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1.
Nature ; 565(7740): 500-504, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30626973

RESUMEN

In mammals, 2'-O-methylation of RNA is a molecular signature by which the cellular innate immune system distinguishes endogenous from exogenous messenger RNA1-3. However, the molecular functions of RNA 2'-O-methylation are not well understood. Here we have purified TAR RNA-binding protein (TRBP) and its interacting partners and identified a DICER-independent TRBP complex containing FTSJ3, a putative 2'-O-methyltransferase (2'O-MTase). In vitro and ex vivo experiments show that FTSJ3 is a 2'O-MTase that is recruited to HIV RNA through TRBP. Using RiboMethSeq analysis4, we identified predominantly FTSJ3-dependent 2'-O-methylations at specific residues on the viral genome. HIV-1 viruses produced in FTSJ3 knockdown cells show reduced 2'-O-methylation and trigger expression of type 1 interferons (IFNs) in human dendritic cells through the RNA sensor MDA5. This induction of IFN-α and IFN-ß leads to a reduction in HIV expression. We have identified an unexpected mechanism used by HIV-1 to evade innate immune recognition: the recruitment of the TRBP-FTSJ3 complex to viral RNA and its 2'-O-methylation.


Asunto(s)
VIH-1/inmunología , VIH-1/patogenicidad , Inmunidad Innata , Metiltransferasas/metabolismo , ARN Helicasas DEAD-box/metabolismo , Células Dendríticas/inmunología , VIH-1/genética , Células HeLa , Humanos , Interferón Tipo I/biosíntesis , Interferón Tipo I/inmunología , Helicasa Inducida por Interferón IFIH1/metabolismo , Metilación , Metiltransferasas/antagonistas & inhibidores , Metiltransferasas/deficiencia , Unión Proteica , ARN Viral/química , ARN Viral/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleasa III/metabolismo
2.
Mol Cell ; 67(3): 387-399.e5, 2017 Aug 03.
Artículo en Inglés | MEDLINE | ID: mdl-28712728

RESUMEN

The DNA-mediated innate immune response underpins anti-microbial defenses and certain autoimmune diseases. Here we used immunoprecipitation, mass spectrometry, and RNA sequencing to identify a ribonuclear complex built around HEXIM1 and the long non-coding RNA NEAT1 that we dubbed the HEXIM1-DNA-PK-paraspeckle components-ribonucleoprotein complex (HDP-RNP). The HDP-RNP contains DNA-PK subunits (DNAPKc, Ku70, and Ku80) and paraspeckle proteins (SFPQ, NONO, PSPC1, RBM14, and MATRIN3). We show that binding of HEXIM1 to NEAT1 is required for its assembly. We further demonstrate that the HDP-RNP is required for the innate immune response to foreign DNA, through the cGAS-STING-IRF3 pathway. The HDP-RNP interacts with cGAS and its partner PQBP1, and their interaction is remodeled by foreign DNA. Remodeling leads to the release of paraspeckle proteins, recruitment of STING, and activation of DNAPKc and IRF3. Our study establishes the HDP-RNP as a key nuclear regulator of DNA-mediated activation of innate immune response through the cGAS-STING pathway.


Asunto(s)
ADN/inmunología , Herpesvirus Humano 8/inmunología , Inmunidad Innata , ARN Largo no Codificante/inmunología , Proteínas de Unión al ARN/inmunología , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/inmunología , Proteínas de Unión al Calcio/metabolismo , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN , Células HEK293 , Células HeLa , Interacciones Huésped-Patógeno , Células Endoteliales de la Vena Umbilical Humana/inmunología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/virología , Humanos , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/inmunología , Factor 3 Regulador del Interferón/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/inmunología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Autoantígeno Ku/genética , Autoantígeno Ku/inmunología , Autoantígeno Ku/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Complejos Multiproteicos , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas Asociadas a Matriz Nuclear/inmunología , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/inmunología , Proteínas Nucleares/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/inmunología , Nucleotidiltransferasas/metabolismo , Factores de Transcripción de Octámeros/genética , Factores de Transcripción de Octámeros/inmunología , Factores de Transcripción de Octámeros/metabolismo , Factor de Empalme Asociado a PTB/genética , Factor de Empalme Asociado a PTB/inmunología , Factor de Empalme Asociado a PTB/metabolismo , Unión Proteica , Interferencia de ARN , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Transducción de Señal , Factores de Transcripción , Transfección
3.
Nucleic Acids Res ; 37(19): 6575-86, 2009 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-19729508

