RESUMEN

The HER-2/Neu oncogene has been implicated in human and mouse breast cancer. Indeed, transgenic MMTV-neu mice expressing this oncogene from the mammary tumor virus long terminal repeat develop spontaneous mammary tumors and die within 1 year of life. We have expressed the class II transactivator (CIITA) and/or the costimulatory molecule CD80 (B7.1) in a mammary carcinoma cell line (MCNeuA) derived from these mice. Class II transactivator directs the expression of MHC class II and the machinery for antigen processing and presentation by this pathway. When injected into MMTV-neu mice, tumor cells expressing CD80 or CD80 and CIITA, were rejected completely. In addition, following the rejection of dual expressing cells, 75% of the mice were protected against the development of subsequent spontaneous tumors. Cells expressing only CD80 or CIITA were not as effective as antitumor vaccines in preventing the development of spontaneous tumors. Thus, converting cancer cells into antigen presenting cells could represent an effective immunotherapy for breast cancer.

Asunto(s)

Antígeno B7-1/genética , Genes MHC Clase II/genética , Neoplasias Mamarias Animales/inmunología , Virus del Tumor Mamario del Ratón/genética , Receptor ErbB-2/genética , Animales , Antígeno B7-1/inmunología , Antígeno B7-1/metabolismo , Western Blotting , Línea Celular , Línea Celular Tumoral , Femenino , Citometría de Flujo , Genes MHC Clase II/inmunología , Humanos , Inmunohistoquímica , Inmunoprecipitación , Linfocitos/inmunología , Neoplasias Mamarias Animales/genética , Neoplasias Mamarias Animales/patología , Ratones , Ratones Transgénicos , Proteínas Nucleares/genética , Proteínas Nucleares/inmunología , Proteínas Nucleares/metabolismo , Ratas , Receptor ErbB-2/inmunología , Receptor ErbB-2/metabolismo , Transactivadores/genética , Transactivadores/inmunología , Transactivadores/metabolismo

RESUMEN

Innate and adaptive immunity are connected via antigen processing and presentation (APP), which results in the presentation of antigenic peptides to T cells in the complex with the major histocompatibility (MHC) determinants. MHC class II (MHC II) determinants present antigens to CD4+ T cells, which are the main regulators of the immune response. Their genes are transcribed from compact promoters that form first the MHC II enhanceosome, which contains DNA-bound activators and then the MHC II transcriptosome with the addition of the class II transactivator (CIITA). CIITA is the master regulator of MHC II transcription. It is expressed constitutively in dendritic cells (DC) and mature B cells and is inducible in most other cell types. Three isoforms of CIITA exist, depending on cell type and inducing signals. CIITA is regulated at the levels of transcription and post-translational modifications, which are still not very clear. Inappropriate immune responses are found in several diseases, including cancer and autoimmunity. Since CIITA regulates the expression of MHC II genes, it is involved directly in the regulation of the immune response. The knowledge of CIITA will facilitate the manipulation of the immune response and might contribute to the treatment of these diseases.

Asunto(s)

Genes MHC Clase II , Antígenos de Histocompatibilidad Clase II/metabolismo , Linfocitos B/inmunología , Regulación de la Expresión Génica , Antígenos de Histocompatibilidad Clase II/genética , Humanos , Inmunidad Innata , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Linfocitos T/inmunología , Transactivadores/química , Transactivadores/genética , Transactivadores/metabolismo

RESUMEN

To stimulate transcriptional elongation of HIV-1 genes, the transactivator Tat recruits the positive transcription elongation factor b (P-TEFb) to the initiating RNA polymerase II (RNAPII). We found that the activation of transcription by RelA also depends on P-TEFb. Similar to Tat, RelA activated transcription when tethered to RNA. Moreover, TNF-alpha triggered the recruitment of P-TEFb to the NF-kappaB-regulated IL-8 gene. While the formation of the transcription preinitiation complex (PIC) remained unaffected, DRB, an inhibitor of P-TEFb, prevented RNAPII from elongating on the IL-8 gene. Remarkably, DRB inhibition sensitized cells to TNF-alpha-induced apoptosis. Thus, NF-kappaB requires P-TEFb to stimulate the elongation of transcription and P-TEFb plays an unexpected role in regulating apoptosis.

