RESUMEN
Automatic tracking of viral structures displayed as small spots in fluorescence microscopy images is an important task to determine quantitative information about cellular processes. We introduce a novel probabilistic approach for tracking multiple particles based on multi-sensor data fusion and Bayesian smoothing methods. The approach exploits multiple measurements as in a particle filter, both detection-based measurements and prediction-based measurements from a Kalman filter using probabilistic data association with elliptical sampling. Compared to previous probabilistic tracking methods, our approach exploits separate uncertainties for the detection-based and prediction-based measurements, and integrates them by a sequential multi-sensor data fusion method. In addition, information from both past and future time points is taken into account by a Bayesian smoothing method in conjunction with the covariance intersection algorithm for data fusion. Also, motion information based on displacements is used to improve correspondence finding. Our approach has been evaluated on data of the Particle Tracking Challenge and yielded state-of-the-art results or outperformed previous approaches. We also applied our approach to challenging time-lapse fluorescence microscopy data of human immunodeficiency virus type 1 and hepatitis C virus proteins acquired with different types of microscopes and spatial-temporal resolutions. It turned out, that our approach outperforms existing methods.
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Algoritmos , Estructuras Virales , Teorema de Bayes , Humanos , Microscopía FluorescenteRESUMEN
PURPOSE: The aim of the present study was to evaluate the reliability and re-producibility of digital shade selection devices, and to correlate the results with conventional human visual shade assessment. MATERIALS AND METHODS: Tooth color gradation and agreement of two different digital shade selection instruments were determined by employing a spectro-photometer (ShadePilot, Degudent; Hanau, Germany, Software V. 2.41) and acolorimeter (ShadeVision, Ammann Girrbach; Pforzheim, Germany; Software V. 1.20). The devices were compared with three human examiners with a negative history of visual color deficiency, looking at 40 subjects under clinical conditions. In one half of the test persons, the visual and digital shade determination was performed on the right maxillary central incisor that was free of any restoration, whereas the other half was carried out in subjects with single implant-supported PFM crowns in the same region. The computer-based readings across the regions (incisal, middle, cervical) were recorded two consecutive times. Differences between the computer-based readings were evaluated with the chi2 test (Chi-square). RESULTS: The same shade was obtained by all three human examiners in 22.5% (9 of 40 cases), by all colorimetric readings in 35% (14 of 40 cases), and by all spectrophotometric readings in 55% (22 of 40 cases). There was considerable agreement between the first and second reading of the investigated spectrophotometric and colorimetric shade instruments. The spectrophotometer demonstrated color matches of the first and second reading in 81.7% of the in vivo measurements (coefficient of contingency = 0.98; chi2 test p = 0.99), while the colorimeter exhibited matches in 70% (coefficient of contingency = 0.96; chi2 test p = 0.96) of the cases. Hence, the spectrophotometer exhibited the highest agreement between the two consecutive readings. While no significant influ-ence of the measuring point (incisal, middle, cervical) on the reproducibility of color results could be ascertained, both the colorimeter and spectrophotometer displayed a statistically significant difference for the frequency distribution of color categories for teeth vs PFM crowns (both chi2 test p < 0.0001). The measuring results of the colorimeter showed distinctly lighter shades than those of the spectrophotometer. CONCLUSIONS: Within the limitations of this study, the results suggest that spectrophotometric shade determination is more reproducible compared to conventional visual shade assessment. It can serve as a reliable addition in color matching and enhances the level of shade analysis, communication, interpretation, and fabrication of dental restorations.
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Colorimetría , Coronas , Espectrofotometría , Diente , Adolescente , Adulto , Anciano , Color , Colorimetría/instrumentación , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reproducibilidad de los Resultados , Espectrofotometría/instrumentación , Percepción Visual , Adulto JovenRESUMEN
T cell antigen receptor (TCR) signaling triggers selective cytokine expression to drive T cell proliferation and differentiation required for immune defense and surveillance. The nuclear signaling events responsible for specificity in cytokine gene expression upon T cell activation are largely unknown. Here, we uncover formation of a dynamic actin filament network in the nucleus that regulates cytokine expression for effector functions of CD4+ T lymphocytes. TCR engagement triggers the rapid and transient formation of a nuclear actin filament network via nuclear Arp2/3 complex, induced by elevated nuclear Ca2+ levels and regulated via N-Wasp and NIK. Specific interference with TCR-induced formation of nuclear actin filaments impairs production of effector cytokines and prevents generation of antigen-specific antibodies but does not interfere with immune synapse formation and cell proliferation. Ca2+-regulated actin polymerization in the nucleus allows CD4+ T cells the rapid conversion of TCR signals into effector functions required for T cell help.
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Actinas/inmunología , Antígenos/inmunología , Linfocitos T CD4-Positivos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Complejo 2-3 Proteico Relacionado con la Actina/inmunología , Traslado Adoptivo , Animales , Línea Celular , Humanos , RatonesRESUMEN
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.
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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 ViralRESUMEN
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.
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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 HumanaRESUMEN
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.
