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1.
Biotechnol Bioeng ; 117(11): 3277-3285, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32648943

RESUMO

Vaccines provide effective protection against many infectious diseases as well as therapeutics for select pathologies, such as cancer. Many viral vaccines require amplification of virus in cell cultures during manufacture. Traditionally, cell cultures, such as VERO, have been used for virus production in bovine serum-containing culture media. However, due to concerns of potential adventitious agents present in fetal bovine serum (FBS), regulatory agencies suggest avoiding the use of bovine serum in vaccine production. Current serum-free media suitable for VERO-based virus production contains high concentrations of undefined plant hydrolysates. Although these media have been extensively used, the lack of chemical definition has the potential to adversely affect cell growth kinetics and subsequent virus production. As plant hydrolysates are made from plant raw materials, performance variations could be significant among different lots of production. We developed a chemically defined, serum-free medium, OptiVERO, which was optimized specifically for VERO cells. VERO cell growth kinetics were demonstrated to be equivalent to EMEM-10% FBS in this chemically defined medium while the plant hydrolysate-containing medium demonstrated a slower doubling time in both two-dimensional (2D) and 3D cultures. Virus production comparisons demonstrated that the chemically defined OptiVERO medium performed at least as good as the EMEM-10%FBS and better than the plant hydrolysate-containing media. We report the success in using recombinant proteins to replace undefined plant hydrolysates to formulate a chemically defined medium that can efficiently support VERO cell expansion and virus production.


Assuntos
Técnicas de Cultura de Células/métodos , Meios de Cultura Livres de Soro , Células Vero , Cultura de Vírus/métodos , Animais , Chlorocebus aethiops , Meios de Cultura Livres de Soro/química , Meios de Cultura Livres de Soro/metabolismo , Preparações de Plantas , Proteínas Recombinantes , Células Vero/citologia , Células Vero/metabolismo , Ensaio de Placa Viral
2.
Microb Pathog ; 107: 81-87, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28330747

RESUMO

Peste des petits ruminants virus (PPRV), belonging to paramyxoviruses, has six structure proteins (such as matrix protein (M), nucleocapsid proteins (N), fusion protein (F) and hemagglutinin protein (H)) and could cause high morbidity and mortality in sheep and goats. Although a vaccine strain of PPRV has been rescued and co-expression of M and N could yield PPRV-like particles, the roles of structure proteins in virion assembly and release have not been investigated in detail. In this study, plasmids carrying PPRV cDNA sequences encoding the N, M, H, and F proteins were expressed in Vero cells. The co-expression of all four proteins resulted in the release of virus-like particles (VLPs) with similar release efficiency to that of authentic virions. Moreover, the co-expression of M together with F also resulted in efficient VLPs release. In the absence of M protein, the expression of no combination of the other proteins resulted in particle release. In summary, a VLPs production system for PPRV has been established and M protein is necessary for promoting the assembly and release of VLPs, of which the predominant protein is M protein. Further study will be focused on the immunogenicity of the VLPs.


Assuntos
Vírus da Peste dos Pequenos Ruminantes/metabolismo , Vírus da Peste dos Pequenos Ruminantes/fisiologia , Células Vero/metabolismo , Proteínas da Matriz Viral/metabolismo , Animais , Anticorpos Antivirais , Chlorocebus aethiops/metabolismo , Chlorocebus aethiops/fisiologia , DNA Complementar , DNA Viral , Hemaglutininas Virais/metabolismo , Hemaglutininas Virais/fisiologia , Camundongos , Proteínas do Nucleocapsídeo/metabolismo , Proteínas do Nucleocapsídeo/fisiologia , Vírus da Peste dos Pequenos Ruminantes/genética , Vírus da Peste dos Pequenos Ruminantes/imunologia , Proteínas Virais de Fusão/metabolismo , Proteínas Virais de Fusão/fisiologia
3.
J Virol ; 86(23): 12891-902, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22993162

RESUMO

We constructed a herpes simplex virus 2 (HSV-2) bacterial artificial chromosome (BAC) clone, bHSV2-BAC38, which contains full-length HSV-2 inserted into a BAC vector. Unlike previously reported HSV-2 BAC clones, the virus genome inserted into this BAC clone has no known gene disruptions. Virus derived from the BAC clone had a wild-type phenotype for growth in vitro and for acute infection, latency, and reactivation in mice. HVEM, expressed on epithelial cells and lymphocytes, and nectin-1, expressed on neurons and epithelial cells, are the two principal receptors used by HSV to enter cells. We used the HSV-2 BAC clone to construct an HSV-2 glycoprotein D mutant (HSV2-gD27) with point mutations in amino acids 215, 222, and 223, which are critical for the interaction of gD with nectin-1. HSV2-gD27 infected cells expressing HVEM, including a human epithelial cell line. However, the virus lost the ability to infect cells expressing only nectin-1, including neuronal cell lines, and did not infect ganglia in mice. Surprisingly, we found that HSV2-gD27 could not infect Vero cells unless we transduced the cells with a retrovirus expressing HVEM. High-level expression of HVEM in Vero cells also resulted in increased syncytia and enhanced cell-to-cell spread in cells infected with wild-type HSV-2. The inability of the HSV2-gD27 mutant to infect neuronal cells in vitro or sensory ganglia in mice after intramuscular inoculation suggests that this HSV-2 mutant might be an attractive candidate for a live attenuated HSV-2 vaccine.


