RESUMO
African swine fever virus (ASFV) causes a devastating hemorrhagic disease with worldwide circulation and no widely available therapeutic prevention. The infectious particle has a multilayered architecture that is articulated upon an endoplasmic reticulum (ER)-derived inner envelope. This membrane acts as docking platform for the assembly of the outer icosahedral capsid and the underlying core shell, a bridging layer required for the formation of the central genome-containing nucleoid. While the details of outer capsid assembly are relatively well understood, those of core formation remain unclear. Here we report the functional characterization of pEP84R, a transmembrane polypeptide embedded in the inner envelope that surrounds the viral core. Using an ASFV recombinant inducibly expressing the EP84R gene, we show that absence of pEP84R results in the formation of non-infectious core-less icosahedral particles displaying a significant DNA-packaging defect. Concomitantly, aberrant core shell-like structures formed by co-assembly of viral polyproteins pp220 and pp62 are mistargeted to non-ER membranes, as also occurs when these are co-expressed in the absence of other viral proteins. Interestingly, co-expression of both polyproteins with pEP84R led to the formation of ER-targeted core shell-like assemblies and co-immunoprecipitation assays showed that pEP84R binds to the N-terminal region of pp220. Altogether, these results indicate that pEP84R plays a crucial role in core assembly by targeting the core shell polyproteins to the inner viral envelope, which enables subsequent genome packaging and nucleoid formation. These findings unveil a key regulatory mechanism for ASFV morphogenesis and identify a relevant novel target for the development of therapeutic tools against this re-emerging threat.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Animais , Suínos , Vírus da Febre Suína Africana/genética , Vírus da Febre Suína Africana/metabolismo , Montagem de Vírus , Proteínas Virais/genética , Proteínas Virais/metabolismo , Poliproteínas/metabolismo , Proteínas de MembranaRESUMO
Hedgehog (Hh) signal molecules play a fundamental role in development, adult stem cell maintenance and cancer. Hh can signal at a distance, and we have proposed that its graded distribution across Drosophila epithelia is mediated by filopodia-like structures called cytonemes. Hh reception by Patched (Ptc) happens at discrete sites along presenting and receiving cytonemes, reminiscent of synaptic processes. Here, we show that a vesicle fusion mechanism mediated by SNARE proteins is required for Ptc placement at contact sites. Transport of Ptc to these sites requires multivesicular bodies (MVBs) formation via ESCRT machinery, in a manner different to that regulating Ptc/Hh lysosomal degradation after reception. These MVBs include extracellular vesicle (EV) markers and, accordingly, Ptc is detected in the purified exosomal fraction from cultured cells. Blockage of Ptc trafficking and fusion to basolateral membranes result in low levels of Ptc presentation for reception, causing an extended and flattened Hh gradient.
Assuntos
Proteínas de Drosophila/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas Hedgehog/metabolismo , Discos Imaginais/metabolismo , Receptores de Superfície Celular/metabolismo , Proteínas SNARE/metabolismo , Asas de Animais , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Proteínas Hedgehog/genética , Transporte Proteico , Receptores de Superfície Celular/genética , Proteínas SNARE/genéticaRESUMO
Apical localization of Intercellular Adhesion Receptor (ICAM)-1 regulates the adhesion and guidance of leukocytes across polarized epithelial barriers. Here, we investigate the molecular mechanisms that determine ICAM-1 localization into apical membrane domains of polarized hepatic epithelial cells, and their effect on lymphocyte-hepatic epithelial cell interaction. We had previously shown that segregation of ICAM-1 into apical membrane domains, which form bile canaliculi and bile ducts in hepatic epithelial cells, requires basolateral-to-apical transcytosis. Searching for protein machinery potentially involved in ICAM-1 polarization we found that the SNARE-associated protein plasmolipin (PLLP) is expressed in the subapical compartment of hepatic epithelial cells in vitro and in vivo. BioID analysis of ICAM-1 revealed proximal interaction between this adhesion receptor and PLLP. ICAM-1 colocalized and interacted with PLLP during the transcytosis of the receptor. PLLP gene editing and silencing increased the basolateral localization and reduced the apical confinement of ICAM-1 without affecting apicobasal polarity of hepatic epithelial cells, indicating that ICAM-1 transcytosis is specifically impaired in the absence of PLLP. Importantly, PLLP depletion was sufficient to increase T-cell adhesion to hepatic epithelial cells. Such an increase depended on the epithelial cell polarity and ICAM-1 expression, showing that the epithelial transcytotic machinery regulates the adhesion of lymphocytes to polarized epithelial cells. Our findings strongly suggest that the polarized intracellular transport of adhesion receptors constitutes a new regulatory layer of the epithelial inflammatory response.