Asunto(s)

FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Polimerasa II/metabolismo , Transcripción Genética/fisiología , Apoptosis/fisiología , Proteínas de Unión al ADN/metabolismo , Diclororribofuranosil Benzoimidazol/farmacología , Productos del Gen tat/metabolismo , Genes Reporteros , VIH-1/genética , VIH-1/metabolismo , Humanos , Interleucina-8/genética , FN-kappa B/genética , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Factor B de Elongación Transcripcional Positiva , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factor de Transcripción ReIA , Activación Transcripcional , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana

RESUMEN

In the infected host, the Nef protein of HIV/SIV is required for high viral loads and thus disease progression. Recent evidence indicates that Nef enhances replication in the T cell compartment after the virus is transmitted from dendritic cells (DC). The underlying mechanism, however, is not clear. Here, we report that a natural variability in the proline-rich motif (R71T) profoundly modulated Nef-stimulated viral replication in primary T cells of immature dendritic cell/T cell cocultures. Whereas both Nef variants (R/T-Nef) downregulated CD4, only the isoform supporting viral replication (R-Nef) efficiently interacted with signaling molecules of the T cell receptor (TCR) environment and stimulated cellular activation. Structural analysis suggested that the R to T conversion induces conformational changes, altering the flexibility of the loop containing the PxxP motif and hence its ability to bind cellular partners. Our report suggests that functionally and conformationally distinct Nef isoforms modulate HIV replication on the interaction level with the TCR-signaling environment once the virus enters the T cell compartment.

Asunto(s)

Productos del Gen nef/genética , VIH-1/fisiología , Linfocitos T/fisiología , Secuencias de Aminoácidos , Antígenos CD4/metabolismo , Antígenos CD8/genética , Antígenos CD8/metabolismo , Línea Celular , Células Dendríticas/fisiología , Productos del Gen nef/química , Productos del Gen nef/metabolismo , VIH-1/patogenicidad , Humanos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Modelos Moleculares , Pruebas de Precipitina , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Receptores de Antígenos de Linfocitos T/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Linfocitos T/virología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana

RESUMEN

Lipid rafts, also known as detergent-resistant membranes (DRM), are microdomains in the plasma membrane enriched in sphingolipids and cholesterol (reviewed in [1, 2]). Human immunodeficiency virus 1 (HIV) buds via lipid rafts [3, 4]. However, the targeting of viral structural components to DRM and its consequences for viral replication are not understood. Moreover, the negative factor Nef from HIV increases viral infectivity (reviewed in [5, 6]). With no apparent differences in structural components and morphology between wild-type and DeltaNef virons, the latter viruses display less efficient reverse transcription in target cells. As Nef is expressed abundantly early in the viral replicative cycle [7], we hypothesized that Nef could affect viral morphogenesis and budding to render viruses more infectious. In this report, we demonstrated first that Nef increases viral budding from lipid rafts. Second, in the presence of Nef, viral envelopes contain more ganglioside (GM1), which is a major component of lipid rafts. This finding correlated directly with the increased infectivity of HIV. Finally, the depletion of exogenous and endogenous cholesterol biochemically and genetically, which disrupted lipid rafts, decreased viral infectivity only in the presence of Nef. Importantly, HIV lacking the nef gene remained unaffected by these manipulations. We conclude that lipids in virions are essential for viral infectivity. Thus, HIV becomes more infectious when it buds from lipid rafts, and Nef plays a major role in this process.

Asunto(s)

Productos del Gen nef/biosíntesis , VIH-1/crecimiento & desarrollo , VIH-1/patogenicidad , Microdominios de Membrana/virología , Colesterol/análisis , Microdominios de Membrana/química , Replicación Viral , Productos del Gen nef del Virus de la Inmunodeficiencia Humana

RESUMEN

The accessory Nef protein of HIV and SIV is essential for viral pathogenesis, yet it is perplexing in its multitude of molecular functions. In this review we analyse the structure-function relationships of motifs recently proposed to play roles in aspects of Nef modification, signalling and trafficking, and thereby to impinge on the ability of the virus to survive in, and to manipulate, its cellular host. Based on the full-length structure assembly of HIV Nef, we correlate surface accessibility with secondary structure elements and sequence conservation. Motifs involved in Nef-mediated CD4 and MHC I downregulation are located in flexible regions of Nef, suggesting that the formation of the transient trafficking complexes involved in these processes depends on the recognition of primary sequences. In contrast, the interaction sites for signalling molecules that contain SH3 domains or the p21-activated kinases are associated with the well folded core domain, suggesting the recognition of highly structured protein surfaces.

