RESUMO
The Naegleria are ubiquitous free-living amoebae and are characterized by the presence of three phases in their biological cycle: trophozoite, cyst and flagellate. Of this genus, only Naegleria fowleri has been reported as pathogenic to humans. The proteasome is a multi-catalytic complex and is considered to be the most important structure responsible for the degradation of intracellular proteins. This structure is related to the maintenance of cellular homeostasis and, in pathogenic microorganisms, to the modulation of their virulence. Until now, the proteasome and its function have not been described for the Naegleria genus. In the current study, using bioinformatic analysis, protein sequences homologous to those reported for the subunits of the 20S proteasome in other organisms were found, and virtual modelling was used to determine their three-dimensional structure. The presence of structural and catalytic subunits of the 20S proteasome was detected by Western and dot blot assays. Its localization was observed by immunofluorescence microscopy to be mainly in the cytoplasm, and a leading role of the chymotrypsin-like catalytic activity was determined using fluorogenic peptidase assays and specific proteasome inhibitors. Finally, the role of the 20S proteasome in the proliferation and differentiation of Naegleria genus trophozoites was demonstrated.
Assuntos
Naegleria fowleri/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Sequência de Aminoácidos , Animais , Diferenciação Celular , Proliferação de CélulasRESUMO
Exosomes are endocytic origin small-membrane vesicles secreted to the extracellular space by most cell types. Exosomes released from virus infected-cells can mediate the cell-to-cell communication to promote or modulate viral transmission. Dengue virus (DENV) is an arbovirus transmitted by Aedes mosquitoes bite to humans. Interestingly, the role of exosomes during the DENV infection in mammalian cells has already been described. However, little is known about exosomes derived from infected mosquito cells. Thus, the exosomes released from DENV-infected C6/36 cells were isolated, purified and analyzed using an antibody against the tetraspanin CD9 from human that showed cross-reactivity with the homologs to human CD9 found in Aedes albopictus (AalCD9). The exosomes from DENV infected cells were larger than the exosomes secreted from uninfected cells, contained virus-like particles, and they were able to infect naïve C6/36 cells, suggesting that exosomes are playing a role in virus dissemination.
Assuntos
Vírus da Dengue/fisiologia , Exossomos/metabolismo , Exossomos/virologia , Mosquitos Vetores/virologia , Aedes , Animais , Linhagem Celular , Dengue/metabolismo , Dengue/virologia , Difusão Dinâmica da Luz , Exossomos/imunologia , Humanos , Proteínas de Insetos/química , Proteínas de Insetos/genética , Proteínas de Insetos/imunologia , Proteínas de Insetos/metabolismo , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Mosquitos Vetores/classificação , Mosquitos Vetores/genética , Mosquitos Vetores/metabolismo , Filogenia , Tetraspaninas/química , Tetraspaninas/genética , Tetraspaninas/imunologia , Tetraspaninas/metabolismo , Replicação ViralRESUMO
Dengue is the most relevant mosquito-borne viral disease in the world. It has been estimated that 390 million infections of dengue occur each year. Dengue virus (DENV) infection can be asymptomatic or can produce a self-limited febrile illness called dengue fever (DF) or a severe form of the infection called severe dengue. In some viruses, the entry and egress from cells, occur in a specific domain of polarized endothelial and epithelial cells. In this study, we investigated whether the entry and release of DENV was polarized in epithelial cells, and evaluated the effect of DENV infection on cellular junctions of epithelial cells. We used MDCK epithelial cells, which serve as an excellent model to study a functional barrier due to the presence of an apical junctional complex (AJC), and showed that entry and release of DENV from the cells, is bipolar. Additionally, we performed paracellular flux, diffusion of membrane lipid, immunofluorescence and immunoblotting assays to evaluate the integrity of the AJC during DENV infection. We observed that at later stages of infection, DENV altered the barrier function causing a decrease in the transepithelial electrical resistance and the degradation and delocalization of TJ and AJ proteins. The present study contributes to understand how DENV traverse epithelia in order to cause a productive infection, and provides insights into the mechanism of DENV pathogenesis.
