RESUMEN
AIMS: To investigate the antiviral potential of the macrolide FK506, produced by Streptomyces tsukubaensis, against Orthopoxvirus infection in cell culture, and determine the replicative stage of viral cycle affected by the treatment. METHODS AND RESULTS: Cell lines were infected with different Orthopoxviruses and treated with FK506. The macrolide inhibited the replication of the prototypic Orthopoxvirus, vaccinia virus strain WR, with an IC50 of 12.05 micromol l(-1). Progeny production of other Orthopoxviruses was also inhibited by FK506 at noncytotoxic concentrations, as evaluated by the neutral-red uptake assay and metabolic labelling of cellular proteins. By Western blot assay, we detected a severe inhibition (approximately 87.6% +/- 2.78%) of VV strain WR post-replicative protein synthesis. A similar reduction of virus DNA accumulation, as observed by slot-blot assay, probably accounts for the subsequent inhibition of virus late proteins. CONCLUSIONS: The macrolide FK506, isolated from S. tsukubaensis, presents a novel anti-poxvirus activity, probably targeting the stage of DNA replication during Orthopoxvirus infection. SIGNIFICANCE AND IMPACT OF THE STUDY: The secondary metabolite FK506, isolated from the culture filtrate of S. tsukubaensis, shows a pleiotropic range of activities, and might be a valuable tool as a lead structure in the generation of non-immunosuppressant analogues with strong anti-poxvirus activity.
Asunto(s)
Antivirales/farmacología , Orthopoxvirus/efectos de los fármacos , Infecciones por Poxviridae/tratamiento farmacológico , Streptomyces/metabolismo , Tacrolimus/farmacología , Animales , Antibiosis , Proteínas Bacterianas/biosíntesis , Western Blotting/métodos , Células Cultivadas , Chlorocebus aethiops , ADN Viral/análisis , Electroforesis en Gel de Poliacrilamida , Orthopoxvirus/genética , Orthopoxvirus/fisiología , Replicación Viral/efectos de los fármacosRESUMEN
AIMS: To investigate the effect of the culture supernatants from three newly isolated Streptomyces strains, 221, 235 and 606 on eukaryotic cells. METHODS AND RESULTS: Cell lines were treated with the culture filtrates and assayed for protein synthesis by metabolic labelling, followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis. RNA synthesis was investigated by [5-3H]uridine incorporation. The three culture filtrates presented a strong inhibitory activity, reducing total protein synthesis of different eukaryotic cell lines by more than 85%. No effect on cellular RNA synthesis was detected. The culture filtrates did not affect the growth of the prokaryotic cells tested. CONCLUSIONS: These new Streptomyces strains, recently isolated from Brazilian tropical soils, produce molecule(s) with inhibitory activity specific to eukaryote protein synthesis. SIGNIFICANCE AND IMPACT OF THE STUDY: Streptomyces strains 221, 235 and 606, probably representing new species, might produce new bioactive compound(s), and can be used as valuable tools to study the protein synthesis pathway in eukaryotes.
Asunto(s)
Inhibidores de la Síntesis de la Proteína/metabolismo , Streptomyces/metabolismo , Células 3T3 , Animales , Brasil , Línea Celular , Medios de Cultivo , Humanos , Ratones , Biosíntesis de Proteínas/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/aislamiento & purificación , Inhibidores de la Síntesis de la Proteína/farmacología , ARN/biosíntesis , Microbiología del Suelo , Streptomyces/efectos de los fármacos , Streptomyces/crecimiento & desarrollo , Streptomyces/aislamiento & purificación , Clima TropicalRESUMEN
It is known that vaccinia core proteins are released into the supernatant fraction when cores are incubated under appropriate conditions. When prepared in the absence of viral transcription this fraction inhibits in vitro protein synthesis. We show here that after incubation, the cores loose the capability to inhibit protein synthesis. Furthermore, we show that no inhibition of translation is observed with supernatant fractions prepared from transcribing cores. SDS-PAGE analysis of the supernatantt fraction of cores obtained in the presence and absence of viral transcription indicated that their protein composiition is similar and approximately 17 peptides are released from the cores and that 4 are phosphorylated. We conclude that the proteins associated with vaccinia cores and released in the presence of ribonucleotides are not involved in protein synthesis inhibition
Asunto(s)
Técnicas In Vitro , Inhibidores de la Síntesis de la Proteína , Transcripción Genética , Virus Vaccinia , Proteínas , RibonucleótidosRESUMEN
When a rabbit reticulocyte lysate is incubated in the presence of vaccina cores, protein synthesis is umpaired at the level of the initiation step and the polyribosomes are depolymerized. However, when the same system is coupled with virus transcription: a) protein synthesis is restored, b) the initiation step is not inhibited, and c) the polyribosomes are not disaggregated. A viral factor activated in the absence of virus transcription and not activated when RNA synthesis occurs may be involved in the early mechanism of protein synthesis inhibition by vaccinia virus