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/fisiologiaRESUMO
Viral-induced apoptosis might be mediated by oxidative stress. It has already been described that cell death in vesicular stomatitis virus (VSV)-infected cells occurs by apoptosis. In this study, oxidative stress parameters present in VSV-infected Vero cells were analyzed. Lipid peroxides (LP) were evaluated in cellular extracts and expressed as thiobarbituric acid-reactive substances. LP levels exhibited a rise at different times post infection, according to the multiplicity of infection (MOI), while the presence of cycloheximide determined a reduction on LP. Also, an increase in protein degradation products and a decrease in polyunsaturated fatty acids content was observed, indicating that cellular proteins and lipids began to be susceptible to degradation during VSV infection. In addition, we analyzed cell viability of VSV-infected Vero cells, which were incubated in the presence of butylated hydroxyanisole. This antioxidant was able to protect Vero cells, at least at MOIs assayed in this study, and to reduce viral yield only when VSV infection was done at MOI 0.05. Further, superoxide dismutases, which occupy the first step within the antioxidant enzyme cascade, also exhibit a rise in VSV-infected Vero cells, at different MOI. These results suggest that both an oxidative stress and an antioxidative cell response precede the induction of apoptosis by VSV.
Assuntos
Estresse Oxidativo , Infecções por Rhabdoviridae/virologia , Vírus da Estomatite Vesicular Indiana/fisiologia , Animais , Chlorocebus aethiops , Peróxidos Lipídicos/metabolismo , Superóxido Dismutase/metabolismo , Fatores de Tempo , Células Vero/metabolismo , Células Vero/patologia , Células Vero/virologiaRESUMO
The control of cell death occurring in high density cultures performed in bioreactors is an important factor in production processes. In this work, medium nutrient removal or feeding was used to determine at which extension apoptosis could be, respectively, involved or prevented in VERO cell cultures on microcarriers. Glutamine and galactose present in the VERO cell culture medium was consumed after, respectively, 6 and 12 days of culture. Kinetics studies showed that fresh medium replacement and, to some extent, galactose or glutamine depleted-fresh medium replacement provided a nutritional environment, allowing the VERO cell cultures to attain high densities. Galactose was shown to be a more critical nutrient when cultures reached a high density. In agreement with that, VERO cell cultures supplemented with galactose and/or glutamine were shown to confirm previous findings and, again at high densities, galactose was shown to be a critical nutrient for VERO cell growth. These observations also indicated that in VERO cell cultures, for feeding purposes, the glutamine could be replaced by galactose. The inverse was not true and led, at high densities, to a decrease of cell viability. In the absence of glutamine and galactose, apoptosis was observed in VERO cell cultures by cytofluorometry, Acridine orange staining or light and electron microscopy, reaching high levels when compared to cultures performed with complete medium. VERO cells apoptosis process could be prevented by the galactose and/or glutamine feeding and, at high densities, galactose was more efficient in protecting the cultures. These cultures, prevented from apoptosis, were shown to synthesize high levels of measles virus following infection. Our data show that apoptosis prevention by glutamine/galactose feeding, led to high productive and metabolic active VERO cell cultures, as indicated by the high cell density obtained and the virus multiplication leading to higher virus titers.
Assuntos
Apoptose/efeitos dos fármacos , Reatores Biológicos , Galactose/farmacologia , Glutamina/farmacologia , Células Vero/metabolismo , Animais , Antígenos Virais/biossíntese , Divisão Celular/efeitos dos fármacos , Chlorocebus aethiops , Meios de Cultura/farmacologia , Citometria de Fluxo , Vírus do Sarampo/crescimento & desenvolvimento , Necrose , Células Vero/citologia , Células Vero/virologia , Replicação ViralRESUMO
The present study was undertaken to investigate the effects of D003, a mixture of very long chain saturated fatty acids isolated and purified from sugar cane wax, on cholesterol biosynthesis in cultured fibroblasts. Cholesterol biosynthesis is regulated through feedback regulation of at least two sequentially acting enzymes, 3-hydroxy-3-methyl coenzyme A (HMG-CoA) synthase and reductase. They are up-regulated when sterol levels fall and down-regulated when sterol levels rise. The exposure of cultured fibroblasts to a lipid-depleted medium (LDM) and D003 (0.05-50 microg ml(-1)) for 12 h inhibited, in a dose-dependent manner, cholesterol biosynthesis from 14C-labelled acetate (33-68%). The addition of D003 at concentrations inhibiting cholesterol biosynthesis from labelled acetate significantly decreased incorporation of radioactivity from 3H2O into sterols, but not from 14C-mevalonate. These data indicate that D003 inhibits cholesterol biosynthesis by interfering with early steps of cholesterol biosynthetic pathway. We reasoned that D003 acts directly on HMG-CoA reductase, the main regulatory enzyme of cholesterol biosynthetic pathway. However, when enzyme activity was measured in cell extracts in the presence of various concentrations of D003 (0.5-50 microg ml(-1)), reductase activity was not inhibited. Thus, there was no evidence for a competitive or non-competitive inhibition of enzyme activity by D003. Treatment with D003 significantly suppressed (68%) the enzyme up-regulation when cells were cultured in LDM, which suggests a depression of de novo synthesis of HMG-CoA reductase and/or a stimulation of its degradation. However, since the suppressive action of D003 on cholesterol biosynthesis was observed in metabolic conditions under which synthase up-regulation was also enhanced, we cannot rule out a possible effect of D003 on HMG-CoA synthase. Thus, further studies are needed to clarify the precise mechanism of the inhibitory effect of D003 on cholesterol biosynthesis.