Isolation and preliminary characterization of temperature-sensitive mutants of vaccinia virus

Abstract

Methods have been developed for rapid isolation and genetic analysis of vaccinia virus mutants. These methods include: (1) monitoring mutagenesis by measuring conversion of wild-type, phosphonoacetic acid-sensitive virus to phosphonoacetic acid-resistant virus, (2) screening for mutants by plaque enlargement, and (3) a qualitative spot test for complementation. Twenty-six temperature-sensitive mutants of vaccinia virus have been isolated. All have reversion indices of 10 −4 or less. One-step growth experiments have been done at 40° and 31° with all the mutants and in all cases the virus yield at 40° is less than 8% of the yield observed at 31°. Complementation analysis has been completed on all 26 mutants, showing that these mutants together comprise 16 complementation groups. Twenty-four of the mutants have been analyzed for their ability to synthesize viral DNA at the nonpermissive temperature. The results show that 3 of the 24 have a DNA-negative phenotype. These three mutants fall into two complementation groups. Twenty-four of the mutants have been analyzed for their ability to synthesize early and late viral proteins at the nonpermissive temperature. From this analysis, four phenotypes appear: (1) normal, (2) a phenotype associated with DNA-negative mutants characterized by prolonged synthesis of early proteins and the absence of late protein synthesis, (3) weak or slow late protein synthesis, (4) abortive late protein synthesis.