Assuntos
Adesão Celular/fisiologia , Células Epiteliais/metabolismo , Hepatócitos/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/metabolismo , Linfócitos T/metabolismo , Linhagem Celular Tumoral , Células Hep G2 , Humanos , Fígado/metabolismo , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/genética , Transcitose/fisiologiaRESUMO
African swine fever virus (ASFV) is a complex nucleocytoplasmic large DNA virus (NCLDV) that causes a devastating swine disease currently present in many countries of Africa, Europe, and Asia. Despite intense research efforts, relevant gaps in the architecture of the infectious virus particle remain. Here, we used single-particle cryo-EM to analyze the three-dimensional structure of the mature ASFV particle. Our results show that the ASFV virion, with a radial diameter of â¼2,080 Å, encloses a genome-containing nucleoid surrounded by two distinct icosahedral protein capsids and two lipoprotein membranes. The outer capsid forms a hexagonal lattice (triangulation number T = 277) composed of 8,280 copies of the double jelly-roll major capsid protein (MCP) p72, arranged in trimers displaying a pseudo-hexameric morphology, and of 60 copies of a penton protein at the vertices. The inner protein layer, organized as a T = 19 capsid, confines the core shell, and it is composed of the mature products derived from the ASFV polyproteins pp220 and pp62. Also, an icosahedral membrane lies between the two protein layers, whereas a pleomorphic envelope wraps the outer capsid. This high-level organization confers to ASFV a unique architecture among the NCLDVs that likely reflects the complexity of its infection process and may help explain current challenges in controlling it.
Assuntos
Vírus da Febre Suína Africana/ultraestrutura , Proteínas do Capsídeo/ultraestrutura , Capsídeo/ultraestrutura , Proteínas do Envelope Viral/ultraestrutura , Vírus da Febre Suína Africana/metabolismo , Animais , Proteínas do Capsídeo/química , Chlorocebus aethiops , Microscopia Crioeletrônica , Lipídeos/química , Multimerização Proteica , Células Vero , Proteínas do Envelope Viral/químicaRESUMO
As many tumor cells synthetize vascular endothelial growth factors (VEGF) that promote neo-vascularization and metastasis, frontline cancer therapies often administer anti-VEGF (α-VEGF) antibodies. To target the oncolytic parvovirus minute virus of mice (MVM) to the tumor vasculature, we studied the functional tolerance, evasion of neutralization, and induction of α-VEGF antibodies of chimeric viruses in which the footprint of a neutralizing monoclonal antibody within the 3-fold capsid spike was replaced by VEGF-blocking peptides: P6L (PQPRPL) and A7R (ATWLPPR). Both peptides allowed viral genome replication and nuclear translocation of chimeric capsid subunits. MVM-P6L efficiently propagated in culture, exposing the heterologous peptide on the capsid surface, and evaded neutralization by the anti-spike monoclonal antibody. In contrast, MVM-A7R yielded low infectious titers and was poorly recognized by an α-A7R monoclonal antibody. MVM-A7R showed a deficient assembly pattern, suggesting that A7R impaired a transitional configuration that the subunits must undergo in the 3-fold axis to close up the capsid shell. The MVM-A7R chimeric virus consistently evolved in culture into a mutant carrying the P6Q amino acid substitution within the A7R sequence, which restored normal capsid assembly and infectivity. Consistent with this finding, anti-native VEGF antibodies were induced in mice by a single injection of MVM-A7R empty capsids, but not by MVM-A7R virions. This fundamental study provides insights to endow an infectious parvovirus with immune antineovascularization and evasion capacities by replacing an antibody footprint in the capsid 3-fold axis with VEGF-blocking peptides, and it also illustrates the evolutionary capacity of single-stranded DNA (ssDNA) viruses to overcome engineered capsid structural restrictions.IMPORTANCE Targeting the VEGF signaling required for neovascularization by vaccination with chimeric capsids of oncolytic viruses may boost therapy for solid tumors. VEGF-blocking peptides (VEbp) engineered in the capsid 3-fold axis endowed the infectious parvovirus MVM with the ability to induce α-VEGF antibodies without adjuvant and to evade neutralization by MVM-specific antibodies. However, these properties may be compromised by structural restraints that the capsid imposes on the peptide configuration and by misassembly caused by the heterologous peptides. Significantly, chimeric MVM-VEbp resolved the structural restrictions by selecting mutations within the engineered peptides that restored efficient capsid assembly. These data show the promise of antineovascularization vaccines using chimeric VEbp-icosahedral capsids of oncolytic viruses but also raise safety concerns regarding the genetic stability of manipulated infectious parvoviruses in cancer and gene therapies.