Asunto(s)

Productos del Gen nef/química , Productos del Gen nef/metabolismo , VIH-1/química , Estructura Terciaria de Proteína , Transducción de Señal , Secuencias de Aminoácidos , Sitios de Unión , VIH-1/metabolismo , Humanos , Modelos Moleculares , Receptores del VIH/química , Receptores del VIH/metabolismo , Relación Estructura-Actividad , Productos del Gen nef del Virus de la Inmunodeficiencia Humana

RESUMEN

Ankyrin repeats are well-known structural modules that mediate interactions between a wide spectrum of proteins. The regulatory factor X with ankyrin repeats (RFXANK) is a subunit of a tripartite RFX complex that assembles on promoters of major histocompatibility complex class II (MHC II) genes. Although it is known that RFXANK plays a central role in the nucleation of RFX, it was not clear how its ankyrin repeats mediate the interactions within the complex and with other proteins. To answer this question, we modeled the RFXANK protein and determined the variable residues of the ankyrin repeats that should contact other proteins. Site-directed alanine mutagenesis of these residues together with in vitro and in vivo binding studies elucidated how RFXAP and CIITA, which simultaneously interact with RFXANK in vivo, bind to two opposite faces of its ankyrin repeats. Moreover, the binding of RFXAP requires two separate surfaces on RFXANK. One of them, which is located in the ankyrin groove, is severely affected in the FZA patient with the bare lymphocyte syndrome. This genetic disease blocks the expression of MHC II molecules on the surface of B cells. By pinpointing the interacting residues of the ankyrin repeats of RFXANK, the mechanism of this subtype of severe combined immunodeficiency was revealed.

Asunto(s)

Repetición de Anquirina/genética , Inmunodeficiencia Combinada Grave/genética , Secuencia de Aminoácidos , Animales , Células COS , Proteínas de Unión al ADN , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Puntual , Conformación Proteica , Inmunodeficiencia Combinada Grave/etiología , Factores de Transcripción/genética

RESUMEN

Human immunodeficiency virus type 1(F12) (HIV-1(F12)) interferes with the replication of other strains of HIV. Its accessory protein, Nef, is sufficient for this phenotype, where the production and infectivity of HIV are impaired significantly. The analysis of three rare mutations in this Nef protein revealed that these effects could be separated genetically. Moreover, the defect in virus production correlated with the lack of processing of the p55(Gag) precursor in the presence of Nef from HIV-1(F12). Importantly, the introduction of one of these mutations (E177G) into Nef from HIV-1(NL4-3) also created a dominant-negative Nef protein. Effects of Nef from HIV-1(F12) on virus production and Gag processing correlated with its altered subcellular distribution. Moreover, the association with two new cellular proteins with molecular masses of 74 and 75 kDa, which do not interact with other Nef proteins, correlated with the decreased virion infectivity. The identification of a dominant-negative protein for the production and infectivity of HIV suggests that Nef plays an active role at this stage of the viral replicative cycle.

Asunto(s)

Productos del Gen nef/fisiología , Infecciones por VIH/virología , VIH-1/fisiología , Antígenos CD8/metabolismo , Línea Celular , Regulación hacia Abajo , Productos del Gen gag/metabolismo , Productos del Gen nef/genética , VIH-1/patogenicidad , Humanos , Mutación , Unión Proteica , Precursores de Proteínas/metabolismo , Proteínas/metabolismo , Transfección , Virulencia , Replicación Viral , Productos del Gen nef del Virus de la Inmunodeficiencia Humana

