RESUMEN

Wild-type p53 (wtp53) is described as a tumour suppressor gene; mutations in this gene occur in many human cancers and promote oncogenic capacity. Here, we establish that the oncogenic activity of mutant p53 (mtp53) is driven by the WASP-interacting protein (WIP). WIP knockdown from mtp53-expressing glioblastoma and breast cancer cells (BCC) greatly reduced proliferation and growth capacity of cancer stem cell (CSC)-like cells and decreased CSC-like markers (CD133, CD44 or YAP/TAZ). mtp53 overexpression in human astrocytes enhanced their proliferative capacity in suspension culture and increased expression of CSC markers and WIP. WIP knockdown compromised tumour glioblastoma and BCC growth capacity in vivo. We show that WIP is phosphorylated by AKT2 and is regulated by mtp53/p63 through enhancement of PI3K/AKT2-mediated integrin/receptor recycling pathways. WIP regulates this oncogenic pathway by controlling YAP/TAZ stability. We thus establish a new CSC signalling pathway downstream of mtp53 in which AKT2 regulates WIP and controls YAP/TAZ stability.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/metabolismo , Neoplasias de la Mama/metabolismo , Proteínas del Citoesqueleto/metabolismo , Glioblastoma/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mutación , Células Madre Neoplásicas/metabolismo , Fosfoproteínas/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Aciltransferasas , Proteínas Adaptadoras Transductoras de Señales/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proteínas del Citoesqueleto/genética , Femenino , Glioblastoma/genética , Glioblastoma/patología , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Células MCF-7 , Masculino , Células Madre Neoplásicas/patología , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas/genética , Fosforilación/genética , Estabilidad Proteica , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factores de Transcripción/genética , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteínas Señalizadoras YAP

RESUMEN

Wiskott-Aldrich syndrome protein (WASP) -interacting protein (WIP) is an actin-binding protein involved in the regulation of actin polymerization in cells, such as fibroblasts and lymphocytes. Despite its recognized function in non-neuronal cells, the role of WIP in the central nervous system has not been examined previously. We used WIP-deficient mice to examine WIP function both in vivo and in vitro. We report here that WIP(-)(/-) hippocampal neurons exhibit enlargement of somas as well as overgrowth of neuritic and dendritic branches that are more evident in early developmental stages. Dendritic arborization and synaptogenesis, which includes generation of postsynaptic dendritic spines, are actin-dependent processes that occur in parallel at later stages. WIP deficiency also increases the amplitude and frequency of miniature excitatory postsynaptic currents, suggesting that WIP(-)(/-) neurons have more mature synapses than wild-type neurons. These findings reveal WIP as a previously unreported regulator of neuronal maturation and synaptic activity.

Asunto(s)

Proteínas Portadoras/metabolismo , Hipocampo/crecimiento & desarrollo , Neurogénesis/fisiología , Neuronas/citología , Neuronas/metabolismo , Sinapsis/metabolismo , Animales , Western Blotting , Proteínas del Citoesqueleto , Potenciales Postsinápticos Excitadores/fisiología , Técnica del Anticuerpo Fluorescente , Regulación del Desarrollo de la Expresión Génica , Hipocampo/citología , Hipocampo/metabolismo , Masculino , Ratones , Ratones Noqueados , Microscopía Confocal

RESUMEN

Podosomes are specialized adhesion sites found in rapidly migrating and invasive cells, most notably in cells from the myeloid lineage that participate in immune surveillance and phagocyte defence mechanisms. In this review, we describe the nature of leukocyte podosomes and the regulation of their turnover during migration by the key regulatory molecules Wiskott-Aldrich syndrome protein and WASP-interacting protein.

Asunto(s)

Movimiento Celular , Proteínas del Citoesqueleto/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Leucocitos/fisiología , Proteína del Síndrome de Wiskott-Aldrich/metabolismo , Leucocitos/diagnóstico por imagen , Modelos Biológicos , Ultrasonografía

RESUMEN

La inflamación es un proceso fisiológico dirigido a prevenir o reparar los daños producidos por agresiones externas, infecciones o intoxicación. Este proceso depende de la coordinación entre la activacióny migración de los leucocitos hacia la región dañada y por tanto está asociado con cambios en la morfología celular y con la reorganización del citoesqueleto de actina. El citoesqueleto de actina tambiéncontribuye a otros cambios morfológicos esenciales necesarios para la vida de la célula. WIP (WASP Interacting Protein) es una proteína que se une a la actina y participa en procesos inflamatorios.Dicha unión estabiliza los microfilamentos de actina y regula su organización especial y temporal. La ausencia de WIP induce la formación de redes de actina deficientes en linfocitos T y B, lo que conducea la modificación de la capacidad linfocitaria de activación y migración y desemboca en alteraciones autoinmunes con infiltración leucocitaria en órganos vitales y muerte del animal