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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 HumanaRESUMEN
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.
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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 ActivadasRESUMEN
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.
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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/genéticaRESUMEN
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.
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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 HumanaRESUMEN
OBJECTIVE: To investigate differences in viral and proviral load between the peripheral blood and the intestinal mucosal immune system in HIV-infected patients. DESIGN: HIV-1 p24 and HIV DNA content were compared in blood samples and intestinal biopsies from HIV-infected patients. METHODS: Intestinal biopsies and peripheral blood were simultaneously obtained from 27 HIV-infected patients undergoing diagnostic endoscopy. The p24 concentrations were measured in serum and homogenized intestinal biopsies by enzyme-linked immunosorbent assay after acid-dissociation of immune complexes. Proviral load was determined in blood and intestinal biopsies by a quantitative competitive polymerase chain reaction amplifying the HIV-1 nef gene from genomic DNA. RESULTS: No significant differences were found in proviral load comparing HIV copies per 1.5 x 10(5) cell equivalents in blood [2650 (600-44000)] and intestinal biopsies [4200 (1325-19 625)]. Paired analysis revealed a strong positive correlation between serum and mucosal proviral load. In contrast, HIV core protein p24 was detected in intestinal biopsies from 18 patients in much higher concentrations than in serum [858 (262-4111) pg/g versus 34 (9-242) pg/g; P < 0.005]. The p24 concentrations in serum and intestinal biopsies did not correlate and no significant correlation was observed in serum or intestinal biopsies between proviral load and p24 concentrations. No clear correlations were observed between clinical parameters and HIV DNA or HIV p24 levels in blood or biopsies. CONCLUSIONS: Our findings demonstrate a homogenous distribution of HIV proviral load in the peripheral blood and the intestinal mucosal immune system. The high viral antigen load in the intestine therefore indicates that mucosal HIV production is upregulated at the transcriptional and/or translational level. The intestinal mucosa is a major reservoir for HIV in HIV-infected patients.
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Proteína p24 del Núcleo del VIH/sangre , Infecciones por VIH/virología , VIH-1/fisiología , Mucosa Intestinal/inmunología , Mucosa Intestinal/virología , Provirus/fisiología , Adulto , Biopsia , Southern Blotting , ADN Viral/sangre , Endoscopía Gastrointestinal , Femenino , Genes nef , Proteína p24 del Núcleo del VIH/análisis , Infecciones por VIH/inmunología , VIH-1/genética , VIH-1/inmunología , Humanos , Masculino , Persona de Mediana Edad , Carga ViralRESUMEN
HIV-Nef protein supports viral infectivity prior to proviral integration. This requires Nef to be present before the expression of viral genes and suggests its delivery as part of the virion. We report here that the Nef proteins of HIV-2-HOM and HIV-2-ROD are associated with the virion. After the separation of pelleted virus in a 20-60% sucrose density gradient, both proteins cosedimented with the virion-associated reverse transcriptase (RT) activity at a density characteristic of retroviral particles. Whereas Nef-2-ROD was present in the virion only as the full-length protein, HIV-2-HOM appeared as 32 and 35 kDa isoforms. The smaller isoform is identical in molecular weight to the protein expected from proteolytic cleavage of full-length Nef-2-HOM by the virion-based protease. Virion-association of Nef helps to explain the recently redefined biological function of this regulatory protein.
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Productos del Gen nef/fisiología , VIH-2/fisiología , Virión/fisiología , Productos del Gen nef/química , Productos del Gen nef/genética , VIH-2/química , VIH-2/genética , Humanos , Immunoblotting , Células Jurkat , Proteínas Recombinantes/química , Virión/química , Virión/genética , Productos del Gen nef del Virus de la Inmunodeficiencia HumanaAsunto(s)
Síndrome de Inmunodeficiencia Adquirida/patología , Proteína p24 del Núcleo del VIH/análisis , Infecciones por VIH/patología , VIH-1/aislamiento & purificación , Mucosa Intestinal/virología , Provirus/aislamiento & purificación , Carga Viral , Síndrome de Inmunodeficiencia Adquirida/sangre , Adulto , ADN Viral/sangre , ADN Viral/aislamiento & purificación , Diarrea/complicaciones , Diarrea/patología , Diarrea/virología , Femenino , Proteína p24 del Núcleo del VIH/sangre , Infecciones por VIH/sangre , VIH-1/crecimiento & desarrollo , Homosexualidad Masculina , Humanos , Mucosa Intestinal/patología , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Provirus/crecimiento & desarrolloRESUMEN
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.
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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 HumanaRESUMEN
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.
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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 HumanaRESUMEN
Nef of primate lentiviruses is critical for high levels of viremia and the progression to AIDS. Nef associates with and activates a serine/threonine kinase (Nef-associated kinase [NAK]) via the small GTPases Rac1 and Cdc42. We identified the protooncogene and guanine nucleotide exchange factor Vav as the specific binding partner of Nef proteins from HIV-1 and SIV. The interaction between Nef and Vav led to increased activity of Vav and its downstream effectors. Both cytoskeletal changes and the activation of c-Jun N-terminal kinase (JNK) were observed. Furthermore, a dominant-negative Vav protein inhibited NAK activation and viral replication. Thus, the interaction between Nef and Vav initiates a signaling cascade that changes structural and physiological parameters in the infected cell.