Assuntos
Herpesvirus Humano 2/genética , Neurônios/virologia , Células Vero/virologia , Proteínas do Envelope Viral/genética , Internalização do Vírus , Animais , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Chlorocebus aethiops , Cromossomos Artificiais Bacterianos/genética , Herpesvirus Humano 2/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Mutagênese , Nectinas , Oligonucleotídeos/genética , Mutação Puntual/genética , Reação em Cadeia da Polimerase , Membro 14 de Receptores do Fator de Necrose Tumoral/metabolismo , Células Vero/metabolismo
4.
J Neurosci Res ; 90(5): 1020-9, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22252837

RESUMO

Herpes simplex virus type 1 (HSV-1) is a neurotropic virus that remains latent in host neurons. Viral DNA replication is a highly structured process in which the redistribution of nuclear proteins plays an important role. Although tau is most widely known as a microtubule-associated protein found in a hyperphosphorylated state in the brains of patients with Alzheimer's disease (AD), this protein has also been detected at other sites such as the nucleolus. Here, we establish that HSV-1 infection gives rise to an increase in tau phosphorylation and that hyperphosphorylated tau accumulates in the nucleus, forming defined structures in HSV-1-infected neuronal cells reminiscent of the common sites of viral DNA replication. When tau expression in human neuroblastoma cells was specifically inhibited using an adenoviral vector expressing a short hairpin RNA to tau, viral DNA replication was not affected, indicating that tau is not required for HSV-1 growth in neuronal cells. Given that HSV-1 is considered a risk factor for AD, our results suggest a new way in which to understand the relationships between HSV-1 infection and the pathogenic mechanisms leading to AD.


Assuntos
Núcleo Celular/metabolismo , Núcleo Celular/virologia , Herpesvirus Humano 1/fisiologia , Proteínas tau/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Antivirais/farmacologia , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Chlorocebus aethiops , Replicação do DNA , DNA Viral/genética , DNA Viral/metabolismo , Inibidores Enzimáticos/farmacologia , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/imunologia , Humanos , Neuroblastoma/patologia , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fatores de Tempo , Células Vero/metabolismo , Células Vero/virologia , Ensaio de Placa Viral
5.
J Appl Toxicol ; 32(6): 446-53, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22161551

RESUMO

The aim of this study was to determine the potential toxic effects of iron(II,III)oxide nanoparticles (IONPs). In in vivo experiments, the toxic effects of IONPs were monitored in adult male Wistar rats by morphological methods after a single intratracheal instillation. For the control group 1 ml of physiological saline per animal was given, and the treatment group received the same volume of a suspension containing 1 and 5 mg kg⁻¹ body weight IONPs. Lungs and internal organs underwent histopathological examination after 1, 3, 7, 14 and 30 days. The mutagenic effect of these nanoparticles was evaluated by the bacterial reverse mutation assay on Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains, and on Escherichia coli WP2uvrA strain, in the presence and absence of the mammalian metabolic activation system S9. The in vitro cytotoxic effect of IONPs was also examined in Vero cells after short-term (4 h) and long-term (24 h) exposure. There were no pathological changes in examined internal organs, except a very weak pulmonary fibrosis developing by the end of the first month in the treated rats. While in vitro MTT assay showed a moderate cytotoxic effect, IONPs proved to be devoid of mutagenic effect in the bacterial systems tested. The results may be a useful extension of our knowledge on the safety of magnetite nanoparticles in view of their possible medical applications, such as in hyperthermia and magnetic resonance imaging.


Assuntos
Compostos Férricos/toxicidade , Nanopartículas Metálicas/toxicidade , Mutagênicos/toxicidade , Animais , Biotransformação , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Dano ao DNA , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Compostos Férricos/administração & dosagem , Compostos Férricos/metabolismo , Exposição por Inalação , Intubação Intratraqueal , Pulmão/efeitos dos fármacos , Pulmão/patologia , Masculino , Nanopartículas Metálicas/administração & dosagem , Nanopartículas Metálicas/ultraestrutura , Testes de Mutagenicidade , Mutagênicos/administração & dosagem , Mutagênicos/metabolismo , Mutação/genética , Tamanho do Órgão/efeitos dos fármacos , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/patologia , Ratos , Ratos Wistar , Proteína S9 Ribossômica , Proteínas Ribossômicas/metabolismo , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética , Células Vero/efeitos dos fármacos , Células Vero/metabolismo , Células Vero/patologia , Aumento de Peso/efeitos dos fármacos
6.
J Membr Biol ; 235(2): 121-9, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20512319

