A point mutation in the poliovirus polymerase gene determines a complementable temperature-sensitive defect of RNA replication

Abstract

We have previously described the isolation of a RNA − temperature-sensitive (ts) mutant of poliovirus type 1, ts035, after chemical mutagenesis by 5-fluorouracil. The is defect of ts035 correlated with defective RNA replication, since the two characters corevert in the case of spontaneous revenants. The alteration of a trans-acting replication function of ts035 was suggested by significant rescue following mixed infection with another is mutant, ts221, or with wild-type virus. Protein synthesis appeared normal at 39° (nonpermissive temperature) in shift-up experiments and no defect of RNA elongation was evidenced when the activity of replication complexes or purified polymerase was measured at 39°. These results provide circumstantial evidence that the initiation of ts035 RNA synthesis at 39° is impaired. Molecular cloning of the ts035 genome allowed us to construct a recombinant virus with the same is phenotype as ts035, by the transfer of a fragment of the mutant polymerase gene into the wild-type genome. Two mutations were present in this region of the ts035 genome but the determination of nucleotide sequences in the case of ts035 revenants indicated that only the substitution from A to G at nucleotide 7256 was necessary for the is phenotype. This mutation replaces Asn 426 by an Asp in polypeptide 3D, the viral polymerase.