Vaccinia Virus: Isolation

more. Virus yield, which decreased only when the drug was added early in infection, was reduced several 100fold by 80 tLM aphidicolin. Viral inhibition was reversed by the suspension of the infected cells in drug-free medium. DNA synthesis in uninfected cells was reduced about 10-fold by 1 ,uM aphidicolin. In infected cells, aphidicolin concentrations over 10 ,uM were needed to reduce DNA synthesis to the same extent as in uninfected cells. Fractionation of infected cells which were incubated with 1 ,uM drug showed that cytoplasmic viral DNA synthesis was resistant to this aphidicolin concentration. The radioactivity associated with crude nuclei from these cells was estimated to be from vaccinia DNA synthesis. Spontaneous virus mutants which were resistant to 80 ,uM aphidicolin did not appear. However, after mutagenesis, mutants were generated which formed large plaques in medium with 80 ,uM drug. In cells with replicating aphidicolin-resistant virus, DNA synthesis was about four times more resistant to 80 ,uM aphidicolin than in cells with replicating wild-type virus. Chromatographic patterns of viral DNA polymerase isolated from cells with wild-type or resistant virus were similar. However, in an in vitro assay, 50% inhibition of enzyme activity was obtained with ca. 75 and 188 ,uM aphidicolin for the wild-type and resistant DNA polymerases, respectively. Viral enzymes were much more resistant to the drug than were the cell polymerases.