Isolation and preliminary characterization of temperature-sensitive mutants of poliovirus type 1

Abstract

Summary Following chemical mutagenesis, four temperature-sensitive mutants of the Mahoney strain of poliovirus type 1 were isolated. These mutants (clones 035, 203, 221 and 247) had a 4 × 102 to 4 × 103-fold lower plaquing efficiency on HeLa cells at 38.5° C than at 32.5° C. Standard growth curves showed that the viral mutants were restricted at 38.5° C but not at 37° C. The virions of clone 247 showed a greatly increased heat sensitivity: infectivity of the mutant stocks decreased by one log after a 1.5-min incubation at 45° C or after 15 min at 38.5° C. In contrast, equivalent heat inactivation of wild type virus or mutant ts 035 required about 6.5 min at 45° C and both viruses were completely stable for 30 min at 38.5° C. The virions of clones 203 and 221 showed partial heat sensitivity at 45° C but none at 38.5° C. Clones 035, 221 and 247 were unable to synthesize viral RNA at 38.5° C. Shift up of HeLa cells infected by any of these mutants from 32.5° C to 38.5° C, during the first 4 h of the virus growth cycle, resulted in prevention or arrest of viral RNA replication. Viral RNA synthesis continued unabated when the infected cells were shifted to non-permissive temperature at 6 h after infection. The RNA− phenotype at non-permissive temperature was therefore only manifest at early times after infection. Genetic recombination between the four mutants was investigated using both the conventional virus yield test and an infectious center assay. Recombinant plaques were found to occur with a significant frequency between ts 035 and ts 203, whereas no recombination was detected when ts 221 or ts 247 were one of the parents.