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Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Citoesqueleto/metabolismo , Productos del Gen nef/metabolismo , VIH-1 , Proteínas Quinasas Activadas por Mitógenos , Proteínas Proto-Oncogénicas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Tamaño de la Célula , Activación Enzimática , Proteínas de Unión al GTP/metabolismo , Productos del Gen nef/química , VIH-1/crecimiento & desarrollo , VIH-1/metabolismo , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , Datos de Secuencia Molecular , Fosforilación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-vav , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia , Transducción de Señal , Replicación Viral , Proteína de Unión al GTP cdc42 , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Quinasas p21 Activadas , Dominios Homologos srcRESUMEN
Human immunodeficiency virus (HIV) Nef functions are thought to be mediated via interactions with cellular proteins. Utilizing zone velocity sedimentation in glycerol gradients we found that recombinant HIV-1 Nef non-covalently associates with actin forming a high-molecular-mass complex of 150-300 kDa. This Nef/actin complex was present in human B and T lymphocytes but not in insect cells and was dependent on the N-terminal myristoylation of Nef, whereas the SH3-binding proline motif of Nef was not involved. Despite being myristoylated, HIV-2 Nef did not associate with actin. This might reflect differences in the subcellular localization of Nef since cell-fractionation experiments revealed that HIV-1 Nef was virtually exclusively localized in the cytoskeletal (detergent-insoluble) fraction whereas HIV-2 Nef had significantly reduced affinity for the cytoskeleton. Colocalization experiments in HIV-1-infected CD4+ fibroblasts revealed that Nef/actin complexes may also exist in HIV-infected cells. This novel interaction of HIV-1 Nef with actin provides insight into the association of Nef with cellular structures and reveals general differences in the interactions of the Nef proteins from HIV-1 and HIV-2.
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Actinas/metabolismo , Productos del Gen nef/metabolismo , VIH-1/metabolismo , VIH-2/metabolismo , Fracciones Subcelulares/metabolismo , Animales , Línea Celular , Productos del Gen nef/química , Peso Molecular , Ácido Mirístico , Ácidos Mirísticos/metabolismo , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Spodoptera , Linfocitos T/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia HumanaRESUMEN
Human immunodeficiency virus type 2 (HIV-2) Nef is proteolytically cleaved by the HIV-2-encoded protease. The proteolysis is not influenced by the absence or presence of the N-terminal myristoylation. The main cleavage site is located between residues 39 and 40, suggesting a protease recognition sequence, GGEY-SQFQ. As observed previously for Nef protein from HIV-1, a large, stable core domain with an apparent molecular mass of 30 kDa is produced by the proteolytic activity. Cleavage of Nef from HIV-1 in two domains by its own protease or the protease from HIV-2 is also independent of Nef myristoylation. However, processing of HIV-1 Nef by the HIV-2 protease is less selective than that by the HIV-1 protease: the obtained core fragment is heterogeneous at its N terminus and has an additional cleavage site between amino acids 99 and 100. Preliminary experiments suggest that the full-length Nef of HIV-2 and the core domain are part of the HIV-2 particles, analogous to the situation reported recently for HIV-1.
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Ácido Aspártico Endopeptidasas/metabolismo , Productos del Gen nef/metabolismo , Proteasa del VIH/metabolismo , VIH-1/metabolismo , VIH-2/metabolismo , Sitios de Unión , Humanos , Ácidos Mirísticos/metabolismo , Especificidad por Sustrato , Productos del Gen nef del Virus de la Inmunodeficiencia HumanaRESUMEN
There is increasing evidence that tumor expressed genes induce immune responses in cancer patients. To identify meningioma expressed antigens, we established a meningioma expression library which was screened with autologous serum. Out of 20 positive cDNA clones eight share high sequence homologies as determined by sequence analysis. These eight clones can be grouped into three classes which differ in length and which are characterized by specific sequence variations. The longest open reading frame was found to be 2412 bp encoding an immunoreactive antigen termed meningioma expressed antigen 6 (MEA6). Using five sequence specific primer pairs, somatic hybrid panel mapping revealed locations of the three classes on several human chromosomes including chromosomes 2, 3, 6, 7, 9, 13 and 14. The mapping results were confirmed by fluorescence in situ hybridization. RT-PCR showed consistent expression of all classes in several meningiomas and additional tissues using the same set of primer pairs as for chromosomal mapping. The expression data were confirmed by northern blot analysis. For the predicted amino acid sequence BLASTX revealed a homology to a human C219-reactive peptide which was previously isolated by an antibody directed against p-glycoprotein. Sequence properties of the MEA protein include an acidic activation domain, a proline-rich region and two coiled-coil domains indicating protein binding and activation functions.