RESUMO
Flaviviruses are major human disease-causing pathogens, including dengue virus (DENV), Zika virus, yellow fever virus and others. DENV infects hundreds of millions of people per year around the world, causing a tremendous social and economic burden. DENV capsid (C) protein plays an essential role during genome encapsidation and viral particle formation. It has been previously shown that DENV C enters the nucleus in infected cells. However, whether DENV C protein exhibits nuclear export remains unclear. By spatially cross-correlating different regions of the cell, we investigated DENV C movement across the nuclear envelope during the infection cycle. We observed that transport takes place in both directions and with similar translocation times (in the ms time scale) suggesting a bidirectional movement of both C protein import and export.Furthermore, from the pair cross-correlation functions in cytoplasmic or nuclear regions we found two populations of C molecules in each compartment with fast and slow mobilities. While in the cytoplasm the correlation times were in the 2-6 and 40-110 ms range for the fast and slow mobility populations respectively, in the cell nucleus they were 1-10 and 25-140 ms range, respectively. The fast mobility of DENV C in cytoplasmic and nuclear regions agreed with the diffusion coefficients from Brownian motion previously reported from correlation analysis. These studies provide the first evidence of DENV C shuttling from and to the nucleus in infected cells, opening new venues for antiviral interventions.
Assuntos
Proteínas do Capsídeo/ultraestrutura , Vírus da Dengue/ultraestrutura , Dengue/virologia , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular , CricetinaeRESUMO
During flavivirus infection, some viral proteins move to the nucleus and cellular components are relocated from the nucleus to the cytoplasm. Thus, the integrity of the main regulator of the nuclear-cytoplasmic transport, the nuclear pore complex (NPC), was evaluated during infection with dengue virus (DENV) and Zika virus (ZIKV). We found that while during DENV infection the integrity and distribution of at least three nucleoporins (Nup), Nup153, Nup98, and Nup62 were altered, during ZIKV infection, the integrity of TPR, Nup153, and Nup98 were modified. In this work, several lines of evidence indicate that the viral serine protease NS2B3 is involved in Nups cleavage. First, the serine protease inhibitors, TLCK and Leupeptin, prevented Nup98 and Nup62 cleavage. Second, the transfection of DENV and ZIKV NS2B3 protease was sufficient to inhibit the nuclear ring recognition detected in mock-infected cells with the Mab414 antibody. Third, the mutant but not the active (WT) protease was unable to cleave Nups in transfected cells. Thus, here we describe for the first time that the NS3 protein from flavivirus plays novel functions hijacking the nuclear pore complex, the main controller of the nuclear-cytoplasmic transport.
Assuntos
Vírus da Dengue/metabolismo , Poro Nuclear/metabolismo , Serina Endopeptidases/metabolismo , Proteínas Virais/metabolismo , Zika virus/metabolismo , Transporte Ativo do Núcleo Celular , Dengue/metabolismo , Dengue/virologia , Vírus da Dengue/ultraestrutura , Immunoblotting , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Membrana Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Zika virus/ultraestrutura , Infecção por Zika virus/metabolismo , Infecção por Zika virus/virologiaRESUMO
BACKGROUND: At the present time, dengue is one of the most important arboviruses affecting man, becoming a serious global public health problem, especially in subtropical and tropical countries, where environmental conditions favor the development and proliferation of the mosquito Aedes aegypti. Dengue is caused by a type of flavivírus, which is an enveloped virus of spherical geometry. Nowadays, it is one of the diseases with the highest incidence in Brazil, reaching the population of all states, regardless of social class. Several papers address the molecular aspects of infection of human cell by the viruses, which are reviewed in this work. CONCLUSION: Analyzing the three-dimensional structures of the fusion peptide of dengue virus protein E, we observed that the fusion peptide presents a region rich in hydrophobic residues and a "collar" of charged, polar residues. Probably, this hydrophilic collar plays an important role in the fusion process between the dengue virus and the cell membrane. In order for this disease to cease being a serious global public health problem, we must deepen our knowledge about the fusion process between the dengue virus and the cell membrane through further experimental and, especially, computational studies to find ways to inhibit the mechanism of virus infection.
Assuntos
Vírus da Dengue/fisiologia , Vírus da Dengue/ultraestrutura , Proteínas do Envelope Viral/química , Internalização do Vírus , Dengue/virologia , Vírus da Dengue/química , Vírus da Dengue/patogenicidade , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Simulação de Dinâmica Molecular , Proteínas do Envelope Viral/metabolismoRESUMO
Dengue virus (DENV) is an arbovirus, which replicates in the endoplasmic reticulum. Although replicative cycle takes place in the cytoplasm, some viral proteins such as NS5 and C are translocated to the nucleus during infection in mosquitoes and mammalian cells. To localized viral proteins in DENV-infected C6/36 cells, an immunofluorescence (IF) and immunoelectron microscopy (IEM) analysis were performed. Our results indicated that C, NS1, NS3 and NS5 proteins were found in the nucleus of DENV-infected C6/36 cells. Additionally, complex structures named strand-like structures (Ss) were observed in the nucleus of infected cells. Interestingly, the NS5 protein was located in these structures. Ss were absent in mock-infected cells, suggesting that DENV induces their formation in the nucleus of infected mosquito cells.
