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

Abstract

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 oliovirion RNA were determined. The binding of purified polymerase to 32P-labeled ribohomopolymeric RNAs was examined, and the order of binding observed was poly(G) ⋙ >>> poly(U)> poly(C) > 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. © 1988 IRL Press Ltd.