RESUMEN
The major structural viral protein, VP1, of the human polyomavirus JC virus (JCV), the causative agent of progressive multifocal leukoencephalopathy (PML), was expressed by using recombinant baculoviruses. Recombinant VP1 formed virus-like particles (VLP) with the typical morphology of empty JCV capsids. Purified VP1 VLP bind to SVG, B, and T cells, as well as to monkey kidney cells. After binding, VP1 VLP were also internalized with high efficiency and transported to the nucleus. Immunization studies revealed these particles as highly immunogenic when administered with adjuvant, while immunization without adjuvant induced no immune response. VP1 VLP hyperimmune serum inhibits binding to SVG cells and neutralizes natural JCV. Furthermore, the potential of VP1 VLP as an efficient transporter system for gene therapy was demonstrated. Exogenous DNA could be efficiently packaged into VP1 VLP, and the packaged DNA was transferred into COS-7 cells as shown by the expression of a marker gene. Thus, VP1 VLP are useful for PML vaccine development and represent a potential new transporter system for human gene therapy.
Asunto(s)
Proteínas de la Cápside , Cápside/inmunología , Vectores Genéticos , Virus JC/fisiología , Ensamble de Virus , Animales , Células COS , Cápside/genética , Cápside/aislamiento & purificación , Línea Celular , Clonación Molecular , Expresión Génica , Terapia Genética/métodos , Humanos , Virus JC/genética , Virus JC/inmunología , Virus JC/ultraestructura , Conejos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/aislamiento & purificación , Spodoptera/citología , Vacunas Sintéticas , Vacunas Virales , Virión/fisiología , Virión/ultraestructuraRESUMEN
The HIV-1 protease is essential for maturation of virus particles and is, therefore, an attractive target for antiviral drugs. The function of this protease depends on the dimerization of two identical subunits. Commonly used protease inhibitors are directed mainly against the active site of the enzyme which often leads to viral resistance. To determine the inhibitory effect of peptides interfering with the dimerization site of the HIV-1 protease, a recombinant bacterial screening assay was established. Escherichia coli was co-transformed with two different plasmids, expressing the 'interface' peptide and an active HIV-1 protease toxic for the bacteria. Co-expression of inhibitory peptides overcomes the incomplete membrane transmission of supplemented inhibitors and leads to a direct interaction of the inhibitory peptide and the HIV-1 protease. The inhibitory effect of co-expressed peptides was measured by an increased growth of co-transformed bacteria, compared with a slowly growing E. coli control culture only expressing the HIV-1 protease. Using this assay several penta- and hexa-peptides were screened for their ability to inhibit HIV-1 protease activity. One of these peptides showed a significant inhibitory effect on co-expressed recombinant HIV-1 protease.
Asunto(s)
Escherichia coli/enzimología , Inhibidores de la Proteasa del VIH/metabolismo , Proteasa del VIH/metabolismo , VIH-1/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , GTP Fosfohidrolasas/farmacología , Vectores Genéticos/genética , Proteasa del VIH/efectos de los fármacos , Inhibidores de la Proteasa del VIH/farmacología , Humanos , Datos de Secuencia Molecular , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Factores de Tiempo , Transformación BacterianaRESUMEN
Guinea pigs were immunized with inactivated measles virus. When challenged with live virus by the intradermal route, most of the animals developed an area of erythema within 24 to 48 hours; however, an unexpected feature of the skin-testing was that some of the guinea pigs developed a very severe necrotic reaction by the sixth or seventh day.
