of viral RNA from treated cells established that retained viral RNA is massively spliced but importantly, normal cellular splicing is unaffected by the drug. Consistently ABX464 is non-toxic in humans and therefore represents a promising complement to current HIV therapies. CONCLUSIONS: ABX464 represents a novel class of anti-HIV molecules with unique properties. ABX464 has a long lasting effect in humanized mice and neutralizes the expression of HIV-1 proviral genome of infected immune cells including reservoirs and it is therefore a promising drug toward a functional cure of HIV.
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Fármacos Anti-VIH/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , VIH-1/aislamiento & purificación , Carga Viral , Adulto , Animales , Fármacos Anti-VIH/farmacología , Modelos Animales de Enfermedad , VIH-1/efectos de los fármacos , Humanos , Ratones SCID , Replicación Viral/efectos de los fármacosRESUMEN
The breakage-fusion-bridge cycle is a classical mechanism of telomere-driven genome instability in which dysfunctional telomeres are fused to other chromosomal extremities, creating dicentric chromosomes that eventually break at mitosis. Here, we uncover a distinct pathway of telomere-driven genome instability, specifically occurring in cells that maintain telomeres with the alternative lengthening of telomeres mechanism. We show that, in these cells, telomeric DNA is added to multiple discrete sites throughout the genome, corresponding to regions regulated by NR2C/F transcription factors. These proteins drive local telomere DNA addition by recruiting telomeric chromatin. This mechanism, which we name targeted telomere insertion (TTI), generates potential common fragile sites that destabilize the genome. We propose that TTI driven by NR2C/F proteins contributes to the formation of complex karyotypes in ALT tumors.
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Inestabilidad Genómica , Neoplasias/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Telómero/metabolismo , Cromosomas Humanos/metabolismo , Roturas del ADN de Doble Cadena , Humanos , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Translocación GenéticaRESUMEN
The HIV-1 Rev protein mediates export of unspliced and singly spliced viral transcripts by binding to the Rev response element (RRE) and recruiting the cellular export factor CRM1. Here, we investigated the recruitment of Rev to the transcription sites of HIV-1 reporters that splice either post- or cotranscriptionally. In both cases, we observed that Rev localized to the transcription sites of the reporters and recruited CRM1. Rev and CRM1 remained at the reporter transcription sites when cells were treated with the splicing inhibitor Spliceostatin A (SSA), showing that the proteins associate with RNA prior to or during early spliceosome assembly. Fluorescence recovery after photobleaching (FRAP) revealed that Rev and CRM1 have similar kinetics as the HIV-1 RNA, indicating that Rev, CRM1, and RRE-containing RNAs are released from the site of transcription in one single export complex. These results suggest that cotranscriptional formation of a stable export complex serves as a means to ensure efficient export of unspliced viral RNAs.
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VIH-1/metabolismo , Carioferinas/metabolismo , Complejos Multiproteicos/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Transcripción Genética/fisiología , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/metabolismo , Empalme Alternativo/fisiología , Sitios de Unión , Células Cultivadas , VIH-1/genética , Humanos , Complejos Multiproteicos/genética , Unión Proteica , Multimerización de Proteína , Estabilidad Proteica , ARN Viral/genética , ARN Viral/metabolismo , Proteína Exportina 1Asunto(s)
Procesamiento de Imagen Asistido por Computador/métodos , Hibridación Fluorescente in Situ/métodos , ARN Mensajero/metabolismo , Transcripción Genética , Actinas , Simulación por Computador , Imagenología Tridimensional/métodos , Modelos Biológicos , Subunidades de Proteína , ARN Polimerasa II/química , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , ARN Mensajero/genética , Reproducibilidad de los ResultadosRESUMEN
Splicing is a key process that expands the coding capacity of genomes. Its kinetics remain poorly characterized, and the distribution of splicing time caused by the stochasticity of single splicing events is expected to affect regulation efficiency. We conducted a small-scale survey on 40 introns in human cells and observed that most were spliced cotranscriptionally. Consequently, we constructed a reporter system that splices cotranscriptionally and can be monitored in live cells and in real time through the use of MS2-GFP. All small nuclear ribonucleoproteins (snRNPs) are loaded on nascent pre-mRNAs, and spliceostatin A inhibits splicing but not snRNP recruitment. Intron removal occurs in minutes and is best described by a model where several successive steps are rate limiting. Each pre-mRNA molecule is predicted to require a similar time to splice, reducing kinetic noise and improving the regulation of alternative splicing. This model is relevant to other kinetically controlled processes acting on few molecules.
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Empalme Alternativo/genética , Modelos Biológicos , Imagen Molecular/métodos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcripción Genética , Línea Celular Tumoral , Humanos , Cinética , Ribonucleoproteína Nuclear Pequeña U1/metabolismoRESUMEN
HIV-1 Gag can assemble and generate virions at the plasma membrane, but it is also present in endosomes where its role remains incompletely characterized. Here, we show that HIV-1 RNAs and Gag are transported on endosomal vesicles positive for TiVamp, a v-SNARE involved in fusion events with the plasma membrane. Inhibition of endosomal traffic did not prevent viral release. However, inhibiting lysosomal degradation induced an accumulation of Gag in endosomes and increased viral production 7-fold, indicating that transport of Gag to lysosomes negatively regulates budding. This also suggested that endosomal Gag-RNA complexes could access retrograde pathways to the cell surface and indeed, depleting cells of TiVamp-reduced viral production. Moreover, inhibition of endosomal transport prevented the accumulation of Gag at sites of cellular contact. HIV-1 Gag could thus generate virions using two pathways, either directly from the plasma membrane or through an endosome-dependent route. Endosomal Gag-RNA complexes may be delivered at specific sites to facilitate cell-to-cell viral transmission.