Assuntos
Vacinas Anticâncer/imunologia , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/metabolismo , Vírus Miúdo do Camundongo/imunologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/genética , Proteínas do Capsídeo/genética , Camundongos Endogâmicos BALB C , Vírus Miúdo do Camundongo/genética , Vírus Miúdo do Camundongo/crescimento & desenvolvimento , Vírus Oncolíticos/genética , Vírus Oncolíticos/crescimento & desenvolvimento , Vírus Oncolíticos/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Carga Viral , Montagem de Vírus , Ligação Viral , Internalização do VírusRESUMO
African swine fever virus (ASFV) is a large and complex DNA virus that causes a highly lethal swine disease for which there is no vaccine available. The ASFV particle, with an icosahedral multilayered structure, contains multiple polypeptides whose identity is largely unknown. Here, we analyzed by mass spectroscopy the protein composition of highly purified extracellular ASFV particles and performed immunoelectron microscopy to localize several of the detected proteins. The proteomic analysis identified 68 viral proteins, which account for 39% of the genome coding capacity. The ASFV proteome includes essentially all the previously described virion proteins and, interestingly, 44 newly identified virus-packaged polypeptides, half of which have an unknown function. A great proportion of the virion proteins are committed to the virus architecture, including two newly identified structural proteins, p5 and p8, which are derived from the core polyproteins pp220 and pp62, respectively. In addition, the virion contains a full complement of enzymes and factors involved in viral transcription, various enzymes implicated in DNA repair and protein modification, and some proteins concerned with virus entry and host defense evasion. Finally, 21 host proteins, many of them localized at the cell surface and related to the cortical actin cytoskeleton, were reproducibly detected in the ASFV particle. Immunoelectron microscopy strongly supports the suggestion that these host membrane-associated proteins are recruited during virus budding at actin-dependent membrane protrusions. Altogether, the results of this study provide a comprehensive model of the ASFV architecture that integrates both compositional and structural information.IMPORTANCE African swine fever virus causes a highly contagious and lethal disease of swine that currently affects many countries of sub-Saharan Africa, the Caucasus, the Russian Federation, and Eastern Europe and has very recently spread to China. Despite extensive research, effective vaccines or antiviral strategies are still lacking, and many basic questions on the molecular mechanisms underlying the infective cycle remain. One such gap regards the composition and structure of the infectious virus particle. In the study described in this report, we identified the set of viral and host proteins that compose the virion and determined or inferred the localization of many of them. This information significantly increases our understanding of the biological and structural features of an infectious African swine fever virus particle and will help direct future research efforts.
Assuntos
Vírus da Febre Suína Africana/fisiologia , Febre Suína Africana/metabolismo , Poliproteínas/metabolismo , Proteoma/análise , Proteínas Virais/metabolismo , Vírion/metabolismo , Febre Suína Africana/virologia , Vírus da Febre Suína Africana/ultraestrutura , Sequência de Aminoácidos , Animais , Chlorocebus aethiops , Microscopia Imunoeletrônica , Suínos , Células Vero , Vírion/crescimento & desenvolvimentoRESUMO
African swine fever virus (ASFV) is a large, multienveloped DNA virus composed of a genome-containing core successively wrapped by an inner lipid envelope, an icosahedral protein capsid, and an outer lipid envelope. In keeping with this structural complexity, recent studies have revealed an intricate entry program. This Gem highlights how ASFV uses two alternative pathways, macropinocytosis and clathrin-mediated endocytosis, to enter into the host macrophage and how the endocytosed particles undergo a stepwise, low pH-driven disassembly leading to inner envelope fusion and core delivery in the cytoplasm.
