RESUMEN
The unprocessed Gag precursor from HIV-1, when expressed in recombinant baculovirus-infected insect cells, is targeted to the plasma membrane and assembles in 100-120 nm particles budding from the cell surface. This process mimics HIV immature particle formation and is dependent on myristoylation of the N-terminal glycine, as deletion of the latter results in particle accumulation in the cytoplasm and, interestingly, in the nucleus, pointing to a potential role of this non-fatty-acid-acylated species in the viral life cycle. Inclusion of the pol gene in the construct results in efficient processing of Pr55gag and a pronounced decrease in particle formation. Deletion of the C terminus (p16) of the Gag precursor, including the finger domains, abolishes particle assembly, but membrane targeting and evagination still occur. Heterologous expression in insect cells may prove very useful for the study of the molecular events leading to retroviral particle morphogenesis.
Asunto(s)
Productos del Gen gag/metabolismo , VIH-1/fisiología , Virus de Insectos/genética , Lepidópteros/genética , Precursores de Proteínas/metabolismo , Replicación Viral , Animales , Productos del Gen gag/biosíntesis , Productos del Gen gag/genética , Genes gag , Virus de Insectos/metabolismo , Virus de Insectos/ultraestructura , Miristatos/metabolismo , Biosíntesis de Proteínas , Precursores de Proteínas/biosíntesis , Precursores de Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/fisiología , Virión/genética , Virión/metabolismo , Virión/fisiologíaRESUMEN
Mutant urokinase-type plasminogen activator (u-PA) genes and hybrid genes between tissue-type plasminogen activator (t-PA) and u-PA have been designed to direct the synthesis of new plasminogen activators and to investigate the structure-function relationship in these molecules. The following classes of constructs were made starting from cDNA encoding human t-PA or u-PA: 1) u-PA mutants in which the Arg156 and Lys158 were substituted with threonine, thus preventing cleavage by thrombin and plasmin; 2) hybrid molecules in which the NH2-terminal regions of t-PA (amino acid residues 1-67, 1-262, or 1-313) were fused with the COOH-terminal region of u-PA (amino acids 136-411, 139-411, or 195-411, respectively); and 3) a hybrid molecule in which the second kringle of t-PA (amino acids 173-262) was inserted between amino acids 130 and 139 of u-PA. In all cases but one, the recombinant proteins, produced by transfected eukaryotic cells, were efficiently secreted in the culture medium. The translation products have been tested for their ability to activate plasminogen after in situ binding to an insolubilized monoclonal antibody directed against urokinase. All recombinant enzymes were shown to be active, except those in which Lys158 of u-PA was substituted with threonine. Recombination of structural regions derived from t-PA, such as the finger, the kringle 2, or most of the A-chain sequences, with the protease part or the complete u-PA molecule did not impair the catalytic activity of the hybrid polypeptides. This observation supports the hypothesis that structural domains in t-PA and u-PA fold independently from one to another.