RESUMEN

Small non-coding RNAs of 18-25 nt in length can regulate gene expression through the RNA interference (RNAi) pathway. To characterize small RNAs in HIV-1-infected cells, we performed linker-ligated cloning followed by high-throughput pyrosequencing. Here, we report the composition of small RNAs in HIV-1 productively infected MT4 T-cells. We identified several HIV-1 small RNA clones and a highly abundant small 18-nt RNA that is antisense to the HIV-1 primer-binding site (PBS). This 18-nt RNA apparently originated from the dsRNA hybrid formed by the HIV-1 PBS and the 3' end of the human cellular tRNAlys3. It was found to associate with the Ago2 protein, suggesting its possible function in the cellular RNAi machinery for targeting HIV-1.


Asunto(s)
VIH-1/genética , ARN Bicatenario/metabolismo , ARN no Traducido/química , ARN Viral/metabolismo , Línea Celular , VIH-1/fisiología , Humanos , ARN de Transferencia de Lisina/metabolismo , ARN no Traducido/metabolismo , Análisis de Secuencia de ARN , Linfocitos T/virología , Replicación Viral
4.
J Clin Invest ; 130(7): 3777-3790, 2020 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-32240121

RESUMEN

Hidradenitis suppurativa (HS) is a chronic, relapsing, inflammatory skin disease. HS appears to be a primary abnormality in the pilosebaceous-apocrine unit. In this work, we characterized hair follicle stem cells (HFSCs) isolated from HS patients and more precisely the outer root sheath cells (ORSCs). We showed that hair follicle cells from HS patients had an increased number of proliferating progenitor cells and lost quiescent stem cells. Remarkably, we also showed that the progression of replication forks was altered in ORSCs from hair follicles of HS patients, leading to activation of the ATR/CHK1 pathway. These alterations were associated with an increased number of micronuclei and with the presence of cytoplasmic ssDNA, leading to the activation of the IFI16/STING pathway and the production of type I IFNs. This mechanistic analysis of the etiology of HS in the HFSC compartment establishes a formal link between genetic predisposition and skin inflammation observed in HS.


Asunto(s)
Daño del ADN , Replicación del ADN , Folículo Piloso/metabolismo , Hidradenitis Supurativa/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Células Madre/metabolismo , Adolescente , Adulto , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Femenino , Folículo Piloso/patología , Hidradenitis Supurativa/patología , Humanos , Inflamación/metabolismo , Inflamación/patología , Masculino , Persona de Mediana Edad , Células Madre/patología
5.
Retrovirology ; 6: 26, 2009 Mar 09.
Artículo en Inglés | MEDLINE | ID: mdl-19272132

RESUMEN

The rate of HIV-1 gene expression is a key step that determines the kinetics of virus spread and AIDS progression. Viral entry and gene expression were described to be the key determinants for cell permissiveness to HIV. Recent reports highlighted the involvement of miRNA in regulating HIV-1 replication post-transcriptionally. In this study we explored the role of cellular factors required for miRNA-mediated mRNA translational inhibition in regulating HIV-1 gene expression. Here we show that HIV-1 mRNAs associate and co-localize with components of the RNA Induced Silencing Complex (RISC), and we characterize some of the proteins required for miRNA-mediated silencing (miRNA effectors). RCK/p54, GW182, LSm-1 and XRN1 negatively regulate HIV-1 gene expression by preventing viral mRNA association with polysomes. Interestingly, knockdown of RCK/p54 or DGCR8 resulted in virus reactivation in PBMCs isolated from HIV infected patients treated with suppressive HAART.


