Isolation of vaccinia virus mutants capable of replicating independently of the host cell nucleus.
alpha-Amanitin-resistant vaccinia virus mutants were isolated after serial viral passages in BSC-40 cells that were carried out in the presence of inhibitory levels (6 micrograms/ml) of alpha-amanitin. One such mutant, alpha-27, was highly refractory (greater than 95%) to alpha-amanitin-mediated inhibition and was selected for further study. In the absence of drug, the phenotypes of alpha-27 and wild-type vaccinia virus were indistinguishable with respect to growth kinetics. DNA synthesis, protein synthesis, and morphogenesis. Infections in the presence of alpha-amanitin revealed two striking differences, however. First, wild-type virus was unable to catalyze proteolytic processing of the two major capsid proteins VP62 and VP60, whereas alpha-27 was most efficient at this process. Second, wild-type viral morphogenesis within the infected cells was arrested by alpha-amanitin at an apparently analogous step to that previously described for enucleated cells. This observation was supported by the ability of alpha-27 virus to replicate in enucleated BSC-40 cells. Restriction enzyme analyses of alpha-27 versus wild-type genomes revealed that a XhoI cleavage site was altered in the alpha-27 DNA molecule, suggesting a possible location for the alpha-amanitin resistance locus.
D000546:Amanitins / Q000633:toxicity; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism* / Q000648:ultrastructure; D002522:Chlorocebus aethiops; D004261:DNA Replication; D004352:Drug Resistance, Microbial; D007668:Kidney; D008854:Microscopy, Electron; D009154:Mutation; D014176:Protein Biosynthesis; D013045:Species Specificity; D014616:Vaccinia virus / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification / Q000648:ultrastructure; D014779:Virus Replication
D000546:Amanitins