Asunto(s)
Células/metabolismo , Quimera , Células Eucariotas/metabolismo , Mutación , Activadores Plasminogénicos/genética , Proteínas Recombinantes/genética , Animales , ADN/análisis , Lisina , Biosíntesis de Proteínas , Conformación Proteica , Relación Estructura-Actividad , Treonina , Activador de Plasminógeno de Tipo Uroquinasa/genética , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
Human recombinant single chain urokinase-type plasminogen activator (recombinant scu-PA) and a hybrid between human tissue-type plasminogen activator (t-PA) and scu-PA, obtained by ligation of cDNA fragments encoding the NH2-terminal region (amino acids 1-67) of t-PA and the COOH-terminal region (amino acids 136-411) of scu-PA, were expressed in a mammalian cell system. The proteins were purified from conditioned culture media containing 2% fetal calf serum by chromatography on zinc chelate-Sepharose, immunoadsorption chromatography on an insolubilized murine monoclonal antibody directed against urokinase, benzamidine-Sepharose chromatography, and Ultrogel AcA 44 gel filtration. Between 180 and 230 micrograms of the purified proteins were obtained per liter of conditioned medium, with a yield of approximately 18% and a purification factor of 720-1900. On sodium dodecyl sulfate gel electrophoresis under reducing conditions, the proteins migrated as single bands with approximate Mr 50,000 for recombinant scu-PA and Mr 43,000 for the t-PA/scu-PA hybrid. Following conversion to urokinase with plasmin, the proteins had a specific amidolytic activity comparable to that of natural scu-PA. Both proteins activated plasminogen directly with Km = 0.53 and 1.4 microM and k2 = 0.0034 and 0.0027 s-1, respectively. Both proteins did not bind specifically to fibrin and had a comparable degree of fibrin selectivity as measured in a system composed of a whole human 125I-fibrin-labeled plasma clot suspended in human plasma. It is concluded that this chimeric protein, consisting of the NH2-terminal "finger-like" domain of t-PA and the COOH-terminal region of scu-PA, has very similar enzymatic properties as compared to scu-PA, but has not acquired the fibrin affinity of t-PA.
Asunto(s)
Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes/genética , Activador de Tejido Plasminógeno/genética , Activador de Plasminógeno de Tipo Uroquinasa/genética , Adenocarcinoma/análisis , Secuencia de Aminoácidos , Aminoácidos/análisis , Animales , Anticuerpos Monoclonales , Fenómenos Químicos , Química Física , Cricetinae , ADN/análisis , Fibrina/metabolismo , Humanos , Cinética , Neoplasias Pulmonares/análisis , Peso Molecular , Proteínas Recombinantes de Fusión/metabolismo , Relación Estructura-Actividad , Especificidad por Sustrato , Activador de Tejido Plasminógeno/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
A normal strain of human foreskin fibroblasts, two SV40-transformed derivatives with finite and infinite life spans, and an established line of SV40-transformed newborn human kidney cells are compared for their susceptibility to infection with parvovirus H-1. H-1 inocula, which do not detectably alter the growth of normal cells, cause a progressive degeneration of all three SV40-transformed cultures. The resistance of normal cells is not a membrane phenomenon since they adsorb and take up H-1 as efficiently as the transformants. Moreover, the fraction of infected cells supporting the synthesis and nuclear migration of H-1 proteins is similar in normal and SV40-transformed cultures. On the other hand, the enhanced H-1 sensitivity of transformed cells correlates with a 5- to 30-fold increase in their accumulation of newly synthesized parvoviral DNA, as compared with normal cultures. This stimulation of H-1 DNA replication is most pronounced for the amplification of duplex replicative forms, although the conversion of parental single-stranded DNA to replicative forms is also enhanced to a smaller extent. In addition, SV40-transformed cells support productive H-1 infection and release a burst of infectious virus, whereas no H-1 production can be detected in the normal cell strain. The latter difference was confirmed for another series of 7 normal and 16 SV40-transformed strains of human skin fibroblasts. Altogether, these results indicate that intracellular limitations on H-1 DNA replication are associated with the abortive nature of the parvoviral life cycle in normal human fibroblasts and are overcome after SV40 transformation, resulting in the selective killing of the transformants. This observation raises the possibility that oncolysis might contribute to the oncosuppressive activity displayed by parvoviruses in vivo.
Asunto(s)
Transformación Celular Viral , Infecciones por Parvoviridae/fisiopatología , Parvoviridae/crecimiento & desarrollo , Supervivencia Celular , Células Cultivadas , ADN Viral/biosíntesis , Endocitosis , Humanos , Masculino , Virus 40 de los Simios , Proteínas Virales/biosíntesis , Replicación ViralRESUMEN
Human skin fibroblasts which are naturally resistant to Parvovirus H-1 can be lysed by this virus after SV40 transformation. This observation raises the possibility that oncosuppression by Parvovirus involves a direct oncolytic effect.