RESUMO

Certain antimicrobial peptides from multicellular animals kill a variety of tumor cells at concentrations not affecting normal eukaryotic cells. Recently, it was reported that also plantaricin A (PlnA), which is a peptide pheromone with strain-specific antibacterial activity produced by Lactobacillus plantarum, permeabilizes cancerous rat pituitary cells (GH(4) cells), whereas normal rat anterior pituitary cells are resistant to the peptide. To examine whether the preferential permeabilization of cancerous cells is a general feature of PlnA, we studied its effect on primary cultures of cells from rat liver (hepatocytes, endothelial, and Kupffer cells) and rat kidney cortex, as well as two epithelial cell lines of primate kidney origin (Vero cells from green monkey and human Caki-2 cells). The Vero cell line is derived from normal cells, whereas the Caki-2 cell line is derived from a cancerous tumor. The membrane effects were studied by patch clamp recordings and microfluorometric (fura-2) monitoring of the cytosolic concentrations of Ca(2+) ([Ca(2+)](i)) and fluorophore. In all the tested cell types except Kupffer cells, exposure to 10-100 microM PlnA induced a nearly instant permeabilization of the membrane, indicated by the following criteria: increased membrane conductance, membrane depolarization, increased [Ca(2+)](i), and diffusional loss of fluorophore from the cytosol. At a concentration of 5 microM, PlnA had no effect on any of the cell types. The Kupffer cells were permeabilized by 500 microM PlnA. We conclude that the permeabilizing effect of PlnA is not restricted to cancerous cells.


Assuntos
Bacteriocinas/metabolismo , Rim/citologia , Lactobacillus plantarum/metabolismo , Fígado/citologia , Animais , Células Cultivadas , Chlorocebus aethiops , Citofotometria , Eletrofisiologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Humanos , Lactobacillus plantarum/crescimento & desenvolvimento , Masculino , Ratos , Ratos Wistar , Células Vero/metabolismo , Células Vero/microbiologia
7.
Virus Res ; 139(1): 39-47, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19014983

RESUMO

In this work we have characterized the virus (RSV(48)) present in passage 48 of a respiratory syncytial virus persistently infected murine macrophage-like cell culture. This virus was noncytopathic in macrophages and had a low-fusogenic activity in RSV-permissive cell lines, although the level of this activity varied among the different cell lines tested. The fusogenic activity of RSV(48) in Vero cells, as evaluated by the number and size (nuclei per syncytium) of syncytia, was lower than that shown in cells H358. However, the syncytia formed by RSV(48) in Vero cells increased significantly when the virus was treated with trypsin previous to cell infection and the protease was left in the medium during the development of polykarions. Moreover, the fusogenic activity of RSV(48) was increased by a brief acidic pH treatment of infected cells. These results imply that the RSV(48) F protein was inefficiently activated by intracellular proteases in Vero cells and exposure to low pH favours membrane fusion. Analysis of the nucleotide and the deduced amino acid sequences of the RSV(48) F protein showed nine amino acid residue differences with respect to the RSV(wt) sequence, some of which mapped to positions that suggest they might be responsible for the low-fusogenic activity observed for the RSV(48) F protein.


Assuntos
Células Gigantes/virologia , Vírus Sinciciais Respiratórios/fisiologia , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Fusão Celular , Linhagem Celular/metabolismo , Linhagem Celular Tumoral , Núcleo Celular , Chlorocebus aethiops , Células Gigantes/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Vírus Sinciciais Respiratórios/genética , Alinhamento de Sequência , Tripsina/metabolismo , Células Vero/metabolismo , Proteínas Virais de Fusão/química , Ativação Viral/fisiologia
8.
Toxicol In Vitro ; 22(5): 1228-33, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18499391

RESUMO

Perfluorooctanoic acid (PFOA) is a perfluorinated compound ubiquitously detected in the environment, including wildlife and humans. Despite the available information, research on the cytotoxicity of PFOA in non-tumoral mammalian cells is relatively limited. In this work, two in vitro toxicity systems were employed to provide further insight into the cytotoxic and mutagenic potential of PFOA. The cytotoxicity of the chemical towards Vero cells was assessed using biochemical and morphological parameters, while mutagenicity was evaluated according to Ames test. High doses of PFOA cause oxidative stress in Vero cells, that was closely linked to cell cycle arrest at the G1 phase and induction of apoptosis. Our results corroborate previous findings in human tumoral cells and suggest that the mode of action of this perfluorinated compound is not a peculiarity among mammalian cell types. On the other hand, the compound was not mutagenic in the Ames test, using four strains of Salmonella typhimurium in the presence or absence of rat S9 metabolic activation system.


Assuntos
Caprilatos/toxicidade , Poluentes Ambientais/toxicidade , Fluorocarbonos/toxicidade , Mutagênicos/toxicidade , Células Vero/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Formazans , Genes Bacterianos/efeitos dos fármacos , Testes de Mutagenicidade , Mutação Puntual/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética , Sais de Tetrazólio , Células Vero/metabolismo , Células Vero/patologia
9.
Biochem Biophys Res Commun ; 367(1): 47-53, 2008 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-18158915

RESUMO

Bimolecular fluorescence complementation (BiFC) is a recently developed technique for detection of protein-protein interactions in living cells. In this study, a new red BiFC system was developed by splitting mCherry, a mutant monomeric red fluorescent protein, into two fragments between amino acids 159-160 and was verified using a pair of interacting proteins, SV40 large T antigen (LTag), and human p53 protein. By combined use of the mCherry-based red BiFC system with a Venus-based yellow BiFC system, the interaction between LTag and p53 as well as the interaction between sp100 and promyelocytic leukemia protein (PML), were detected simultaneously in Vero cells. The brilliant redness, short maturation time, and the long excitation and emission wavelengths (587/610 nm) of mCherry make the new BiFC system an excellent candidate for analyzing protein-protein interactions in living cells and for studying multiple protein-protein interactions when coupled with other BiFC systems.