Asunto(s)
Regulación Viral de la Expresión Génica , VIH-1/fisiología , MicroARNs/metabolismo , ARN Viral/metabolismo , Replicación Viral/fisiología , Proteínas Argonautas , Línea Celular , Células Cultivadas , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Polirribosomas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Interferencia de ARN/fisiología , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Latencia del Virus/fisiología
6.
Eur J Cell Biol ; 87(12): 947-62, 2008 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-18760861

RESUMEN

The human immunodeficiency virus (HIV) transactivating Tat protein is not only critical for viral replication but also affects the host immune system by inducing the production of cytokines such as IL-10. This anti-inflammatory cytokine is upregulated during the course of HIV infection, representing an important pathway by which HIV may induce immunodeficiency. Here, we show that, by acting at the membrane, Tat induces IL-10 expression in primary monocytes and promonocytic U937 cells by NF-kappaB-dependent pathways. The trans-dominant negative mutants of NF-kappaB-inducing kinase (NIK), IKKalpha and IKKbeta expressed in our transactivation model, in accordance with the nuclear binding of p65 and p52 NF-kappaB subunits to the IL-10 promoter, suggest the involvement of both classical and alternative NF-kappaB pathways. In inactivated cells, IKKalpha is localized predominantly in the cytoplasm. Interestingly, Tat stimulates IKKalpha translocation from the cytoplasm to the nucleus in monocytes. Chromatin immunoprecipitation (ChIP) assay experiments, after Tat treatment, revealed IKKalpha and CBP/p300 recruitment to the IL-10 promoter and histone H3 phosphorylation (Ser 10) and acetylation (Lys 14) in this region, presumably leading to chromatin remodeling. We demonstrate that, upstream of NF-kappaB, PKC, ERK1/2 and p38 MAP kinases are involved in Tat-induced IKKalpha nuclear translocation and histone H3 modifications on the IL-10 promoter in accordance with the role of these three kinases in IL-10 production. As a whole, the study demonstrates that Tat activates at least three signaling pathways concurrently, including the classical, alternative and IKKalpha pathways, to promote production of IL-10.


Asunto(s)
VIH-1 , Interleucina-10/biosíntesis , Monocitos/metabolismo , FN-kappa B/metabolismo , Transducción de Señal , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Acetilación , Núcleo Celular/metabolismo , Células Cultivadas , Inmunoprecipitación de Cromatina , Citoplasma/metabolismo , Histonas/metabolismo , Humanos , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , FN-kappa B/genética , Fosforilación , Regiones Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de Proteínas , Transcripción Genética , Factores de Transcripción p300-CBP/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Quinasa de Factor Nuclear kappa B
7.
Cell Immunol ; 253(1-2): 45-53, 2008.
Artículo en Inglés | MEDLINE | ID: mdl-18541226

RESUMEN

HIV-1 Tat protein stimulates the production of both TNF-alpha and IL-10 in human monocytes. Taking into account the ability of TNF-alpha to induce IL-10 production, we evaluated the link between Tat, TNF-alpha and IL-10 and the implication of PKC and p38 MAP kinase pathways. Our data showed that (i) in the presence of neutralizing anti-TNF-alpha antibodies, IL-10 production is only partially inhibited; (ii) in a calcium-free medium, while TNF-alpha production is totally inhibited, Tat continues to induce IL-10; (iii) under these conditions, Tat-mediated IL-10 production is associated with PKC-delta activation; and (iv) downstream of PKC, p38 MAP kinase is crucial for TNF-alpha independent IL10 production. Overall, our data suggest a new mechanism, implicating Tat protein, by which HIV-1 may maintain a constant production of the immunosuppressive IL-10 cytokine, even in the absence of TNF-alpha production. In consequence, HIV-1 may escape immune surveillance and thus promote the establishment of an immunosuppressive state.


Asunto(s)
Interleucina-10/inmunología , Monocitos/inmunología , Proteína Quinasa C-delta/metabolismo , Factor de Necrosis Tumoral alfa/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/inmunología , Calcio/metabolismo , Activación Enzimática , Humanos , Isoenzimas/metabolismo , Monocitos/citología , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Proteína Quinasa C beta , Proteína Quinasa C-delta/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal/fisiología , Fracciones Subcelulares/enzimología , Factor de Necrosis Tumoral alfa/genética , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética
8.
Curr Med Chem ; 14(2): 191-7, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-17266578

RESUMEN

MicroRNAs (miRNAs) are non-coding small RNAs that play important roles in a variety of biological pathways including cellular proliferation and apoptosis. Recent studies have linked the expression of selected miRNAs to carcinogenesis and viral pathogenesis. Here, we will discuss examples of roles served by cellular miRNAs and virus-encoded miRNAs in the development of cancers and viral diseases.


