Asunto(s)
Vectores Genéticos , Hemaglutininas Virales/inmunología , Virus de la Influenza A/inmunología , Vacunas contra la Influenza , Infecciones por Orthomyxoviridae/prevención & control , ARN/genética , Proteínas Recombinantes de Fusión/inmunología , Vacunas Sintéticas , Animales , Anticuerpos Antivirales/biosíntesis , Glicoproteínas Hemaglutininas del Virus de la Influenza , Hemaglutininas Virales/biosíntesis , Hemaglutininas Virales/genética , Inmunización Secundaria , Virus de la Influenza A/genética , Virus de la Influenza A/aislamiento & purificación , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/inmunología , Inyecciones Intramusculares , Ratones , Infecciones por Orthomyxoviridae/inmunología , Proteínas Recombinantes de Fusión/biosíntesis , Sistema Respiratorio/virología , Vacunación , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunologíaRESUMEN
We have established a stable, continuous culture Drosophila Schneider 2 cell line that efficiently expresses a secreted, truncated form of the HIV envelope gp120 protein in a regulated manner. The Drosophila produced recombinant gp120 protein is highly glycosylated, is recognized by gp120-specific monoclonal antibodies, binds to the CD4 receptor and has the ability to inhibit syncytia formation between uninfected CD4+ cells and HIV infected cells. We conclude that this recombinant Drosophila envelope protein is an appropriate mimic of the authentic viral envelope protein. Thus, the Drosophila cell provides a continuous, stable culture system for the efficient expression of secreted forms of complex surface glycoproteins in quantities sufficient for detailed analyses.
Asunto(s)
Drosophila/genética , Regulación de la Expresión Génica/genética , Proteína gp120 de Envoltorio del VIH/biosíntesis , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Antígenos CD4/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Línea Celular , Clonación Molecular , Glicosilación , Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/crecimiento & desarrollo , Humanos , Datos de Secuencia Molecular , Plásmidos , Proteínas Recombinantes/biosíntesisRESUMEN
Sera, from HIV-1 and HIV-2 seropositive individuals, were tested for the presence of antibodies able to inhibit the binding (BI) of HIV-IIIB gp 160 (produced in mammalian cells using a vaccinia expression system) to the extracellular portion of the CD4 receptor. A competition enzyme immunoassay (EIA) with soluble CD4 (sCD4) was used. BI antibodies were highly prevalent among HIV-1 seropositives but not in HIV-2 infected individuals. Attempts to localize the binding site for these BI antibodies on the primary sequence of gp 120 by using synthetic peptides encompassing the putative CD4 binding site on gp 120 (aa 397-439) were not successful. This study did not reveal a significant correlation between the presence of BI antibodies and disease evolution. BI antibody titres correlated less well with anti-gp 160 titres (r = 0.51, P less than or equal to 0.011) than with neutralizing antibody (NA) titres using either the isolates HIV-SF2 (SF2) (r = 0.77, P less than or equal to 0.000) and HIV-MN (MN) (r = 0.61, P less than or equal to 0.002) or the isolate HIV-IIIB (HX10) (r = 0.89, P less than or equal to 0.000) of which the gp 160 for the assays was derived. An HIV-IIIB neutralizing serum, elicited in a rabbit by immunization with a 17-mer synthetic peptide derived from the third variable domain (V3) of gp 120, did bind gp 160 without inhibiting the subsequent attachment of sCD4 to gp 160.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Anticuerpos Anti-VIH , Infecciones por VIH/inmunología , VIH-1/inmunología , Unión Competitiva , Antígenos CD4/inmunología , Epítopos , Productos del Gen env/inmunología , Proteínas gp160 de Envoltorio del VIH , Seropositividad para VIH/inmunología , Humanos , Masculino , Pruebas de Neutralización , Precursores de Proteínas/inmunologíaRESUMEN
Mouse mAb reactive to the HIV-1 envelope glycoprotein precursor gp160 of the HTLVIII(B) isolate were characterized in radioimmunoprecipitation and immunoblot tests with the use of HTLVIII(B) isolate as Ag. The reactivities of these mAb were also measured in a capture enzyme immunoassay and in radioimmunoprecipitation assay by using gp160 and gp120 expressed as vaccinia recombinants. Striking differences in exposure of specific epitopes were noted between the gp120 component of the gp160 precursor and the fully processed gp120 in both tests. These conformational rearrangements affecting the gp120 moiety of the HIV-1 envelope glycoprotein might have important implications on its immunogenicity.
Asunto(s)
Epítopos/análisis , Antígenos VIH/análisis , VIH-1/inmunología , Precursores de Proteínas/análisis , Proteínas de los Retroviridae/análisis , Proteínas del Envoltorio Viral/análisis , Animales , Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Antivirales/análisis , Anticuerpos Antivirales/biosíntesis , Reacciones Antígeno-Anticuerpo , Epítopos/inmunología , Antígenos VIH/inmunología , Proteína gp120 de Envoltorio del VIH , Ratones , Ratones Endogámicos BALB C , Precursores de Proteínas/inmunología , Proteínas Recombinantes/análisis , Proteínas Recombinantes/inmunología , Proteínas de los Retroviridae/inmunología , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
Recent studies indicate that human immunodeficiency virus type 1 (HIV) gene expression can be dramatically enhanced by certain heterologous viral and chemical agents, implicating these as potential reactivating agents of latent virus infection. A common denominator shared by these agents is their ability to cause stress responses in cells. In an effort to determine whether stress responses affect HIV gene expression, we examined the effects of ultraviolet light (UV) and mitomycin C, on HIV gene expression as well as on viral growth and development. We demonstrate that these agents enhance HIV gene expression up to 150-fold. These levels are similar to those obtained by the tat gene product, the HIV trans-activating factor responsible for enhancing viral gene expression. The increase in gene expression after UV irradiation appears to require transcription but not de novo protein synthesis, and correlates with an accumulation of stable mRNA. Most importantly, UV irradiation of human T-cells prior to viral infection significantly shortens the viral growth cycle. Apparently, UV-induced cellular stress is highly conducive for viral replication and growth. We further demonstrate that even direct sunlight can activate HIV gene expression. These results demonstrate that DNA damaging agents, and perhaps other agents which elicit SOS-like stress responses in mammalian cells, can activate HIV expression thereby enhancing viral replication and development.
Asunto(s)
Daño del ADN , VIH/crecimiento & desarrollo , Activación Viral , Acetiltransferasas/genética , Línea Celular , Cloranfenicol O-Acetiltransferasa , VIH/genética , Células HeLa/efectos de los fármacos , Células HeLa/efectos de la radiación , Humanos , Mitomicina , Mitomicinas/farmacología , Linfocitos T/efectos de la radiación , Rayos Ultravioleta , Activación Viral/efectos de los fármacos , Activación Viral/efectos de la radiaciónRESUMEN
Rat hepatoma cells that do not synthesize the hepatic enzyme ornithine carbamoyl transferase spontaneously give rise to producing cells at a low frequency. Reexpression of this differentiation trait is strongly increased by 5-azacytidine treatment, suggesting that hypermethylation plays a critical role in the impaired expression of the ornithine carbamoyl transferase gene in hepatoma cells.