Assuntos
Técnicas Biossensoriais/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/química , Proteínas Luminescentes/química , Sequência de Aminoácidos , Animais , Antígenos Transformantes de Poliomavirus/química , Antígenos Transformantes de Poliomavirus/metabolismo , Sequência de Bases , Células Cultivadas/metabolismo , Células Cultivadas/patologia , Chlorocebus aethiops , Humanos , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patologia , Dados de Sequência Molecular , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , Células Vero/metabolismo , Células Vero/patologia , Proteína Vermelha Fluorescente
10.
Life Sci ; 80(7): 650-8, 2007 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-17125799

RESUMO

Cadmium (Cd) is an environmental and industrial pollutant that affects various organs in humans and animals. A body of evidence has accumulated implicating the free radical generation with subsequent oxidative stress in the biochemical and molecular mechanisms of Cd toxicity. Since kidney is the critical target of Cd toxicity, we carried out this study to investigate the effects of diallyl tetrasulfide (DTS), an organosulfur compound derived from garlic on Cd induced toxicity in the kidney of rats and also in the kidney cell line (vero cells). In experimental rats, subcutaneous administration of Cd (3 mg/kg bw/day) for 3 weeks induced renal damage, which was evident from significantly increased levels of serum urea and creatinine with significant decrease in creatinine clearance. A markedly increased levels of lipid peroxidation markers (thiobarbituric acid reactive substances and lipid hydroperoxides) and protein carbonyl contents with significant decrease in nonenzymic antioxidants (total sulphydryl groups, reduced glutathione, vitamin C and vitamin E) and enzymic antioxidants (superoxide dismutase, catalase, glutathione peroxidase and glutathione-S-transferase) as well as glutathione metabolizing enzymes (glutathione reductase, and glucose-6-phosphate dehydrogenase) were also observed in Cd intoxicated rats. Coadministration of DTS (40 mg/kg bw/day) and Cd resulted in the reversal of the kidney function accompanied by a significant decrease in lipid peroxidation and increase in the antioxidant defense system. In vitro studies with vero cells showed that incubation of DTS (5-50 microg/ml) with Cd (10 microM) significantly reduced the cell death induced by Cd. DTS at 40 microg/ml effectively blocked the cell death and lipid peroxidation induced by Cd (10 microM) indicating its cytoprotective property. Further, the flow cytometric assessment on the level of intracellular reactive oxygen species using a fluorescent probe 2', 7'-dichlorofluorescein diacetate (DCF-DA) confirmed the Cd induced intracellular oxidative stress in vero cells, which was significantly suppressed by DTS (40 microg/ml). The histopathological studies in the kidney of rats also showed that DTS (40 mg/kg bw/day) markedly reduced the toxicity of Cd and preserved the architecture of renal tissue. The present study suggests that the cytoprotective potential of DTS in Cd toxicity might be due to its antioxidant and metal chelating properties, which could be useful for achieving optimum effects in Cd induced renal damage.


Assuntos
Compostos Alílicos/farmacologia , Antioxidantes/farmacologia , Cloreto de Cádmio/toxicidade , Nefropatias/prevenção & controle , Rim/efeitos dos fármacos , Estresse Oxidativo , Sulfetos/farmacologia , Animais , Antioxidantes/análise , Antioxidantes/metabolismo , Cloreto de Cádmio/análise , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Creatinina/sangue , Citoproteção/efeitos dos fármacos , Modelos Animais de Doenças , Poluentes Ambientais/toxicidade , Enzimas , Rim/metabolismo , Rim/patologia , Nefropatias/induzido quimicamente , Nefropatias/patologia , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Ratos , Ratos Wistar , Ureia/sangue , Células Vero/efeitos dos fármacos , Células Vero/metabolismo , Células Vero/patologia
11.
J Virol ; 80(15): 7354-63, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16840316

RESUMO

Via careful control of multiple kinases that inactivate the critical translation initiation factor eIF2 by phosphorylation of its alpha subunit, the cellular translation machinery can rapidly respond to a spectrum of environmental stresses, including viral infection. Indeed, virus replication produces a battery of stresses, such as endoplasmic reticulum (ER) stress resulting from misfolded proteins accumulating within the lumen of this organelle, which could potentially result in eIF2alpha phosphorylation and inhibit translation. While cellular translation is exquisitely sensitive to ER stress-inducing agents, protein synthesis in herpes simplex virus type 1 (HSV-1)-infected cells is notably resistant. Sustained translation in HSV-1-infected cells exposed to acute ER stress does not involve the interferon-induced, double-stranded RNA-responsive eIF2alpha kinase PKR, and it does not require either the PKR inhibitor encoded by the Us11 gene or the eIF2alpha phosphatase component specified by the gamma(1)34.5 gene, the two viral functions known to regulate eIF2alpha phosphorylation. In addition, although ER stress potently induced the GADD34 cellular eIF2alpha phosphatase subunit in uninfected cells, it did not accumulate to detectable levels in HSV-1-infected cells under identical exposure conditions. Significantly, resistance of translation to the acute ER stress observed in infected cells requires HSV-1 gene expression. Whereas blocking entry into the true late phase of the viral developmental program does not abrogate ER stress-resistant translation, the presence of viral immediate-early proteins is sufficient to establish a state permissive of continued polypeptide synthesis in the presence of ER stress-inducing agents. Thus, one or more previously uncharacterized viral functions exist to counteract the accumulation of phosphorylated eIF2alpha in response to ER stress in HSV-1-infected cells.


