RESUMEN
The PE2 cleavage signal in a full-length cDNA clone of the alphavirus Venezuelan equine encephalitis virus (VEE) was ablated by site-directed mutagenesis. RNA transcripts derived from the resulting plasmids programmed the production of nonviable particles upon transfection of baby hamster kidney (BHK) cells. However, the mutant RNAs also gave rise to a small proportion of viable revertants. Analysis of these biological revertants and their molecularly cloned homologs demonstrated that second-site suppressor mutations at either E2 position 243 or E1 position 253 were able to restore viability to PE2 cleavage signal mutants. The viable revertants incorporated unprocessed PE2 into particles which showed normal infectivity for BHK cells, but reduced ability to grow in C6/36 mosquito cells. A mutant carrying a lethal PE2 cleavage signal mutation in combination with a suppressor at E1 253 was either avirulent or highly attenuated in adult mice when inoculated by the subcutaneous, intracerebral, or intranasal route and conferred complete protection against both intraperitoneal and intranasal challenge with virulent VEE. These results indicate the close functional association of the E2 and E1 proteins in the alphavirus spike. They also have implications for the design of recombinant live virus vaccines for VEE, for other alphaviruses, and for other viruses that use a similar mechanism for glycoprotein maturation.
Asunto(s)
Virus de la Encefalitis Equina Venezolana/genética , Proteínas del Envoltorio Viral/genética , Secuencia de Aminoácidos , Animales , Virus de la Encefalitis Equina Venezolana/inmunología , Femenino , Genes Letales , Genes Supresores , Glicoproteínas/genética , Glicoproteínas/metabolismo , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Peso Molecular , Mutagénesis Sitio-Dirigida , Procesamiento Proteico-Postraduccional , Vacunas Atenuadas/genética , Proteínas del Envoltorio Viral/metabolismo , Vacunas Virales/genética , Replicación ViralRESUMEN
Remote afterloading high-dose-rate brachytherapy (RAHDRB) was used endobronchially for the management of malignant airway obstruction in 82 patients, 72 of whom had primary disease in the lung. Treatment was palliative (n = 58) or definitive (n = 24). The extent of airway compromise was determined at bronchoscopy and with symptoms of hemoptysis, dyspnea, or cough or with radiographic evidence of atelectasis. RAHDRB doses were 1,000-4,700 cGy in one to five fractions. External beam radiation was used in previously unirradiated patients. A substantial reduction ,N airway disease and an improvement in symptoms were seen in 82% of patients. Obstruction scores showed an overall 74% improvement. Complications occurred in only 10 patients (two of whom died). Median survival was short (palliative group, 5 months; definitive group, 12 months); however, symptoms remained palliated in 62 patients (76%) until death or the last follow-up examination. RAHDRB is effective and can be applied with equal success in all patients with malignant airway obstruction, even those whose disease has recurred after external beam irradiation.