Asunto(s)
MicroARNs/fisiología , Neoplasias/etiología , Virosis/etiología , Apoptosis , Proliferación Celular , Humanos , Neoplasias/genética , Virosis/genética
9.
BioDrugs ; 21(1): 17-22, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-17263586

RESUMEN

Inside eukaryotic cells, small RNA duplexes, called small interfering RNAs (siRNAs), activate a conserved RNA interference (RNAi) pathway which leads to specific degradation of complementary target mRNAs through base-pairing recognition. As with other viruses, studies have shown that replication of the HIV-1 in cultured cells can be targeted and inhibited by synthetic siRNAs. The relative ease of siRNA design and the versatility of RNAi to target a broad spectrum of mRNAs have led to the promise that drug discovery in the RNAi pathway could be effective against pathogens. This review discusses the current experimental principles that guide the application of RNAi against HIV and describes challenges and limitations that need to be surmounted in order for siRNAs to become practical antiviral drugs. The practical use of RNAi therapy for HIV infection will depend on overcoming several challenges, including the ability to establish long-term expression of siRNA without off-target effects and the capacity to counteract mutant escape viruses.


Asunto(s)
Infecciones por VIH/tratamiento farmacológico , ARN Interferente Pequeño/uso terapéutico , Humanos , Interferencia de ARN , ARN Interferente Pequeño/efectos adversos , ARN Interferente Pequeño/farmacología , Replicación Viral/efectos de los fármacos
10.
Retrovirology ; 3: 95, 2006 Dec 20.
Artículo en Inglés | MEDLINE | ID: mdl-17181864

RESUMEN

Dicer is an RNase III which processes two classes of cellular small RNAs: the microRNAs (miRNA) and short interfering RNAs (siRNA). Previously, we observed that over-expressed HIV-1 Tat protein can suppress the processing of small RNAs inside cells. Here, we have investigated the requirements for Tat interaction with Dicer. We report that Tat-Dicer interaction depends on RNA, requires the helicase domain of Dicer, and is independent of Tat's transactivation domain.


Asunto(s)
Productos del Gen tat/metabolismo , VIH-1/metabolismo , ARN Viral/metabolismo , Ribonucleasa III/metabolismo , Proteínas Argonautas , Factor 2 Eucariótico de Iniciación , Productos del Gen tat/genética , VIH-1/enzimología , VIH-1/genética , Humanos , MicroARNs/genética , Factores de Iniciación de Péptidos/genética , Estructura Terciaria de Proteína , ARN Interferente Pequeño/genética , ARN Viral/genética , Complejo Silenciador Inducido por ARN/genética , Ribonucleasa III/genética , Activación Transcripcional , Productos del Gen tat del Virus de la Inmunodeficiencia Humana
11.
Methods Mol Biol ; 342: 241-53, 2006.
Artículo en Inglés | MEDLINE | ID: mdl-16957379

RESUMEN

Initially reported for Caenorhabditis elegans, microRNA (miRNA) has been shown to regulate gene expression in plants, flies, and mammals . Here, we report on our approaches to investigate the role of miRNAs in human immunodeficiency virus (HIV)-1 infection. Using computer-directed foldings, we first identify potential sequences in HIV-1 that putatively encode miRNAs. Subsequently, we use Northern blotting of RNAs isolated from HIV-infected cells to confirm expression of predicted miRNA sequences. Finally, we use a scanning algorithm to search 3' untranslated regions (UTRs) of human messenger RNAs (mRNAs) in the attempt to predict potential sites targeted by HIV-1 miRNAs.


Asunto(s)
Infecciones por VIH/virología , VIH-1/genética , MicroARNs/genética , Algoritmos , Biología Computacional/métodos , Humanos , MicroARNs/química , MicroARNs/metabolismo , Análisis por Micromatrices/métodos
12.
Retrovirology ; 2: 81, 2005 Dec 28.
Artículo en Inglés | MEDLINE | ID: mdl-16381609

RESUMEN

MicroRNAs (miRNAs) are small RNAs of 18-25 nucleotides (nt) in length that play important roles in regulating a variety of biological processes. Recent studies suggest that cellular miRNAs may serve to control the replication of viruses in cells. If such is the case, viruses might be expected to evolve the ability to modulate the expression of cellular miRNAs. To ask if expression of HIV-1 genes changes the miRNA profiles in human cells, we employed a high throughput microarray method, termed the RNA-primed Array-based Klenow Enzyme (RAKE) assay. Here, we describe the optimization of this assay to quantify the expression of miRNAs in HIV-1 transfected human cells. We report distinct differences in miRNA profiles in mock-transfected HeLa cells versus HeLa cells transfected with an infectious HIV-1 molecular clone, pNL4-3.