Assuntos
Retículo Endoplasmático/fisiologia , Herpes Simples/virologia , Herpesvirus Humano 1/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , Animais , Antígenos de Diferenciação/metabolismo , Antivirais/farmacologia , Proteínas de Ciclo Celular/metabolismo , Chlorocebus aethiops , Herpes Simples/genética , Herpesvirus Humano 1/patogenicidade , Herpesvirus Humano 1/fisiologia , Humanos , Iniciação Traducional da Cadeia Peptídica , Fosforilação/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Proteína Fosfatase 1 , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Tunicamicina/farmacologia , Células Vero/efeitos dos fármacos , Células Vero/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral , eIF-2 Quinase/fisiologia
12.
J Virol ; 80(15): 7555-68, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16840335

RESUMO

Rinderpest virus (RPV) is a paramyxovirus closely related to the human pathogen Measles virus. It causes severe disease in cattle, buffalo, and some wild animals; although it can infect humans, it does not cause disease. Here, we demonstrate that RPV blocks the action of both type I (alpha) and type II (gamma) interferons (IFNs) by blocking the phosphorylation and nuclear translocation of STAT1 and STAT2 and that this block is not related to species specificity. In addition, both wild-type virulent and vaccine strains of the virus blocked IFN action. Unlike the case with some other paramyxoviruses, neither STAT1 nor STAT2 is degraded upon virus infection. STAT1 is bound by both the viral structural protein P, and thereby recruited to concentrations of viral protein in the cell, and the nonstructural protein V. Although both P and V proteins bind to STAT1 and can block IFN action when expressed in transfected cells, the IFN antagonist activity of the P protein is weaker than that of the V protein. The viral C protein also seems to weakly block IFN-induced activation of STAT1 in transfection experiments. However, studies with knockout viruses showed that the viral V protein appears to be the dominant inhibitor of IFN signaling in the context of virus infection, since prevention of viral V expression restored the IFN sensitivity of infected cells. Although a change in the distribution pattern of STAT2 was observed in virus-infected cells, STAT2 was not bound by any viral protein.


Assuntos
Antivirais/farmacologia , Interferon-alfa/farmacologia , Interferon gama/farmacologia , Vírus da Peste Bovina/fisiologia , Proteínas não Estruturais Virais/fisiologia , Proteínas Estruturais Virais/fisiologia , Animais , Bovinos , Núcleo Celular , Chlorocebus aethiops , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/virologia , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/virologia , Fosforilação , Transporte Proteico , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Pele/citologia , Pele/metabolismo , Pele/virologia , Células Vero/metabolismo , Células Vero/virologia , Replicação Viral
13.
Antiviral Res ; 68(1): 36-42, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16115693

RESUMO

The 3C-like protease (3CLpro) of SARS-coronavirus mediates the proteolytic processing of replicase polypeptides 1a and 1ab into functional proteins, becoming an important target for the drug development. In this study, Isatis indigotica root extract, five major compounds of I. indigotica root, and seven plant-derived phenolic compounds were tested for anti-SARS-CoV 3CLpro effects using cell-free and cell-based cleavage assays. Cleavage assays with the 3CLpro demonstrated that IC50 values were in micromolar ranges for I. indigotica root extract, indigo, sinigrin, aloe emodin and hesperetin. Sinigrin (IC50: 217 microM) was more efficient in blocking the cleavage processing of the 3CLpro than indigo (IC50: 752 microM) and beta-sitosterol (IC50: 1210 microM) in the cell-based assay. Only two phenolic compounds aloe emodin and hesperetin dose-dependently inhibited cleavage activity of the 3CLpro, in which the IC50 was 366 microM for aloe emodin and 8.3 microM for hesperetin in the cell-based assay.


Assuntos
Endopeptidases/efeitos dos fármacos , Isatis/química , Fenóis/farmacologia , Proteínas Virais/efeitos dos fármacos , Animais , Antraquinonas , Chlorocebus aethiops , Corantes/química , Corantes/farmacologia , Proteases 3C de Coronavírus , Cisteína Endopeptidases , Emodina/química , Emodina/farmacologia , Endopeptidases/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Glucosinolatos/química , Glucosinolatos/farmacologia , Hesperidina/química , Hesperidina/farmacologia , Índigo Carmim , Indóis/química , Indóis/farmacologia , Extratos Vegetais/farmacologia , Raízes de Plantas/química , Células Vero/metabolismo , Proteínas Virais/metabolismo
14.
Med Microbiol Immunol ; 194(1-2): 55-9, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14624358