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Inflammation refers to a physiological process aimed at preventing or repairing the damage induced by infection, injury or intoxication. This process requires the coordinated activation and migration of leukocytes to the damaged area and therefore it is associatedwith changes in cell morphology and actin cytoskeleton reorganization. Actin cytoskeleton is also required for many other changes in cellular morphology that take place during cell life. WIP (WASP InteractingProtein) is an actin-binding protein involved in inflammation. WIP stabilizes actin filaments and regulates their temporal and spatial organization. Absence of WIP leads to formation of defective actinnetworks in T and B lymphocytes (that entail defective lymphocyte activation and migration) and ultimately results in autoimmunity and leukocyte infiltration in vital organs resulting in a fatal outcome

Asunto(s)

Humanos , Inflamación/inmunología , Autoinmunidad/inmunología , Síndrome de Wiskott-Aldrich/inmunología , Mediadores de Inflamación/inmunología , Trastornos Leucocíticos/inmunología , Proteínas del Citoesqueleto/inmunología , Enfermedades Autoinmunes/inmunología

RESUMEN

The Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency syndrome caused by mutations in the WAS protein (WASP). This participates in signalling and cytoskeletal homoeostasis, and some of its activities are regulated by its binding to the WASP interacting protein (WIP). WIP deficiency, however, has not yet been shown to be of pathological significance in humans. Here we show that, in WIP null (WIP(-/-)) mice, it produces haematological alterations and anatomical abnormalities in several organs, most probably as a consequence of autoimmune attacks. Granulocytosis and severe lymphopenia are associated with a proportional increase in segmented cells and fewer bone marrow erythrocytes and lymphocytes. Splenomegaly is accompanied by an increase of haematopoietic tissue and red pulp, reduction of the white pulp, and fewer B (B220(+)) lymphocytes (also apparent in the lymph nodes and Peyer's patches). Ulcerative colitis, interstitial pneumonitis, glomerular nephropathy with IgA deposits, autoantibodies, and joint inflammation are also evident. These progressive immunological disorders closely mimic those seen in WAS. WIP deficiency may thus be implicated in some cases in which mutations in the gene encoding WASP are not detected.

Asunto(s)

Proteínas Portadoras/genética , Síndrome de Wiskott-Aldrich/genética , Animales , Artritis/genética , Autoanticuerpos/sangre , Linfocitos B/inmunología , Proteínas Portadoras/metabolismo , Colitis Ulcerosa/genética , Colitis Ulcerosa/patología , Proteínas del Citoesqueleto , Recuento de Eritrocitos , Femenino , Citometría de Flujo , Glomerulonefritis/genética , Glomerulonefritis/patología , Intestinos/patología , Riñón/patología , Pulmón/patología , Enfermedades Pulmonares Intersticiales/genética , Enfermedades Pulmonares Intersticiales/patología , Ganglios Linfáticos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Animales , Recuento de Plaquetas , Bazo/inmunología , Síndrome de Wiskott-Aldrich/inmunología , Síndrome de Wiskott-Aldrich/patología , Proteína del Síndrome de Wiskott-Aldrich/metabolismo

RESUMEN

Induction of filopodia is dependent on activation of the small GTPase Cdc42 and on neural Wiskott-Aldrich-syndrome protein (N-WASP). Here we show that WASP-interacting protein (WIP) interacts directly with N-WASP and actin. WIP retards N-WASP/Cdc42-activated actin polymerization mediated by the Arp2/3 complex, and stabilizes actin filaments. Microinjection of WIP into NIH 3T3 fibroblasts induces filopodia; this is inhibited by microinjection of anti-N-WASP antibody. Microinjection of anti-WIP antibody inhibits induction of filopodia by bradykinin, by an active Cdc42 mutant (Cdc42(V12)) and by N-WASP. Our results indicate that WIP and N-WASP may act as a functional unit in filopodium formation, which is consistent with their role in actin-tail formation in cells infected with vaccinia virus or Shigella.