Asunto(s)
Perfilación de la Expresión Génica , VIH-1/genética , MicroARNs/análisis , Células HeLa , Humanos , Reacción en Cadena de la Polimerasa , Transfección
13.
Cell Res ; 15(11-12): 935-46, 2005.
Artículo en Inglés | MEDLINE | ID: mdl-16354572

RESUMEN

Small interfering RNA (siRNA) and microRNA (miRNA) are small RNAs of 18-25 nucleotides (nt) in length that play important roles in regulating gene expression. They are incorporated into an RNA-induced silencing complex (RISC) and serve as guides for silencing their corresponding target mRNAs based on complementary base-pairing. The promise of gene silencing has led many researchers to consider siRNA as an anti-viral tool. However, in long-term settings, many viruses appear to escape from this therapeutical strategy. An example of this may be seen in the case of human immunodeficiency virus type-1 (HIV-1) which is able to evade RNA silencing by either mutating the siRNA-targeted sequence or by encoding for a partial suppressor of RNAi (RNA interference). On the other hand, because miRNA targeting does not require absolute complementarity of base-pairing, mutational escape by viruses from miRNA-specified silencing may be more difficult to achieve. In this review, we discuss stratagems used by various viruses to avoid the cells' antiviral si/mi-RNA defenses and notions of how viruses might control and regulate host cell genes by encoding viral miRNAs (vmiRNAs).


Asunto(s)
Infecciones por VIH/metabolismo , VIH/fisiología , MicroARNs/fisiología , ARN Interferente Pequeño/fisiología , ARN Viral/fisiología , VIH/genética , Infecciones por VIH/genética , Humanos , MicroARNs/aislamiento & purificación , ARN Interferente Pequeño/aislamiento & purificación , ARN Viral/aislamiento & purificación
14.
FASEB J ; 16(6): 546-54, 2002 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-11919157

RESUMEN

In HIV-infected patients, production of interleukin-10 (IL-10), a highly immunosuppressive cytokine, is associated with the disease progression toward AIDS. We have previously shown that HIV-1 Tat induces IL-10 production by human monocytes via a protein kinase C (PKC) -dependent pathway. Here we show that PKC activation by Tat is essential for IL-10 induction. Among the eight PKC isoforms present in human monocytes, we investigated which isoform(s) plays this crucial role in Tat-mediated IL-10 production and show that 1) Tat can activate PKC-alpha, PKC-betaII, PKC-delta, and PKC-epsilon, 2) of these four potential candidates, only PKC-betaII, PKC-delta, and PKC-epsilon are activated by the active domain Tat 1-45, which is responsible for IL-10 production and depleted by long-term exposure to PMA, which abolishes Tat-mediated IL-10 production, 3) whereas selective inhibition of PKC-alpha and PKC-epsilon by specific antisense oligonucleotides has no effect on Tat-mediated IL-10 induction, inhibition of either PKC-betaII or PKC-delta partially inhibits IL-10 production; and 4) the simultaneous inhibition of PKC-betaII and PKC-delta totally inhibits Tat-mediated IL-10. Altogether, these results suggest that the induction of IL-10 by Tat is strictly dependent on the PKC-delta and -betaII isoforms.


Asunto(s)
Productos del Gen tat/farmacología , VIH-1 , Interleucina-10/biosíntesis , Monocitos/inmunología , Proteína Quinasa C/metabolismo , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Cinética , Monocitos/efectos de los fármacos , Oligonucleótidos Antisentido/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C beta , Proteína Quinasa C-delta , Transporte de Proteínas , Productos del Gen tat del Virus de la Inmunodeficiencia Humana
15.
Mol Immunol ; 38(8): 607-18, 2002 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-11792429