Assuntos
Vírus da Febre Suína Africana/fisiologia , Febre Suína Africana/virologia , Internalização do Vírus , Febre Suína Africana/imunologia , Febre Suína Africana/metabolismo , Animais , Transporte Biológico , Endocitose , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/virologia , Fusão de Membrana , Suínos , Desenvelopamento do VírusRESUMO
African swine fever virus (ASFV) is a nucleocytoplasmic large DNA virus (NCLDV) that causes a highly lethal disease in domestic pigs. As other NCLDVs, the extracellular form of ASFV possesses a multilayered structure consisting of a genome-containing nucleoid successively wrapped by a thick protein core shell, an inner lipid membrane, an icosahedral protein capsid and an outer lipid envelope. This structural complexity suggests an intricate mechanism of internalization in order to deliver the virus genome into the cytoplasm. By using flow cytometry in combination with pharmacological entry inhibitors, as well as fluorescence and electron microscopy approaches, we have dissected the entry and uncoating pathway used by ASFV to infect the macrophage, its natural host cell. We found that purified extracellular ASFV is internalized by both constitutive macropinocytosis and clathrin-mediated endocytosis. Once inside the cell, ASFV particles move from early endosomes or macropinosomes to late, multivesicular endosomes where they become uncoated. Virus uncoating requires acidic pH and involves the disruption of the outer membrane as well as of the protein capsid. As a consequence, the inner viral membrane becomes exposed and fuses with the limiting endosomal membrane to release the viral core into the cytosol. Interestingly, virus fusion is dependent on virus protein pE248R, a transmembrane polypeptide of the inner envelope that shares sequence similarity with some members of the poxviral entry/fusion complex. Collective evidence supports an entry model for ASFV that might also explain the uncoating of other multienveloped icosahedral NCLDVs.
Assuntos
Vírus da Febre Suína Africana/patogenicidade , Febre Suína Africana/virologia , Internalização do Vírus , Desenvelopamento do Vírus/fisiologia , Animais , Western Blotting , Capsídeo/metabolismo , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Endocitose , Endossomos/ultraestrutura , Endossomos/virologia , Citometria de Fluxo , Técnicas de Silenciamento de Genes , Microscopia Eletrônica , Microscopia de Fluorescência , Corpos Multivesiculares/metabolismo , Corpos Multivesiculares/ultraestrutura , Suínos , Proteínas do Envelope Viral/metabolismoRESUMO
The base of the primary cilium contains a zone of condensed membranes whose importance is not known. Here, we have studied the involvement of MAL, a tetraspanning protein that exclusively partitions into condensed membrane fractions, in the condensation of membranes at the ciliary base and investigated the importance of these membranes in primary cilium formation. We show that MAL accumulates at the ciliary base of epithelial MDCK cells. Knockdown of MAL expression resulted in a drastic reduction in the condensation of membranes at the ciliary base, the percentage of ciliated cells and the length of the cilia, but did not affect the docking of the centrosome to the plasma membrane or produce missorting of proteins to the pericentriolar zone or to the membrane of the remaining cilia. Rab8 (for which there are two isoforms, Rab8A and Rab8b), IFT88 and IFT20, which are important components of the machinery of ciliary growth, were recruited normally to the ciliary base of MAL-knockdown cells but were unable to elongate the primary cilium correctly. MAL, therefore, is crucial for the proper condensation of membranes at the ciliary base, which is required for efficient primary cilium extension.
Assuntos
Membrana Celular/fisiologia , Centrossomo/fisiologia , Cílios/fisiologia , Cílios/ultraestrutura , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/metabolismo , Animais , Western Blotting , Células Cultivadas , Cães , Imunofluorescência , Humanos , Células Madin Darby de Rim Canino , Microscopia Eletrônica , Morfogênese , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/antagonistas & inibidores , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Exosomes secreted by T cells play an important role in coordinating the immune response. HIV-1 Nef hijacks the route of exosome secretion of T cells to modulate the functioning of uninfected cells. Despite the importance of the process, the protein machinery involved in exosome biogenesis is yet to be identified. In this study, we show that MAL, a tetraspanning membrane protein expressed in human T cells, is present in endosomes that travel toward the plasma membrane for exosome secretion. In the absence of MAL, the release of exosome particles and markers was greatly impaired. This effect was accompanied by protein sorting defects at multivesicular endosomes that divert the exosomal marker CD63 to autophagic vacuoles. Exosome release induced by HIV-1 Nef was also dependent on MAL expression. Therefore, MAL is a critical element of the machinery for exosome secretion and may constitute a target for modulating exosome secretion by human T cells.