Assuntos
Vírus da Dengue/fisiologia , Células Epiteliais/citologia , Células Epiteliais/virologia , Internalização do Vírus , Animais , Dengue/virologia , Cães , Células Madin Darby de Rim CaninoRESUMO
Fourteen pools of Aedes aegypti larvae collected within the urban area of Culiacán, Sinaloa, were analyzed by RT-PCR. The results demonstrate, for the first time, the vertical infection of serotype-2 dengue virus (DENV-2) in Sinaloa, Mexico, suggesting that Ae. aegypti acts as a natural reservoir of DENV-2 in this region.
Assuntos
Aedes/virologia , Vírus da Dengue , Dengue/transmissão , Transmissão Vertical de Doenças Infecciosas , Aedes/crescimento & desenvolvimento , Animais , Larva/virologia , México , RNA ViralRESUMO
Immunological factors, such as cytokines, have been proposed as a cause of changes in the lipid profile of dengue patients. We studied whether serum lipid levels and serum TNF-α levels are associated in a group of dengue patients from an endemic region in the Northwest of Mexico. We found statistically important differences in the serum lipid profile and the TNF-α levels of dengue patients compared with the control group, were observed. However, TNF-α levels did not correlate with the lipid profile of dengue patients.
Assuntos
Dengue/patologia , Lipídeos/sangue , Fator de Necrose Tumoral alfa/sangue , Adulto , Idoso , Doadores de Sangue , Estudos de Casos e Controles , Feminino , Humanos , Masculino , México , Pessoa de Meia-Idade , Soro/químicaRESUMO
The role of Ca2+ during dengue virus (DENV) replication is unknown; thus, changes in Ca2+ homeostasis in DENV infected human hepatic HepG2 and Huh-7 cells were analyzed. Infected HepG2 cells, but not Huh-7 cells, showed a significant increase in plasma membrane permeability to Ca2+, while both cell lines showed marked reduced levels of Ca2+ stored in the endoplasmic reticulum. While the expression levels of STIM1 and ORAI1 showed no changes, STIM1 and ORAI1 were shown to co-localized in infected cells, indicating activation of the store-operated Ca2+ entry (SOCE) pathway. Finally, manipulation in the infected cells of the intra and extracellular Ca2+ levels by chelators (BAPTA-AM and EGTA), SOC inhibitor (SKF96365), IP3 Receptor antagonist (2APB) or increase of extracellular [Ca2+], significantly reduced DENV yield, but not vesicular stomatitis virus yield, used as a control. These results show that DENV infection alters cell Ca2+ homeostasis and that such changes favor viral replication.
Assuntos
Quelantes de Cálcio/farmacologia , Cálcio/metabolismo , Vírus da Dengue/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Interações Hospedeiro-Patógeno , Replicação Viral/efeitos dos fármacos , Animais , Compostos de Boro/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Membrana Celular/virologia , Permeabilidade da Membrana Celular , Chlorocebus aethiops , Vírus da Dengue/fisiologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Retículo Endoplasmático/virologia , Expressão Gênica , Células Hep G2 , Humanos , Imidazóis/farmacologia , Receptores de Inositol 1,4,5-Trifosfato/antagonistas & inibidores , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Transporte de Íons , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/antagonistas & inibidores , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/antagonistas & inibidores , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo , Células Vero , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Vírus da Estomatite Vesicular Indiana/fisiologia , Replicação Viral/genéticaRESUMO
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
Dengue virus NS1 is a glycoprotein of 46-50 kDa that is conserved among flaviviruses, associates as a dimer to cell membranes and is secreted as a hexamer to the extracellular milieu. Recent evidence showed that NS1 is secreted efficiently from infected mosquito cells. To explore the secretory route of NS1 in mosquito cells, infected cells were treated with brefeldin A (BFA) and methyl-beta-cyclodextrin (MßCD). The results showed that MßCD, but not BFA, significantly reduced the release of NS1. Moreover, silencing the expression of caveolin-1 (CAV1; a key component of the caveolar system that transports cholesterol inside the cell), but not SAR1 (a GTPase that participates in the classical secretory pathway), also results in a significant reduction of the secretion of NS1. These results indicate that NS1 is released from mosquito cells via an unconventional secretory route that bypasses the Golgi complex, with the participation of CAV1. In agreement with this notion, differences were observed in the glycosylation status between secreted NS1 and E proteins. Classical mechanics and docking simulations suggested highly favoured interactions between the caveolin-binding domain present in NS1 and the scaffolding domain of CAV1. Finally, proximity ligation assays showed direct interaction between NS1 and CAV1 in infected mosquito cells. These findings are in line with the lipoprotein nature of secreted NS1 and provide new insights into the biology of NS1.