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Endosomas/metabolismo , VIH-1/metabolismo , ARN Viral/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Transporte Biológico/efectos de los fármacos , Western Blotting , Cloruro de Calcio/farmacología , Línea Celular Tumoral , Cloroquina/farmacología , Endosomas/ultraestructura , Endosomas/virología , Técnica del Anticuerpo Fluorescente , VIH-1/genética , VIH-1/aislamiento & purificación , Humanos , Hibridación Fluorescente in Situ , Ionóforos/farmacología , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Electrónica , Monensina/farmacología , Nocodazol/farmacología , Transporte de ARN/efectos de los fármacos , ARN Interferente Pequeño/genética , ARN Viral/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Factores de Tiempo , Transfección , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
RNA polymerase II (RNAPII) is a fundamental enzyme, but few studies have analyzed its activity in living cells. Using human immunodeficiency virus (HIV) type 1 reporters, we study real-time messenger RNA (mRNA) biogenesis by photobleaching nascent RNAs and RNAPII at specific transcription sites. Through modeling, the use of mutant polymerases, drugs, and quantitative in situ hybridization, we investigate the kinetics of the HIV-1 transcription cycle. Initiation appears efficient because most polymerases demonstrate stable gene association. We calculate an elongation rate of approximately 1.9 kb/min, and, surprisingly, polymerases remain at transcription sites 2.5 min longer than nascent RNAs. With a total polymerase residency time estimated at 333 s, 114 are assigned to elongation, and 63 are assigned to 3'-end processing and/or transcript release. However, mRNAs were released seconds after polyadenylation onset, and analysis of polymerase density by chromatin immunoprecipitation suggests that they pause or lose processivity after passing the polyA site. The strengths and limitations of this kinetic approach to analyze mRNA biogenesis in living cells are discussed.
Asunto(s)
Regulación Viral de la Expresión Génica , VIH-1/genética , Transcripción Genética , Línea Celular Tumoral , Supervivencia Celular , Simulación por Computador , Recuperación de Fluorescencia tras Fotoblanqueo , Genes Reporteros , Humanos , Hibridación in Situ , Cinética , Modelos Genéticos , Mutación/genética , Fotoblanqueo , Poliadenilación , Procesamiento de Término de ARN 3' , ARN Polimerasa II/metabolismo , ARN Mensajero/biosíntesis , ARN Viral/biosíntesis , Factores de TiempoRESUMEN
HIV-1 transcription is tightly regulated: silent in long-term latency and highly active in acutely-infected cells. Transcription is activated by the viral protein Tat, which recruits the elongation factor P-TEFb by binding the TAR sequence present in nascent HIV-1 RNAs. In this study, we analyzed the dynamic of the TAR:Tat:P-TEFb complex in living cells, by performing FRAP experiments at HIV-1 transcription sites. Our results indicate that a large fraction of Tat present at these sites is recruited by Cyclin T1. We found that in the presence of Tat, Cdk9 remained bound to nascent HIV-1 RNAs for 71s. In contrast, when transcription was activated by PMA/ionomycin, in the absence of Tat, Cdk9 turned-over rapidly and resided on the HIV-1 promoter for only 11s. Thus, the mechanism of trans-activation determines the residency time of P-TEFb at the HIV-1 gene, possibly explaining why Tat is such a potent transcriptional activator. In addition, we observed that Tat occupied HIV-1 transcription sites for 55s, suggesting that the TAR:Tat:P-TEFb complex dissociates from the polymerase following transcription initiation, and undergoes subsequent cycles of association/dissociation.
Asunto(s)
Productos del Gen tat/metabolismo , Duplicado del Terminal Largo de VIH , VIH-1/genética , Factores de Elongación de Péptidos/metabolismo , ARN Viral/metabolismo , Transcripción Genética , Línea Celular Tumoral , Quinasa 9 Dependiente de la Ciclina/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo , VIH-1/fisiología , Humanos , Cinética , Unión Proteica , Activación Transcripcional , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
Retroviral assembly is driven by Gag, and nascent viral particles escape cells by recruiting the machinery that forms intralumenal vesicles of multivesicular bodies. In this study, we show that the clathrin adaptor complex AP-1 is involved in retroviral release. The absence of AP-1mu obtained by genetic knock-out or by RNA interference reduces budding of murine leukemia virus (MLV) and HIV-1, leading to a delay of viral propagation in cell culture. In contrast, overexpression of AP-1mu enhances release of HIV-1 Gag. We show that the AP-1 complex facilitates retroviral budding through a direct interaction between the matrix and AP-1mu. Less MLV Gag is found associated with late endosomes in cells lacking AP-1, and our results suggest that AP-1 and AP-3 could function on the same pathway that leads to Gag release. In addition, we find that AP-1 interacts with Tsg101 and Nedd4.1, two cellular proteins known to be involved in HIV-1 and MLV budding. We propose that AP-1 promotes Gag release by transporting it to intracellular sites of active budding, and/or by facilitating its interactions with other cellular partners.