Richard C. Condit

Isolation, characterization, and physical mapping of temperature-sensitive mutants of vaccinia virus

Thirty-nine new temperature-sensitive mutants of vaccinia virus have been isolated, expanding a previously reported collection of mutants (R. C. Condit and A. Motyczka, Virology 113, 224-241, 1981) to a total of 65. The 65 mutants have been assigned to 32 complementation groups, based primarily on a qualitative spot test described previously (Condit and Motyczka, 1981). Representatives of each complementation group have been assayed for DNA and protein synthesis at the nonpermissive temperature, revealing one new DNA-negative complementation group, three new groups which contain mutants defective in late protein synthesis, and ten new groups containing mutants which synthesize DNA and protein in a normal fashion. Marker rescue has been achieved with 29 of the 65 mutants using cloned DNA fragments from wild-type virus. These 29 mutants together represent 20 of the 32 complementation groups. A preliminary physical map of the mutants is presented.

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.

Marker rescue mapping of vaccinia virus temperature-sensitive mutants using overlapping cosmid clones representing the entire virus genome

A set of 11 overlapping cosmid clones of wild type (wt) vaccinia virus DNA was constructed. The clones together span almost the entire vaccinia virus genome. The clones were used to map temperature-sensitive (ts) mutants of vaccinia virus by marker rescue. Map positions were obtained for mutants representing 29 of 32 complementation groups tested.

Selection for temperature-sensitive mutations in specific vaccinia virus genes: Isolation and characterization of a virus mutant which encodes a phosphonoacetic acid-resistant, temperature-sensitive DNA polymerase

Seven temperature-sensitive mutants of vaccinia virus have been isolated after preselection for virus resistant to phosphonoacetic acid (PAA). In all seven mutants, the PAA-resistant (PAAr) and ts lesions represent separate mutations. In one mutant, NG26, the PAAr (NG26-PAAr) and ts (NG26-ts) mutations are very closely linked. Both NG26-ts and NG26-PAAr map in the HindIII E DNA fragment. NG26 has a DNA-negative phenotype at 40 degrees. NG26-ts is in the same complementation group as ts42, another DNA-negative mutant which maps in the HindIII E DNA fragment (R. C. Condit, A. Motyczka, and G. Spizz, Virology 128, 000-000, 1983). The order of the mutations is (NG26-ts)-(NG26-PAAr)-ts42. The virus-coded DNA polymerase has been partially purified from wt- and NG26-infected cells. The DNA polymerase encoded by NG26 is temperature sensitive and PAA resistant in vitro as compared to the wt enzyme.

PHENOTYPIC CHARACTERIZATION OF A VACCINIA VIRUS TEMPERATURE-SENSITIVE COMPLEMENTATION GROUP AFFECTING A VIRION COMPONENT

Genetic and biochemical evidence is presented which shows that the product of the vaccinia virus gene 18R is a virion protein. Western blot analysis of virion proteins using anti-18R serum detects a 78,000-Da protein, localized in the virus core. Of five ts mutants which map to gene 18R, two mutants, ts 10 and ts 44, possess thermolabile virions. Temperature shifts performed during single-step growth of ts 44 suggest that precursors required for virion maturation accumulate during nonpermissive infections with ORF 18R mutants and that protein synthesis is required for recovery from nonpermissive condition.