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

Abstract

Summary Eighty temperature-sensitive(ts) mutants of vaccinia virus were examined for defects in synthesis of DNA. Ninets mutants were incapable of synthesizing DNA at the restrective temperature of 39.5° 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 6ts 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 5ts mutants capable of synthesizing DNA at the elevated temperature, but this DNA failed to form infectious virions. Thesets mutants were designated as DNAf⁻ mutants. Pulse-chase experiments for the DNAf⁻ mutant 1877 revealed that viral DNA produced at 39.5° C was incapable of entering into mature virions or any subviral particles. Based on the data for recombination amongts mutants with the DNA⁻ and DNAf⁻ phenotype a tentative genetic map was constructed.