Assuntos
Infecções por HIV/imunologia , HIV-1/imunologia , Corpos Multivesiculares/metabolismo , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/metabolismo , Linfócitos T/imunologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/imunologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Humanos , Células Jurkat , Corpos Multivesiculares/imunologia , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/genética , Tetraspanina 30/imunologiaRESUMO
Amphibian-like ranaviruses include pathogens of fish, amphibians, and reptiles that have recently evolved from a fish-infecting ancestor. The molecular determinants of host range and virulence in this group are largely unknown, and currently fish infection models are lacking. We show that European sheatfish virus (ESV) can productively infect zebrafish, causing a lethal pathology, and describe a method for the generation of recombinant ESV, establishing a useful model for the study of fish ranavirus infections.
Assuntos
Infecções por Vírus de DNA/veterinária , Modelos Animais de Doenças , Doenças dos Peixes/virologia , Ranavirus/genética , Peixe-Zebra/virologia , Animais , Sequência de Bases , Infecções por Vírus de DNA/patologia , Infecções por Vírus de DNA/virologia , Doenças dos Peixes/patologia , Engenharia Genética , Genótipo , Larva/virologia , Dados de Sequência Molecular , Filogenia , Ranavirus/classificação , Ranavirus/patogenicidade , VirulênciaRESUMO
Collective evidence argues that two members of the nucleocytoplasmic large DNA viruses (NCLDVs) acquire their membrane from open membrane intermediates, postulated to be derived from membrane rupture. We now study membrane acquisition of the NCLDV African swine fever virus. By electron tomography (ET), the virion assembles a single bilayer, derived from open membrane precursors that collect as ribbons in the cytoplasm. Biochemically, lumenal endoplasmic reticulum (ER) proteins are released into the cytosol, arguing that the open intermediates are ruptured ER membranes. ET shows that viral capsid assembles on the convex side of the open viral membrane to shape it into an icosahedron. The viral capsid is composed of tiny spikes with a diameter of â¼5 nm, connected to the membrane by a 6 nm wide structure displaying thin striations, as observed by several complementary electron microscopy imaging methods. Immature particles display an opening that closes after uptake of the viral genome and core proteins, followed by the formation of the mature virion. Together with our previous data, this study shows a common principle of NCLDVs to build a single internal envelope from open membrane intermediates. Our data now provide biochemical evidence that these open intermediates result from rupture of a cellular membrane, the ER.
Assuntos
Vírus da Febre Suína Africana/fisiologia , Retículo Endoplasmático/metabolismo , Montagem de Vírus , Vírus da Febre Suína Africana/ultraestrutura , Animais , Células COS , Capsídeo/metabolismo , Capsídeo/ultraestrutura , Chlorocebus aethiops , Tomografia com Microscopia Eletrônica , Retículo Endoplasmático/ultraestrutura , Microscopia Eletrônica , Células VeroRESUMO
OBJECTIVE: Angiogenesis and inflammation are closely related processes. Gremlin is a novel noncanonical vascular endothelial growth factor receptor-2 (VEGFR2) ligand that induces a proangiogenic response in endothelial cells (ECs). Here, we investigated the role of the cyclic adenosine monophosphate-response element (CRE)-binding protein (CREB) in mediating the proinflammatory and proangiogenic responses of ECs to gremlin. APPROACH AND RESULTS: Gremlin induces a proinflammatory response in ECs, leading to reactive oxygen species and cyclic adenosine monophosphate production and the upregulation of proinflammatory molecules involved in leukocyte extravasation, including chemokine (C-C motif) ligand-2 (Ccl2) and Ccl7, chemokine (C-X-C motif) ligand-1 (Cxcl1), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1). Accordingly, gremlin induces the VEGFR2-dependent phosphorylation, nuclear translocation, and transactivating activity of CREB in ECs. CREB activation mediates the early phases of the angiogenic response to gremlin, including stimulation of EC motility and permeability, and leads to monocyte/macrophage adhesion to ECs and their extravasation. All these effects are inhibited by EC transfection with a dominant-negative CREB mutant or with a CREB-binding protein-CREB interaction inhibitor that competes for CREB/CRE binding. Also, both recombinant gremlin and gremlin-expressing tumor cells induce proinflammatory/proangiogenic responses in vivo that are suppressed by the anti-inflammatory drug hydrocortisone. Similar effects were induced by the canonical VEGFR2 ligand VEGF-A165. CONCLUSIONS: Together, the results underline the tight cross-talk between angiogenesis and inflammation and demonstrate a crucial role of CREB activation in the modulation of the VEGFR2-mediated proinflammatory/proangiogenic response of ECs to gremlin.