RESUMEN

The class II transactivator (CIITA) regulates not only the transcription of HLA-DR, -DQ, -DP, but also invariant chain, DMA and DMB genes. A hybrid mutant CIITA protein, which contained residues from positions 302 to 1130 in CIITA fused to the enhanced green fluorescent protein (EdCIITA), inhibited the function of the wild-type protein. EdCIITA extinguished the inducible and constitutive expression of MHC II genes in epithelial cells treated with IFN-gamma and B lymphoblastoid cells respectively. Also, it blocked T cell activation by superantigen. This inhibition correlated with the localization of EdCIITA but not CIITA in the cytoplasm of cells. However, when EdCIITA was co-expressed with a dominant-negative form of the nucleoporin Nup214/CAN, it also accumulated in the nucleus. These data suggest that EdCIITA not only competes with the wild-type protein for the binding to MHC II promoters but sequesters a critical co-factor of CIITA in the cytoplasm. CIITA also recruits the histone acetyltransferase cAMP responsive element binding protein (CREB) binding protein and positive transcription elongation factor b (p-TEFb) for the transcription of MHC II genes. Dominant-negative p300 (DNp300) or CDK9 (DNCDK9) proteins inhibited the function of CIITA and of the DRA promoter. Thus, combinations of EdCIITA and DNp300 and/or DNCDK9 proteins extinguished the transcription of MHC II genes. They might become useful for future genetic therapeutic approaches in organ transplantation and autoimmune diseases.

Asunto(s)

Quinasas Ciclina-Dependientes/metabolismo , Genes MHC Clase II , Proteínas Nucleares/metabolismo , Transactivadores/metabolismo , Transcripción Genética , Animales , Células COS , Chlorocebus aethiops , Quinasa 9 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/genética , Citoplasma/metabolismo , Expresión Génica , Productos del Gen tat/metabolismo , Células HeLa , Humanos , Interferón gamma/metabolismo , Proteínas Nucleares/genética , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transactivadores/genética , Activación Transcripcional

RESUMEN

The accessory Nef protein from human and simian immunodeficiency viruses is critical for efficient viral replication and pathogenesis. Here we present an assembly of the full length structure of HIV-1 Nef, allele NL4-3, based on the previously solved anchor and core domain structures. The center part of the 33 residue encompassing flexible loop at the C-terminus of Nef, involved in Nef internalization and CD4 endocytosis, has been modelled. The degree of sequence conservation in HIV-1 Nef proteins was determined using a total of 186 different strains from five different subtypes. The sequence conservation has been correlated with the accessible surface area and with secondary structure features for individual residues. The high amount of flexible regions in Nef accounts for the large surface and the multiple interaction sites the protein exhibits.

Asunto(s)

Productos del Gen nef/química , VIH-1/química , Alelos , Secuencia de Aminoácidos , Animales , Antígenos CD4/química , Secuencia Conservada , Endocitosis , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Especificidad de la Especie , Productos del Gen nef del Virus de la Inmunodeficiencia Humana

RESUMEN

The accessory protein negative factor (Nef) from human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) is required for optimal viral infectivity and the progression to acquired immunodeficiency syndrome (AIDS). Nef interacts with the endocytic machinery, resulting in the down-regulation of cluster of differentiation antigen 4 (CD4) and major histocompatibility complex class I (MHCI) molecules on the surface of infected cells. Mutations in the C-terminal flexible loop of Nef result in a lower rate of internalization by this viral protein. However, no loop-dependent binding of Nef to adaptor protein-2 (AP-2), which is the adaptor protein complex that is required for the internalization of proteins from the plasma membrane, could be demonstrated. In this study we investigated the relevance of different motifs in Nef from SIV(mac239) for its internalization, CD4 down-regulation, binding to components of the trafficking machinery, and viral infectivity. Our data suggest that the binding of Nef to the catalytic subunit H of the vacuolar membrane ATPase (V-ATPase) facilitates its internalization. This binding depends on the integrity of the whole flexible loop. Subsequent studies on Nef mutant viruses revealed that the flexible loop is essential for optimal viral infectivity. Therefore, our data demonstrate how Nef contacts the endocytic machinery in the absence of its direct binding to AP-2 and suggest an important role for subunit H of the V-ATPase in viral infectivity.

