Poliovirus RNA Polymerase Mutation 3D-M394T Results in a Temperature-Sensitive Defect in RNA Synthesis

AbstractMutant ts10 is an RNA-negative temperature-sensitive mutant of Mahoney type 1 poliovirus. Mutant ts10 3Dpolwas purified from infected cells and was shown to be rapidly heat-inactivated at 45° when compared to wild-type polymerase. Sequencing of mutant ts10 genomic RNA revealed a U to C transition at nt 7167 resulting in an amino acid change of methionine 394 of 3Dpolto threonine. The 3D-M394T mutation was engineered into a wild-type infectious clone of poliovirus type 1. The resultant mutant virus, 3D-105, had a temperature-sensitive phenotype in plaque assays. The translation and replication of wild-type, ts10, and 3D-105 virion RNAs were all characterized in HeLa S10 translation-RNA replication reactionsin vitro.The optimum temperatures for the replication of the wild-type and mutant viral RNAs in the HeLa S10 translation-replication reactions were 37 and 34°, respectively. To characterize the temperature-sensitive defect in the replication of the mutant RNA, we used preinitiation RNA replication complexes which were formed in HeLa S10in vitroreactions containing guanidine HCl. Negative-strand RNA synthesis in 3D-M394T mutant preinitiation replication complexes was normal at 34° but was rapidly and irreversibly inhibited at 39.5°. To differentiate between the initiation and elongation steps in RNA replication, we compared the elongation rates in mutant and wild-type replication complexes at 39.5°. The results showed that the elongation rates for nascent negative strands in both the mutant and wild-type replication complexes were identical. Therefore, the results indicate that the heat-sensitive step in negative-strand synthesis exhibited by the 3D-M394T replication complexes is in the initiation of RNA synthesis and not in the elongation of nascent chains

Measurement of poliovirus RNA polymerase binding to poliovirion and nonviral RNAs using a filter-binding assay.

The binding of the purified poliovirus RNA-dependent RNA polymerase to viral and nonviral RNAs was studied using a protein-RNA nitrocellulose filter binding assay. A cellular poly(A)-binding protein was found in viral polymerase preparations, but was easily separated from the polymerase by chromatography on poly(A) Sepharose. Optimal conditions for the binding of purified polymerase (fraction 5-PAS) to 32P-labeled poliovirion RNA were determined. The binding of purified polymerase to 32P-labeled ribohomopolymeric RNAs was examined, and the order of binding observed was poly(G) much much greater than poly(U) greater than poly(C) greater than poly(A). In competitive binding studies, the polymerase bound with equal efficiency to virion RNA and to a subgenomic transcript which contained the 3' end of the genome. The polymerase bound to 18S ribosomal RNA and to globin mRNA equally well, but with a five-fold lower affinity than to virus-specific RNAs. The results suggest that the polymerase exhibits sequence specificity in binding and that polymerase binding sites in poliovirus RNA may contain (G- and/or U)-rich sequences