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Células Endoteliais/metabolismo , Mediadores da Inflamação/metabolismo , Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neovascularização Fisiológica , Transporte Ativo do Núcleo Celular , Inibidores da Angiogênese/farmacologia , Animais , Anti-Inflamatórios/farmacologia , Permeabilidade Capilar , Adesão Celular , Movimento Celular , Quimiocina CCL2/metabolismo , Quimiocina CCL7/metabolismo , Quimiocina CXCL1/metabolismo , Embrião de Galinha , Técnicas de Cocultura , Meios de Cultivo Condicionados/metabolismo , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Citocinas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/imunologia , Células Endoteliais da Veia Umbilical Humana/imunologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Hidrocortisona/farmacologia , Inflamação/genética , Inflamação/imunologia , Inflamação/prevenção & controle , Molécula 1 de Adesão Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Ligantes , Células MCF-7 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Neovascularização Fisiológica/efeitos dos fármacos , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Fatores de Tempo , Transfecção , Molécula 1 de Adesão de Célula Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Hedgehog (Hh) moves from the producing cells to regulate the growth and development of distant cells in a variety of tissues. Here, we have investigated the mechanism of Hh release from the producing cells to form a morphogenetic gradient in the Drosophila wing imaginal disk epithelium. We describe that Hh reaches both apical and basolateral plasma membranes, but the apical Hh is subsequently internalized in the producing cells and routed to the basolateral surface, where Hh is released to form a long-range gradient. Functional analysis of the 12-transmembrane protein Dispatched, the glypican Dally-like (Dlp) protein, and the Ig-like and FNNIII domains of protein Interference Hh (Ihog) revealed that Dispatched could be involved in the regulation of vesicular trafficking necessary for basolateral release of Hh, Dlp, and Ihog. We also show that Dlp is needed in Hh-producing cells to allow for Hh release and that Ihog, which has been previously described as an Hh coreceptor, anchors Hh to the basolateral part of the disk epithelium.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Epitélio/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas de Membrana/metabolismo , Animais , Animais Geneticamente Modificados , Membrana Celular/metabolismo , Vesículas Citoplasmáticas/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Epitélio/crescimento & desenvolvimento , Epitélio/ultraestrutura , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Hedgehog/genética , Imuno-Histoquímica , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Microscopia Confocal , Microscopia Imunoeletrônica , Morfogênese , Mutação , Transporte Proteico , Proteoglicanas/genética , Proteoglicanas/metabolismo , Interferência de RNA , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Asas de Animais/crescimento & desenvolvimento , Asas de Animais/metabolismo , Asas de Animais/ultraestruturaRESUMO
Bluetongue virus (BTV) is the causative agent of the important livestock disease bluetongue (BT), which is transmitted via Culicoides bites. BT causes severe economic losses associated with its considerable impact on health and trade of animals. By reverse genetics, we have designed and rescued reporter-expressing recombinant (r)BTV expressing NanoLuc luciferase (NLuc) or Venus fluorescent protein. To generate these viruses, we custom synthesized a modified viral segment 5 encoding NS1 protein with the reporter genes located downstream and linked by the Porcine teschovirus-1 (PTV-1) 2A autoproteolytic cleavage site. Therefore, fluorescent signal or luciferase activity is only detected after virus replication and expression of non-structural proteins. Fluorescence or luminescence signals were detected in cells infected with rBTV/Venus or rBTV/NLuc, respectively. Moreover, the marking of NS2 protein confirmed that reporter genes were only expressed in BTV-infected cells. Growth kinetics of rBTV/NLuc and rBTV/Venus in Vero cells showed replication rates similar to those of wild-type and rBTV. Infectivity studies of these recombinant viruses in IFNAR(-/-) mice showed a higher lethal dose for rBTV/NLuc and rBTV/Venus than for rBTV indicating that viruses expressing the reporter genes are attenuated in vivo. Interestingly, luciferase activity was detected in the plasma of viraemic mice infected with rBTV/NLuc. Furthermore, luciferase activity quantitatively correlated with RNAemia levels of infected mice throughout the infection. In addition, we have investigated the in vivo replication and dissemination of BTV in IFNAR (-/-) mice using BTV/NLuc and non-invasive in vivo imaging systems.IMPORTANCEThe use of replication-competent viruses that encode a traceable fluorescent or luciferase reporter protein has significantly contributed to the in vitro and in vivo study of viral infections and the development of novel therapeutic approaches. In this work, we have generated rBTV that express fluorescent or luminescence proteins to track BTV infection both in vitro and in vivo. Despite the availability of vaccines, BTV and other related orbivirus are still associated with a significant impact on animal health and have important economic consequences worldwide. Our studies may contribute to the advance in orbivirus research and pave the way for the rapid development of new treatments, including vaccines.