Assuntos
Culicidae/virologia , Vírus da Dengue/ultraestrutura , Dengue/virologia , Proteínas não Estruturais Virais/ultraestrutura , Animais , Linhagem Celular , Núcleo Celular/ultraestrutura , Núcleo Celular/virologia , Retículo Endoplasmático/ultraestrutura , Retículo Endoplasmático/virologia , Humanos , Camundongos Endogâmicos BALB C , RNA Helicases/ultraestrutura , Serina Endopeptidases/ultraestrutura , Replicação ViralRESUMO
In cell culture, cell structures suffer strong impact due to centrifugation during processing for electron microscope observation. In order to minimise this effect, a new protocol was successfully developed. Using conventional reagents and equipments, it took over one week, but cell compression was reduced to none or the lowest deformation possible.
Assuntos
Aedes/ultraestrutura , Vírus da Dengue/ultraestrutura , Microscopia Eletrônica de Transmissão/métodos , Aedes/virologia , Animais , Técnicas de Cultura de Células , Centrifugação/métodos , Chlorocebus aethiops , Fixadores , Indicadores e Reagentes , Células Vero/ultraestruturaRESUMO
In cell culture, cell structures suffer strong impact due to centrifugation during processing for electron microscope observation. In order to minimise this effect, a new protocol was successfully developed. Using conventional reagents and equipments, it took over one week, but cell compression was reduced to none or the lowest deformation possible.
Assuntos
Animais , Aedes/ultraestrutura , Vírus da Dengue/ultraestrutura , Microscopia Eletrônica de Transmissão/métodos , Aedes/virologia , Técnicas de Cultura de Células , Centrifugação/métodos , Chlorocebus aethiops , Fixadores , Indicadores e Reagentes , Células Vero/ultraestruturaRESUMO
O vírus Dengue (DENV) codifica uma poliproteína que é clivada em três proteínas estruturais (capsídeo,envelope e membrana) e sete proteínas não estruturais (NS1, NS2A, NS2B, NS3,NS4A, NS4B e NS5). Durante a infecção viral, a NS1 é encontrada no interior de células hospedeiras, associada à membrana plasmática e também secretada para a circulação como um multímero solúvel. [...] Considerando que o fígado é normalmente afetado pela infecção por DENV, este trabalho teve como objetivo estudar o efeito da expressão isolada da proteína NS1 em células de hepatocarcinoma humano,HepG2. Anteriormente, foram construídos em nosso laboratório plasmídeos contendo ogene ns1: pcENS1, que codifica a região C-terminal da proteína E (peptídeo sinal da proteína NS1) e NS1; pcENS1ANC, similar ao pcENS1 com a adição da porção Nterminalda NS2A (ANC), importante para a associação da NS1 à membrana plasmática; e pcTPANS1, que contém a sequência que codifica o peptídeo sinal do ativador de plasminogênio de tecido humano (t-PA) fusionada à sequência NS1. Neste trabalho, nós inicialmente padronizamos um protocolo de transfecção eficiente para aHepG2, utilizando o plasmídeo pcENS1. Avaliamos três métodos de transfecção distintos: com os lipossomos Lipofectamina e FuGENE 6, e a eletroporação no aparelho NucleofectorTM. A produção da NS1 recombinante foi detectada e quantificada por imunofluorescência e citometria de fluxo...
The dengue virus (DENV) encodes a polyprotein,which is cleaved in three structural proteins (capsid, envelope and membrane) and sevennon-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5). During virusinfection, the NS1 is found inside host cells, associated with cell plasma membrane andalso secreted into the circulation as a soluble multimer. [...] Considering that the liver is normally affected by DENV infection, thiswork aimed to study the effect of isolated expression of the NS1 protein in humanhepatocellular carcinoma cells, HepG2. Previously, plasmids containing the ns1 genesequence were constructed in our laboratory: pcENS1, which encodes the C-terminalend of E protein (signal peptide of NS1 protein) and the NS1; pcENS1ANC, similar topcENS1 with the addition of the N-terminal portion of NS2A (ANC), important for NS1association with the plasma membrane; and pcTPANS1, encoding the human tissueplasminogen activator signal sequence (t-PA) fused to the NS1 sequence. In this work,we initially standardized an efficient protocol of HepG2 transfection, using the plasmidpcENS1. We evaluated three different transfection methods: with the liposomesLipofectamine and FuGENE 6 and the electroporation in NucleofectorTM Device. Therecombinant NS1 production was detected and quantified by immunofluorescence andflow cytometry. We observed a reduced number of cells producing NS1 in transfectionswith Lipofectamine (3.9 percent), and FuGENE 6 (6.8 percent)...