RESUMO

Intracellular glutathione (GSH) plays an important regulatory role in the host response to viral infections. Replenishment of intracellular GSH is a desirable yet challenging goal, since systemic GSH supplementation is rather inefficient due to a short half-life of GSH in blood plasma. Further, GSH is not taken up by cells directly, but needs to be broken down into amino acids and resynthesized to GSH intracellularly, this process often being impaired during viral infections. These obstacles may be overcome by a novel glutathione derivative S-acetylglutathione (S-GSH), which is more stable in plasma and taken up directly by cells with subsequent conversion to GSH. In the present study, in vitro effects of supplementation with S-GSH or GSH on intracellular GSH levels, cell survival and replication of human herpes simplex virus type 1 (HSV-1) were studied in human foreskin fibroblasts. In addition, in vivo effects of supplementation with S-GSH or GSH on HSV-1-induced mortality were studied in hr/hr mice. In cell culture, viral infection resulted in a significant decrease of intracellular GSH levels. S-GSH efficiently and dose-dependently (5 and 10 mM tested) restored intracellular GSH, and this replenishment was more efficient than with GSH supplementation. In mice, S-GSH, but not GSH, significantly decreased HSV-1-induced mortality ( P<0.05). The data suggest that S-GSH is a suitable antiviral agent against HSV-1 both in vitro and in vivo, indicating that this drug may be of benefit in the adjunctive therapy of HSV-1 infections.


Assuntos
Antivirais/farmacologia , Glutationa/análogos & derivados , Glutationa/farmacologia , Herpes Simples/tratamento farmacológico , Herpes Simples/virologia , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 1/patogenicidade , Animais , Animais não Endogâmicos , Células Cultivadas , Chlorocebus aethiops , Fibroblastos/metabolismo , Fibroblastos/virologia , Glutationa/metabolismo , Herpes Simples/mortalidade , Humanos , Camundongos , Camundongos Pelados , Células Vero/metabolismo , Células Vero/virologia , Replicação Viral
15.
Genes Dev ; 18(6): 660-72, 2004 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-15075293

RESUMO

Although the activity of the translation initiation factor eIF4F is regulated in part by translational repressors (4E-BPs) that prevent incorporation of eIF4E, the cap-binding protein, into the initiation complex, the contribution of eIF4E phosphorylation to translational control remains controversial. Here, we demonstrate that the herpes simplex virus-1 (HSV-1) ICP0 gene product, a multifunctional transactivator of viral gene expression with ubiquitin E3 ligase activity that is important for vegetative replication and reactivation of latent infections, is required to stimulate phosphorylation of eIF4E as well as 4E-BP1, and promote assembly of eIF4F complexes in infected cells. Furthermore, 4E-BP1 is degraded by the proteasome in an ICP0-dependent manner, establishing that the proteasome can control 4E-BP1 steady-state levels. Preventing eIF4E phosphorylation by inhibiting the eIF4E kinase mnk-1 dramatically reduced viral replication and the translation of viral polypeptides in quiescent cells, providing the first evidence that phosphorylation of eIF4E by mnk-1 is critical for viral protein synthesis and replication. Thus, in marked contrast to many viruses that inactivate eIF4F, HSV-1 stimulates eIF4F complex assembly in quiescent, differentiated cells; moreover, this is important for viral replication, and may be crucial for HSV-1 to initiate its productive growth cycle in resting cells, such as latently infected neurons.


Assuntos
Fator de Iniciação 4E em Eucariotos/metabolismo , Herpes Simples/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Simplexvirus/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Chlorocebus aethiops , Cisteína Endopeptidases/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Complexos Multienzimáticos/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Fosfotransferases/metabolismo , Complexo de Endopeptidases do Proteassoma , Simplexvirus/genética , Células Vero/metabolismo
16.
J Gen Virol ; 83(Pt 7): 1601-1612, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12075078

RESUMO

The human cytomegalovirus (HCMV) tegument phosphoprotein pp71 activates viral immediate early (IE) transcription and thus has a role in initiating lytic infection. Protein pp71 stimulates expression from a range of promoters in a sequence-independent manner, and in this respect behaves similarly to the herpes simplex virus type 1 (HSV-1) IE protein ICP0. The intracellular localization of pp71 was investigated after its expression from transfected plasmids or from HSV-1 mutants constructed to produce pp71 transiently. The protein colocalized with the cell promyelocytic leukaemia (PML) protein at nuclear domain 10 (ND10) structures but, unlike ICP0, pp71 did not induce disruption of ND10. The activity of pp71 in mouse sensory neurons in vivo was investigated after co-inoculation of animals with pairs of HSV-1 mutants, one expressing pp71 and the second containing the E. coli lacZ gene controlled by various promoters. In this system, pp71 stimulated beta-galactosidase expression from a range of viral IE promoters when mice were analysed at 4 days postinoculation. At later times, expression of pp71 resulted in a reduction in numbers of neurons containing beta-galactosidase, indicating cytotoxicity or promoter shutoff. The HSV-1 latency-active promoter was not responsive to pp71, demonstrating specificity in the activity of the protein. Pp71 was as active in mice lacking both copies of the PML gene (PML-/-) as in control animals, and in PML-/- fibroblasts pp71 stimulated gene expression as effectively as in other cell types. Therefore, neither the PML protein nor the normal ND10 structure is necessary for pp71 to stimulate gene expression.