Asunto(s)

Actinas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas del Citoesqueleto , Proteínas del Tejido Nervioso/metabolismo , Seudópodos/metabolismo , Células 3T3 , Proteína 2 Relacionada con la Actina , Proteína 3 Relacionada con la Actina , Animales , Western Blotting , Bradiquinina/farmacología , Línea Celular , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Glutatión Transferasa/metabolismo , Ratones , Microscopía Fluorescente , Mutación , Unión Proteica , Estructura Terciaria de Proteína , Conejos , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/metabolismo , Shigella/metabolismo , Transducción de Señal , Factores de Tiempo , Técnicas del Sistema de Dos Híbridos , Proteína Neuronal del Síndrome de Wiskott-Aldrich , Proteína de Unión al GTP cdc42/metabolismo

RESUMEN

WIP, the Wiskott-Aldrich syndrome protein-interacting protein, is a human protein involved in actin polymerization and redistribution in lymphoid cells. The mechanism by which WIP reorganizes actin cytoskeleton is unknown. WIP is similar to yeast verprolin, an actin- and myosin-interacting protein required for polarized morphogenesis. To determine whether WIP and verprolin are functional homologues, we analyzed the function of WIP in yeast. WIP suppresses the growth defects of VRP1 missense and null mutations as well as the defects in cytoskeletal organization and endocytosis observed in vrp1-1 cells. The ability of WIP to replace verprolin is dependent on its WH2 actin binding domain and a putative profilin binding domain. Immunofluorescence localization of WIP in yeast cells reveals a pattern consistent with its function at the cortical sites of growth. Thus, like verprolin, WIP functions in yeast to link the polarity development pathway and the actin cytoskeleton to generate cytoskeletal asymmetry. A role for WIP in cell polarity provides a framework for unifying, under a common paradigm, distinct molecular defects associated with immunodeficiencies like Wiskott-Aldrich syndrome.

Asunto(s)

Proteínas Portadoras/genética , Polaridad Celular/genética , Proteínas Contráctiles , Citoesqueleto/fisiología , Proteínas Fúngicas/genética , Proteínas de Microfilamentos/genética , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Sitios de Unión , Proteínas Portadoras/aislamiento & purificación , Compartimento Celular , Proteínas del Citoesqueleto , Endocitosis/fisiología , Evolución Molecular , Prueba de Complementación Genética , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas de Microfilamentos/metabolismo , Datos de Secuencia Molecular , Mutación Missense , Profilinas , Unión Proteica , Homología de Secuencia de Aminoácido , Supresión Genética , Síndrome de Wiskott-Aldrich , Levaduras

RESUMEN

Wiskott-Aldrich syndrome (WAS) is an inherited immune deficiency that is marked by eczema, bleeding and recurrent infections. The lymphocytes and platelets of WAS patients display cytoskeletal abnormalities, and their T lymphocytes show a diminished proliferative response to stimulation through the T-cell receptor-CD3 complex (TCR-CD3). The product of the WAS gene, WAS protein (WASP), binds to the small GTPase Cdc42. Small GTPases of the Rho family are crucial for the regulation of the actin-based cytoskeleton. WASP and its relative NWASP might play an important role in regulating the actin cytoskeleton. Since both WASP and NWASP have the potential to bind to multiple proteins, they might serve as a hub to coordinate the redistribution of many cellular signals to the actin cytoskeleton. In this review, the authors discuss the possible role of WASP/NWASP and of the newly described protein WIP, which interacts with WASP and NWASP, in coupling signals from the T-cell receptor to the actin-based cytoskeleton.

Asunto(s)

Proteínas/metabolismo , Síndrome de Wiskott-Aldrich/metabolismo , Actinas , Animales , Proteínas Portadoras/metabolismo , Proteínas del Citoesqueleto , Citoesqueleto , GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Linfocitos , Complejo Receptor-CD3 del Antígeno de Linfocito T/metabolismo , Transducción de Señal , Proteína del Síndrome de Wiskott-Aldrich , Proteínas de Unión al GTP rho

RESUMEN

Coronaviruses have been described as pleomorphic, round particles with a helical nucleocapsid as the unique internal structure under the virion envelope. Our studies on the organization of the transmissible gastroenteritis coronavirus (TGEV) have shown that the structure of these viruses is more complex. Different electron microscopy techniques, including cryomicroscopy of vitrified viruses, revealed the existence of an internal core, most probably icosahedral, in TGEV virions. Disruption of these cores induced the release of elongated ribonucleoprotein complexes. Ultrastructural analysis of freeze-substituted TGEV-infected swine testis (ST) cells showed characteristic intracellular budding profiles as well as two types of virions. While large virions with an electron-dense internal periphery are seen at perinuclear regions, smaller viral particles exhibiting compact internal cores of poligonal contours are more abundant in areas closer to the plasma membrane of the cell. These data strongly suggest that maturation events following the budding process are responsible for the formation of the internal core shell, the new structural element that we have recently described in extracellular infectious TGEV virions.

