Biogenesis of poxviruses: Preliminary characterization of conditional lethal mutants of vaccinia virus defective in DNA synthesis

Characterization of six is mutants of the DNA^- phenotype of vaccinia 1HD-W is described. Complementation analysis revealed that four of these map into different complementation groups while one was able to complement, albeit inefficiently, with only one among the other ts mutants tested. Judging by the rate of viral DNA synthesis it became evident that all DNA^- mutants except ts 6389 became more "leaky" for DNA synthesis at the restrictive temperature in single-cycle infection if adsorption was carried out at 4° when compared with values obtained after initiation of infection at 39.5°. This suggested that these mutants are defective in some function required very early in the virus life cycle. This notion was further supported by the observation that multiple rounds of viral DNA synthesis could occur at 39.5° provided that the temperature was raised after initiating the infection at a lower temperature. Employing density-shift experiments coupled with analysis of the size of replicative intermediates by means of alkaline sucrose gradients, it was found that progeny DNA synthesized by all but ts 6389 was indistinguishable from wild-type DNA. By contrast, DNA synthesis in ts 6389-infected cytoplasm was is throughout the period of viral DNA replication, regardless of the time when temperature was shifted up, relegating this mutant to the "fast-stop" category. This idea is supported by evidence that all of the DNA^- isolates except ts 6389 are deficient at 39.5° in some of the virus-specified DNA-binding proteins. Therefore, ts 6389 is the only isolate at hand with the characteristics expected of a mutant with a defective protein related to the growing fork of the vaccinia DNA replication apparatus.