RESUMEN

We have characterized the humoral and cellular immune responses of BALB/c mice immunized with HIV-1 Nef regulatory protein encapsulated in poly(DL-lactide-co-glycolide) PLG particles. Three groups of mice were immunized with Nef PLG, Nef in the presence of complete Freund's adjuvant (CFA) or Nef alone in PBS. When titers were compared 7 months after the last injection, anti-Nef titers in mice immunized with Nef PLG were still close to the maximum, whereas a significant decrease was observed in mice immunized with Nef alone (five times lower) or with Nef in CFA (three times lower). These results indicate that Nef PLG is at least a similar or better vector/adjuvant than Nef in CFA concerning the duration of the humoral immune response. The analysis of cytokine profiles (IL-5 and IL-10) and the isotypic patterns of anti-Nef antibodies (predominantly IgG1), in the three groups of mice, indicated a predominant Th2 immune response. Using synthetic peptides covering the entire sequence of Nef, we identified at least three linear epitopes within sequences 32-64, 118-167 and 185-205 in the sera of mice immunized with Nef PLG or Nef CFA. In contrast, anti-Nef antibodies against Nef alone failed to recognize synthetic peptides, indicating that the majority of anti-Nef antibodies were primarily directed against conformational epitopes. We then examined the ability of Nef PLG to prime for the antigen-specific proliferative responses in vitro. The data obtained indicate the presence of both B-cell and T-cell epitopes in the C-terminal fragment of the protein after immunization of mice with Nef encapsulated in PLG particles.


Asunto(s)
Productos del Gen nef/inmunología , Anticuerpos Anti-VIH/biosíntesis , Antígenos VIH/inmunología , VIH-1/inmunología , Ácido Láctico/administración & dosificación , Ácido Poliglicólico/administración & dosificación , Polímeros/administración & dosificación , Animales , Células Cultivadas , Citocinas/biosíntesis , Epítopos de Linfocito B/inmunología , Femenino , Adyuvante de Freund/farmacología , Productos del Gen nef/administración & dosificación , Antígenos VIH/administración & dosificación , Inmunización , Inmunoglobulina G/biosíntesis , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Microesferas , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Bazo/inmunología , Linfocitos T/inmunología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana
16.
Retrovirology ; 1: 43, 2004 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-15601472

RESUMEN

MicroRNAs (miRNAs) are small RNAs of 21-25 nucleotides that specifically regulate cellular gene expression at the post-transcriptional level. miRNAs are derived from the maturation by cellular RNases III of imperfect stem loop structures of ~ 70 nucleotides. Evidence for hundreds of miRNAs and their corresponding targets has been reported in the literature for plants, insects, invertebrate animals, and mammals. While not all of these miRNA/target pairs have been functionally verified, some clearly serve roles in regulating normal development and physiology. Recently, it has been queried whether the genome of human viruses like their cellular counterpart also encode miRNA. To date, there has been only one report pertaining to this question. The Epstein-Barr virus (EBV) has been shown to encode five miRNAs. Here, we extend the analysis of miRNA-encoding potential to the human immunodeficiency virus (HIV). Using computer-directed analyses, we found that HIV putatively encodes five candidate pre-miRNAs. We then matched deduced mature miRNA sequences from these 5 pre-miRNAs against a database of 3' untranslated sequences (UTR) from the human genome. These searches revealed a large number of cellular transcripts that could potentially be targeted by these viral miRNA (vmiRNA) sequences. We propose that HIV has evolved to use vmiRNAs as a means to regulate cellular milieu for its benefit.


Asunto(s)
VIH-1/genética , MicroARNs/genética , ARN Viral/genética , Regiones no Traducidas 3'/genética , Secuencia de Bases , Genoma Humano , Genoma Viral , Humanos , Procesamiento Postranscripcional del ARN
17.
Microbes Infect ; 6(13): 1182-90, 2004 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-15488737

RESUMEN

The effect of HIV-1 Tat protein on the production of IL-10, an immunosuppressive cytokine, was examined in human primary monocytes obtained from healthy HIV-1-negative blood donors. As expected and in agreement with our previous data, a dose-dependent induction of IL-10 was observed. In addition, we showed that this induction is mediated by the PKC pathway: in the presence of Ro 31-8220, an inhibitor of all PKC isozymes, or after 48 h of PMA treatment, Tat protein becomes unable to stimulate IL-10 production. Among the 11 PKC isozymes, eight (PKC alpha, beta(I), beta(II), delta, epsilon, eta, zeta, mu) are expressed in monocytes. In this study, by analyzing the translocation to the membrane after Tat stimulation, we showed that PKC alpha, beta(I), beta(II), delta and epsilon isozymes are activated by Tat. Moreover, by combining different approaches including selective PKC inhibitors (Gö6983, Gö6976, hispidin and rottlerin), we showed that PKC beta(II) and delta isozymes are essential for the activation of IL-10 production in human monocytes following stimulation by HIV-1 Tat protein.