Assuntos
Citomegalovirus/metabolismo , Regulação Viral da Expressão Gênica , Proteínas Nucleares , Proteínas Virais/metabolismo , Animais , Núcleo Celular/metabolismo , Chlorocebus aethiops , Citomegalovirus/química , Feminino , Genes Precoces , Vetores Genéticos , Herpesvirus Humano 1/genética , Humanos , Óperon Lac , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Neurônios/metabolismo , Plasmídeos , Proteína da Leucemia Promielocítica , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Transcrição Gênica , Proteínas Supressoras de Tumor , Células Vero/metabolismo , Proteínas Virais/análise , beta-Galactosidase/metabolismo
17.
BMC Infect Dis ; 2: 7, 2002 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-12019036

RESUMO

BACKGROUND: The thymidine kinase (tk) mutagenesis assay is often utilized to determine the frequency of herpes simplex virus (HSV) replication-mediated mutations. Using this assay, clinical and laboratory HSV-2 isolates were shown to have a 10- to 80-fold higher frequency of spontaneous mutations compared to HSV-1. METHODS: A panel of HSV-1 and HSV-2, along with polymerase-recombinant viruses expressing type 2 polymerase (Pol) within a type 1 genome, were evaluated using the tk and non-HSV DNA mutagenesis assays to measure HSV replication-dependent errors and determine whether the higher mutation frequency of HSV-2 is a distinct property of type 2 polymerases. RESULTS: Although HSV-2 have mutation frequencies higher than HSV-1 in the tk assay, these errors are assay-specific. In fact, wild type HSV-1 and the antimutator HSV-1 PAAr5 exhibited a 2-4 fold higher frequency than HSV-2 in the non-HSV DNA mutatagenesis assay. Furthermore, regardless of assay, HSV-1 recombinants expressing HSV-2 Pol had error rates similar to HSV-1, whereas the high mutator virus, HSV-2 6757, consistently showed significant errors. Additionally, plasmid DNA containing the HSV-2 tk gene, but not type 1 tk or LacZ DNA, was shown to form an anisomorphic DNA structure. CONCLUSIONS: This study suggests that the Pol is not solely responsible for the virus-type specific differences in mutation frequency. Accordingly, it is possible that (a) mutations may be modulated by other viral polypeptides cooperating with Pol, and (b) the localized secondary structure of the viral genome may partially account for the apparently enhanced error frequency of HSV-2.


Assuntos
DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Herpesvirus Humano 1/enzimologia , Herpesvirus Humano 2/enzimologia , Mutação/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Animais , Bioensaio , Linhagem Celular , Chlorocebus aethiops , DNA Polimerase II/biossíntese , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/genética , DNA Recombinante/genética , DNA Recombinante/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , DNA Polimerase Dirigida por DNA/biossíntese , Exodesoxirribonucleases/biossíntese , Genoma Viral , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/metabolismo , Herpesvirus Humano 2/genética , Humanos , Mutagênese/efeitos dos fármacos , Mutagênese/genética , Mutação/efeitos dos fármacos , Conformação de Ácido Nucleico/efeitos dos fármacos , Plasmídeos/biossíntese , Plasmídeos/genética , Timidina Quinase/genética , Timidina Quinase/metabolismo , Transfecção , Células Vero/química , Células Vero/metabolismo , Proteínas Virais/biossíntese
18.
Cancer Gene Ther ; 9(3): 289-95, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11896446

RESUMO

BACKGROUND: Systemic IL-2 has shown some activity in metastatic melanoma, but its use is severely limited by toxicity. TG2001 is a product in which the human IL-2 cDNA was incorporated into the genome of Vero cells, a monkey fibroblast cell line. The goal of this intratumorally applied therapy was to create an antitumor immune response stimulated by xeno-antigens and local production of IL-2 in the close vicinity of tumor-specific antigens. TG2001 was reported to have a good safety profile in two previous dose-escalating phase I studies performed in 18 patients with various solid tumors, with encouraging clinical responses in three patients. The objectives of this study were to evaluate the tolerance and incidence of tumor regression in patients with metastatic melanoma, following repeated administration of Vero-IL-2 cells. PATIENTS AND METHODS: This was on open-label, randomized phase II study comparing two doses of Vero-IL-2, 5x10(5) and 5x10(6) cells. Twenty-eight patients with metastatic melanoma were enrolled in the study, 14 in each treatment group. Patients received TG2001 by intratumoral injection on days 1, 3, and 5 every 4 weeks for four cycles, and every 8 weeks thereafter, until evidence of progressive disease (PD). Criteria for patient selection included histologically proven metastatic melanoma, with one tumor accessible for product administration, and at least another tumor site for response assessment. Evaluation included tumor measurements, humoral and T cell-mediated local and systemic immune response, humoral response to Vero cells, adverse events and standard laboratory parameters. RESULTS: None of the patients achieved a confirmed objective response. Stable disease (SD) was seen in six (43%) and eight patients (57%) at the 5x10(5) and the 5x10(6) dose level, respectively. Two patients, one in each group, died during the study (i.e., within 1 month after the last injection) due to PD. Three patients exhibited antibody responses to Vero cells. T-cell immunity, serum cytokine levels and cytokine mRNA expression in tumor biopsies did not show meaningful alterations after therapy, except for a trend toward an increase in intratumoral TH2 cytokine (IL-4 and/or IL-10) levels. The study drug was well tolerated at both dose levels and side effects mainly consisted of injection site pain and erythema, and pyrexia. CONCLUSION: The intratumoral administration of TG2001 was generally well tolerated in patients with metastatic melanoma, and transient disease stabilization was observed in 50% of patients.


