RESUMEN
We investigated the effects of alpha1-antitrypsine Portland variant (alpha1-PDX) and decanoylRVKRchloromethylketone (decRVKRcmk) on HIV-2(ROD) replication in the Jurkat lymphoblastoid cell line. To this end, cells were stably transfected with the alpha1-PDX (J-PDX) and used as targets for HIV-2(ROD) infection. Controls were prepared with the empty vector (J-pcDNA3). HIV-2(ROD) and HIV-1(LAI) replications were significantly inhibited and delayed in the presence of the alpha1-PDX protein. When decRVKRcmk was used at 35 microM, inhibition rates were 70-80% for HIV-2(ROD) and HIV-1(LAI), while total inhibition occurred at 70 microM. Control peptides consisting of decanoylRVKR and acetylYVADcmk had no effect. In the presence of the alpha1-PDX or the decRVKRcmk at 35 microM, the infectivity of HIV-2(ROD) and HIV-1(LAI) produced was 3-4-fold lower. Both molecules inhibited syncytium formation by HIV-2(ROD) and HIV-1(LAI) to a considerable extent. Finally, the inhibition of viral replication was correlated with the ability of the decRVKRcmk at 35 and 70 microM and of the alpha1-PDX, to inhibit the processing of envelope glycoprotein precursors. The alpha1-PDX protein and the decRVKRcmk peptide at 35 microM inhibited HIV-2 and HIV-1 to a similar level suggesting that identical or closely related endoproteases are involved in the maturation of their envelope glycoprotein precursors into surface and transmembrane glycoproteins. The significant inhibition observed with alpha1-PDX indicates that furin or furin-like endoproteases appear to play a major role in the maturation process.
Asunto(s)
Fármacos Anti-VIH/farmacología , VIH-1/efectos de los fármacos , VIH-2/efectos de los fármacos , VIH-2/fisiología , Péptidos/farmacología , Replicación Viral/efectos de los fármacos , alfa 1-Antitripsina/farmacología , Clorometilcetonas de Aminoácidos , Secuencia de Aminoácidos , Relación Dosis-Respuesta a Droga , Productos del Gen env/metabolismo , Células Gigantes/efectos de los fármacos , Células Gigantes/virología , Proteínas gp160 de Envoltorio del VIH/metabolismo , VIH-1/fisiología , Humanos , Células Jurkat , Péptidos/química , Precursores de Proteínas/metabolismo , Ensamble de Virus/efectos de los fármacos , Productos del Gen env del Virus de la Inmunodeficiencia HumanaRESUMEN
T-cell epitopes within viral polypeptide VP4 of the capsid protein of foot-and-mouth disease virus were analyzed using 15-mer peptides and peripheral blood mononuclear cells (PBMC) from vaccinated outbred pigs. An immunodominant region between VP4 residues 16 and 35 was identified, with peptide residues 20 to 34 (VP4-0) and 21 to 35 (VP4-5) particularly immunostimulatory for PBMC from all of the vaccinated pigs. CD25 upregulation on peptide-stimulated CD4(+) CD8(+) cells-dominated by Th memory cells in the pig-and inhibition using anti-major histocompatibility complex class II monoclonal antibodies indicated recognition by Th lymphocytes. VP4-0 immunogenicity was retained in a tandem peptide with the VP1 residue 137 to 156 sequential B-cell epitope. This B-cell site also retained immunogenicity, but evidence is presented that specific antibody induction in vitro required both this and the T-cell site. Heterotypic recognition of the residue 20 to 35 region was also noted. Consequently, the VP4 residue 20 to 35 region is a promiscuous, immunodominant and heterotypic T-cell antigenic site for pigs that is capable of providing help for a B-cell epitope when in tandem, thus extending the possible immunogenic repertoire of peptide vaccines.
Asunto(s)
Aphthovirus/inmunología , Proteínas de la Cápside , Cápside/inmunología , Epítopos Inmunodominantes , Complejo Mayor de Histocompatibilidad/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Secuencia de Aminoácidos , Animales , Antígenos Virales/química , Antígenos Virales/inmunología , Cápside/química , Cápside/genética , Fiebre Aftosa/prevención & control , Activación de Linfocitos , Datos de Secuencia Molecular , Pruebas de Neutralización , Péptidos/síntesis química , Péptidos/inmunología , Porcinos , Vacunación , Vacunas Virales/inmunologíaRESUMEN
DKP formation is a serious side reaction during the solid-phase synthesis of peptide acids containing either Pro or Gly at the C-terminus. This side reaction not only leads to a lower overall yield, but also to the presence in the reaction crude of several deletion peptides lacking the first amino acids. For the preparation of protected peptides using the Fmoc/tBu strategy, the use of a ClTrt-Cl-resin with a limited incorporation of the C-terminal amino acid is the method of choice. The use of resins with higher loading levels leads to more impure peptide crudes. The use of HPLC-ESMS is a useful method for analysing complex samples, such as those formed when C-terminal Pro peptides are prepared by non-optimized solid-phase strategies.