Asunto(s)

Antígenos CD4/metabolismo , ATPasas de Translocación de Protón/metabolismo , Virus de la Inmunodeficiencia de los Simios/metabolismo , Virus de la Inmunodeficiencia de los Simios/patogenicidad , ATPasas de Translocación de Protón Vacuolares , Proteínas Reguladoras y Accesorias Virales/metabolismo , Proteínas Adaptadoras del Transporte Vesicular , Secuencia de Aminoácidos , Dominio Catalítico , Células Cultivadas/virología , Regulación hacia Abajo , Humanos , Datos de Secuencia Molecular , Mutación , Proteínas del Tejido Nervioso/metabolismo , Fosfoproteínas/metabolismo , Proteínas Reguladoras y Accesorias Virales/genética

RESUMEN

Molecular biological investigations of HIV have made fundamental contributions to our understanding of eukaryotic biology. These studies elucidated new paradigms in transcription, RNA and protein export from the nucleus to the cytoplasm, cellular activation, morphology and vesicular trafficking.

Asunto(s)

Transporte Activo de Núcleo Celular/fisiología , VIH/metabolismo , Proteínas de los Retroviridae/metabolismo , Endocitosis/fisiología , Productos del Gen rev/metabolismo , Productos del Gen tat/metabolismo , VIH/genética , Humanos , Modelos Biológicos , Proteínas de los Retroviridae/genética , Transcripción Genética/fisiología , Productos del Gen rev del Virus de la Inmunodeficiencia Humana , Productos del Gen tat del Virus de la Inmunodeficiencia Humana

RESUMEN

Enveloped viruses enter target cells by membrane fusion or endocytosis. In the latter case, fusion of the viral envelope is induced by the acidic pH of the endocytic vesicle [1]. As with most other retroviruses, entry of the human immunodeficiency virus (HIV) is thought to be exclusively by pH-independent membrane fusion after interaction of its envelope with CD4 and a chemokine co-receptor on the target cell [2,3]. Expression of CD4 on the virus-producing cell impairs the release and infectivity of HIV-1(NL4-3) particles [4-6]. In sharp contrast, we found that the infectivity of another HIV isolate, HIV-1SF2, was enhanced by expression of CD4 on the producer cells, which correlated with significantly increased amounts of viral proteins in the vesicular fraction of target cells. Endocytic inhibitors decreased infectivity of HIV-1SF2 but enhanced that of HIV-1 NL4-3. Expression of CD4 in the producer cell did not remove gp41 from HIV-1SF2 virions. With these cells, the formation of syncytia could be induced by acidic medium. Thus, HIV-1SF2 can enter the cytoplasm by an endocytic route after activation of gp41 by the acidic pH of endocytic vesicles. Endocytic entry might expand the range of cells that HIV could infect and should be considered in antiviral strategies against AIDS.

Asunto(s)

Endocitosis , Endosomas/virología , VIH-1/fisiología , Antígenos CD4/metabolismo , Línea Celular , Células Gigantes , Proteína gp41 de Envoltorio del VIH/metabolismo , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Células Jurkat , Fusión de Membrana , Replicación Viral

RESUMEN

Flavopiridol (L86-8275, HMR1275) is a cyclin-dependent kinase (Cdk) inhibitor that is in clinical trials as a cancer treatment because of its antiproliferative properties. We found that the flavonoid potently inhibited transcription by RNA polymerase II in vitro by blocking the transition into productive elongation, a step controlled by P-TEFb. The ability of P-TEFb to phosphorylate the carboxyl-terminal domain of the large subunit of RNA polymerase II was inhibited by flavopiridol with a K(i) of 3 nm. Interestingly, the drug was not competitive with ATP. P-TEFb composed of Cdk9 and cyclin T1 is a required cellular cofactor for the human immunodeficiency virus (HIV-1) transactivator, Tat. Consistent with its ability to inhibit P-TEFb, flavopiridol blocked Tat transactivation of the viral promoter in vitro. Furthermore, flavopiridol blocked HIV-1 replication in both single-round and viral spread assays with an IC(50) of less than 10 nm.

Asunto(s)

Antineoplásicos/farmacología , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Ciclinas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , VIH-1/efectos de los fármacos , Piperidinas/farmacología , Replicación Viral/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Ciclina T , Quinasa 9 Dependiente de la Ciclina , VIH-1/genética , VIH-1/fisiología , Humanos , Regiones Promotoras Genéticas , Transcripción Genética/efectos de los fármacos