Asunto(s)

Virus de la Gastroenteritis Transmisible/fisiología , Ensamble de Virus , Animales , Línea Celular , Porcinos , Virus de la Gastroenteritis Transmisible/ultraestructura

RESUMEN

Nck is a ubiquitous adaptor molecule composed of three Src homology 3 (SH3) domains followed by a single SH2 domain. Nck links, via its SH2 domain, tyrosine-phosphorylated receptors to effector proteins that contain SH3-binding proline-rich sequences. In this report, we demonstrate that recombinant Nck precipitates endogenous WIP, a novel proline-rich protein that interacts with the Wiskott-Aldrich syndrome protein (WASP), from BJAB cell lysates. Nck binds through its second SH3 domain to WIP, and Nck binds to WIP at a site (amino acids 321-415) that differs from the WASP-binding site (amino acids 416-488). WIP has been shown to associate with the actin polymerization regulatory protein profilin and to induce actin polymerization and cytoskeletal reorganization in lymphoid cells. We demonstrate the presence of profilin in Nck precipitates suggesting that Nck may couple extracellular signals to the cytoskeleton via its interaction with WIP and profilin.

Asunto(s)

Proteínas Oncogénicas/metabolismo , Proteínas/metabolismo , Síndrome de Wiskott-Aldrich/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Sitios de Unión , Línea Celular , Citoesqueleto/metabolismo , Receptores de Superficie Celular/metabolismo , Proteína del Síndrome de Wiskott-Aldrich , Dominios Homologos src

RESUMEN

Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency caused by mutations that affect the WAS protein (WASP) and characterized by cytoskeletal abnormalities in hematopoietic cells. By using the yeast two-hybrid system we have identified a proline-rich WASP-interacting protein (WIP), which coimmunoprecipitated with WASP from lymphocytes. WIP binds to WASP at a site distinct from the Cdc42 binding site and has actin as well as profilin binding motifs. Expression of WIP in human B cells, but not of a WIP truncation mutant that lacks the actin binding motif, increased polymerized actin content and induced the appearance of actin-containing cerebriform projections on the cell surface. These results suggest that WIP plays a role in cortical actin assembly that may be important for lymphocyte function.

Asunto(s)

Actinas/metabolismo , Proteínas Portadoras/genética , Linfocitos/metabolismo , Proteínas/metabolismo , Síndrome de Wiskott-Aldrich/genética , Secuencia de Aminoácidos , Proteínas Portadoras/metabolismo , Células Cultivadas , Proteínas del Citoesqueleto , Dimerización , Humanos , Péptidos y Proteínas de Señalización Intracelular , Linfocitos/ultraestructura , Datos de Secuencia Molecular , Proteínas/genética , Síndrome de Wiskott-Aldrich/metabolismo , Proteína del Síndrome de Wiskott-Aldrich

RESUMEN

Following infection of haplotype defined NIH-miniswine with virulent transmissible gastroenteritis coronavirus (TGEV), isolated mesenteric lymph node CD4+ T-cells mounted a specific proliferative response against infectious or inactivated purified virus in secondary in vitro stimulation. A specific, dose-dependent response to the three major recombinant viral proteins: spike (S), membrane (M), and nucleoprotein (N), purified by affinity chromatography, was characterized. Induction of in vitro antibody synthesis was analyzed. The purified recombinant viral proteins induced the in vitro synthesis of neutralizing TGEV-specific antibodies when porcine TGEV-immune cells were stimulated with each of the combinations made with two of the major structural proteins: S + N, S + M, and to a minor extent with M + N, but not by the individual proteins. S-protein was dissociated from purified virus using NP-40 detergent and then micellar S-protein oligomers (S-rosettes) were formed by removing the detergent. These occurred preferentially by the association of more than 10 S-protein trimmers. These S-rosettes in collaboration with either N or M-proteins elicited TGEV-specific antibodies with titers up to 84 and 60%, respectively, of those induced by the whole virus. N-protein could be partially substituted by a 15-mer peptide that represents a T helper epitope previously identified in N-protein (Antón et al. (1995)). These results indicate that the induction of high levels of TGEV-specific antibodies requires stimulation by at least two viral proteins, and that optimum responses are induced by a combination of S-rosettes and the nucleoprotein.

