POLIOVIRUS RNA-POLYMERASE MUTATION 3D-M394T RESULTS IN A TEMPERATURE-SENSITIVE DEFECT IN RNA-SYNTHESIS

Mutant ts10 is an RNA-negative temperature-sensitive mutant of Mahoney type 1 poliovirus. Mutant ts10 3D(pol) was purified from infected cel ls and was shown to be rapidly heat-inactivated at 45 degrees when com pared to wild-type polymerase. Sequencing of mutant ts10 genomic RNA r evealed a U to C transition at nt 7167 resulting in an amino acid chan ge of methionine 394 of 3D(pol) to threonine. The 3D-M394T mutation wa s engineered into a wild-type infectious clone of poliovirus type 1. T he resultant mutant virus, 3D-105, had a temperature-sensitive phenoty pe in plaque assays. The translation and replication of wild-type, ts1 0, and 3D-105 virion RNAs were all characterized in HeLa S10 translati on-RNA replication reactions in vitro. The optimum temperatures for th e replication of the wild-type and mutant viral RNAs in the HeLa S10 t ranslation-replication reactions were 37 and 34 degrees, respectively. To characterize the temperature-sensitive defect in the replication o f the mutant RNA, we used preinitiation RNA replication complexes whic h were formed in HeLa S10 in vitro reactions containing guanidine HCI. Negative-strand RNA synthesis in 3D-M394T mutant preinitiation replic ation complexes was normal at 34 degrees but was rapidly and irreversi bly inhibited at 39.5 degrees. To differentiate between the initiation and elongation steps in RNA replication, we compared the elongation r ates in mutant and wild-type replication complexes at 39.5 degrees. Th e results showed that the elongation rates for nascent negative strand s in both the mutant and wild-type replication complexes were identica l. Therefore, the results indicate that the heat-sensitive step in neg ative-strand synthesis exhibited by the 3D-M394T replication complexes is in the initiation of RNA synthesis and not in the elongation of na scent chains. (C) 1995 Academic Press, Inc.