RESUMEN

Human immunodeficiency virus type 1 (HIV-1) is unique in that it encodes its own transcriptional activator Tat, which specifically binds to the viral mRNA sequence TAR (transactivation response) element and activates viral transcription at the step of elongation as well as initiation. We recently reported that fluoroquinoline derivatives inhibited HIV-1 replication most likely by blocking viral transcription. In this report, we investigated the mechanism of action of one such compound 7-(3, 4-dehydro-4-phenyl-1-piperidinyl)-1, 4-dihydro-6-fluoro-1-methyl-8-trifluoromethyl-4-oxoquinoline-3-carbox ylic acid (K-37). We demonstrated that K-37 inhibited not only Tat but also other RNA-dependent transactivators. No effect was observed with DNA-dependent transactivators such as p65 (NF-kappaB) and Gal4VP16. Moreover, K-37 did not inhibit carboxyl-terminal domain (CTD)-kinase activities of CDK-activating kinase (CAK) and positive transcription elongation factor b (P-TEFb), which are known to be involved in Tat-mediated transactivation at the step of transcriptional elongation. It is suggested that RNA-mediated transactivation may involve a common unknown factor to which K-37 directly interacts. Since K-37 did not appear to block DNA-mediated transactivation and thus did not show strong nonspecific cytotoxicity as reported previously, K-37 and its derivative compounds are considered to be feasible candidates for a novel AIDS therapy.

Asunto(s)

Fármacos Anti-VIH/farmacología , Antiinfecciosos/farmacología , Fluoroquinolonas , VIH-1/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Animales , Línea Celular , ADN Viral/fisiología , Productos del Gen tat/antagonistas & inhibidores , Duplicado del Terminal Largo de VIH/efectos de los fármacos , VIH-1/enzimología , VIH-1/genética , Humanos , Inhibidores de Proteínas Quinasas , Proteínas Quinasas/metabolismo , ARN Viral/fisiología , Activación Transcripcional/genética , Células U937 , Replicación Viral/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana

RESUMEN

Transcriptional transactivators (Tat) from many lentiviruses interact with their cognate transactivation response RNA structures (TAR) to increase rates of elongation rather than initiation of transcription. For several of them, the complex of Tat and a species-specific cyclin T1 must be formed before the binding to TAR can occur with high affinity and specificity. In sharp contrast, Tat from the bovine immunodeficiency virus (BIV) binds to its TAR without the help of the cyclin T1. This binding depends on the upper stem and 5' bulge, but not the central loop in TAR. Moreover, cyclins T1 from different species can mediate effects of this Tat in cells. Unlike the situation with other lentiviruses, Tat transactivation can be rescued simply by linking a heterologous promoter to TAR in permissive cells. Thus, lentiviruses have evolved different strategies to recruit Tat and the positive transcription elongation factor b to their promoters, and interactions between Tat and TAR are independent from those between Tat and the cyclin T1 in BIV.

Asunto(s)

Productos del Gen tat/metabolismo , Virus de la Inmunodeficiencia Bovina/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Viral/metabolismo , Animales , Células COS , Bovinos , Medio de Cultivo Libre de Suero , Ciclina T , Ciclinas/metabolismo , Perros , Células HeLa , Humanos , Virus de la Inmunodeficiencia Bovina/genética , Factor B de Elongación Transcripcional Positiva , Unión Proteica , Especificidad de la Especie , Secuencias Repetidas Terminales , Activación Transcripcional

RESUMEN

The activation of the HIV-1 long terminal repeat (LTR) by the viral transcriptional transactivator Tat is an essential step in the viral replication cycle. To increase the processivity of RNA polymerase II, Tat interacts with the positive transcription elongation factor b (P-TEFb) and cyclin-dependent kinase (CDK)-activating kinase (CAK). In this study, we demonstrate that a pseudo-substrate peptide for CDK7, mC2p, inhibits HIV-1 replication as well as Tat transactivation. Specifically, mC2p blocks only the activity of CAK and not that of P-TEFb. Moreover, mC2p inhibits Tat transactivation and HIV replication. Therefore, the activation of CDK7 by Tat is considered a critical step of Tat transactivation and mC2p and related compounds represent potential candidates for novel anti-HIV therapeutics.