Assuntos
Aedes/metabolismo , Aedes/virologia , Caveolina 1/metabolismo , Vírus da Dengue/metabolismo , Proteínas de Insetos/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Linhagem Celular , Vírus da Dengue/genética , Ligação Proteica , Via Secretória , Proteínas não Estruturais Virais/genéticaRESUMO
Dengue virus (DENV) replicative cycle occurs in the endoplasmic reticulum where calcium ions play an important role in cell signaling. Calmodulin (CaM) is the primary sensor of intracellular Ca2+ levels in eukaryotic cells. In this paper, the effect of the calmodulin antagonist W-7 in DENV infection in Huh-7 cells was evaluated. W7 inhibited viral yield, NS1 secretion and viral RNA and protein synthesis. Moreover, luciferase activity, encoded by a DENV replicon, was also reduced. A decrease in the replicative complexes formation was clearly observed in W7 treated cells. Docking simulations suggest 2 possible mechanisms of action for W7: the direct inhibition of NS2B-NS3 activity and/or inhibition of the interaction between NS2A with Ca2+-CaM complex. This last possibility was supported by the in vitro interaction observed between recombinant NS2A and CaM. These results indicate that Ca2+-CaM plays an important role in DENV replication.
Assuntos
Antivirais/farmacologia , Calmodulina/antagonistas & inibidores , Vírus da Dengue/efeitos dos fármacos , Dengue/virologia , Sulfonamidas/farmacologia , Calmodulina/metabolismo , Linhagem Celular Tumoral , Dengue/metabolismo , Vírus da Dengue/genética , Vírus da Dengue/fisiologia , Humanos , Ligação Proteica , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
Dengue virus NS1 is a glycoprotein of 46-50kDa which associates as a dimer to internal and cytoplasmic membranes and is also secreted, as a hexamer, to the extracellular milieu. However, the notion exist that NS1 is secreted only from infected vertebrate and not mosquito cells. In this work, evidence is presented showing that NS1 is secreted efficiently by infected mosquito cells. NS1 was detected in cell supernatants starting at 6hpi with a continuous concentration increase up to 24hpi. Nevertheless, cell viability showed an average cell survival of 97%. At variance with observations with vertebrate cells, NS1 does not seems to associate with the cytoplasmic membrane of insect cells. Finally, evidence is presented indicating that NS1 is secreted from insect cells as a barrel-shaped hexamer. These findings provide new insights into the biology of NS1 and open questions about the role of secreted NS1 in the vector mosquito.
Assuntos
Culicidae/virologia , Vírus da Dengue/fisiologia , Proteínas não Estruturais Virais/metabolismo , Animais , Linhagem Celular , Meios de Cultura/química , Fatores de TempoRESUMO
Given dengue virus (DENV) genome austerity, it uses cellular molecules and structures for virion entry, translation and replication of the genome. NS1 is a multifunctional protein key to viral replication and pathogenesis. Identification of cellular proteins that interact with NS1 may help in further understanding the functions of NS1. In this paper we isolated a total of 64 proteins from DENV infected human hepatic cells (Huh-7) that interact with NS1 by affinity chromatography and immunoprecipitation assays. The subcellular location and expression levels during infection of the ribosomal proteins RPS3a, RPL7, RPL18, RPL18a plus GAPDH were determined. None of these proteins changed their expression levels during infection; however, RPL-18 was redistributed to the perinuclear region after 48hpi. Silencing of the RPL-18 does not affect cell translation efficiency or viability, but it reduces significantly viral translation, replication and viral yield, suggesting that the RPL-18 is required during DENV replicative cycle.