Assuntos
Vírus Bluetongue , Vacinas , Chlorocebus aethiops , Animais , Camundongos , Vírus Bluetongue/genética , Genes Reporter , Células Vero , Proteínas Virais/genética , Luciferases/genéticaRESUMO
Epithelial intercellular adhesion molecule (ICAM)-1 is apically polarized, interacts with, and guides leukocytes across epithelial barriers. Polarized hepatic epithelia organize their apical membrane domain into bile canaliculi and ducts, which are not accessible to circulating immune cells but that nevertheless confine most of ICAM-1. Here, by analyzing ICAM-1_KO human hepatic cells, liver organoids from ICAM-1_KO mice and rescue-of-function experiments, we show that ICAM-1 regulates epithelial apicobasal polarity in a leukocyte adhesion-independent manner. ICAM-1 signals to an actomyosin network at the base of canalicular microvilli, thereby controlling the dynamics and size of bile canalicular-like structures. We identified the scaffolding protein EBP50/NHERF1/SLC9A3R1, which connects membrane proteins with the underlying actin cytoskeleton, in the proximity interactome of ICAM-1. EBP50 and ICAM-1 form nano-scale domains that overlap in microvilli, from which ICAM-1 regulates EBP50 nano-organization. Indeed, EBP50 expression is required for ICAM-1-mediated control of BC morphogenesis and actomyosin. Our findings indicate that ICAM-1 regulates the dynamics of epithelial apical membrane domains beyond its role as a heterotypic cell-cell adhesion molecule and reveal potential therapeutic strategies for preserving epithelial architecture during inflammatory stress.
Assuntos
Actomiosina , Molécula 1 de Adesão Intercelular , Animais , Camundongos , Humanos , Actomiosina/metabolismo , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Células Epiteliais/metabolismo , Hepatócitos/metabolismo , Fígado/metabolismo , Citoesqueleto de Actina/metabolismo , Leucócitos/metabolismo , Polaridade CelularRESUMO
SUMMARY: This is a descriptive summary of the case of a patient with Axenfeld-Rieger syndrome associated with a congenital malformation of the iris and consequent pupillary morphological alteration of an atypical characteristic reported. This anomaly is unique in scientific literature and exhibits a peculiarity that we have called pseudoacorea: Hidden pupil. Other associated abnormal clinical findings were posterior embryotoxon, astigmatism, amblyopia, and exotropia. Diagnosis was achieved by instilling ocular mydriatics into the cul-de-sac that revealed this peculiarity. It is necessary to make a differential diagnosis with other pupillary pathologies such as corectopia, acorea and microcoria. Early detection of pathology and surgical management is necessary, since it would lead to a better visual prognosis for both amblyopia and strabismus. BACKGROUND: Among the malformations of the pupil, we can find polycoria (more than one pupil), dyscoria (abnormal pupil shape), corectopia (abnormal pupil position) and acorea (absence of pupil). In addition, morphologically normal pupils can denote other anomalies such as the microcoria described by Holth in 1923. Acorea is a rare anomaly, congenital or acquired, characterized by an absolute absence of the pupil both at rest and in mydriasis. In our case we prefer to differentiate it and name it pseudoacorea, since although there is a total absence of the pupil at rest thanks to the application of ocular mydriatics, a micropupil with discoric and corectopic characteristics is achieved. It is worth noting that we have not detected in the scientific literature any case described as the one that we will develop here. CONCLUSION: The case of a patient with Axenfeld-Rieger syndrome associated with a congenital malformation of the iris and consequent atypical pupillary morphological alteration is presented. This anomaly is unique in the scientific literature and presents a peculiarity that we have called pseudoacorea: Hidden pupil. Early detection of pathology and surgical management is necessary, since it would lead to a better visual prognosis for both amblyopia and strabismus.