Asunto(s)

Anticuerpos Antivirales/biosíntesis , Virus de la Gastroenteritis Transmisible/química , Virus de la Gastroenteritis Transmisible/inmunología , Proteínas Estructurales Virales/inmunología , Proteínas Estructurales Virales/fisiología , Animales , Anticuerpos Bloqueadores/farmacología , Anticuerpos Monoclonales/farmacología , Antígenos de Superficie/inmunología , Línea Celular , Epitelio , Haplotipos/inmunología , Activación de Linfocitos , Masculino , Micelas , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Especificidad de la Especie , Porcinos , Porcinos Enanos , Linfocitos T/inmunología , Testículo , Virus de la Gastroenteritis Transmisible/genética , Proteínas Virales/aislamiento & purificación , Proteínas Estructurales Virales/genética

RESUMEN

Coronaviruses are enveloped RNA viruses involved in a variety of pathologies that affect animals and humans. Existing structural models of these viruses propose a helical nucleocapsid under the virion envelope as the unique internal structure. In the present work, we have analyzed the structure of the transmissible gastroenteritis coronavirus. The definition of its organization supports a new structural model for coronaviruses, since a spherical, probably icosahedral, internal core has been characterized. Disruption of these cores induces the release of N-protein-containing helical nucleocapsids. Immunogold mapping and protein analysis of purified cores showed that they consist of M and N proteins, M being the main core shell component. This surprising finding, together with the fact that M protein molecules are also located in the virion envelope, indicates that a reconsideration of the assembly and maturation of coronaviruses, as well as a study of potential M-protein subclasses, is needed.

Asunto(s)

Cápside/análisis , Virus de la Gastroenteritis Transmisible/química , Proteínas del Núcleo Viral/análisis , Proteínas de la Matriz Viral/análisis , Animales , Línea Celular , Octoxinol , Polietilenglicoles/farmacología , Porcinos , Virus de la Gastroenteritis Transmisible/ultraestructura , Virión/química , Virión/ultraestructura

RESUMEN

Antigenic site D from the spike protein of transmissible gastroenteritis virus (TGEV), which is a continuous epitope critical in neutralization, has been expressed as a fusion protein with E. coli heat-labile toxin B subunit (LT-B) in attenuated S. typhimurium. Synthetic peptides containing the sequence of site D induced TGEV neutralizing antibodies when inoculated subcutaneously in both rabbits and swine. A synthetic oligonucleotide encoding residues 373-398 of TGEV S protein, including antigenic site D, was cloned in frame with the 3' end of LT-B gene, into a plasmid used to transform S. typhimurium delta asd chi 3730. A collection of 6 recombinant plasmids designated pYALTB-D I-VI encoding LTB-site D fusions with a variable number of site D sequences were selected. Four of the 6 LTB-site D fusion products expressed in S. typhimurium chi 3730 formed oligomers (pentamers) that dissociated at > 70 degrees. S. typhimurium chi 3730 (pYALTB-D) V and VI expressed the oligomer forming products with higher antigenicity. Partially purified LTB-site D fusion product expressed from S. typhimurium chi 3730 (pYALTB-D) V induced anti-TGEV neutralizing antibodies in rabbits. Recombinant vaccine strain S. typhimurium delta cya delta crp delta asd chi 3987 transformed with plasmid pYALTB-D V expressed constitutively products that formed oligomers presumably containing 20 copies of site D, and showed a high stability in vitro. This recombinant strain was orally inoculated in rabbits and induced TGEV specific antibodies in both serum and intestinal secretion.