Asunto(s)
Productos del Gen tat/metabolismo , VIH-1/patogenicidad , Interleucina-10/biosíntesis , Monocitos/inmunología , Acetofenonas/farmacología , Benzopiranos/farmacología , Carbazoles/farmacología , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica , Humanos , Indoles/farmacología , Maleimidas , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Proteína Quinasa C beta , Proteína Quinasa C-delta , Pironas/farmacología , Productos del Gen tat del Virus de la Inmunodeficiencia Humana
19.
Nat Struct Mol Biol ; 18(3): 323-7, 2011 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-21297638

RESUMEN

MicroRNAs (miRNAs) are a class of small, noncoding RNAs that function by regulating gene expression post-transcriptionally. Alterations in miRNA expression can strongly influence cellular physiology. Here we demonstrated cross-regulation between two components of the RNA interference (RNAi) machinery in human cells. Inhibition of exportin-5, the karyopherin responsible for pre-miRNA export, downregulated expression of Dicer, the RNase III required for pre-miRNA maturation. This effect was post-transcriptional and resulted from an increased nuclear localization of Dicer mRNA. In vitro assays and cellular RNA immunoprecipitation experiments showed that exportin-5 interacted directly with Dicer mRNA. Titration of exportin-5 by overexpression of either pre-miRNA or the adenoviral VA1 RNA resulted in loss of Dicer mRNA-exportin-5 interaction and reduction of Dicer level. This saturation also occurred during adenoviral infection and enhanced viral replication. Our study reveals an important cross-regulatory mechanism between pre-miRNA or viral small RNAs and Dicer through exportin-5.


Asunto(s)
ARN Helicasas DEAD-box/genética , Regulación de la Expresión Génica , Carioferinas/metabolismo , MicroARNs/metabolismo , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Ribonucleasa III/genética , Adenoviridae/genética , Adenoviridae/fisiología , Infecciones por Adenoviridae/virología , ARN Helicasas DEAD-box/metabolismo , Células HeLa , Humanos , Carioferinas/genética , MicroARNs/genética , Unión Proteica , Interferencia de ARN , ARN Mensajero/genética , ARN Viral/genética , Ribonucleasa III/metabolismo , Replicación Viral
20.
Cancer Res ; 68(21): 8976-85, 2008 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-18974142

RESUMEN

A role for microRNAs (miRNA) in human T-cell leukemia virus 1 (HTLV-1)-mediated cellular transformation has not been described. Here, we profiled miRNA expression in HTLV-1-transformed human T-cell lines and primary peripheral blood mononuclear cells from adult T-cell leukemia patients. Analyses of 11 different profiles revealed six miRNAs that were consistently up-regulated. Two of the up-regulated miRNAs (miR-93 and miR-130b) target the 3' untranslated region (3'UTR) of the mRNA for a tumor suppressor protein, tumor protein 53-induced nuclear protein 1 (TP53INP1). A low expression level of TP53INP1 protein was found in HTLV-1-transformed cells. Additionally, when antagomirs were used to knock down miR-93 and miR-130b in these cells, the expression of TP53INP1 was increased, suggesting that the latter is regulated inside cells by the former. A role for TP53INP1 in regulating cell growth was established by experiments that showed that enhanced TP53INP1 expression increased apoptosis. Collectively, the findings implicate a miR-93/miR-130b-TP53INP1 axis that affects the proliferation and survival of HTLV-1-infected/transformed cells.


Asunto(s)
Proteínas Portadoras/fisiología , División Celular/fisiología , Proteínas de Choque Térmico/fisiología , Virus Linfotrópico T Tipo 1 Humano/fisiología , MicroARNs/fisiología , Regiones no Traducidas 3' , Secuencia de Bases , Proteínas Portadoras/genética , Línea Celular Transformada , Cartilla de ADN , Proteínas de Choque Térmico/genética , Etiquetado Corte-Fin in Situ , Leucemia/genética , MicroARNs/genética , Reacción en Cadena de la Polimerasa
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