Assuntos
Vírus da Dengue/fisiologia , Hepatócitos/virologia , Interações Hospedeiro-Patógeno , Biossíntese de Proteínas , Proteínas Ribossômicas/metabolismo , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Linhagem Celular , Cromatografia de Afinidade , Humanos , Imunoprecipitação , Ligação ProteicaRESUMO
Dengue virus (DENV) is the causative agent of dengue fever. In recent years, patients with more severe form of the disease with acute heart failure or progression to cardiogenic shock and death have been reported. However, the pathogenesis of myocardial lesions and susceptibility of cardiomyocytes to DENV infection have not been evaluated. Under this perspective, the susceptibility of the myoblast cell line H9c2, obtained from embryonic rat heart, to DENV infection was analyzed. Our findings indicate that H9c2 cells are susceptible to the infection with the four DENV serotypes. Moreover, virus translation/replication and viral production in this cell line is as efficient as in other susceptible cell lines, supporting the idea that DENV may target heart cells as evidenced by infection of H9c2 cells. This cell line may thus represent an excellent model for the study and characterization of cardiac physiopathology in DENV infection.
Assuntos
Vírus da Dengue/fisiologia , Miócitos Cardíacos/virologia , Animais , Linhagem Celular , Vírus da Dengue/crescimento & desenvolvimento , Modelos Biológicos , RatosRESUMO
MicroRNAs (miRNAs) constitute an important class of non-coding RNA implicated in gene expression regulation. More than 1900 miRNA molecules have been identified in humans and their modulation during viral infection and it is recognized to play a role in latency regulation or in establishing an antiviral state. The liver cells are targets during DENV infection, and alteration of liver functions contributes to severe disease. In this work the miRNAs expression profile of the human hepatoma cell line, Huh-7, infected with DENV-2 was determined using microarray and real-time PCR. Let-7c is one of the miRNAs up-regulated during DENV infection in the hepatic Huh-7 as well as in the macrophage-monocytic cell line U937-DC-SIGN. Let-7c overexpression down-regulates both DENV-2 and DENV-4 infection. Additionally, we found that the transcription factor BACH1, a let-7c target, is also down-regulated during DENV infection. In accordance with this finding, HO-1, the main responsive factor of BACH1 was found up-regulated. The up-regulation of HO-1 may contribute to the stress oxidative response in infected cells.
Assuntos
Vírus da Dengue/genética , Expressão Gênica , MicroRNAs/genética , Interferência de RNA , Replicação Viral/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Sítios de Ligação , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/virologia , Linhagem Celular Tumoral , Vírus da Dengue/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Genoma Viral , Heme Oxigenase-1/genética , Interações Hospedeiro-Patógeno/genética , Humanos , RNA Viral , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Reprodutibilidade dos Testes , Fatores de Tempo , Transfecção , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
Dengue is the most common mosquito borne viral disease in humans. The infection with any of the 4 dengue virus serotypes (DENV) can either be asymptomatic or manifest in two clinical forms, the mild dengue fever or the more severe dengue hemorrhagic fever that may progress into dengue shock syndrome. A DENV replicative cycle relies on host lipid metabolism; specifically, DENV infection modulates cholesterol and fatty acid synthesis, generating a lipid-enriched cellular environment necessary for viral replication. Thus, the aim of this work was to evaluate the anti-DENV effect of the Nordihydroguaiaretic acid (NDGA), a hypolipidemic agent with antioxidant and anti-inflammatory properties. A dose-dependent inhibition in viral yield and NS1 secretion was observed in supernatants of infected cells treated for 24 and 48 h with different concentrations of NDGA. To evaluate the effect of NDGA in DENV replication, a DENV4 replicon transfected Vero cells were treated with different concentrations of NDGA. NDGA treatment significantly reduced DENV replication, reiterating the importance of lipids in viral replication. NDGA treatment also led to reduction in number of lipid droplets (LDs), the neutral lipid storage organelles involved in DENV morphogenesis that are known to increase in number during DENV infection. Furthermore, NDGA treatment resulted in dissociation of the C protein from LDs. Overall our results suggest that NDGA inhibits DENV infection by targeting genome replication and viral assembly.
