Biochemical and genetic characterization of vaccinia virus temperature-sensitive mutants with DNA- and DNAf- phenotypes

Eighty temperature-sensitive (ts) mutants of vaccinia virus were examined for defects in synthesis of DNA. Nine ts mutants were incapable of synthesizing DNA at the restrictive temperature of 39.5 degrees C (DNA- mutants). Biochemical and genetic data indicate that all 9 DNA- mutants carry mutations in different genes. Temperature shift-up experiments have shown that 6 ts mutants with the DNA- phenotype have mutations in the genes coding for the proteins directly associated with vaccinia DNA synthesis. Temperature shift-down experiments in the presence of cytosine arabinoside revealed 5 ts mutants capable of synthesizing DNA at the elevated temperature, but this DNA failed to form infectious virions. These ts mutants were designated as DNAf- mutants. Pulse-chase experiments for the DNAf- mutant 1877 revealed that viral DNA produced at 39.5 degrees C was incapable of entering into mature virions or any subviral particles. Based on the data for recombination among ts mutants with the DNA- and DNAf- phenotype a tentative genetic map was constructed.