Asunto(s)

Toxinas Bacterianas/inmunología , Enterotoxinas/inmunología , Epítopos de Linfocito B/inmunología , Proteínas de Escherichia coli , Glicoproteínas de Membrana/inmunología , Virus de la Gastroenteritis Transmisible/inmunología , Proteínas del Envoltorio Viral/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales/inmunología , Toxinas Bacterianas/genética , Secuencia de Bases , ADN Viral , Enterotoxinas/genética , Epítopos de Linfocito B/genética , Expresión Génica , Glicoproteínas de Membrana/genética , Datos de Secuencia Molecular , Pruebas de Neutralización , Conejos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Salmonella typhimurium , Glicoproteína de la Espiga del Coronavirus , Porcinos , Virus de la Gastroenteritis Transmisible/genética , Proteínas del Envoltorio Viral/genética

RESUMEN

Four strong T cell epitopes have been identified studying the blastogenic response of lymphocytes from haplotype-defined transmissible gastroenteritis virus (TGEV) immune miniswine to sixty-one 15-mer synthetic peptides. Three of these epitopes are located on the nucleoprotein (N46, amino acids 46 to 60; N272, amino acids 272 to 286; and N321, amino acids 321 to 335), and one on the membrane protein (M196, amino acids 196 to 210). N321 peptide induced the highest T cell response and was recognized by immune miniswine lymphocytes with haplotypes dd, aa, and cc. T lymphocytes from peptide N321-immune miniswine reconstituted the in vitro synthesis of TGEV-specific antibodies by complementing CD4- TGEV-immune cells. This response was directed at least against the three major structural proteins. The synthesized antibodies specific for S protein preferentially recognized discontinuous epitopes and neutralized TGEV infectivity. These results show that peptide N321 defines a functional T helper epitope eliciting T cells capable of collaborating with B cells specific for different proteins of TGEV.

Asunto(s)

Anticuerpos Antivirales/biosíntesis , Epítopos de Linfocito T/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Virus de la Gastroenteritis Transmisible/inmunología , Proteínas Estructurales Virales/inmunología , Secuencia de Aminoácidos , Animales , Formación de Anticuerpos , Especificidad de Anticuerpos , Haplotipos , Activación de Linfocitos , Complejo Mayor de Histocompatibilidad , Datos de Secuencia Molecular , Pruebas de Neutralización , Péptidos/síntesis química , Péptidos/inmunología , Porcinos , Proteínas Estructurales Virales/química

RESUMEN

The binding domains of four monoclonal antibodies (MAbs) specific for the M protein of the PUR46-MAD strain of transmissible gastroenteritis coronavirus (TGEV) have been located in the 46 carboxy-terminal amino acids of the protein by studying the binding of MAbs to recombinant M protein fragments. Immunoelectron microscopy using these MAbs demonstrated that in a significant proportion of the M protein molecules, the carboxy terminus is exposed on the external surface both in purified viruses and in nascent TGEV virions that recently exited infected swine testis cells. The same MAbs specifically neutralized the infectivity of the PUR46-MAD strain, indicating that the C-terminal domain of M protein is exposed on infectious viruses. This topology of TGEV M protein probably coexists with the structure currently described for the M protein of coronaviruses, which consists of an exposed amino terminus and an intravirion carboxy-terminal domain. The presence of a detectable number of M protein molecules with their carboxy termini exposed on the surface of the virion has relevance for viral function, since it has been shown that the carboxy terminus of M protein is immunodominant and that antibodies specific for this domain both neutralize TGEV and mediate the complement-dependent lysis of TGEV-infected cells.

Asunto(s)

Virus de la Gastroenteritis Transmisible/metabolismo , Proteínas de la Matriz Viral/análisis , Virión/metabolismo , Animales , Anticuerpos Monoclonales , Reacciones Antígeno-Anticuerpo , Antígenos Virales/análisis , Células Cultivadas , Clonación Molecular , Masculino , Ratones/inmunología , Microscopía Inmunoelectrónica , Modelos Estructurales , Pruebas de Neutralización , Conformación Proteica , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/inmunología , Porcinos , Testículo , Virus de la Gastroenteritis Transmisible/ultraestructura , Proteínas de la Matriz Viral/química , Proteínas de la Matriz Viral/inmunología , Virión/ultraestructura

Asunto(s)

Infecciones por Coronavirus/inmunología , Coronavirus/inmunología , Vacunas Virales , Animales , Antígenos Virales/inmunología , Linfocitos B/inmunología , Coronavirus/genética , Infecciones por Coronavirus/prevención & control , Diseño de Fármacos , Epítopos/inmunología , Humanos , Linfocitos T/inmunología , Vacunas Sintéticas/inmunología

Asunto(s)