Assuntos
Antivirais/farmacologia , Vírus da Dengue/efeitos dos fármacos , Dengue/virologia , Masoprocol/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Linhagem Celular , Replicação do DNA/efeitos dos fármacos , Dengue/tratamento farmacológico , Vírus da Dengue/genética , Vírus da Dengue/crescimento & desenvolvimento , Vírus da Dengue/fisiologia , Genoma Viral/efeitos dos fármacos , Humanos , Proteínas Virais/genética , Proteínas Virais/metabolismo , Montagem de Vírus/efeitos dos fármacosRESUMO
Higher levels of viremia and circulating nonstructural protein 1 (NS1) have been associated with dengue disease severity. In this study, viremia and circulating NS1 levels were determined in 225 serum samples collected from patients in Mexico infected with dengue virus serotypes 1 and 2 (DENV-1 and DENV-2). Patients with dengue hemorrhagic fever (DHF) who were infected with DENV-1 showed higher levels of circulating NS1 than patients with dengue fever (DF) (P = 0.0175). Moreover, NS1 levels were higher in patients with primary infections with DENV-1 than in patient infected with DENV-2 (P < 0.0001) and in patients with primary infections with DENV-2 than in patients with secondary infections with DENV-2 (P = 0.0051). Unexpectedly, viremia levels were higher in patients with DF than in those with DHF infected with either DENV-1 or DENV-2 (P = 0.0019 and P = 0.001, respectively) and in patients with primary infections than those with secondary DENV-2 infections (P < 0.0001). Results indicate that levels of circulating NS1 vary according to the infecting serotype, immunologic status (primary or secondary infection), and dengue disease severity.
Assuntos
Vírus da Dengue/classificação , Vírus da Dengue/fisiologia , Dengue/epidemiologia , Proteínas não Estruturais Virais/sangue , Viremia , Dengue/sangue , Dengue/virologia , Feminino , Humanos , Masculino , México/epidemiologia , RNA Viral/sangue , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adulto JovemRESUMO
The 3' untranslated region (3'UTR) of the dengue virus (DENV) genome contain several sequences required for translation, replication and cyclization processes. This region also binds cellular proteins such as La, polypyrimidine tract-binding protein (PTB), Y box-binding protein 1, poly(A)-binding protein and the translation initiation factor eEF-1 alpha. PTB is a cellular protein that interacts with the regulatory sequences of positive-strand RNA viruses such as several picornaviruses and hepatitis C virus. In the present report, it was demonstrated that PTB translocates from the nucleus to the cytoplasm during DENV infection. At 48 h post-infection, PTB, as well as the DENV proteins NS1 and NS3, were found to co-localize with the endoplasmic reticulum marker calnexin. Silencing of PTB expression inhibited virus translation and replication, whilst overexpression of PTB augmented these processes. Thus, these results provide evidence that, during infection, PTB moves from the nucleus to the cytoplasm and plays an important role in the DENV replicative cycle.
Assuntos
Citoplasma/metabolismo , Vírus da Dengue/patogenicidade , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Animais , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Chlorocebus aethiops , Vírus da Dengue/genética , Vírus da Dengue/fisiologia , Humanos , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Células Vero , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação ViralRESUMO
The molecules involved in dengue virus entry into human cells are currently unknown. We have previously shown that two surface heat shock proteins (Hsps), Hsp90 and Hsp70 are part of a receptor complex in monocytic cells. In the present report, the effect of heat shock (HS) on dengue virus infection is analyzed. We have documented a more than twofold increase in dengue virus infectivity after HS treatment in monocytic cells U937; this effect correlates mainly with an increase in viral entry due to a major presence of both Hsps on the surface of monocytic cells, particularly in membrane microdomains. Interestingly, since heat shock treatment at 6h post-infection also increased viral yields, it is likely that HS also modulates positively dengue virus replication.
