Biochemical characterization of poliovirus type 1 temperature-sensitive mutants

Four temperature-sensitive mutants selected upon chemical mutagenesis of the poliovirus type 1 Mahoney strain [H. Agut, T. Matsukura, C. Bellocq, M. Dréano, J. C. Nicolas, and M. Girard, Ann. Virol. (Inst. Pasteur), 132E, 445-460 (1981)]. were analyzed by T1 oligonucleotide mapping. Three mutants (ts 203, ts 221, and ts 035) had T1 fingerprints identical to that of wild-type virus while mutant ts 247 exhibited two differences on oligonucleotides that mapped in the region of the genome coding for the replicase (polypeptide 4b) and for the protease (polypeptide 7c) of the virus. Cells were infected with each of the four mutants separately, labeled with [35S]methionine, and the labeled polypeptides were analyzed by SDS-polyacrylamide gel electrophoresis. Mutants and wild-type virus polypeptides showed a similar electrophoretic pattern except for the replicase and the protease of ts 247 which showed abnormal apparent molecular weights. The labeled proteins were subjected to two-dimensional isoelectrofocusing and SDS-polyacrylamide gel electrophoresis. Polypeptides 4b (replicase) and 2 (the common precursor to polypeptides 7c and 4b) of ts 247 and ts 035 exhibited distinct charge alterations when compared to the corresponding wild-type polypeptides. These alterations were also found on polypeptide 6a in the case of ts 247 and on polypeptide 6b in the case of ts 035, both polypeptides resulting from an alternate cleavage of polypeptide 2.