Conditional lethal mutants of vesicular stomatitis virus: I. Phenotypic characterization of single and double mutants exhibiting host restriction and temperature sensitivity

Abstract

Chemically mutagenized populations of vesicular stomatitis virus (VSV) were screened for two general classes of conditional lethal viruses. Host-restricted ( hr) clones that were unable to elicit productive infections in nonpermissive HeLa or HEp-2 cells were selected from small plaques formed on mixed indicator cultures of chick embryo fibroblasts and HeLa cells. Most of these hr mutants also displayed a temperature-sensitive ( ts) phenotype in permissive CEF cells. Evidence for the existence of spontaneous VSV hr mutants was obtained. The majority of the ts clones selected in CEF cells by conventional methods lacked the hr marker. Specific hr and ts mutants could be distinguished from wild-type virus on the basis of increased thermolability of their infectivity or diminished in vitro activity of their virion-associated transcriptase. Tests for the ability of these mutants to promote virus-specific RNA synthesis under nonpermissive conditions in cells treated with actinomycin D established the following relationships: (a) both “RNA +” and “RNA −” classes of mutants were found when infections were carried out in permissive cells incubated at temperatures restrictive for growth of ts phenotypes; (b) all clones possessing the hr marker failed to induce significant uridine incorporation (“RNA −”) in nonpermissive HEp-2 cells incubated at high or low temperatures; (c) all mutants scored as “RNA −” types when infections were carried out in nonpermissive HEp-2 cells at restrictive temperature irregardless of their phenotype for the hr and ts markers.