Assuntos
Vírus da Dengue/fisiologia , Dengue/fisiopatologia , Resposta ao Choque Térmico , Monócitos/fisiologia , Replicação Viral , Membrana Celular/metabolismo , Membrana Celular/virologia , Dengue/metabolismo , Dengue/virologia , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Monócitos/virologia , Células U937 , Internalização do VírusRESUMO
The endocytic pathway followed by dengue virus to infect the mosquito cells C6/36 HT was analyzed. Using DIL-labeled virions and real-time imaging it was determined that viral entry into C6/36 HT takes approximately 5 to 7 min. Pretreatment of C6/36 HT cells with sucrose and bafilomycin A, but not filipin, inhibited dengue virus infection up to 80%. Furthermore, the overexpression of dominant-negative mutants of Eps15, a molecule required for the formation of clathrin-coated vesicles, reduced dengue infection up to 50%, indicating that dengue virus entry is through clathrin-mediated endocytosis and is pH-dependent. By double-immunofluorescence assays, DIL-labeled particles were colocalized with early endosomes at 5 min and with lysosomes mainly at 30 min post-infection. Finally, disruption of the microtubule and microfilaments by nocodazole and by cytochalasin D reduced viral infection by more than 80%. Taken together these results indicate that dengue virions enter into C6/36 HT cells by clathrin-mediated endocytosis, using the endosomal pathway from early endosomes to acidic lysosomes before viral RNA is released into the cytoplasm.
Assuntos
Aedes/citologia , Aedes/virologia , Clatrina/metabolismo , Vírus da Dengue/patogenicidade , Endocitose/fisiologia , Aedes/classificação , Animais , Linhagem Celular , Células Cultivadas , Cricetinae , Vírus da Dengue/genética , Vírus da Dengue/metabolismo , Endossomos/virologia , Imunofluorescência , Lisossomos/virologia , Microscopia Confocal , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Vírion/patogenicidade , Internalização do VírusRESUMO
Current methods for dengue virus quantitation are either time consuming, technically demanding or costly. As an alternative, the commercial enzyme immunoassay Platelia Dengue NS1 AG (Bio-Rad Laboratories) was used to monitor semiquantitatively dengue virus replication in cultured cells. The presence of NS1 protein was evaluated in supernatants from Vero and C6/36 HT cells infected with dengue virus. The amount of NS1 detected in the supernatants of infected cells was proportional to the initial MOI used and to the time of post infection harvest. This immunoassay was also able to detect the presence of NS1 in the supernatants of infected human macrophages. Inhibition of dengue virus replication in C6/36 HT cells treated with lysosomotropic drugs was readily monitored with the use of this assay. These results suggest that the Platelia Dengue NS1 AG kit can be used as a fast and reliable surrogate method for the relative quantitation of dengue virus replication in cultured cells.
Assuntos
Vírus da Dengue/isolamento & purificação , Técnicas Imunoenzimáticas/métodos , Replicação Viral , Aedes , Cloreto de Amônio/farmacologia , Animais , Linhagem Celular , Células Cultivadas , Chlorocebus aethiops , Cricetinae , Vírus da Dengue/efeitos dos fármacos , Vírus da Dengue/fisiologia , Humanos , Macrófagos/metabolismo , Macrófagos/virologia , Camundongos , Células Vero , Proteínas não Estruturais Virais/análise , Ensaio de Placa Viral , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
Dengue virus (DENV) is transmitted to humans by mosquitoes of the genus Aedes. Although several molecules have been described as part of DENV receptor complex in mosquito cells, none of them have been identified. Our group characterized two glycoproteins (40 and 45 kD) as part of the DENV receptor complex in C6/36 cells. Because identification of the mosquito cell receptor has been unsuccessful and some cell receptors described for DENV in mammalian cells are heat-shock proteins (HSPs), the role of HSPs in DENV binding and infection in C6/36 cells was evaluated. Our results indicate that gp45 and a 74-kD molecule (p74), which interact with DENV envelope protein, are immunologically related to HSP90. Although p74 is induced by heat shock, gp45 apparently is not. However, these proteins are relocated to the cell surface after heat-shock treatment, causing an increase in virus binding without any effect on virus yield.