Assuntos
Imunoterapia/métodos , Interleucina-2/biossíntese , Melanoma/terapia , Neoplasias/terapia , Células Vero/metabolismo , Adulto , Idoso , Animais , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Feminino , Humanos , Imunidade , Injeções Intralesionais , Interleucina-2/genética , Masculino , Melanoma/diagnóstico por imagem , Melanoma/secundário , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Cintilografia , Linfócitos T/imunologia , Transplante Heterólogo , Resultado do Tratamento , Células Vero/imunologia
19.
Exp Cell Res ; 269(1): 117-29, 2001 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-11525645

RESUMO

Inefficient nuclear incorporation of foreign DNA remains a critical roadblock in the development of effective nonviral gene delivery systems. DNA delivered by traditional protocols remains within endosomal/lysosomal vesicles, or is rapidly degraded in the cytoplasm. Verotoxin I (VT), an AB(5) subunit toxin produced by enterohaemorrhagic Escherichia coli, binds to the cell surface glycolipid, globotriaosylceramide (Gb(3)) and is internalized into preendosomes. VT is then retrograde transported to the Golgi, endoplasmic reticulum (ER), and nucleus of highly VT-sensitive cells. We have utilized this nuclear targeting of VT to design a unique delivery system which transports exogenous DNA via vesicular traffic to the nucleus. The nontoxic VT binding subunit (VTB) was fused to the lambda Cro DNA-binding repressor, generating a 14-kDa VTB-Cro chimera. VTB-Cro binds specifically via the Cro domain to a 25-bp DNA fragment containing the consensus Cro operator. VTB-Cro demonstrates simultaneous specific binding to Gb(3). Treatment of Vero cells with fluorescent-labeled Cro operator DNA in the presence of VTB-Cro, results in DNA internalization to the Golgi, ER, and nucleus, whereas fluorescent DNA alone is incorporated poorly and randomly within the cytoplasm. VTB-Cro mediated nuclear DNA transport is prevented by brefeldin A, consistent with Golgi/ER intracellular routing. Pretreatment with filipin had no effect, indicating that caveoli are not involved. This novel VTB-Cro shuttle protein may find practical applications in the fields of intracellular targeting, gene delivery, and gene therapy.


Assuntos
Núcleo Celular/efeitos dos fármacos , Proteínas de Ligação a DNA , DNA/metabolismo , Vetores Genéticos/metabolismo , Imunotoxinas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/metabolismo , Toxina Shiga I/metabolismo , Triexosilceramidas/metabolismo , Animais , Antivirais/farmacologia , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Brefeldina A/farmacologia , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Chlorocebus aethiops , DNA/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Filipina/farmacologia , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Proteínas Repressoras/genética , Vesículas Transportadoras/efeitos dos fármacos , Vesículas Transportadoras/metabolismo , Células Vero/citologia , Células Vero/efeitos dos fármacos , Células Vero/metabolismo , Proteínas Virais de Fusão/síntese química , Proteínas Virais de Fusão/metabolismo , Proteínas Virais , Proteínas Virais Reguladoras e Acessórias
20.
J Immunol ; 165(8): 4528-36, 2000 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-11035093

RESUMO

The envelope surface glycoprotein C (gC) of HSV-1 interferes with the complement cascade by binding C3 and activation products C3b, iC3b, and C3c, and by blocking the interaction of C5 and properdin with C3b. Wild-type HSV-1 is resistant to Ab-independent complement neutralization; however, HSV-1 mutant virus lacking gC is highly susceptible to complement resulting in > or =100-fold reduction in virus titer. We evaluated the mechanisms by which complement inhibits HSV-1 gC null virus to better understand how gC protects against complement-mediated neutralization. C8-depleted serum prepared from an HSV-1 and -2 Ab-negative donor neutralized gC null virus comparable to complement-intact serum, indicating that C8 and terminal lytic activity are not required. In contrast, C5-depleted serum from the same donor failed to neutralize gC null virus, supporting a requirement for C5. EDTA-treated serum did not neutralize gC null virus, indicating that complement activation is required. Factor D-depleted and C6-depleted sera neutralized virus, suggesting that the alternative complement pathway and complement components beyond C5 are not required. Complement did not aggregate virus or block attachment to cells. However, complement inhibited infection before early viral gene expression, indicating that complement affects one or more of the following steps in virus replication: virus entry, uncoating, DNA transport to the nucleus, or immediate early gene expression. Therefore, in the absence of gC, HSV-1 is readily inhibited by complement by a C5-dependent mechanism that does not require viral lysis, aggregation, or blocking virus attachment.


Assuntos
Anticorpos Antivirais/fisiologia , Herpesvirus Humano 1/imunologia , Adulto , Animais , Anticorpos Antivirais/sangue , Chlorocebus aethiops , Complemento C5/fisiologia , Complemento C8/fisiologia , Via Alternativa do Complemento/imunologia , Dissacarídeos/imunologia , Regulação Viral da Expressão Gênica/imunologia , Genes Precoces/imunologia , Células HeLa/imunologia , Células HeLa/metabolismo , Células HeLa/virologia , Herpes Simples/genética , Herpes Simples/imunologia , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/fisiologia , Herpesvirus Humano 1/ultraestrutura , Humanos , Testes de Neutralização , Receptores Virais/antagonistas & inibidores , Receptores Virais/imunologia , Células Vero/imunologia , Células Vero/metabolismo , Células Vero/virologia , Proteínas do Envelope Viral/deficiência , Proteínas do Envelope Viral/genética
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