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The differentiation of B cells into antibody-secreting cells is fundamental for the generation of humoral immunity. In mammals, this process involves a series of metabolic and intracellular changes, not studied to date in teleost fish, where a clear distinction between naive B cells and plasmablasts/plasma cells (PCs) is still missing. Thus, in the current study, we have established that upon activation, teleost B cells undergo an expansion of the endoplasmic reticulum (ER) but experience no significant changes in mitochondria content. In parallel, the transcription of genes implicated in B cell differentiation increases, while that of mitochondrial genes decreases. In this context, ER monitoring has allowed us to distinguish between small cells with low amounts of ER (FSCloERlo B cells), that correspond to undifferentiated cells, and large cells with expanded ER (FSChiERhi B cells), characterized as plasmablasts. The results shed new light on the B cell differentiation process in teleosts and provide us with novel tools to study B cell function in these species.
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The first morphological evidence of African swine fever virus (ASFV) assembly is the appearance of precursor viral membranes, thought to derive from the endoplasmic reticulum, within the assembly sites. We have shown previously that protein p54, a viral structural integral membrane protein, is essential for the generation of the viral precursor membranes. In this report, we study the role of protein p17, an abundant transmembrane protein localized at the viral internal envelope, in these processes. Using an inducible virus for this protein, we show that p17 is essential for virus viability and that its repression blocks the proteolytic processing of polyproteins pp220 and pp62. Electron microscopy analyses demonstrate that when the infection occurs under restrictive conditions, viral morphogenesis is blocked at an early stage, immediately posterior to the formation of the viral precursor membranes, indicating that protein p17 is required to allow their progression toward icosahedral particles. Thus, the absence of this protein leads to an accumulation of these precursors and to the delocalization of the major components of the capsid and core shell domains. The study of ultrathin serial sections from cells infected with BA71V or the inducible virus under permissive conditions revealed the presence of large helicoidal structures from which immature particles are produced, suggesting that these helicoidal structures represent a previously undetected viral intermediate.
Assuntos
Vírus da Febre Suína Africana/fisiologia , Proteínas Estruturais Virais/fisiologia , Montagem de Vírus , Animais , Células COS , Chlorocebus aethiops , Produtos do Gene env/metabolismo , Microscopia Eletrônica de Transmissão , Microscopia Imunoeletrônica , Processamento de Proteína Pós-Traducional , Células Vero , Vírion/ultraestruturaRESUMO
OBJECTIVE: To evaluate stricture recurrence and urinary incontinence (UI) rates in patients who underwent bulbomembranous anastomosis for management of short (≤ 2cm) bulbomembranous urethral stricture (BMS) after benign prostatic hyperplasia (BPH) surgical treatment. In addition, we studied if there was any relation between post urethroplasty UI and the method employed for BPH surgical treatment. MATERIALS AND METHODS: A retrospective study was conducted between January 2011 and October 2019. We included all patients who developed BMS after undergoing Transurethral Resection of the Prostate, Holmium Laser Enucleation of the Prostate or Open Simple Prostatectomy (OSP). We excluded patients with UI after BPH surgical treatment as well as patients who underwent a dorsal or ventral onlay oral graft urethroplasty for longer proximal bulbar strictures, and also patients with associated bladder neck contracture or other strictures locations. We defined failure as the need for any intervention to restore the urethral caliber. RESULTS: Overall, 77 patients were included in the study with mean age 70 years (sd 8). Median BMS length was 1.5 cm (IQR 1-2). Median follow-up was 53 months (IQR 24 to 82). Of the patients, 74/77 (96.1%) were classified as success and 3/77 (3.9%), as failure. Out of the 6/77 (7.8%) patients who had postoperative UI, 5 of them had been treated for their BPH with OSP (p 0.001). CONCLUSIONS: Bulbomembranous anastomosis is a suitable reconstructive option for short proximal bulbar urethral strictures after BPH surgical treatment. OSP was associated with postoperative UI more frequently than endoscopic treatments modalities.