Adenovirus Humanos , Antígenos Virales/inmunología , Infecciones por Coronavirus/prevención & control , Epítopos/inmunología , Vectores Genéticos , Intestinos/microbiología , Glicoproteínas de Membrana/inmunología , Proteínas Recombinantes de Fusión/inmunología , Salmonella typhimurium , Virus de la Gastroenteritis Transmisible/inmunología , Vacunación , Vacunas Sintéticas/inmunología , Proteínas del Envoltorio Viral/inmunología , Vacunas Virales/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales/biosíntesis , Clonación Molecular , Infecciones por Coronavirus/inmunología , Cricetinae , Intestinos/inmunología , Datos de Secuencia Molecular , Especificidad de Órganos , Ganglios Linfáticos Agregados/inmunología , Ganglios Linfáticos Agregados/microbiología , Glicoproteína de la Espiga del Coronavirus

RESUMEN

The antigenic structure of the S glycoprotein of transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV) has been determined and correlated with the physical structure. Four antigenic sites have been defined (A, B, C, and D). The sites involved in the neutralization of TGEV are: A, D, and B, sites A and D being antigenically dominant for TGEV neutralization in vitro. These two sites have specific properties of interest: site A is highly conserved and is present in coronaviruses of three animal species, and site D can be represented by synthetic peptides. Both sites might be relevant in protection in vivo. PRCV does not have sites B and C, due to a genomic deletion. Complex antigenic sites, i.e., conformation and glycosylation dependent sites, have been represented by simple mimotopes selected from a library expressing recombinant peptides with random sequences, or by anti-idiotypic internal image monoclonal antibodies. An epidemiological tree relating the TGEVs and PRCVs has been proposed. The estimated mutation fixation rate of 7 +/- 2 x 10(-4) substitutions per nucleotide and year indicates that TGEV related coronaviruses show similar variability to other RNA viruses. In order to induce secretory immunity, different segments of the S gene have been expressed using a virulent forms of Salmonella typhimurium and adenovirus. These vectors, with a tropism for Peyer's patches may be ideal candidates in protection against TGEV.

Asunto(s)

Antígenos Virales/inmunología , Virus de la Gastroenteritis Transmisible/inmunología , Proteínas Virales/inmunología , Secuencia de Aminoácidos , Animales , Antígenos Virales/genética , Sitios de Unión , Clonación Molecular , Epítopos/análisis , Epítopos/genética , Regulación Viral de la Expresión Génica , Vectores Genéticos , Glicoproteínas/genética , Glicoproteínas/inmunología , Filogenia , Plásmidos , Homología de Secuencia de Aminoácido , Porcinos , Virus de la Gastroenteritis Transmisible/genética , Proteínas Virales/genética

RESUMEN

Monoclonal antibody (MAb) 6A.C3 neutralizes transmissible gastroenteritis coronavirus (TGEV) and is specific for a conserved epitope within subsite Ac of the spike (S) glycoprotein of TGEV. Six hybridomas secreting anti-idiotypic (Ab2) MAbs specific for MAb 6A.C3 (Ab1) have been selected. All six MAbs inhibited the binding of Ab1 to TGEV and specifically cross-linked MAb1-6A.C3. Four of these hybridomas secreted gamma-type anti-idiotypic MAbs. The other two Ab2s (MAbs 9A.G3 and 9C.E11) were recognized by TGEV-specific antiserum induced in two species. This binding was inhibited by viruses of the TGEV group but not by serologically unrelated coronaviruses. These results indicate that MAb2-9A.G3 and MAb2-9C.E11 mimic an antigenic determinant present on the TGEV surface, and they were classified as beta-type ("internal-image") MAbs. TGEV-binding Ab3 antiserum was induced in 100% of mice immunized with the two beta-type MAb2s and in 25 to 50% of mice immunized with gamma-type MAb2. Both beta- and gamma-type Ab2s induced neutralizing Ab3 antibodies in mice that were mainly directed to antigenic subsite Ac of the S protein.

Asunto(s)

Anticuerpos Antiidiotipos/inmunología , Anticuerpos Monoclonales/inmunología , Antígenos Virales/inmunología , Coronaviridae/inmunología , Epítopos/análisis , Virus de la Gastroenteritis Transmisible/inmunología , Animales , Especificidad de Anticuerpos , Complejo Antígeno-Anticuerpo , Antígenos Virales/análisis , Coronavirus Felino/inmunología , Humanos , Hibridomas/inmunología , Cinética , Virus de la Hepatitis Murina/inmunología , Pruebas de Neutralización , Especificidad de la Especie