Isolation of vaccinia virus mutants capable of replicating independently of the host cell nucleus

J Virol. 1984 Aug;51(2):359-66. doi: 10.1128/JVI.51.2.359-366.1984.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amanitins / toxicity
  • Animals
  • Cell Line
  • Cell Nucleus / metabolism*
  • Cell Nucleus / ultrastructure
  • Chlorocebus aethiops
  • DNA Replication*
  • Drug Resistance, Microbial
  • Kidney
  • Microscopy, Electron
  • Mutation*
  • Protein Biosynthesis
  • Species Specificity
  • Vaccinia virus / drug effects
  • Vaccinia virus / genetics*
  • Vaccinia virus / isolation & purification
  • Vaccinia virus / ultrastructure
  • Virus Replication

Substances

  • Amanitins