Asunto(s)

Productos del Gen tat/fisiología , VIH-1/fisiología , Replicación Viral/fisiología , Secuencia de Aminoácidos , Regulación Viral de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Péptidos/genética , Péptidos/metabolismo , Unión Proteica , Especificidad por Sustrato , Activación Transcripcional , Productos del Gen tat del Virus de la Inmunodeficiencia Humana

RESUMEN

Nef is a myristoylated protein of 27 to 35 kDa that is conserved in primate lentiviruses. In vivo, Nef is required for high viral load and full pathological effects. In vitro, Nef has at least four activities: induction of CD4 and major histocompatibility complex (MHC) class I downregulation, enhancement of viral infectivity, and alteration of T-cell activation pathways. We previously reported that the Nef protein from human immunodeficiency virus type 1 interacts with a novel human thioesterase (hTE). In the present study, by mutational analysis, we identified a region of the Nef core, extending from the residues D108 to W124, that is involved both in Nef-hTE interaction and in Nef-induced CD4 downregulation. This region of Nef is located on the oligomer interface and is in close proximity to the putative CD4 binding site. One of the mutants carrying a mutation in this region, targeted to the conserved residue D123, was also found to be defective in two other functions of Nef, MHC class I downmodulation and enhancement of viral infectivity. Furthermore, mutation of this residue affected the ability of Nef to form dimers, suggesting that the oligomerization of Nef may be critical for its multiple functions.

Asunto(s)

Antígenos CD4/biosíntesis , Secuencia Conservada , Regulación hacia Abajo/inmunología , Productos del Gen nef/inmunología , VIH-1/inmunología , Antígeno HLA-A2/biosíntesis , Tioléster Hidrolasas/inmunología , Secuencia de Aminoácidos , Membrana Celular/inmunología , Dimerización , Productos del Gen nef/química , Productos del Gen nef/genética , VIH-1/fisiología , Células HeLa , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligopéptidos/química , Oligopéptidos/genética , Oligopéptidos/inmunología , Palmitoil-CoA Hidrolasa , Unión Proteica , Conformación Proteica , Productos del Gen nef del Virus de la Inmunodeficiencia Humana

RESUMEN

The regulatory factor X (RFX) complex, which contains RFXANK(B), RFXAP, and RFX5, binds to X and S boxes in major histocompatibility complex class II (MHC II) promoters. In the bare lymphocyte syndrome (BLS), which is a human severe combined immunodeficiency, MHC II promoters are neither occupied nor transcribed. Thus, the absence of any one subunit prevents the formation of the RFX complex. Nevertheless, except for a weak binding between RFX5 and RFXAP, no other interactions between RFX proteins have been described. In this study, we demonstrate that RFXANK(B) binds to RFXAP to form a scaffold for the assembly of the RFX complex, which then binds to DNA. Moreover, mutant RFXANK(B) and RFXAP proteins from complementation groups B and D of BLS, respectively, cannot support this interaction. Our data elucidate an intriguing medical situation, where a genetic disease targets two different surfaces that are required for the nucleation of a multisubunit DNA-protein complex.

Asunto(s)

Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Genes MHC Clase II , Inmunodeficiencia Combinada Grave/sangre , Inmunodeficiencia Combinada Grave/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Regulación de la Expresión Génica , Humanos , Mutación , Regiones Promotoras Genéticas , Factores de Transcripción del Factor Regulador X

RESUMEN

The activation of Nef-associated kinase (NAK) by Nef from human and simian immunodeficiency viruses is critical for efficient viral replication and pathogenesis. This induction occurs via the guanine nucleotide exchange factor Vav and the small GTPases Rac1 and Cdc42. In this study, we identified NAK as p21-activated kinase 1 (PAK1). PAK1 bound to Nef in vitro and in vivo. Moreover, the induction of cytoskeletal rearrangements such as the formation of trichopodia, the activation of Jun N-terminal kinase, and the increase of viral production were blocked by an inhibitory peptide that targets the kinase activity of PAK1 (PAK1 83-149). These results identify NAK as PAK1 and emphasize the central role its kinase activity plays in cytoskeletal rearrangements and cellular signaling by Nef.

Asunto(s)

Productos del Gen nef/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Células COS , Línea Celular , Citoesqueleto/metabolismo , Técnica del Anticuerpo Fluorescente , Proteínas Fluorescentes Verdes , VIH-1/metabolismo , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , Proteínas Luminiscentes , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fragmentos de Péptidos/farmacología , Unión Proteica , Transducción de Señal , Transfección , Proteínas Virales/biosíntesis , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Quinasas p21 Activadas