Assuntos
Humanos , Dengue , Proteômica , Vírus da Dengue/ultraestrutura , ImunofluorescênciaRESUMO
Dengue virus is the most significant virus transmitted by arthropods worldwide and may cause a potentially fatal systemic disease named dengue hemorrhagic fever. In this work, dengue virus serotype 4 was detected in the tissues of one fatal dengue hemorrhagic fever case using electron immunomicroscopy and molecular methods. This is the first report of dengue virus polypeptides findings by electron immunomicroscopy in human samples. In addition, not-previously-documented virus-like particles visualized in spleen, hepatic, brain, and pulmonary tissues from a dengue case are discussed.
Assuntos
Vírus da Dengue/genética , Vírus da Dengue/ultraestrutura , Dengue Grave/diagnóstico , Adulto , Anticorpos Antivirais/sangue , Encéfalo/ultraestrutura , Encéfalo/virologia , Cuba , DNA Viral/análise , Vírus da Dengue/isolamento & purificação , Ensaio de Imunoadsorção Enzimática , Evolução Fatal , Feminino , Coração/virologia , Humanos , Imunoglobulina M/sangue , Rim/ultraestrutura , Rim/virologia , Fígado/ultraestrutura , Fígado/virologia , Microscopia Eletrônica de Transmissão/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Dengue Grave/virologia , Baço/ultraestrutura , Baço/virologiaRESUMO
The entry of dengue virus-1 (DENV-1) strain Hawaii into mosquito C6/36 cells was analyzed using a variety of biochemical inhibitors together with electron microscopy. The treatment with ammonium chloride, chlorpromazine, dansylcadaverine and dynasore inhibited virus yields, determined by infectivity titrations, whereas nystatin and methyl-ß-cyclodextrin did not have any effect. The effect of the clathrin and dynamin inhibitors on DENV-1 entry was corroborated by detection of internalized virions using immunofluorescence staining. Furthermore, electron micrographs showed the incoming virions attached to electron-dense invaginations of the plasma membrane and within coated vesicles that resembled clathrin-coated pits and vesicles, respectively. The susceptibility to clathrin and dynamin inhibitors of clinical isolates from recent outbreaks was comparable to that shown by the cell culture-adapted reference strain. Similarly, DENV-3 strain H87 and DENV-4 strain 8124 were also inhibited in the presence of ammonium chloride, chlorpromazine and dynasore, allowing conclude that the infectious entry of DENV serotypes to mosquito cells occurs by low pH-dependent clathrin-mediated endocytosis.
Assuntos
Vírus da Dengue/fisiologia , Internalização do Vírus , Animais , Linhagem Celular , Vesículas Revestidas por Clatrina/virologia , Culicidae , Vírus da Dengue/crescimento & desenvolvimento , Vírus da Dengue/ultraestrutura , Endocitose , Microscopia EletrônicaRESUMO
Os insetos possuem mecanismos imunes que fornecem proteção contra diversos microorganismos. Estas barreiras anatômicas e fisiológicas incluem as respostas de defesa celulares e humorais. A maior parte dos estudos de imunidade de insetos concentra-se na resposta humoral. Entretanto, os hemócitos são também conhecidos por estarem envolvidos nas respostas imunes celulares contra muitos tipos de microorganismos. Inicialmente, nós caracterizamos os hemócitos dos mosquitos Aedes aegypti e Aedes albopictus revelando seis diferentes subpopulações celulares: prohemócito, adipohemócito, granulócito, plasmatócito, oenocitóide e trombocitóide. Nós demonstramos que estes mosquitos têm aproximadamente 3.000 hemócitos/μl de hemolinfa e eles exibem diversos padrões no reconhecimento de diferentes lectinas (Bandeiraea simplicifolia, Canavalis ensifonnis, Helix pomatia, Arachis hypogea, Ulex europaeus, Triticum vulgaris, Limulus polyphemus, Ricinus communis).
Os ensaios de endocitose demonstraram que, em A. albopictus, o granulócito e o plasmatócito apresentaram atividade fagocítica contra as partículas de látex. Além disso, nestas células e no oenocitóide foi observada a presença de diversos lisossomos através da utilização de anticorpos específicos. Em A. aegypti, o prohemócito, o granulócito e o oenocitóide foram os tipos celulares que endocitaram a albumina. Entretanto, o plasmatócito e o oenocitóide foram as células marcadas como possuindo lisossomos. Os estudos de interação do A. aegypti com Dengue virus demonstraram que no 5º e 7º dia após a alimentação oral houve um aumento significante no número total de hemócitos onde os plasmatócitos, oenocitóides e granulócitos foram os tipos celulares que aumentaram neste período. Os estudos por microscopia exibiram alterações morfológicas características nos plasmatócitos e nos oenocitóides de A. aegypti devido à presença do vírus na hemolinfa. Entretanto, os plasmatócitos, oenocitóides e granulócitos estão hábeis para endocitar os vírus, como demonstrado pela imonumarcação específica visualizada na microscopia confocal.