RESUMEN
Recombinant human procollagen C-proteinase enhancer (rPCPE) was expressed using a baculovirus system and purified to homogeneity using a three-step procedure including heparin affinity chromatography. Heparin binding was dependent on the C-terminal netrin-like domain. The recombinant protein was found to be active, increasing the activity of procollagen C-proteinase/bone morphogenetic protein-1 on type I procollagen in a manner comparable to the native protein. Enhancing activity was dependent on intact disulfide bonding within the protein. By circular dichroism, the observed secondary structure of rPCPE was consistent with the known three-dimensional structures of proteins containing homologous domains.
Asunto(s)
Proteínas Morfogenéticas Óseas/química , Proteínas Morfogenéticas Óseas/metabolismo , Metaloendopeptidasas/química , Metaloendopeptidasas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Animales , Baculoviridae/metabolismo , Proteína Morfogenética Ósea 1 , Proteínas Morfogenéticas Óseas/aislamiento & purificación , Línea Celular , Cromatografía de Afinidad , Dicroismo Circular , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Matriz Extracelular/metabolismo , Heparina/química , Heparina/metabolismo , Humanos , Insectos , Metaloendopeptidasas/aislamiento & purificación , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Rayos UltravioletaRESUMEN
A local strain of Helicoverpa armigera baculovirus was isolated from infected H. armigera larvae. Infectivity to Helicoverpa cells, restriction enzyme analysis and electron microscopy allowed its identification as a single embedded nucleopolyhedrovirus, designated HaSNPV-IS. Analysis of DNA replication, protein synthesis and polyhedrin expression in HaSNPV-infected cells located the late and very late phases of the viral cycle at 24 and 48 h after infection, respectively. The viral polyhedrin gene was isolated and characterized. It encoded for a polypeptide of 246 amino acid residues. A 32 kDa polypeptide was identified by immunoblot analysis using antipolyhedrin antiserum. The HaSNPV-IS polyhedrin DNA sequence revealed 99.4% of homology to the HzSNPV polyhedrin. The availability of this efficient replication system and the above knowledge paves the way to future genetic engineering of the HaSNPV.
Asunto(s)
Genes Virales/genética , Mariposas Nocturnas/virología , Nucleopoliedrovirus/aislamiento & purificación , Proteínas Virales/genética , Proteínas Estructurales Virales/genética , Replicación Viral , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Viral/química , ADN Viral/genética , ADN Viral/metabolismo , Expresión Génica , Israel , Datos de Secuencia Molecular , Nucleocápside , Nucleopoliedrovirus/genética , Nucleopoliedrovirus/ultraestructura , Proteínas de la Matriz de Cuerpos de Oclusión , Análisis de Secuencia de ADN , Proteínas Virales/biosíntesisRESUMEN
Apoptosis was postulated as the main barrier to replication of the Autographa californica nuclear polyhedrosis virus (AcMNPV) in a Spodoptera littoralis SL2 cell line (N. Chejanovsky and E. Gershburg, Virology 209:519-525, 1995). Thus, we hypothesized that the viral apoptotic suppressor gene p35 is either poorly expressed or nonfunctional in AcMNPV-infected SL2 cells. These questions were addressed by first determining the steady-state levels of the p35 product, P35, in AcMNPV-infected SL2 cells. Indeed, very low levels of P35 were found in infected SL2 cells in comparison with those in SF9 cells. Overexpression of p35, in transient-transfection and recombinant-virus infection experiments, inhibited actinomycin D- and AcMNPV-induced apoptosis, as determined by reduced cell blebbing and release of oligonucleosomes and increased cell viability of SL2. However, SL2 budded-virus (BV) titers of a recombinant AcMNPV which highly expressed p35 did not improve significantly. Also, injection of S. littoralis larvae with recombinant and wild-type AcMNPV BVs showed similar 50% lethal doses. These data suggest that apoptosis is not the only impediment to AcMNPV replication in these nonpermissive S. littoralis cells, and probably in S. littoralis larvae, so p35 may not be the only host range determinant in this system.
Asunto(s)
Apoptosis , Genes Supresores , Genes Virales , Nucleopoliedrovirus/fisiología , Proteínas Virales/biosíntesis , Replicación Viral , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Fragmentación del ADN , Dactinomicina/farmacología , Cinética , Nucleopoliedrovirus/genética , Spodoptera , TransfecciónRESUMEN
The Leiurus quinquestriatus hebraeus alpha anti-insect toxin (Lqh alpha IT) cDNA was engineered into the Autographa californica Nuclear Polyhedrosis Virus (AcNPV) genome. Insect cells infected with the recombinant virus secreted a functional Lqh alpha IT polypeptide. Spodoptera littoralis and Heliothis armigera larvae injected with recombinant budded virus, showed typical intoxication symptoms. This recombinant virus showed enhanced insecticidal potency against H. armigera larvae compared with wild type AcNPV. The present expression system will facilitate: (1) the future elucidation of structural elements involved in its prominent anti-insect toxicity; and (2) the future design of genetically modified alpha toxins with improved anti-insect selectivity.